Week 2

Histological considerations of a skin graft rejection

Day 2‐4: Graft first appears pale, then becomes vascularized and appears pink as though graft is being accepted. However, recognition and differentiation phases are proceeding with no effect on the graft. Day 4‐7: Graft becomes cyanotic and eventually necrotic as destruction phase has commenced. Microscopic examination reveals perivascular infiltration by mononuclear cells and vascular thrombosis. Day 10‐12: Macroscopic signs of rejection become obvious with graft finally falling off. Attempted engraftment using same donor‐recipient combination would result in memory responses and accelerated acute rejection (as seen in third panel of figure above).

Anti-receptor diseases

Depending on its exact epitope specificity, bound receptor‐reactive antibody may act as either an agonist or an antagonist.

Which of the following immunodeficiency disorders is the result of a 22q11 gene deletion? X-linked agammaglobulinemia Wiskott-Aldrich syndrome Ataxia-telangiectasia DiGeorge syndrome Chronic granulomatous disease

DiGeorge syndrome

Syndromes of type II hypersensitivity

Drug‐induced hemolytic anemia Blood transfusion reactions Hemolytic Disease of the Newborn (HDN) Autoimmune diseases involving autoreactive antibodies

Maintenance Immunosuppression: Anti-proliferatives

*AZATHIOPRINE* *MoA* - 6-mercaptopurine (6-MP) prodrug - 6-MP is a purine analog; acts as anE-metabolite - Inhibits DNA synthesis --> Results in T cell depletion *Hepatically metabolized* a. Active metabolite: 6-TG (thioguanine) b. Inactive via - TPMT (thiopurine methyltransferase)- Genetic polymorphisms - Xanthine oxidase *Drug interactions* - Xanthine oxidase inhibitors (allopurinol, febuxostat) *Adverse effects* - Myelosuppression (Primarily leukopenia & thrombocytopenia; Dose-dependent; Dose-limiting) - Hepatotoxicity - N/V - Alopecia - Hepatotoxicity - Hypersensitivity - Pancreatitis *MYCOPHENOLATE* - formulations not interchangeable *MoA* - Prodrug of mycophenolic acid (MPA) - MPA inhibits inosine monophosphate dehydrogenase: Rate limiting step in de-novo purine synthesis *Drug interactions* - Nausea, vomiting, diarrhea (similar with enteric coated product) - Myelosuppression *Adverse effects* (see table) - Most due to reductions in intesEnal absorpEon - Proton pump inhibitor & antacids (aluminum, magnesium, calcium) --> Decrease absorption *Preferred v. AZATHIOPRINE because MMF has more specific T-cell inhibition*

Therapy for severe eosinophilic asthma

*Anti-IgE- Omalizumab* a. MoA: recombinant (IgG) anti-IgE antibody - Binds circulating IgE - Prevents binding to mast cells and basophils decreases release of inflammatory mediators b. For ≥ 12 years of age with severe, persistent allergic asthma that is not well controlled by corticosteroids c. Dosage/administration - Subcutaneous every 2 to 4 weeks - Dose based on serum IgE level and weight (performed at clinic) d. Adverse effects: Anaphylaxis (0.1% incidence); up to 24 hour post injection (70% within 2 hours) *IL-5 Receptor Antagonists- Mepolizumab and Reslizumab* a. MoA: bind to and neutralize IL-5, thus blocking eosinophil signal b. Administration: Subcutaneous (M): IV (R) c. Adverse effects - Mepolizumab: Injection site reaction; headache - Reslizumab: Increased creatine phosphokinase; myalgia

Biological chemotherapy: Target-specific inflammatory markers

*Antibody therapy* a. Cancer adjunctives - ID cancerous cells as foreign - Stimulate immune system to attack b. Autoimmune diseases - Decrease immune response

Maintenance Immunosuppression: Calcineurin Inhibitors

*Calcineurin Inhibitors (CNI)* NOTE: formulations not interchangeable *MoA* - bind cytoplasmic proteins (cyclophilin and FK-binding protein, Cyclosporine and Tacrolimus, respectively) and inhibit calcineurin phosphate (1) downregulation of IL-2 gene transcription (2) downregulation of T-cell activation *Metabolized via CYP3A4 and P-Glycoprotein* *CYP3A4/PGP inhibitors (increase CNI levels)* • Statins • Macrolide antibiotics • Azole antifungals • Calcium channel blockers • Protease inhibitors • Grapefruit juice *CYP3A4/PGP inducers (decrease CNI levels)* • Rifampin • Carbamazepine, phenobarbital, phenytoin • Efavirenz • St. John's wort *Synergistic nephrotoxicity* - NSAIDs - Aminoglycosides - Amphotericin-B - Acyclovir, ganciclovir *Trough concentration* - Correlated to efficacy & toxicity: Individualize target - Transplanted organ - Patient condition - Time since transplant *Adverse effects* (See table)

B Cell and Antibody Deficiencies: 1) Clinical Presentation, 2) B Cell Maturation, 3) B-cell Receptor Signaling, 4) X-Linked Agammaglobulinemia (XLA)

*Clinical Presentation* - Recurrent bacterial sinopulmonary infections (sinusitis, bronchitis, pneumonia, ear infections). The most common bacteria include Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis - Development of chronic lung disease because of recurrent infections and inflammation - Recurrent or persistent GI infections with protozoa, e.g., Giardia lamblia and Cryptosporidium *B Cell Maturation* - Pro-/Pre-BI: CD117 - Large/Small Pre-BII: CD25 - Immature/Transitional B: CD93, IgM - Constant markers: CD19 *BCR signaling* - Signaling domains of CD45, Igα, Igβ --> Fyn, Syk --> BTK/PLCy2/BLNK --> PIP2 (three outcomes) (1) PIP2 --> IP3 --> Calmodulin/Calcineurin --> Nuclear factor of activated T-cells (NFAT) (2) PIP2 --> DAG --> PKCβ --> CARMA1/MALT/BCL-10 --> IKK --> Iκβ/NFκβ (3) PIP2 --> DAG and PKCβ --> RAS-GRP --> RAS --> RAF --> MEK --> ERK *X-Linked Agammaglobulinemia (XLA)* - Bruton's Tyrosine Kinase (BTK) deficiency - B cell development blocked at Pro-B to Pre-B transition due to absence of BTK protein kinase - B cells are very low or absent in the circulation as a result of the developmental block

Deficiencies in cell-mediated immunity

*DiGeorge Syndrome* - Associated with congenital thymic aplasia due to grossly abnormal embryonic development during about the 12th week of gestation at which time the thymus, the parathyroid glands, the aortic arch of the heart, and other tissues of the upper chest cavity are differentiating and migrating into correct position. The developmental defect involves the 3rd and 4th pharyngeal pouches of the embryo, likely due to absence of Tbx-1 transcription factor. - *Without a thymus, mature T cells cannot be generated*, although pre-T cells are present (administration of T cell growth factors can induce their maturation which may be therapeutic). B cells are present but demonstrate variable function due to improper regulation in the absence of functional T cells. - *Hypocalcaemia*, probably due to *parathyroid impairment*, causes influxes of sodium into muscle and dysregulated muscle action potential. This together with increased phosphorus results in multiple action potentials sent to muscle and causing spasms (tetany). Tetanic episodes can be treated with intravenous calcium gluconate together with a low phosphorus diet, calcium supplements, and high doses of vitamin D (important for calcium absorption). - Proper development of upper chest tissues may also affect tissues the face, creating a striking set of facial features that are very useful for diagnosis. How do you think this disorder would affect antibody immunity? *Chronic Mucocutaneous Candidiasis* - Selective dysfunction of cell-mediated immunity (evidence pointing to defective TH17 cells), and is associated with Candida (yeast) infections. Humoral immunity appears normal. *Acquired Immunodeficiency Syndrome (AIDS)* - NOT a congenital disorder, but instead is transmitted by an infectious virus, HUMAN IMMUNODEFICIENCY VIRUS (HIV). Becoming HIV-infected is the first step in a multi-step process that leads to AIDS (in other words, being HIV positive does not mean the same as having AIDS). Asymptomatic HIV+ individuals can transmit infectious virus.

Diagnosis of B cell or Ab Deficiency, Ab Levels by Age, and Ig Replacement Therapy for Ab Deficiency

*Diagnosis* 1. Quantify B cells and Ab - CBC/Differential - Quantify T/B/NK cells by flow cytometry - Quantify IgG, IgM, IgA in serum 2. Evaluate B cell and Ab function - Measure Ab titers to vaccine (protein and carbohydrate vaccine) *Ab Levels Vary by Age in Childhood* - Maternal IgG actively transported across placenta during pregnancy - Physiologic "nadir" of IgG at 2-3 months of age - Normal "adult level" of IgG reached ~ 5 years old *Ig Replacement Therapy for Antibody Deficiency* - Aka "Gamma globulin shots" - Enriched IgG from 1000s of donors -- plasma centers, blood donors, etc. - Can be given IV (IVIG), subcutaneously (SCIG), or intramuscularly (IMIG)

1) The Classical Pathway (Early v. Late Pathways) and 2) their Effects

*Early Pathway Defects* - C1-C4 - present in one of two ways: (1) Invasive infections (sepsis, pneumonia, meningitis) with encapsulated organisms (Strep. pneumoniae, Haemophilus influenzae, Neisseria meningitidis, etc.) (2) Lupus or glomerulonephritis (autoimmune kidney disease that occurs in Lupus) *Late Pathway Defects* - C5-C9 (MAC) - present almost exclusively with invasive infections (sepsis, meningitis, etc.) with Neisseria species - Late pathway defects increase the risk of *meningococcal disease approximately 8000-fold* - Typically, the first Neisserial infections occurs in the 2nd decade of life

Humoral and Cellular Allograft Rejection

*Humoral rejection* a. Hyperacute- pre-existing Abs b. Acute- Ab formed against donor organ after transplant c. Chronic- Anti-donor antibodies d. Implicated cells and factors: - B cells - Abs - Complement - Platelets - Coagulation factors *Cellular rejection* a. Acute- T cell primary means for acute cellular rejection b. Implicated cells and factors - T cells- CD4+ Th2 and CD8+ CTLs - APCs- macrophages, dendritic cells, plasma cells - Cytokines - Adhesion molecules - NK cells

Mechanisms responsible for T cell autoimmunity

*IMMUNOLOGICAL TOLERANCE* to self: The goal of positive and negative selection in the thymus is to create a population of T cells that can respond to all foreign antigens, on the one hand, and are unable to respond to self-antigens, on the other (i.e., immunologically tolerant to self). This is referred to as CENTRAL TOLERANCE. However, it is not a perfect mechanism, allowing some self‐reactive T cells to escape. Additional mechanisms involve the induction and maintenance of PERIPHERAL TOLERANCE. Two such mechanisms include induction of ANERGY and induction of SUPPRESSIVE REGULATORY T CELLS (TREG CELLS). *Regarding Central Tolerance of T cells*: ‐ Central T cell tolerance is produced by one primary mechanism: *Negative selection* (see T cell lecture) ‐ In the absence of infection or immunization with foreign antigens, MHC binding sites are filled with various peptides derived from *host proteins*. These self‐antigen peptides are presented as *stable complexes* with Class I or II MHC presenting molecules to immature autoreactive T cells as they pass through the thymus. Thymic T cells that attempt to react against self-peptides are triggered into apoptosis, perhaps due to receiving Signal 1 in the absence of an effective co‐stimulatory Signal 2. This is sometimes referred to as "activation‐induced cell death". - *Autoimmune Regulator (AIRE)*: A transcriptional activator that allows epithelial cells in the thymus to express numerous proteins that are usually restricted to expression by particular tissues out in the body. This allows a broader effect of negative selection that goes beyond only those proteins produced by, or brought into the thymus by, dendritic cells. ‐ *Effectiveness of negative selection during early T cell development in the thymus (i.e., tolerogenesis)* is dependent on the same qualities that allow foreign peptides to activate mature T cells in the periphery, including having the correct antigen peptide anchor residues in relation to polymorphic contact residues in MHC molecules. ‐ There are at least two opportunities for things to go wrong with Central Tolerance: (1) *Certain self‐peptides may not be available in the thymus for negative selection* The *spectrum of self‐peptides* available in the thymus is not broad enough to guarantee that all T cell reactivities against self are eliminated. Negative selection of a T cell reactivity for a particular self‐peptide cannot occur in the absence of that peptide. (2) *MHC may not present certain self‐peptides effectively for negative selection* - Depending on exactly *which MHC alleles* that an individual carries (which encode MHC proteins with particular polymorphic peptide contact points and therefore the ability to bind particular peptides with certain anchor residues), different individuals may demonstrate differences in the *ability to bind a particular self‐peptide* and a subsequently different degree of immune tolerance (non‐reactivity) for that self‐antigen. - *RELATIVE RISK*: There is an increased *probability* for particular autoimmune diseases in certain individuals carrying certain MHC alleles. Relative Risk is a numerical value that population geneticists assign to relationships between the risk of getting a disease and carrying a certain gene (see examples at the end of this handout). *Risk is not the same as certainty*; that is, since many diseases are caused by defective interactions of *multiple gene products*, carrying one particular gene, such as for an MHC molecule, may increase the risk, but doesn't guarantee onset of the disease. The risk for getting certain autoimmune diseases increases when individuals carry particular MHC alleles (different alleles are related to different diseases). *Regarding Peripheral Tolerance of T cells*: - Peripheral T cell tolerance, required to regulate the activities of self‐reactive T cells that escape central tolerance in the thymus, is maintained by a variety of mechanisms: ‐ *IMMUNOLOGICALLY PRIVILEGED SITES*: Autoimmune T cell responses are limited by sequestering, during early embryological development before the immune system has developed, some self‐antigens in immunologically privileged sites (e.g., CNS). However, injury may disturb tissue boundaries, thereby releasing sequestered antigens. Having never "seen" these self‐antigens before, the immune system perceives them as foreign. ‐ *RESTRICTED DISTRIBUTION OF CLASS II MHC*: Autoimmune TH1, TH17, and TH2 responses are limited by the restricted tissue/cell distribution of Class II MHC (i.e., on professional APCs only). Inappropriate expression of Class II MHC will increase the probability of autoimmune T cell responsiveness. ‐ *ANERGY DEPENDENT ON RESTRICTED DISTRIBUTION OF CO‐STIMULATORY MOLECUES*: Autoimmune T cells that escape the thymus may encounter a variety of host tissues that express MHC complexed to self‐peptides (particularly Class I MHC, since it is distributed in the body on virtually all cells/tissues). However, most cells, other than professional antigen presenting cells (macrophages, dendritic cells, B cells), do not have the ability to produce co‐ stimulatory molecules such as CD80/86 (B7). The result of providing Signal 1 to a T cell in the absence of Signal 2 is either apoptosis, or a state of subsequent unresponsiveness called anergy. Inappropriate expression of co‐stimulatory molecules will increase the probability of autoimmune T cell responsiveness. ‐ *REGULATORY (SUPPRESSIVE) T CELLS (TREG)*: A small subset (1‐3%) of CD4+ CD25high T cells helps regulate the delicate balance of cytokines that can steer inflammatory and autoimmune responses away from potentially harmful effector activities. It is known that TReg cells require: 1) direct contact with their target T cells, and that they 2) constitutively express a co‐ stimulatory molecule other than CD28 known as CTLA‐4 that can compete for the CD80/86 (B7) on the APC. The exact mechanisms that allow TReg cells to recognize autoreactive T cells is unclear at this time, but probably provide a major strategy for preserving immunological self‐ tolerance. Example of shutting down potentially pathological autoimmune TH1 cells: (see image) *EPITOPE SPREADING* - *CRYPTIC EPITOPES are weakly immunogenic* regions of a protein due to inefficient presentation to T cells. This inefficiency may be due to how the protein is processed (cut) or how the resulting peptides bind MHC. Although these epitopes may not initially activate T cells, repeated (albeit inefficient) presentation may allow these epitopes to eventually become prominent later in the response... i.e., epitope spreading.

Immunodeficiency Diseases

*Immunodeficiency Diseases* are the result of missing or impaired components of the immune system. These diseases are frequently associated with chronic or recurrent infections; infections may involve microbial pathogens that are common to the surrounding environment, or the normal microbiota of the patient (referred to as "opportunistic" pathogens), and are rarely pathogenic in immunologically healthy individuals. Characteristics associated with, but not diagnostic of, immunodeficiency include recurrent skin rashes or abscesses, chronic diarrhea, abnormal growth, hepatosplenomegaly, thrombocytopenia, or endocrinopathies. During your studies, you must learn to identify relationships between normal immune functions and key immune defects that lead to immunodeficiency diseases; understanding these relationships will allow extrapolation to an understanding of other similar clinical scenarios. There are a variety of developmental levels/compartments of the immune system in which immunodeficiency can reside. It is important to note that defects in one compartment of the immune system may have dramatic effects on functions in another compartment (for example, defective complement components will likely affect phagocytic cell function, or defects in the T cell compartment may affect B cell function): - Complement abnormalities that can affect several or individual complement components make up ∼2-4% of immunodeficiency syndromes. - Phagocytic cell dysfunctions represent ∼15% of immunodeficiency syndromes. *Leukocyte Adhesion Deficiency* Defective integrin (e.g., *CD18* ... a subunit of LFA-1) results in inability of leukocytes to move from the blood stream to the site of infection. Individuals with LAD suffer from bacterial infections beginning at birth. Infections such as omphalitis, pneumonia, gingivitis, abscesses, and peritonitis are common and often life-threatening. *Chronic Granulomatous Disease* Relatively rare genetic mutations affecting the activity of NADPH resulting in *defective superoxide production*; decreased ability of phagocytes to destroy ingested *bacteria and fungi*. *Downstream Infections* Infections may be local or disseminate to numerous organ systems (most frequently skin, lungs, GI tract, lymph nodes, liver, and spleen). Infections are frequently associated with abscess formation, often caused by Staphylococcus aureus; abscesses are prominently associated with the skin and lungs, although hepatic, splenic, and perianal/perirectal abscesses also occur. Granulomas may develop. A common laboratory test is based on the inability of *affected neutrophils to reduce nitroblue tetrazolium dye (NBT)*. For those who have access to the necessary equipment, another more quantifiable and therefore preferred test requires flow cytometry for analysis. In this test, Dihydrorhodamine (DHR) that is reduced by NADPH in phagocytes is converted to the highly fluorescent species, rhodamine. *Granulocyte/Macrophage Colony Stimulating Factor (GM-CSF)* is a cytokine that functions as a white blood cell growth factor. GM-CSF stimulates stem cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes. Monocytes mature into macrophages. Therapies for a variety of diseases caused by deficits in these cell types are based on the administration of GM-CSF. *Lymphocyte deficiencies* Generalized and selective B cell (antibody) immunodeficiency diseases (∼50%)* AND *Generalized and selective T cell immunodeficiency diseases (∼30-40%)* - T cell defects resulting in effects on cell-mediated immunity, or can have indirect effects via requirements for T cell-dependent regulatory activities - Immunodeficiencies can also be caused by or become secondary to other immune dysfunctions - Examples: acquired as a result of lymphotrophic infection ( e.g., AIDS); neoplasia (e.g., MYELOMA, LYMPHOMA); autoimmunity

Effectors v. Regulatory Defects: Outcomes of immunodeficiency and immune dysregulation.

*Immunodeficiency* - Recurrent infections - Severe infections - Frequent need for antibiotics - Infectious organisms can be rare or common - Autoimmunity less common - Onset can be early or late *Immune Dysregulation* - Recurrent or severe autoimmunity - Any organ can be affected - Infections less common - Onset can be early or late

Transplant Pharmacology: Induction & Rescue Therapy

*Immunosuppresion* a. Induction therapy - For high-risk patients; have to consider risk v. benefit in others - Used to prevent acute rejection (improved 1-year outcomes) - Peri-transplant initiated intra-operatively - Intense, potent b. Rescue - Treatment for acute rejection - Short course c. Maintenance - All transplant recipients need maintenance - Long-term combination therapy - Used to prevent acute rejection - Patient-specific follow-up *Induction of immunosuppresion* a. Lymphocyte depleting (Anti-thymocyte globulins) - used for induction or rescue - lymphopenia may persist for > 1 year b. Non-lymphocyte depleting - Basiliximab *Induction therapy* a. Alemtuzumab: Humanized monoclonal antibody - MoA: Binds CD52 on T and B cells --> (1) Causes Ab-dependent lysis; (2) Rapid lymphocyte depletion - Adverse effects: (1) Flu-like effects- nausea, vomiting, diarrhea, headache; (2) Myelosuppression- infection, malignancy b. Basiliximab: Chimeric monoclonal antibody - MoA: IL-2 receptor antagonist --> Inhibits IL-2 mediated T cell activation - Adverse effects: similar to placebo - Used for induction therapy only

Interferons: Enhance immune system to fight infection/disease

*Interferons (IFN)* - Cytokines produced by infected cells- interferes with pathogen reproduction - Anti-proliferative - Immune-regulating - Activate NK and macrophages *Type I IFN*- produced by virus-infected cells 1. Type I IFN (α and β) - Different affinity - Similar biological effects - Induce resistance to viral replication in all cells - Activate NK cells to attack virus-infected cells - Stimulated IFNγ (Type II IFN) 2. Type II IFN a. Produced by T cells, NK cells b. Type II IFN (γ) - Potent macrophage activator - Immunoregulatory > anti-tumor > antiviral 3. IFN α a. PEGylating (protein protection) used to extend protein life (and effect) b. Different drugs can treat - chronic hepatitis B, C, D - chronic myeloid leukemia - renal cell carcinoma - malignant melanoma 4. IFN β - Precise mechanism unknown - Alters expression and response to surface Ag and enhances immune cell activity - Indications: Multiple sclerosis 5. IFN γ a. MoA: immunoregulation - Enhances oxidative metabolism of macrophages - Increased Ab-dependent cytotoxicity - Activates NK cells - Changes expression of FcR and MHC b. Indications: - Chronic granulomatous disease - Osteoporosis *Common IFN adverse effects* a. Acute injection reaction - Flu-like sx - Pre-medicate with antipyretics b. Myelosuppression - Neutropenia - Anemia - thrombocytopenia c. Fatigue/lethargy d. Neurotoxicity - Depression - Somnolence - Change in behavior - Seizures (rare)

Myasthenia Gravis

*Mechanism*: Antibody that acts as an acetylcholine receptor antagonist; inhibition of neuromuscular transmission due to impaired/destroyed receptor. *Presentation*: Weakness and rapid onset fatigue of skeletal muscles, particularly during repetitive activity; any skeletal muscles may be affected, but ocular muscles are often observed at very early stages, resulting in double vision (diplopia), and drooping eyelids (ptosis) that is often symmetrical. Other early signs resulting from impaired muscle function include slurred speech and difficulty in swallowing. Large limb and trunk muscles may then become affected; myasthenic crisis is indicated when respiratory function is affected.

Grave's Disease

*Mechanism*: Autoantibody that acts as a Thyroid Stimulating Hormone (TSH) receptor agonist; Thyroid hormones (triiodothyronine [T3] and thyroxine [T4]) are responsible for regulation of metabolism, and pituitary‐derived thyroid stimulating hormone (TSH) regulates the production of T3 and T4. Agonist TSHR antibody binding results in a hyper‐metabolic state by stimulating abnormally high levels of thyroid hormones. *Presentation*: Enlarged non-tender thyroid; Hyperthyroidism and metabolic imbalance may result in tachycardia, hypertension, diarrhea, and muscle weakness. Patients often experience insomnia, nervousness, irritability, tremors, excessive sweating, heat intolerance, and weight loss.

Multiple Sclerosis

*Mechanism*: Demyelinating disease of the CNS; Inductive event unknown; Mixed and complex immune effectors; Antibody and cell‐mediated responses probably involving Type II and Type IV hypersensitivities. Exact antigenic target unclear; Both myelin and myelin‐ producing cells (oligodendrocyte) are damaged. Presence of oligoclonal antibodies found in CSF is common although significance is unclear. *Presentation*: Many neurological symptoms are possible including: fatigue, numbness (hypoesthesia), tingling, muscle weakness or spasms, difficulties with coordination and balance (ataxia); problems with speech (dysarthria), swallowing (dysphagia), and bladder/bowel control. Optic neuritis is common. Cognitive and emotional symptoms of depression or unstable mood are also common.

Insulin-dependent (type 1) diabetes mellitus

*Mechanism*: TH1 and CD8 autoreactive T cells and antibody with reactivity against insulin‐ producing pancreatic beta cells (and sometimes insulin); the result is diminished insulin production and persistent hyperglycemia. *Presentation*: Typical presentation includes fatigue, increased thirst, polyuria, and weight loss.

Maintenance Immunosuppression: Co-stimulatory inhibitor

*MoA* - Binds CD80 & CD86 (B7) on APC - Blocks co-stimulation of CD28 on inactive T cells (signal 2) *Uses* - Used with basiliximab, MMF, corticosteroids in place of CNI • May preserve renal function by avoiding CNI • Improved blood pressure & lipids • Improve renal funcEon • Increased acute rejection *Adverse effects* - Most associated to infecEon - GI - Headache - Electrolytes (Hypokalemia) - CNS

Maintenance Immunosuppression: mTOR inhibitors

*MoA* - Binds mammalian target of rapamycin (mTOR) - suppress cellular response to IL-2 and other cytokines - Sirolimus has LONG half life - Everolimus, a sirolimus derivative, has a SHORTER half life *Drug interactions* - CYP3A4 substrate and inhibitor *Uses* - May be used with CNIs to replace AZA or MFF - Alternative if cannot tolerate CNI *Adverse effects* - Myelosuppression (Leukopenia, Thrombocytopenia) - Hyperlipidemia - Impaired wound healing

Maintenance Immunosuppression: Corticosteroids

*MoA* - Exact immunosuppressive action NOT defined - Suppress cell-mediated immunity - ↓ cytokines, interleukins, TNF γ - Inhibit leukocyte access to inflammatory site - High dose may be directly lymphotoxic *Most common* - Methylprednisolone - Prednisone *Adverse effects* (see attached)

NADPH Oxidase Complex and Chronic Granulomatous Disease (CGD), Diagnosis,

*NADPH Oxidase Complex* - gp91 and p22 subunits - present in the membrane - other subunits in cytoplasm - assemble with gp91/p22 when cells are activated by pathogens, etc. to generate active NADPH oxidase complex - makes bleach *Chronic Granulomatous Disease (CGD)* - Caused by defects in components of NADPH oxidase complex - gp91 mutations (X-linked CGD) ~ 2/3 of patients - p22, p47, and p67 (Autosomal Recessive CGD) - Cells do everything normally EXCEPT they cannot degrade what they have ingested - Thus, patients have skin and soft tissue infections or lymphadenitis with catalase(+) organisms (Staphylococcus, Aspergillus, Burkholderia, cepacia, etc.) *Diagnosis of CGD* - Measurement of neutrophil oxidative burst using dihydrorhodamine 123 (DHR) dye in flow cytometry

Neutropenia, Diagnosis

*Neutropenia* - Low PMN count - Congenital or acquired - Acquired (most common) due to chemotherapy, drug-induced, autoimmune, etc. - Symptoms: infections/sepsis, fever, mouth sores, peri-rectal pain and abscesses, skin infections, swelling/inflammation of the gums, etc. *Diagnosis* - CBC with a differential - Neutropenia: < 1500 neutrophils/µL - Severe neutropenia: < 500 neutrophils/µL

Enhance immune therapy (passive immunity): Immunoglobulin Therapy

*Passive immunity* - Temporary protection - General protection: immune globulin (IVIg)- IgG and trace IgA, IgM - Multiple mechanisms of action: provide passive immunity by increasing Ab titer *IVIg* - Replacement therapy: primary and secondary immunodeficiencies; cytopenias - Immunomodulatory effect: bacterial/viral infections; multiple MoA, e.g., (1) modulates complement activation and suppresses inflammation, (2) blocks macrophage FcR *Targeted protection (specific)* - Ig from vaccinated human donors - High Ab titers to specific Ag - NOT a vaccine *Toxin exposure (specific)* - Rabies immune globulin: Provides protection until active immunity from vaccination takes effect - Tetanus immune globulin: Wound management- unvaccinated persons or those with unknown vaccination status - Botulism immune globulin: Infant botulism- reduces average hospitalization from 5.5 weeks to 2.5 weeks - Antivenin (antivenom): From human or equine Ig *Viral exposure (specific)* - Hep A, B Ig - post-exposure PPX - Varicella-zoster Ig: Post-exposure PPX for high risk groups, e.g., (1) Immunocompromised; (2) Pregnant women- Reduced severity of maternal infection in pregnant women *Rh- mother; Rh+ fetus (specific)* - Rho(D) immune globulin- prevent active Ab formation - Antepartum in Rh- mother - Post-partum if Rh+ birth - Low risk to later pregnancy

Phagocyte Activation & Migration, 4-step Model of Leukocyte Extravasation, and Leukocyte Adhesion Deficiency (LAD),

*Phagocyte Activation & Migration* 1. Bacteria 2. Chemokines/Inflammatory mediators in tissue 3. Leukocytes in blood vessels start rolling against endothelial lining 4. Extravasation 5. Leukocyte phagocytosis bacteria, processes it 6. Leukocyte travels to lymph node *4-step Model of Leukocyte Extravasation* 1. Tethering/Rolling (L-, E-, P-Selectin, Ligands) 2. Integrin Activation (Chemokines) 3. Form Adhesion (Integrins, CAMs) 4. Transmigration (Chemokines and other chemoattractants) Note: *CD11/CD18 = Complement Receptor 3 (CR3)* *Leukocyte Adhesion Deficiency (LAD)* - Different types caused by genetic defects that affect either the "Tethering/Rolling" step or the "Firm Adhesion" step of leukocyte extravasation - All affect the ability of phagocytes to traffic into tissues normally - As a result: (1) Circulating PMNs high (2) Patients get recurrent skin and soft tissue infections (abscesses and boils BUT NO PUS, because no neutrophils in tissue)

Transplant Infections

*Pre-transplant screening* - Donor - Recipient *Vaccinations* - Avoid live vaccines post-transplant

1) Primary (Congenital) Immunodeficiencies, 2) Secondary (Acquired) Immunodeficiencies, 3)

*Primary (Congenital) Immunodeficiencies* - Often caused by mutations in specific genes - Often there is a family history of immunodeficiency - Clinical symptoms often begin early in life but can begin later, even in adulthood - Can affect the function of *either the effector or regulatory arms* of the immune system *Secondary (Acquired) Immunodeficiencies* - Not caused by a genetic defect - Occurs secondary to something else, e.g., (a) T cell deficiency secondary to HIB (b) Neutropenia secondary to certain drugs (c) B cell deficiency after Rituximab - May resolve if underlying problem is treated

1) Purposes of Vaccines, 2) Terminology (Vaccine Antigen, Adjuvant, Vaccine Titer), and 3) 5 Different Types of Vaccines

*Purposes of Vaccines* - Generate protective immunity response against an organism that can cause severe illness, disability, or death by administering a safe vaccine - By preventing life-threatening infectious diseases, vaccination is estimated to avert 2-3 million deaths/year worldwide *Terminology* - Vaccine antigen: the molecule (protein or carbohydrate) in the vaccine that an antibody response should be generated against - Adjuvant: substance that non-specifically activates the immune system -- added to vaccines to enhance immune response - Vaccine titer: a measure of the amount of Ab made to a vaccine antigen *5 Different Types of Vaccines* 1. *Inactive/Killed* - Polio (IPV), Hepatitis A, Rabies - Made by inactivating the whole pathogen by heat/chemicals (e.g., formaldehyde) - Relatively easy to generate -- can make a new formulation of a vaccine relatively quick. Safe -- no risk of virus re-activating 2. *Toxoid (inactive toxin)* - Tetanus, Diphtheria - Some bacterial disease is not caused directly by the bacterium but by a bacterial toxin (e.g., tetanospasmin, neurotoxin made by C. tetani) - Toxoid vaccines are made from toxin that has been inactivated with heat or chemical treatment (formalin) - Typically requires intermittent boosters to maintain a protective Ab level (~ every 10 years for the Td-tetanus/diphtheria booster) 3. *Subunit* - Hep B, HPV, Pertussis, Influenza (shot), Pneumococcal polysaccharide, Meningococcal polysaccharide - Made by isolating or making a specific component of a pathogen that can be given as a vaccine - isolated antigen + adjuvant --> increase response to vaccine - Hepatitis B: Hep B vaccine is a portion of the surface antigen (HBsAg) expressed recombinantly in yeast - Pneumococcal Polysaccharide: Vaccine is mixed polysaccharides isolated from the outer capsule of 23 different Streptococcus pneumoniae serotypes 4. *Conjugate* - Haemophilus influenzae (HiB), pneumococcal conjugate, meningococcal conjugate - Vaccines intended to prevent infections with encapsulated organisms that are thickly coated in polysaccharide - But some patients (infants) don't make good immune responses to purified carbohydrates. Can be enhanced by conjugating carbohydrates to protein (Diphtheria toxoid used most commonly) 5. *Live, attenuated* - Measles, Mumps, Rubella (MMR), Varicella (Chickenpox), Rotavirus, Influenza (nasal spray), Polio (oral-OPV), yellow fever, Bacille Calmette Guerin (BCG) - Vaccine consists of live, attenuated organisms that are either injected, ingested, or sprayed into nose - Pathogens are attenuated by growing in cells from other animals (fertilized check embryos). Over time, the organism adapts to grow better in those cells and becomes worse at growing/dividing in human cells. Still recognized by human immune system - If immune system is compromised, attenuated pathogen can become pathogenic - Over time, attenuated pathogen growing in human can revert back to wild-type, fully pathogenic form (e.g., Oral polio vaccine, OPV, replicates in gut mucosa, reverts to virulent strain within weeks of vx) - Bacille Calmette Guerin (BCG): Live, attenuated Mycobacterium bovis (bovine Tuberculosis) is injected to immunize against TB. Variable effect. Not used in the U.S. Common elsewhere. Causes disseminated TB-like disease in px with compromised immune system

Three phases involved in allograft rejection - Example; Kidney rejection

*RECOGNITION PHASE* Following transplantation, graft antigens shed from the graft are taken up, processed, and displayed on the surface of ANTIGEN PRESENTING CELLS (APC) such as dendritic cells. Antigen‐reactive lymphocytes, primarily in draining lymph nodes, become activated. Alternatively, circulating lymphoid cells are exposed to the graft directly as it becomes vascularized. Early immune recognition of the graft is primarily by CD4+ CELLS. *PROLIFERATION AND DIFFERENTIATION PHASE* Following contact with antigen, CD4+ T cells and/or macrophages in regional draining lymph nodes mediate the induction of proliferation and differentiation of effector cell precursors (T and/or B cells). Effector T cells leave the lymph node and reach the graft via the blood. *DESTRUCTION PHASE* Activated CYTOTOXIC CD8+ T LYMPHOCYTES (CTL) are the primary destructive force in the rejection process, although CD4+ T cells and B cells/antibody also play a role. CTL are capable of directly lysing engrafted cells as well as of releasing various toxic and chemotactic factors.

1) Response to T-cell Independent (Carbohydrate) Antigens and 2) Carbohydrate Vaccine Problems

*Response to T-cell Independent (Carbohydrate) Antigens * - Carbohydrate reaches marginal zone of spleen/lymph nodes through blood/lymph. Directly cross-links BCRs on MZ B cells 1. Repetitive structure of carbohydrates binds and cross-links BCRs on surface of MZ B cells 2. Cross-linking of BCRs activates the B cells 3. During the week after immunizations, activated MZ B cells directly undergo class-switching to IgG and IgA and differentiate into plasma cells without T cell help 4. After extrafollicular differentiation, polysaccharide-specific plasma cells migrate to the red pulp in the spleen where they persist secreting antibody for some time 5. After a few months, the polysaccharide-specific cells die by apoptosis and the antibody response wanes 6. Bona fide memory B cells are not generated because there is NO germinal center reaction *Carbohydrate Vaccine Problems* - Children under the age of 2 are unable to reliably mount responses to carbohydrate antigens. Can be enhanced by conjugating carbohydrate to a carrier protein, e.g., Diphtheria toxoid

Therapy to LOWER hyper-activated immune system, i.e., autoimmune disease

*Rheumatoid arthritis (RA)* o Glucocorticoids o DMARDs o Biologicals *Inflammatory bowel disease (CD) - ulcerative colitis (UC) and Crohn's disease (CD)* *Systemic lupus erythematosus (SLE)* o Glucocorticoids o Antibody therapy *Multiple sclerosis* o Glucocorticoids o Interferon therapy *Guillian-Barre Syndrome*: acute immune-mediated polyneuropathy o IVIg o Plasma exchange *Neoplasia*: use antineoplastic o Methotrexate- antifolate o Cyclophosphamide- immunosuppressant and alkylating agent *T-cell co-stimulation blocker (abatacept)* *TNF blocker (etanercept, adalimumab infliximab)* *Same drugs, different effects* a. Multiple sclerosis - Type 1 IFN effective - Anti-CD20 effective - TNF blockade ineffective b. Rheumatoid arthritis - TNF blockade effective - Anti-CD20 effective - Type 1 IFN ineffective

T Cell and Combined Defects: 1) Severe Combined Immune Deficiency (SCID), 2) Diagnosis, 3) Screening

*SCID* - Inability to generate effective Ab responses (CD4+ help problem) - Severe viral infections (CMV, EBV, Adenovirus, etc.) - (CD8+ CTL problem) - Severe fungal infections (Pneumocystis jirovecii pneumonia) - (CD8+ CTL problem) - Autoimmunity (Treg problem) *Causes of SCID* - 21 genetic defects - All lack T cells: most lack all T cells but some lack only specific subsets - SCID px may also lack B or NK cells - Phenotype of SCID = defined by whether T, B, and NK cells present/absent -- depends on whether mutant signaling molecule is required for cell development *Diagnosis* 1. Quantify T cells - CBC/Differential - Quantify T/B/NK cells by flow cytometry - TREC analysis 2. Evaluate T cell Function - T cell proliferation after growth stimulation - Immune responses to vaccines 3. Genetics *Newborn Screening for SCID* - TRECS present in naive T cells do not replicate, and become dilute as T cells replicate and divide - qPCR of conserved segments can be used to quantify the number of TRECs in a blood sample

Specific active immunity: Allergy desensitization

*Subcutaneous allergen immunotherapy* *IgE-mediated allergies* o Allergic rhinitis o Allergic asthma o Insect venom *Indication* o Not well controlled with conventional therapy o Adverse effects to drugs o Cannot easily avoid trigger *ID specific allergen* o "vaccine" with small amount of allergen mild immune response without triggering allergic response o Increase amount over time - Desensitize system - Tolerance builds - Reaction to allergen lessens *Resuscitation therapy on-site & long-term benefits*

B Cell and Antibody Deficiencies: 1) T Cell Required for Normal Ab Production, 2) X-Linked Hyper-IgM Syndrome (CD40 Ligand Deficiency)

*T Cell Required for Normal Ab Production* 1. Ag binds to BCR and ingested by B cells 2. Ingested Ag degraded and peptides presented in MHC II 3. CD4+ Th bind via TCR/MHC II interactions 4. CD40L on activated T cells interacts with CD40 on B cell and induces class switching on B cell from IgM to IgG. IL-4, IL-5 provides additional signal. *X-Linked Hyper-IgM Syndrome (CD40L Deficiency)* - Cause: mutations in CD40L - Result: B cells make IgM but cannot get help needed to class-switch to make IgG, IgA. Thus high IgM, low IgG - Symptoms: Recurrent bacterial sinopulmonary infections, Cryptosporidium bowel infections, liver inflammation, malignancies

Strategies to avoid graft rejection

*TISSUE MATCHING* involves the identification and matching of cell surface antigens (primarily HLA molecules) expressed by donor and recipient tissues for purposes of avoiding rejection responses. *MAJOR HISTOCOMPATIBILITY COMPLEX (MHC)*. Cluster of genes that encodes transplantation antigens; in humans, these are the HLA antigens. There are CLASS I ANTIGENS (HLA‐A, B, and C) that serve as the primary CTL targets; CLASS II ANTIGENS (HLA‐D [DP,DQ,DR]) often serve as the targets of CD4+ T cells. Genes of the MHC are tightly linked: *TISSUE‐TYPING*. Determination of the exact collection of HLA antigens expressed by graft donors/recipients; performed with panels of monoclonal antibodies reactive with individual HLA antigens; alternatively, PCR techniques can be used to identify HLA antigens at the gene level. Determination of mismatches does not necessarily predict the intensity of biological rejection responses. That is, multiple HLA mismatches may result in mild and manageable rejection responses, while minimal mismatching may lead to intense graft rejection. Even a perfect MHC match determined by tissue typing does not guarantee graft acceptance; difficult to define/detect MINOR HISTOCOMPATIBILITY ANTIGEN mismatches may not be accounted for that can result in graft rejection. Therefore, a functional test of graft compatibility may need to be performed. *MIXED LEUKOCYTE CULTURE (MLC) TEST* ‐ Mimics proliferation phase of graft rejection by measuring stimulation of recipients CD4+ T cells against allogeneic donor cells. Primary antigenic targets in the test are Class II (HLA‐D) alloantigens found mainly on B cells or macrophages [T cells express very small amounts of Class II MHC (HLA‐D) on their surface and serve as poor MLC stimulators]. Class I antigens may serve as secondary stimuli.

Transplant Pharmacology: Maintenance Immunosuppression

*Typical Maintenance Regime* a. Calcineurin inhibitor - Cornerstone of therapy - Tacrolimus, cyclosporine - (+) b. Anti-proliferative - Mycophenolate, azathioprine, sirolimus, mTOR inhibitors - (+/-) c. Corticosteroids - Methylprednisolone, prednisone d. T-cell co-stimulation blocker - Belatacept

Specific active immunity: Vaccination

*Vaccination- active immunity --> expose immune system to infectious agent* a. Antibodies form against weakened infectious agent • Does not cause disease b. Subsequent exposure to disease • Recognizes infectious agents • Mounts immediate attack c. Requires intact immune system

C1 Inhibitor Deficiency, Kinin-Kallikrein System, and Hereditary Angioedema

- *C1 inhibitor (C1-INH)* binds covalently to C1r and C1s. Binding of C1-INH dissociates the C1 complex and inhibits the enzymatic activity of C1r and C1s - C1-INH is also important for controlling part of the clotting cascade known as the *kinin-kallikrein system* that is involved in controlling blood vessel dilation, vascular permeability, and blood pressure - *Bradykinin* is a small peptide that causes blood vessel dilation and increased vascular permeability. Causes fluid leak into tissues (edema) and decreases BP - C1-INH blocks generation of Bradykinin at 2 levels (see image) - *Hereditary Angioedema* is a type of C1-INH Deficiency, characterized by attacks of rapid swelling in various tissues - Attacks can be triggered by trauma, surgery, emotional stress, etc. Dental procedures are particularly dangerous because they can cause life-threatening swelling of the airways.

Factor H/Factor I/MCP Deficiency, Hemolytic Uremic Syndrome (HUS), and Atypical Hemolytic Uremic Syndrome (aHUS)

- *Factor H* works mainly in the alternative pathway, where it competes with Factor B for binding to hydrolyzed C3 (C3b) to prevent formation of the C3 convertase complex (C3bBb). Also displaces Bb from C3b once the convertase if formed - *Factor I* (soluble) works together with MCP (membrane-bound) to cleave and inactivate active C3b to an inactive form (iC3b) - *Hemolytic Uremic Syndrome (HUS)* results from abnormal, premature destruction of RBCs and inappropriate clotting/platelet destruction in the vasculature. - Post-diarrheal HUS is cased by Shiga toxin produced in bacterial (E. coli, etc.) bowel infection from undercooked hamburgers, etc. Often accompanied by bloody diarrhea - *Atypical Hemolytic Uremic Syndrome (aHUS)* arises from mutations in Factor H (FH), Factor I (FI), or MCP - Decreased FH, FI, or MCP leads to uncontrolled C3 convertase activity --> increased levels of active C3b --> damage to endothelial cells --> inflammation --> clotting --> more damage

Severe Combined Immunodeficiency (SCID)

- A family of disorders; defective differentiation of early progenitor cells of the immune system. Different forms of SCID may follow either autosomal or sex-linked patterns of inheritance. - Some forms of SCID result in a complete absence of T cells and B cells; in some forms, B cells may actually be present but dysfunctional due to a lack of T helper cells. - Without therapeutic intervention, death associated with SCID may occur during the first year after birth due to overwhelming infection. Therapeutic strategies can include combinations of antibiotic treatments, gammaglobulin administration, and transplantation (bone marrow/stem cell and/or thymus transplantation). SCIDS is also likely to be the first human disease for which gene therapy is the treatment of choice. *--Autosomal SCID--* Approximately 50% of autosomal recessive SCID cases is associated with enzyme deficiency (ADENOSINE DEAMINASE [ADA] DEFICIENCY or PURINE NUCLEOSIDE PHOSPHORYLASE [PNP] DEFICIENCY) in which toxic intermediaries of metabolism accumulate and poison early progenitors of the B cell, T cell, and NK cell lineages. *Bare-Leukocyte Syndrome* - A family of disorders involving deficient expression or function of Class I or Class II HLA molecules; results in impaired development, or impaired activation of subsets of T cells that are dependent on antigen presentation via these molecules (review previous T cell lectures). - A variation on Class I MHC deficiency involves mutated TAP GENES resulting in impaired antigen presentation to CD8+ T cells. *RAG Deficiencies* - Involve the impaired expression of recombination activation genes (RAG1/RAG2) necessary for the proper genetic rearrangements required for production of the antigen receptors on B and T lymphocytes. *Artemis Mutations* - Defective ubiquitous non-homologous end-joining protein that is of great importance in DNA repairs and during gene rearrangement necessary for the expression of immunoglobulin and T cell receptor (i.e., VDJ recombination, VJ recombination, etc.). *--X-linked SCID--* - X-LINKED SCID is frequently caused by mutation and defective expression of the common gamma common chain associated with receptors for several important cytokines (IL-2, 4, 7, 9, 15, 21). This is the most frequent form of SCID (SCID-X1). Defective IL-7 receptor may be of exceptional significance, due to its importance with regard to the development of early lymphocyte progenitors, particularly those for T cells; NK cell deficiency in SCID-X1 appears to result from defective IL-15 signaling through its receptor. - Since signaling upon cytokine binding to gamma common chain receptors is mediated mostly by Janus kinase-3 (JAK-3) kinase, a tyrosine kinase, JAK-3 gene mutations result in a SCID phenotype indistinguishable from SCID-X1, but inheritance is autosomal recessive.

Type IV (delayed-type cell-mediated) hypersensitivity

- Although expressed with exaggerated intensity and/or periods of time, immune mechanisms involved in Type IV hypersensitivities are similar to cell‐mediated mechanisms involved in resistance to intracellular infectious disease. EXAMPLES: Contact dermatitis (poison ivy, metals), responses to certain infectious agents (prominent in Leprosy, Tuberculosis, Schistosomiasis), associated with certain autoimmune diseases (Crohn's disease). SENSITIZATION: - *Activation of CD4+ TH1/TH17 cells* is key to initiating Type IV hypersensitivity reactions. Sensitization occurs over a 10‐14 day period when foreign antigen, often a small molecular weight chemical, associates covalently with host membrane proteins. Eventually, these "hapten‐carrier conjugates" are degraded and come into association with class II MHC molecules. The usual progression leading to activation of CD4+ T cells is then initiated against the foreign antigen, but also focuses effector activities on the host tissue to which the foreign antigen is attached. - *Activation of CD8+ cytotoxic T cells* Some foreign antigens may be lipid soluble, allowing them to cross the cell membrane and modify cytoplasmic proteins that are eventually transported to the surface in association with class I MHC molecules. Sensitization then occurs over a 10‐14 day period via the usual progression leading to the activation of CD8+ T cells (CTL) with the ability to produce inflammatory cytokines and to lyse host tissues that express foreign antigen in this way. ELICITATION: Type IV hypersensitivity is an exaggerated form of cell‐mediated immunity, the consequence of activating antigen‐specific T cells to foreign molecules associated with host tissue (distinguishes from Type I, II, and III antibody‐mediated hypersensitivities described above). Symptoms in previously sensitized individuals appear within about 24‐48 hours following antigen exposure. Many of the symptoms are caused by T cell‐derived cytokines including: 1) CHEMOKINES; 2) IFN‐y; 3) TNF‐a and TNF‐B; and IL‐3/GM‐CSF. The result of heavy production of these cytokines is the recruitment and activation of CD4+ T cells that may not be antigen‐ specific. These non‐specific T cells become prominent (>99%) early in the response, but as the response progresses, macrophages eventually become the most prominent cell type overall. Resulting influx and activation of cells produces significant EDEMA (swelling) and INDURATION (firm to the touch) in area surrounding antigen expression/deposition (distinguishes from Type I hypersensitivity described above in which swelling is soft to the touch, due mainly to fluid, not cellular influx). Three variants of Type IV hypersensitivity are recognized (not mutually exclusive): CONTACT HYPERSENSITIVITY Primarily an epidermal reaction with the Langerhans' cell being the principal cell that presents antigen to T cells. Infiltrating T cells are almost exclusively CD4+ (very few CD8+ T cells). Occurs within 24‐48 hours of exposure of tissue to allergen. Must take care in making diagnoses to distinguish from symptoms resulting from exposure to non‐immunogenic tissue‐damaging irritants. TUBERCULIN HYPERSENSITIVITY Primarily an intradermal reaction with macrophages being the principal cell that presents antigen to T cells. Monocytes make up 80‐90% of the cellular infiltrate. Infiltrating CD4+ T cells outnumber CD8+ T cells by about 2:1. Occurs within 24‐48 hours. Classic example of this reaction is seen during tuberculin skin testing with the purified protein derivative (PPD) of tubercule bacilli. GRANULOMATOUS HYPERSENSITIVITY Occurs slowly (21‐28 days). Associated with persistence of difficult‐to‐degrade antigen within macrophages, including intracellular pathogens. Inorganic materials can also stimulate these reactions, but can be distinguished due to lack of participation by lymphocytes. GRANULOMA FORMATION, the hallmark of this form of hypersensitivity, is due mainly to recruitment, proliferation, and activation of TH1 lymphocytes to the site of monocytes/macrophages that are responding to the chronic presence/production of antigen. T cells form a "cuff" around a core of macrophages and macrophages fused into what is referred to as MULTI‐NUCLEATED GIANT CELLS. In addition to macrophages, infiltrating cells in the core of the granuloma include EPITHELIOID CELLS (a highly developed form of macrophage that secretes TNF‐a at a very high rate). In addition to damage caused by inflammatory activities themselves, as cells continue to be recruited, the mass of the granuloma increases, eventually causing physical disruption/damage to surrounding tissue. Considerable fibrosis may be observed, due to the presence of increased collagen synthesis by fibroblasts. GRANULOMA FORMATION IN THE CONTEXT OF MICROBIAL INFECTION Some microorganisms are able to survive ingestion into phagocytes such as macrophages. Chronic maintenance of a microbial pathogen in macrophage endosomes leads to continuous exogenous processing and Class II MHC presentation of microbial antigens. Upon activation of CD4+ T cells, particularly of the TH1 subset, production of IFN‐y and TNF‐α will, in turn, cause a chronic activation of the same infected macrophages and surrounding uninfected macrophages. Recruitment (in response to macrophage‐derived chemokines) and activation of additional TH1 cells will occur resulting in additional rounds of macrophage activation which will activate additional TH1 cells ... and on and on and on. At first, this process results in an effective host defense in which the pathogen is "walled off" and prevented from spreading. Over time, however, large masses in sensitive tissues may cause significant damage. For example, infection with Mycobacterium tuberculosis (the causative agent of tuberculosis) often results in granuloma formation in the lungs that can become life‐threatening.

Relative Incidence of Primary Immunodeficiencies

- Antibody deficiency (53%) - Combined immunodeficiency (23%) - Neutrophil dysfunction (14%) - Cellular immunodeficiency (7%) - Other (2%) - Complement deficiency (1%)

C3 Nephritic Factor (C3NeF)

- Autoantibody directed toward C3bBb (C3 convertase of alternative pathway). *C3NeF prolongs the half-life of C3bBb so more C3 is activated (cleaved)* - Causes *Membranoproliferative Glomerulonephritis Type II (MPGN II)* that causes kidney dysfunction due to deposits of immune complexes in the glomeruli

Major infectious susceptibilities when compartment is absent. Innate (Complement, Phagocytes) & Adaptive (B cells, T cells)

- Complement: encapsulated bacteria - Phagocytes: bacteria, fungi, molds - B cells: bacteria - T cells: viruses, fungi, bacteria

B and T cell immunodeficiencies relate to lymphocyte differentiation and function.

- Direct influences (blocks in development) - Indirect influences (blocks in regulation) *Sign-posts of immune deficits* a. Antibody-mediated immunity - Frequent extracellular bacterial infections - Reduced *SERUM ANTIBODY LEVELS*; particular isotype levels - Reduced circulating B cells; decreased percent of *SURFACE Ig POSITIVE CELLS* (normal levels of B cells in blood usually ∼20-30% of lymphocytes) - Reduced functional B cells; responsiveness to B cell specific mitogens such as *STAPHYLOCOCCAL PROTEIN A* b. Cell-mediated immunity - Frequent viral, fungal, and intracellular bacterial infections - Reduced circulating T cells; decreased percent of T cells such as those that express CD3 (all mature T cells), CD4 (helper/inducer T cells), or CD8 (cytotoxic T cells) - Reduced functional T cells; responsiveness (proliferation and/or cytokine production) to T cell specific mitogens such as *PHYTOHEMAGGLUTININ* (PHA); negative skin testing against antigens associated with microorganisms commonly found in our environment

Which of the following laboratory assays can be used to detect the number and types of immune cells in the peripheral blood? Immunofixation electrophoresis Flow cytometry EIA Immunoblot

Flow cytometry

Syndromes of type IV hypersensitivity

Contact dermatitis (poison ivy, metals) TB skin test granuloma (tuberculin hypersensitivity and granulomas, mild and grave, respectively)

Graft T-Cell Signaling

- Signal 1: Ag triggers TRCs - Signal 2: Co-stimulatory between ligands (B7/CD80/6-CD28) - Signal 3: Cytokines trigger receptors (IL-2 and IL-2R/CD25)

Effector mechanisms leading to graft rejection

Cytotoxic T lymphocyte (CTL) activation leads to the primary destructive force in graft rejection. Activation follows the same basic 2‐signal pathway described previously: 1) Signal 1 provided via T cell receptor bound to APC‐antigen peptide complex; 2) Signal 2 provided via CD28 on T cells that have engaged B7 on APC. Note: Co‐stimulatory molecules are required only for activation; once activated, the CTL can perform its effector functions in the absence of B7. CYTOLYSIS of target cells by CTLs is a multi‐step process (refer to T cell presentation). Following binding to target cells, cytoplasmic granules in CTL move to points adjacent to target cell contact (granules contain PERFORINS that are capable of intercalating and polymerizing in the target cell membrane to form disruptive channels), and the induction of GRANZYME expression/release (thought to have serine protease activity that enters target cells and engages caspase pathway to trigger APOPTOSIS); Fas/Fas ligand interaction may also play a role in triggering apoptosis. ACTIVATED B CELLS differentiate into Ab‐secreting plasma cells. Anti‐graft Ab may result in: 1) Complement mediated lysis, 2) Activation of chemoattractant complement components (e.g., C5a) which attract polymorphonuclear leukocytes (PMN) that cause graft destruction by proteolytic enzymes and vasoactive peptides which enhance infiltration by monocytes and other PMN, and 3) ANTIBODY‐DEPENDENT CELL‐MEDIATED CYTOTOXICITY (ADCC) via K/NK cells.

Hypersensitivity

- i.e., allergy - represents exaggerated manifestations of normal immune responsiveness and reactivity; reactions typically exceed healthy limits of intensity and/or are directed at tissues that are particularly sensitive to the products of such responsiveness/reactivity. Individuals with a predisposition for allergy are said to be ATOPIC.

Autoimmunity

- reflects the loss of regulation designed to prevent immune reactivity against the host's own antigens. Under certain circumstances, autoimmunity may result in the generation of effector activities with disease‐causing potential, that is, autoimmune disease. - reflects the loss of *IMMUNE TOLERANCE* to self‐tissues and cellular antigens. *CLONAL DELETION/ABORTION* theories originally postulated that "forbidden", self‐reactive clones were *physically* eliminated during ontogeny. We have since discovered that many autoreactive lymphocytes exist in our immune system's specificity repertoire. Although these autoreactive lymphocytes are generally of no pathological consequence (often because they are functionally inactive, that is, *ANERGIC*), there is, nonetheless, a significant *potential* for autoimmune disease. *Therefore, mechanisms of both physical and functional clonal elimination exist that fully explains the maintenance of self‐ tolerance*

How might tests of responsiveness to PHA and Staph Protein A distinguish between these two possibilities?

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In addition to innate immunity, are there circumstances in which defective phagocytic cells might also have negative effects on specific adaptive immunity?

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MLC protocol

1. Obtain cells (from potential graft recipient) to be tested for expression of alloreactivity, referred to as the RESPONDER CELLS, and cells (from potential graft donor) to be tested as the targets of potential alloreactivity, referred to as the STIMULATOR CELLS. 2. Stimulator cells receive a lethal dose of irradiation or mitomycin C (DNA synthesis inhibitor) so that proliferative activity in the culture can be attributed to only the responder cell (ONE‐WAY MLC). 3. Mix and culture stimulator cells with responder cells for several days at 37o C. Following incubation, radioisotope‐labeled DNA synthetic precursor (i.e., 3H‐ thymidine [TdR]) is added to the culture and incorporation into DNA is measured to determine the rate of cell division/proliferation, and as a reflection of the intensity of activation of the responder cells against the stimulators. 4. Calculate the Stimulation Index (SI) = Counts per minute (CPM) of [3H]‐TdR incorporated into stimulated responder cells divided by CPM incorporated into unstimulated (control) responder cells. The higher the SI, the more proliferation is indicated, and the less attractive that donor‐recipient combination become

Concepts of Health & Disease Revealed by Primary Immune Deficiency Diseases (PIDD)

1. The immune system's ability to assemble (leukocyte adhesion deficiency, LAD) and disassemble (autoimmune lymphoproliferative disease, ALPS) as needed is a unique property. 2. The immune system is one of only two body systems (the brain is the other) that has a mechanism for specifically responding to individual stimuli (adaptive immune system), as well as a system for responding more broadly to general classes of stimuli (innate immune system). 3. Deficiency in one part of the immune system not only results in absence of function of the missing part, but often affects the remaining parts of the system in a negative way (certain forms of severe combined immune deficiency syndrome, SCIDS; complement component deficiencies, et al.). 4. Defects in a system or pathway can lead to similar clinical expression of defects in different molecules (SCIDS); likewise, expression of a particular deficiency often depends on the precise molecular mutation involved, i.e., genotype-phenotype correlation (x-linked agammaglobulinemia, XLA). 5. In many cases, one normal gene copy is enough to provide normal function, especially when the normal cells can outcompete the abnormal ones (x-linked SCIDS); in other cases, especially when the defect is manifest in terminally differentiated cells, gene dosage makes a significant difference (x-linked chronic granulomatous disease, CGD). 6. Some illnesses can be adequately treated when clinical disease expression becomes apparent (antibody deficiency disorders) while others are best treated when recognized as early as possible; prenatal or newborn screening is the ultimate early detection (SCIDS). 7. There are often multiple corrective strategies for a given deficiency disorder; each strategy has advantages and disadvantages: replace the missing functional activity (immunoglobulin replacement for antibody deficiency), replace the precursor that generates the functional activity (hematopoietic stem cell transplantation for SCIDS), correct the underlying defect (gene therapy for SCIDS).

Incompatible ABO blood groups can cause serious transfusion reactions. Why is an ABO mismatch between a pregnant woman and her developing baby rarely if ever a serious threat to the baby? ABO-reactive antibodies are of the IgM isotype ABO-reactive antibodies are specific for a conserved protein antigen ABO-reactive T cells are of the TH1 subset ABO-reactive T cells do not receive co-stimulatory signal 2 ABO-reactive antibodies are of the IgG isotype

ABO-reactive antibodies are of the IgM isotype

Syndromes of type I hypersensitivity

Allergic rhinitis Allergic asthma Venom reactions Food allergies Drug allergies Anaphylactic shock

Diagnostic Testing: CH50 Test

Amount of complement (serum dilution) that causes lysis of 50% of antibody-coated sheep RBCs. *Tests only the classical pathway*

Allergen

An antigen (harmless in most individuals under most circumstances) that stimulates allergic responses in atopic individuals (particularly antigens responsible for IgE‐associated allergy).

Graft versus Host Disease (GVHD)

GVHD is a potential complication of any transplantation or transfusion procedure that results in hematopoietic elements from the donor, primarily mature T cells and monocytes, that are transferred into the recipient. Immune responsiveness of the donor cells is generated against antigenic differences (MHC) expressed by the recipients tissues. Primary manifestations of GVHD result from activation of large numbers of donor CD4+ T cells and their subsequent production of inflammatory cytokines. Activated mononuclear cells infiltrating affected tissues is prominent. Although activation of donor cells is the initiating step in a complicated, incompletely understood progression, inflammatory cells from the recipient may become an additional source of immune mediators as the response progresses. Chances of anti‐recipient immune reactivity/GVHD greatly increase when immunocompetent donor lymphoid cells are transferred into an immunoincompetent recipient. Examples: ‐ NEWBORNS ‐ Adult recipients rendered immunoincompetent by IMMUNOSUPPRESSIVE THERAPY ‐ IMMUNODEFICIENCY patients who are already immunocompromised Acute and chronic forms of GVHD are observed with the chronic form generally becoming apparent more than 3 months post transplant and can affect a wider range of organs. Pathology, particularly associated with acute GVHD, often results in skin, liver and gut involvements that produce, respectively, symptoms of: Rash (diffuse maculopapular sunburn‐like rash, sometimes in a lacy pattern, beginning on the hands, face and feet); Jaundice (reflecting liver damage and increased levels of bilirubins in the blood); and Diarrhea (often accompanied by nausea and vomiting). Pathology may become life‐threatening; sloughing of intestinal and bronchial mucosa may be observed.

Corticosteroids

Glucocorticoid action o Metabolic effects o Permissive actions- increases epinephrine, norepinephrine o Adaptation to stress o Anti-inflammatory o Immunosuppression Mineralocorticoid action o Essential for life o BP o Na+, K+, water regulation o Regulates: RAAS, Adrenal cortex ([K+])

Glucocorticoids

Glucocorticoids allow for adaptation to stress, such as (1) protection during fasting- increased glycogen stores, and (2) protein breakdown- frees amino acids for repair They also have metabolic effects, which increase blood glucose. The mechanisms include (1) increases hepatic gluconeogenesis, (2) Protein catabolism, (3) Lipolysis, (4) Inhibit peripheral glucose use (save glucose for brain) Glucocorticoids have immune suppressant effects, more specifically they suppress cell-mediated immunity via a decrease cytokines TNF-y and IL. Some of the permissive actions include (1) Catecholamine vasoconstriction, (2) Glucagon, epinephrine, hepatic gluconeogenesis. *Glucocorticoids have important anti-inflammatory effects via a DECREASE in cyclooxygenase 2 (COX2)* *MoA*: Inhibition of COX2 decreases inflammation by decreasing prostaglandin formation, which then decreases leukotriene and platelet-activation factor synthesis. Ultimately these effects cause: (1) suppression of inflammatory humoral factors, and (2) inhibition of tissue macrophage and other APC-- markers: low TNF, low IL-1, low metalloproteinases, low plasminogen activator (3) Low IL-12 and INF, thus low T-4-effector cells Th1 activity and decreased cell immunity (Th1 --> activate macrophages and NK)

Hemolytic disease of the newborn

HDN results from an incompatibility between the Rh BLOOD GROUP ANTIGENS of a developing fetus, and the Rh antigens of the pregnant mother. Typically, the fetus is Rh positive, while the mother is Rh negative. Upon delivery of her first baby, antigen is introduced into the mother's circulation. An antibody response (dominated by IgG antibodies) develops. During a second pregnancy, these or newly produced antibodies cross the placenta (remember that IgG antibodies have this ability while IgM antibodies cannot), causing severe anemia if the fetus is Rh positive. Administration of RhoGam has been helpful in attempting to avoid these complications.

Sensitization v. elicitation

Hypersensitivity diseases often requires initial sensitizing exposure to allergen, followed by subsequent exposures that elicit symptoms. In some instances, the sensitization phase may be completed before allergen is cleared from the body, therefore allowing elicitation to begin during the initial allergen exposure.

Immunofixation Electrophoresis (IFE)

IFE is used to detect the presence of a particular immunoglobulin isotype in serum, urine, and cerebrospinal fluid. Traditionally, multiple myeloma is classified according to the heavy‐chain class and light‐chain type of the monoclonal antibody that it produces. In addition, light chains may be overproduced relative to heavy chains and enter the blood in the form of light chain dimers (in the absence of heavy chains). These light chain dimers are easily filtered by the glomeruli because of their low molecular weight. Thus, free light chains can be found in the urine and are called BENCE‐JONES PROTEIN. IFE is useful in the detection and classification of both monoclonal antibody (i.e., heavy‐chain class and light‐chain type) in serum, as well as Bence Jones protein in urine from patients with multiple myeloma.

Which one of the following laboratory assays is considered to be an in vitro counterpart of the type IV hypersensitivity reactions seen in the TB skin test? Immunoblot for TB antigen IFN-γ production by white blood cell (WBC) treated with TB antigen Immunofluorescence assay for TB antibody EIA analysis of sera from TB patient

IFN-γ production by white blood cell (WBC) treated with TB antigen

Which cytokine listed below promotes differentiation of B cells, enhances class switching to IgA, and stimulates growth and differentiation of eosinophils? IL-2 IL-1 IL-5 IL-4 TNF-alpha

IL-5

MHC interactions in transplantation

In many ways, responses and reactions associated with transplant rejection follow the same rules of immune responsiveness as has been discussed many times in this module (review, in particular, the T cell presentation). One notable challenge to our understanding is that the target antigen is a molecule encoded by genes of the major histocompatibility complex (i.e., HLA); the intriguing outcome is that we must consider scenarios in which MHC molecules on APCs of the transplant recipient acting as presenting molecules must present MHC molecules from the transplant donor acting as antigen, thereby allowing activation of T cells from the transplant recipient.

Serum Protein Electrophoresis (SPE)

In the clinical laboratory, the electrophoretic separation of serum proteins based on their charge can be performed in order to determine the relative concentrations of the various normally occurring protein fractions present in a patient's serum. This method is useful for detecting abnormally high or low levels of serum proteins. SPE separates 5 major fractions of serum based on their electrophoretic mobility (rates of movement) as they migrate through an electric field. In order of highest rate of migration to lowest rate of migration toward a positive electrode (therefore, highest negative charge to lowest negative charge of serum components), these fractions are: Albumin, alpha‐1 globulins (α1), alpha‐2 globulins (α2), beta globulins (), and gamma globulins (). Each serum fraction is actually composed of a family of similarly charged proteins (except the albumin fraction composed of a single member). A partial list of components contained in the five protein fractions of serum include: (see imagine) Method: Major protein fractions of serum, in a pH 8.6 buffer that gives each protein a characteristic charge, are separated on a cellulose acetate strip by allowing them to migrate through an electric field (electrophoresis). After electrophoresis, locations of separated protein bands are visualized by staining the strip. The strip is then scanned by a densitometer which allows the amount of dye bound to each protein band to be determined and converted into a graph referred to as its densitometer tracing. NOTE: The concentration of each peak (g/dl) is determined by multiplying the total protein concentration of serum times the fraction that a particular peak represents in relation to the entire serum profile (i.e., total areas under all peaks). Verification of polyclonality or monoclonality can be performed in the laboratory in a number of ways. At a genetic level, SOUTHERN BLOT ANALYSIS using the proper genetic probes can reveal the number of different gene rearrangements yielding the antibodies produced by a population of B cells; one VDJ product = one antibody product = monoclonality. Multiple different VDJ products = multiple antibody products = polyclonality. Monoclonality can also be verified by characterization of antigenic epitopes associated with the antibody(s) produced. The IDIOTYPE of an antibody is a unique serologically defined region roughly corresponding to the antigen‐combining site (can include sub‐regions [CDRs] on both H and L chains). Each antibody, and its B cell of origin, has one unique antigen combining site, and therefore one unique idiotype.

Which one of the following cell types expresses receptors for IgE on its cell surface that stimulate the cell to mount a response to parasites such as worms? B cells NK cells Mast cells T cells Dendritic cells

Mast cells

Hypogammaglobulinemia

Maturation or regulatory defects in which B cells are either physically missing or are present but dysfunctional. May be generalized (affecting all B cells) or restricted to B cells responsible for production of particular isotypes. Defects in antibody production may also be the indirect result of faulty regulation by helper T cells. *--X-linked Congenital Agammaglobulinemia--* - (previously known as *Bruton's agammaglobulinemia*) results from a defective tyrosine kinase (btk) necessary for appropriate developmental activities; pre-B cells may be present but subsequent maturation to immunocompetent B cells is defective. Generally presents itself in young boys. *--Common Variable Immunodeficiency (CVID), Autosomal--* - Resembles X-linked hypogammaglobulinemia (above) in that immunoglobulin levels are decreased. However, CVID may affect older patient population, B cells may be present, and frequencies of affected males and females may be equal due to some forms demonstrating autosomal inheritance. Most cases of CVID are probably not inherited in a straightforward Mendelian fashion and mechanisms responsible for this collection of syndromes are varied and sometimes poorly understood, involving regulatory/signaling defects that interfere with B cell activation or antibody class switching (selective isotype deficiencies of any isotype can occur). *Selective IgA Deficiency* The most common of any immunodeficiency (1 in 400 individuals have it), although the vast majority remain asymptomatic. *X-linked Hyper-IgM Syndrome* Defective CD40 ligand (CD154) on activated T cells resulting in an accumulation of B cells unable to switch from IgM to IgG or other isotypes. These patients often have B cells with defective ability to demonstrate class switching and affinity maturation nodes *lacking germinal centers*. *Activation-Induced Cytidine Deaminase (AID) Deficiency* B cells with defective ability to demonstrate class switching and affinity maturation.

Goodpasture's Syndrome (T2H)

Mechanism: Autoantibodies against Type IV collagen of the basement membrane, primarily react with renal glomeruli, but can also react with pulmonary alveoli. Presentation: Vague early symptoms such as fatigue, nausea, burning upon urination, or difficulty breathing; difficult to make an early diagnosis. Can progress rapidly to glomerulonephritis with hematuria, proteinuria. If lung basement membrane becomes involved, patient might cough up blood (hemoptysis). Because of the vagueness of early symptoms and rapid progression of the disease, diagnosis is often not reached until very late in the course of the disease. Demonstration of circulating antibodies against Type IV collagen may aid in diagnosis; also, kidney biopsy may confirm by allowing the demonstration of linear IgG deposits along basement membrane.

Rheumatoid arthritis

Mechanism: Mixed antibody and CMI effectors involving Type III and IV hypersensitivities; prominent immune complex disease in joints. Presentation: Typical presentation includes signs of inflammation in affected joints that are swollen, warm, painful and stiff, particularly following prolonged inactivity such as first thing in the morning.

Systemic Lupus Erythematosus

Mechanism: Systemic autoimmune attack with a single antigenic stimulus is unlikely. More likely a complex immunoregulatory impairment is responsible. Although a specific inducing event is unknown, sex hormone influences are likely to be important (note the greater incidence in women during child‐bearing years). Multiple tissue pathologies associated with virtually any organ/tissue of the body may be observed. Although specific cytolytic antibody attack is observed (i.e., Type II hypersensitivities), prominent immune complex disease (Type III hypersensitivity) is responsible for much of the life‐threatening pathology. Anti‐ nuclear antibodies (ANA) and DNA‐reactive antibodies are common components of immune complexes. Numerous circulating engorged phagocytic cells (lupus erythematosus [LE] cells) can be observed attempting to clear immune complexes. Presentation: There is no "typical" SLE presentation, and virtually any organ/tissue of the body may be affected. SLE is one of numerous autoimmune diseases that are observed more frequently (5‐10X) in women of child‐bearing age, than in men or post‐menopausal women. Many initial and chronic complaints may resemble a variety of infectious and non‐ infectious diseases such as fever, rash, malaise, joint pains, myalgia, fatigue, and temporary loss of cognitive abilities. Therefore, making a diagnosis may be difficult and sometimes missed. Disease course includes periods of remissions and unpredictable flare‐ups.

Immune response v. immune reaction

Most hypersensitivity diseases require sensitization involving an immune response that produces potentially harmful soluble or cellular factors. Pathology will be noted when these factors combine with and elicit damage to host tissues (immune reactions). Depending on the nature of the hypersensitivity involved, the time‐frame of the immune response in relation to when the pathology is observed may vary

Acquired Immunodeficiency Syndrome (AIDS)

NOT a congenital disorder, but instead is transmitted by an infectious virus, HUMAN IMMUNODEFICIENCY VIRUS (HIV). Becoming HIV-infected is the first step in a multi-step process that leads to AIDS (in other words, being HIV positive does not mean the same as having AIDS). Asymptomatic HIV+ individuals can transmit infectious virus. *Common modes of transmission* - Transmission due to unprotected sexual contact - Transmission in contaminated blood or blood products - Transmission to babies of AIDS mothers - There is no evidence for "casual contact" transmission. *Certain behaviors are considered "high risk" for HIV transmission* - Frequent unprotected sexual contact with multiple partners - Sharing syringes and needles (i.v. drug users) - Frequent blood or blood product transfusions To what extent do you think there is a risk of HIV infection for blood bank donors? *HIV and the Immune System* - HIV infection of CD4+ cells is initiated when HIV gp120 attaches to DC-SIGN, a receptor on dendritic cells, followed by interactions between DCs and CD4+ T cells or macrophages. HIV uses CD4 as a receptor for entry into the cell. Certain chemokine receptors (e.g., CCR5 on T cells and macrophagHIV infection of CD4+ cells is initiated when HIV gp120 attaches to DC-SIGN, a receptor on dendritic cells, followed by interactions between DCs and CD4+ T cells or macrophages. HIV uses CD4 as a receptor for entry into the cell. Certain chemokine receptors (e.g., CCR5 on T cells and macrophages and CXCR4 on only T cells) act as "co-receptors" for the virus. HIV is carried as a provirus that is integrated into the genome of T cells or macrophages. This so-called "latent infection" allows the dormant virus to evade immune defenses. - HIV reactivation results in loss of CD4+ cells, and symptoms ranging from relatively asymptomatic to AIDS related complex, which consists of fever, fatigue, persistent lymphadenopathy, and weight loss. The terminal stages of AIDS generally involve opportunistic infections which are often the cause of patient death.es and CXCR4 on only T cells) act as "co-receptors" for the virus. HIV is carried as a provirus that is integrated into the genome of T cells or macrophages. This so-called "latent infection" allows the dormant virus to evade immune defenses. - HIV reactivation results in loss of CD4+ cells, and symptoms ranging from relatively asymptomatic to AIDS related complex, which consists of fever, fatigue, persistent lymphadenopathy, and weight loss. The terminal stages of AIDS generally involve opportunistic infections which are often the cause of patient death. - Since macrophages/dendritic cells and CD4+ T cells can be infected, the decline of the immune system can involve both non-adaptive and adaptive (humoral and cellular) immunity resulting in dramatically increased susceptibility to infection by a variety of microbes (viral, bacterial, and fungal), as well as increased cancer incidence (KAPOSI'S SARCOMA is prominent). *As CD4+ T cells decline, a chain of reaction of lost immune function can occur* See image *Tools for monitoring the course of HIV infection* - Screening for HIV seropositivity (ELISA assay)... Problems: (1) Danger of false positives (confirm by Western blot analysis) (2) Danger of false negatives (consider kinetics of antibody response in relation to time of HIV infection) - Tests for presence of HIV genome (polymerase chain reaction [PCR]) - Tests for circulating CD4+ cells (immunofluorescence with monoclonal antibodies) *CD4+ T cell count per microliter* - Normal numbers ∼1000 - Numbers in HIV-infected asymptomatic individuals ∼800 - Numbers as opportunistic infections first begin to emerge <500 - Full blown AIDS <200

Addison's disease

Organs affected: Adrenal glands Pathology: Anti-adrenal cell membranes and lymphocyte/monocyte cortical infiltration

Goodpasture's Syndrome

Organs affected: Basement membranes of renal glomeruli (and in some cases of pulmonary alveoli) Pathology: Kidney/respiratory failure

Multiple Sclerosis

Organs affected: CNS (oligodendrocytes) Pathology: Demyelination

Pemphigus vulgaris

Organs affected: Epidermis Pathology: Blistering of skin

Autoimmune hemolytic anemia

Organs affected: Erythrocytes Pathology: Erythrocyte destruction and anemia

Chronic atrophic gastritis; Pernicious anemia

Organs affected: Gastric mucosa Pathology: Loss of gastric secretory function; Failure of vitamin B12 absorption due to loss of intrinsic factor or intrinsic factor producing cells

Rheumatic fever

Organs affected: Heart Pathology: Myocarditis; Scarring of heart valves

Rheumatoid arthritis

Organs affected: Initially joints, spreading to cartilage and neighboring bone and muscle Pathology: Immune complex disease in synovium; Antibodies (RF) against IgG prominent inflammatory T cells noted

Intestinal granulomatous disease (Crohn's disease)

Organs affected: Mucosal membrane of terminal ileum Pathology: Mucosal ulceration; Obstructive granuloma

Myasthenia gravis

Organs affected: Neuromuscular junction; AChR Pathology: Destruction or blocking of ACh receptor; impaired transmission

Acute Idiopathic Polyneuritis (Guillian‐Barré syndrome)

Organs affected: Peripheral nerves Pathology: Demyelination

Thrombocytopenias

Organs affected: Platelets or megakaryocytes Pathology: Platelet destruction/abnormal bleeding

Grave's disease

Organs affected: Thyroid (hyperthyroidism) Pathology: Anti-TSH receptor

Hashimoto's thyroiditis

Organs affected: Thyroid (hypothyroidism) Pathology: Anti-thyroglobulin; anti-thyroid epithelium

Systemic lupus erythematosus

Organs affected: Variety of organs Pathology: Numerous specificities of autoreactivity; Acute immune complex disease prominent

Type 1 (insulin-dependent) diabetes mellitus

Organs affected: pancreatic islets (beta cells) Pathology: Beta cell destruction by Ab, Th1 and CD8+ cells

Sjögren's syndrome

Organs affected: salivary glands Pathology: Glandular inflammation

A 14-year-old girl demonstrated polyuria, excessive thirst, and a weight loss of nearly 6 pounds within a matter of weeks. Tests demonstrated the presence of circulating autoantibodies indicative of insulin-dependent diabetes (IDDM). Additional tests for autoantibodies to intrinsic factor were performed and found to be positive. Insulin-dependent diabetes (IDDM) is caused by autoreactivities directed at which of the following? Insulin receptors Pancreatic islet cells Glucose Acetylcholine receptors Insulin

Pancreatic islet cells

Mechanisms responsible for B cell autoimmunity

Regarding Central Tolerance of B cells: ‐ Clonal deletion of immature (IgM‐only) B cells occurs in bone marrow via negative selection during the sensitive immature B cell stage of development (see previous B cell development lecture). ‐ At least three opportunities for things to go wrong are observed: (1) *Incomplete clonal deletion of self‐reactive B cells due to limited availability of self-antigens in marrow*. Self‐reactive B cells are allowed to migrate to periphery. (2) *Somatic mutations may occur in B cells activated to foreign antigen, allowing the expression of receptors for antigen with new reactivities against self in periphery*. (3) *ANTIGEN MIMICRY* is a structural similarity between a foreign antigen and a self‐antigen that allows *IMMUNE CROSS REACTIVITY*. This phenomenon is thought to be responsible for many autoimmune scenarios, but usually without significant proof. A classic, and proven, example is Streptococcal M protein antibodies cross-reacting with structural proteins in heart valve to cause *RHEUMATIC FEVER/RHEUMATIC HEART DISEASE*. Evidence for additional examples is weak at best. ‐ Examples: Streptococcal M protein (heart valve tissue) Measles virus P3 antigen (myelin basic protein) Polio virus VP2 antigen (acetylcholine receptor) Cytomegalovirus IE2 antigen (HLA‐DR antigens) Papillomavirus E2 antigen (insulin receptor) Note: Mechanisms are needed to prevent activation of autoreactive B cells that escape Central Tolerance. *Regarding Peripheral Tolerance of B cells* - Peripheral B cell tolerance, required to regulate the activities of self‐reactive B cells that escape central tolerance, is maintained by a variety of mechanisms: ‐ *IMMUNOLOGICALLY PRIVILEGED SITES*: Autoimmune B cell responses are limited by sequestering, during early embryological development before the immune system has developed, some self-antigens in immunologically privileged anatomical sites (e.g., CNS). However, injury may disturb tissue boundaries, thereby releasing sequestered antigens. Having never "seen" these self-antigens before, the immune system perceives them as foreign. - *Peripheral B cell tolerance maintained by* ‐ *SPLIT TOLERANCE*: Autoimmune B cell responses are limited by a lack of T cell helper activity resulting from central and peripheral T cell tolerance mechanisms. This results in restricted expansion or maturation of autoreactive B cell populations. In other words, potentially harmful effector B cells that have escaped the central tolerance mechanism are kept quiet due to the absence (via clonal deletion or anergy) of helper T cells necessary for their activation. This is sometimes referred to as split tolerance... Two cell types needed for a response, one is tolerant, the other is not but dependent on the first... net result, no response. If, however, there is a mechanism provided that allows either *1) the activation of anergic T cells as described in the previous section*, or *2) the by‐pass of necessary autoreactive T cells*, as described below, the result may be activation of pathological autoreactive B cells. - In the absence of autoantigen‐specific T cells: Two main mechanisms have been observed that will allow self‐reactive B cells to be activated. These are sometimes referred to as *T CELL BYPASS* mechanisms. B cell autoimmunity would reflect the activating influence of alternative populations of helper T cells which do not express specificity for the self‐antigen in question; alternatively, the need for inducer T cells may be by‐passed altogether. (1) *Nonspecific, T inducer‐independent* induction of effector functions - *MITOGENS*; Certain products derived from infectious agents are *polyclonal B lymphocyte activators (mitogens)* (e.g., Staphylococcal Protein A, Epstein‐Barr virus, etc.). When exposed to a mitogen, polyclonal B cell proliferation and differentiation will occur, regardless of the antigen specificity of the B cell's antigen receptor (even if self‐reactive), and resulting in the production of antibody. (2) *Nonspecific, inducer‐dependent* induction of effector functions (see Type II hypersensitivity) - Physical linkage of a self-antigen with a newly acquired, extrinsic (foreign) antigen, creates a scenario in which antibodies are produced against a usually non‐immunogenic self‐antigen. As shown in the diagram below, this is the result of linked recognition and activation of B cells with reactivity against self‐antigen (circles) that are usually quiet (due to a lack of self‐reactive TH); when allowed to interact with TH cells that have become activated against epitopes on the linked foreign antigen (triangles), Signal 2 is provided to the B cells. (See image) - Alternatively, *new TH cell targets may be provided by newly expressed, intrinsic antigenic determinants (neo‐epitopes)* created as the result of partial degradation of self‐antigens during injury or trauma. The self‐antigen has literally been deformed and now possesses "foreign" character. Partial degradation may also expose "cryptic" antigenic determinants that are normally sequestered from the immune system by the three‐dimensional structure of that antigen. TH cells with reactivity against neo‐epitopes can provide Signal 2 for B cells reactive with self‐epitopes associate with the same antigen molecule.

Type I (immediate) hypersensitivity

SENSITIZATION - Exposure to antigen (allergen) resulting in antigen‐specific antibody production dominated by the IgE isotype. Multiple possible routes of exposure (skin, oral, etc.). - Initial allergen exposure generally comes by way of transmucosal presentation of very low doses of antigen under circumstances that preferentially promote the activation of TH2 responses, followed by activation of antigen‐specific B cells. ‐ allergen can enter via intravenous, oral or respiratory routes ‐ most allergens are small soluble molecules; effective activators of TH2 responses. ‐ *IL‐4 production by TH2 cells promotes selective switching to IgE antibody production* - During sensitization, allergen‐specific IgE binds tail (Fc)‐down onto mast cells, basophils, and eosinophils associated with mucosal and epithelial tissues; these cells express FcRI. Specific IgE may remain associated with these tissues for a long time (even years). ELICITATION - Symptoms are a consequence of released pharmacologically active factors that cause increased vascular permeability, increased blood flow, decreased blood pressure, increased mucous secretions, and smooth muscle contraction. Dependent on crosslinking of allergen‐specific IgE antibody fixed onto mast cells or basophils, and observed within minutes of allergen exposure. ‐ Reactions can vary in intensity depending on the dose and route of allergen exposure. Symptoms range in severity from minor localized irritations (such as ALLERGIC RHINITIS) to serious localized reactions (such as ALLERGIC ASTHMA) to life‐threatening systemic circulatory collapse (ANAPHYLACTIC SHOCK). DEGRANULATION - Release of pharmacologically active mediators from the cytoplasmic granules of mast cells or basophils. The process begins with the simultaneous binding (bridging) of allergen to adjacent IgE molecules fixed to specialized *Fc-epsilon RECEPTORS* on the surface membranes of the mast cells or basophils. Bridging between IgE molecules triggers an *INFLUX OF Ca2+* ions into the cell, which mediates a *decreased production of CYCLIC AMP* that results in membrane destabilization prior to granule release. Metabolic membrane activities triggered during degranulation by increased Ca++ also result in the production of *arachidonic acid* and very important lipid mediators, the *leukotrienes and prostaglandins*. Released products are circulating at pharmacologically significant levels within minutes, and have direct effects on blood vessels, smooth muscle, and other target cells. Mediators include: 1) HISTAMINE causes increased vascular permeability and local blood flow. 2) LEUKOTRIENES and PROSTAGLANDINS cause smooth muscle contraction, and increased vascular permeability and mucous secretion. 3) CHEMOKINES that attract leukocytes. 4) ENZYMES that break down tissue matrix proteins. 5) CYTOKINES that promote inflammatory activities, amplification of the TH2 responses, and stimulate the growth and activities of eosinophils. SITES OF REACTIVITY Skin ‐ Localized WHEAL‐AND‐FLARE REACTIONS observed following local intracutaneous exposure to allergen, such as insect bite; SKIN TESTS for antigen‐specific IgE hypersensitivity mimic and measure this type of localized reaction. A more disseminated form of this reaction often associated with allergen ingestion and migration to the skin is URTICARIA (HIVES). Gut ‐ Cramping, vomiting, diarrhea due to smooth muscle contraction. If allergen is found systemically, URTICARIA and/or ANAPHYLAXIS can occur. Lungs ‐ ALLERGIC RHINITIS associated with nasopharnyx and upper airway; more serious reactions occur following activation of mast cells of the submucosa of the lower airways resulting in ALLERGIC ASTHMA. Systemic ‐ Exposure to significant doses of allergen can result in catastrophic consequences associated with widespread increased vascular permeability, decreased blood pressure, and contracted smooth muscles that, among other things, constricts the airways (while surrounding tissues swell due to abnormal shifts in fluids). This form of immediate hypersensitivity is referred to as SYSTEMIC ANAPHYLAXIS that may progress into its most serious form, ANAPHYLACTIC SHOCK. TREATMENT ‐ AVOIDANCE - Avoid exposure to allergen (if you know what it is). ‐ DESENSITIZATION - Goal is to shift IgE‐dominated antibody responses to IgG production (IgG much less effectively associates with mast cells). Strategy involves administration of multiple very small, and then escalating, doses of allergen. Although a wide‐spread clinical strategy, effectiveness is highly variable from patient‐to‐patient, effects are temporary, and the potential danger of accidental induction of anaphylaxis is always present. ‐ INHIBITION OF MEDIATOR RELEASE - Agents designed to either inhibit calcium influxes or to raise levels of cAMP may provide relief. ‐ INHIBITION OF MEDIATOR EFFECTS - Designed to treat symptoms by reversing target tissue activities stimulated by the mediators of the allergic response. Examples of such strategies include administration of EPINEPHRINE (ADRENALIN) (promotes the re‐forming of endothelial tight junctions, relaxation of smooth muscle, and stimulation of the heart), inhalation of BRONCHODIALATORS, and administration of ANTIHISTAMINES (H receptor antagonists). Inflammatory activities, particularly associated with late‐phase responses, can be treated with administration of CORTICOSTEROIDS.

Type III hypersensitivity (immune complex disease)

SENSITIZATION - Initial antigen exposure results in activation of antigen‐specific B cells. Specific antibodies are reactive with antigen that is typically soluble and freely circulating. ELICITATION - Symptoms occur when concentrations of circulating soluble antigen and specific antibody are appropriate (generally of equi‐molar concentrations); many molecules of antibody bind to many molecules of antigen, forming multiple crosslinks to each other, resulting in large insoluble antibody‐antigen complexes. Normally, immune complexes are cleared by FcR+, CR+ phagocytic cells. However, if the amount or location of immune complex formation exceeds that which can be handled by phagocytes, deposition of the complexes may occur on nearby tissues (frequently in 'filtering' organs such as kidneys or areas rich in capillary beds such as the lungs). - Symptoms caused by non‐specific deposition of immune complexes onto host tissues, followed by intense complement activation and further aggravated by FcR‐bearing effector cells. *THE SPECIFICITY OF THE ANTIBODY INVOLVED IS TYPICALLY UNRELATED TO ANTIGENS EXPRESSED BY THE TISSUE BEING ATTACKED* (distinguishes from Type II hypersensitivity described above). - Deposition of antigen/antibody immune complexes detected in biopsy specimens by immunofluorescent staining, occur as irregular aggregates (distinguishes from Type II hypersensitivity described above). Inflammatory processes focused onto the tissue: 1) Activation of the complement system, including both lytic activities and the release of *ANAPHYLATOXINS (C5a and C3a)*; 2) Recruitment of neutrophils, basophils, and eosinophils by C5a; 3) Mast cell and basophil degranulation stimulated by anaphylotoxins, as well as by deposition of immune complexes on their surface; 4) Macrophage activation. ARTHUS REACTION is the basis for tests indicative of Type III hypersensitivity; injection of antigen into skin that attracts circulating IgG, thereby forming immune complexes, activating the complement cascade, attracting phagocytic cells (C5a is prominent), and resulting in a local inflammatory response. SERUM SICKNESS was recognized long ago when it was common to treat infections or intoxications (snake‐bite, etc.) with large doses of horse antiserum, which is itself a source of foreign antigen. Infections and toxins would be neutralized, but immune complex disease could become life threatening. Similar scenarios can also develop following intravenous large‐dose administration of drugs, etc.

Type II (cytotoxic antibody hypersensitivity)

SENSITIZATION Production of antibodies reactive with EXTRINSIC (FOREIGN) ANTIGEN or reactive with INTRINSIC ANTIGEN produced by the target tissue itself. Time of onset and severity of symptoms varies based on kinetics and exact characteristics (e.g., specificity, affinity, isotype) of antibodies produced. ELICITATION Initial antigen exposure may result in concomitant sensitizing responses and elicitation of symptoms with no need for additional antigen exposures (although memory responses will intensify both the response and the symptoms). Symptoms usually caused by binding of antigen‐reactive antibody (usually IgG) specifically to antigen‐coated host cells, followed by direct lysis due to complement activation and further aggravated by release of active by‐products of the complement cascade (ANAPHYLATOXINS C5a, C3a). FcR‐bearing and CR‐bearing leukocytes are also recruited into the area. Examples.. HEMOLYTIC DISEASE OF THE NEWBORN HDN results from an incompatibility between the Rh BLOOD GROUP ANTIGENS of a developing fetus, and the Rh antigens of the pregnant mother. Typically, the fetus is Rh positive, while the mother is Rh negative. Upon delivery of her first baby, antigen is introduced into the mother's circulation. An antibody response (dominated by IgG antibodies) develops. During a second pregnancy, these or newly produced antibodies cross the placenta (remember that IgG antibodies have this ability while IgM antibodies cannot), causing severe anemia if the fetus is Rh positive. Administration of RhoGam has been helpful in attempting to avoid these complications. GOODPASTURE'S SYNDROME Caused by the production and binding of antibodies reactive with the basement membranes of renal glomeruli (and in some cases of pulmonary alveoli). Primary antibody specificity is for the alpha-3 chain of type IV collagen of the basement membrane. Antibody binding triggers the complement cascade, as well as the activation of monocytes and neutrophils via their Fc receptors. Kidney biopsy specimens generally demonstrate a smooth layer of antibody/complement coating host tissue (detectable by immunofluorescent staining) that follows the paths of the various tubules (distinguishes from Type III hypersensitivity described below).

Define: Syngeneic Allogeneic Xenogeneic Autograft Isograft Allograft Xenograft MHC

SYNGENEIC ‐ identical genetic constitution (as between individuals of an inbred mouse strain or identical twins) ALLOGENEIC ‐ differing genetic constitution between members of the same species (as between different inbred mouse strains or humans who are not identical twins) XENOGENEIC ‐ major genetic differences between different species AUTOGRAFT ‐ tissue grafted back onto the original donor ISOGRAFT ‐ tissue grafted between syngeneic individuals (e.g., between identical twins or between mice of the same strain) ALLOGRAFT ‐ tissue grafted between allogeneic individuals XENOGRAFT ‐ tissue grafted between xenogeneic individuals (e.g., ape to man) MAJOR HISTOCOMPATIBILITY COMPLEX ‐ closely linked family of polymorphic genes which encode for cell surface histocompatibility antigens that are targets of transplant rejection reactions between genetically dissimilar individuals.

Syndromes of type III hypersensitivity

Serum sickness Farmers lung syndrome Rheumatoid arthritis Systemic lupus erythematosus

Examples of antigen mimicry

Streptococcal M protein (heart valve tissue) Measles virus P3 antigen (myelin basic protein) Poliovirus VP2 antigen (acetylcholine receptor) Cytomegalovirus IE2 antigen (HLA‐DR antigens) Papillomavirus E2 antigen (insulin receptor)

Classifications of hypersensitivity

TYPE I Immune reactant: Antigen‐specific IgE antibody. Mast cell, basophil, and eosinophil degranulation involved with release of pharmacologically active agents. TYPE II Immune reactant: Target tissue‐specific IgG antibody. Antibody binding is specific for intrinsic or extrinsic cell‐bound molecules and subsequent activation of the complement cascade. TYPE III Immune reactant: IgG antibody not specific for target tissue (soluble Ags). Associated with the formation and deposition of antibody‐antigen complexes in sensitive tissues and activation of the complement cascade. TYPE IV Immune reactant: T lymphocytes (CTL and/or TH1) and sometimes macrophages. Associated with effector T cells that destroy antigen‐coated target cells either 1) directly, by cytolytic attack of target cells or 2) indirectly, by activating macrophages that then damage target cells.

A few comments about serological methods for identifying and monitoring multiple myeloma...

The following case involving MYELOMA (malignant monoclonal plasma cells, sometimes referred to as plasmacytoma) will be used as an opportunity to explore additional ways of monitoring antibody production. Since over‐production of monoclonal antibody is characteristic of myeloma cells, being able to demonstrate elevation and monoclonality of the myeloma's antibody product is, in part, central to an accurate diagnosis, and to distinguish myeloma from polyclonal gammopathies. In addition to serum, antibodies can be quantified in urine, saliva, cerebrospinal fluid, and synovial fluid. Methods of analysis to be explored in this discussion include: IMMUNONEPHELOMETRY (for quantification of serum immunoglobulins), SERUM PROTEIN ELECTROPHORESIS (for separation and semi‐quantification of major protein fractions of serum), and IMMUNOFIXATION ELECTROPHORESIS (to analyze abnormal patterns of specific immunoglobulins).

Bone marrow/stem cell transplantation

Therapeutic strategies useful for replacing hematopoietic elements that are damaged or missing due to immunopathologic disease (e.g., immunodeficiency disease) or to immunoablative therapies (e.g., immunosuppressive cancer therapies). HEMATOPOIESIS - Differentiation and development of cells of the blood system originating from a common self‐renewing, multipotential progenitor (STEM CELL). A rich source of stem cells (CD34+ cells) are found in the bone marrow of the adult, but also at low frequency in the blood. Each cell type in the blood is derived from stem cells via unique programs of cytokine‐driven DIFFERENTIATION that dictate which genes are on or off in a cell. Bone marrow/stem cell transplant strategies are of great value when defects in hematopoiesis and blood cell functions occur due to bone marrow damage. Stem cell damage, congenital or due to exposure to drugs or irradiation, can be catastrophic. The earlier in the hematopoietic pathways that a defect occurs, the more wide‐spread the effects. Examples of bone marrow/stem cell transplant strategies: - Replace defective progenitors of immune system due to tumor‐ablative drug or irradiation treatments resulting in defective progenitors of immune/blood system. - Replace defective progenitors of immune/blood system due to immunodeficiency disease. - - Replace defective specific progenitors and/or their products via gene transfer therapies

A man in his twenties presents in the emergency center with shortness of breath and fatigue. He is also very pale. Two days earlier he was given penicillin for an infection. He had penicillin previously without problems and stated that he had "no allergy" to penicillin. Laboratory testing shows that antibodies to penicillin are present in the patient's serum and that he is breaking down his own red blood cells. He is diagnosed with immune hemolytic anemia. The patient has which type of hypersensitivity reaction? Type IV Type II Type I Type III

Type II

Goodpasture's syndrome

Type II hypersensitivity. Caused by the production and binding of antibodies reactive with the basement membranes of renal glomeruli (and in some cases of pulmonary alveoli). Primary antibody specificity is for the alpha-3 chain of type IV collagen of the basement membrane. Antibody binding triggers the complement cascade, as well as the activation of monocytes and neutrophils via their Fc receptors. Kidney biopsy specimens generally demonstrate a smooth layer of antibody/complement coating host tissue (detectable by immunofluorescent staining) that follows the paths of the various tubules (distinguishes from Type III hypersensitivity described below).

A 14-year-old girl presents to her pediatrician complaining of an itching, erythematous rash over her ear lobes that began after wearing a new pair of earrings she received as a gift. What is the most likely type of hypersensitivity response in this patient? Type III Type IV Type I Type II

Type IV

A 23‑year‑old woman demonstrated periodic episodes of fever and weakness. A facial rash was present at the time of diagnosis. There was tenderness in the joints of her fingers, wrists, and knees. Lab tests showed significant proteinuria. Analysis of her serum antibodies revealed high titers of autoantibodies against DNA. Blood smears demonstrated PMN leukocytes presenting displaced nuclei due to large quantities of ingested debris. The proteinuria and engorged PMNs are probably each the result of the most prominent characteristic of this disease that is best described by which of the following? kidney-reactive antibodies muscle-reactive antibodies antibody-antigen complexes complement depletion

antibody-antigen complexes

Cyclosporine and Tacrolimus are highly nephrotoxic agents used for transplant rejection prophylaxis that share a common mechanism of action. Which of the following is the mechanism of action of these immunosuppressant drugs? NF-kB inhibitor blocks nucleotide synthesis mTOR inhibitor blocks IL-2 receptor calcineurin inhibitor

calcineurin inhibitor

ECF (eosinophil chemotactic factor)

eosinophil chemotactic factor of anaphylaxis, leukotriene B4, complement complex (C5-C6-C7), interleukin 5, and histamine (though this has a narrow range of concentration)

Multiple Myeloma

the result of the malignant monoclonal transformation of a plasma cell. Transformed cells demonstrate unregulated growth, primarily in bone marrow; and they secrete, at a significant rate, the antibody for which they were programmed to produce due to the immunoglobulin gene rearrangements that occurred during progression through the early B cell developmental pathway. It is commonly thought that subsequent errors that occur during antibody isotype switch recombination may be intimately involved in the pathogenesis of multiple myeloma in a significant subset of patients by allowing translocation of DNA from another chromosome into the highly transcriptionally active switch region of the immunoglobulin heavy chain (IgH) locus (the 14q32 translocation appears to be one of the most frequent). Thus, if resulting inappropriate insertion involves coding elements associated with growth regulation, malignant transformation may result. The high frequency with which such translocations are observed in myeloma cells supports this model. Of note, since myeloma cells generally express a functional heavy chain, any illegitimate recombination accompanied by chromosomal translocations most likely occurs on the alternate chromosome. Typically, the overgrowth of monoclonal plasma cells in the bone marrow of multiple myeloma patients can crowd out normal hematopoietic activities, leading to anemia and resulting pallor, weakness, and fatigue. In addition, thrombocytopenia can lead to increased bleeding and bruising, while leukopenia can lead to immunocompromise and increased susceptibility to infection. In addition to impaired hematopoiesis, myeloma cells also interfere with cells that participate in bone remodeling and repair. Normally, there is a delicate balance provided by osteoclasts that break down old bone and osteoblasts that lay down new bone. Myeloma cells create circumstances that tip the balance in such a way as to inhibit the development and activation of osteoblasts, and to enhance the development and activation of osteoclasts. The result is the weakening of bones, making them highly susceptible to fracture. Accelerated bone decay can be monitored by measuring increased levels of calcium in the blood, and as fractures occur, elevated alkaline phosphatase may be observed indicating increased osteoblast activity attempting to provide new bone formation.

Kinetics of rejection are determined by

‐ Degree of GENETIC DIFFERENCE between graft donor/recipient (i.e., HLA differences) ‐ QUANTITY of tissue engrafted ‐ TYPE of tissue engrafted; immunogenicity and vascularization of various tissues may differ. ‐ IMMUNE STATUS of recipient ‐ PREVIOUS EXPOSURE of recipient to graft With no previous sensitization, FIRST SET REJECTION KINETICS is anticipated resulting in rejection within 10‐12 days; if recipient has been previously sensitized, accelerated acute rejection with SECOND SET REJECTION KINETICS is anticipated. DONOR‐RECIPIENT CROSSMATCHING (a test for serum antibodies in the recipient that react with donor transplant antigens) is a way to determine if previous exposure to transplant antigens has occurred.

Other Factors Associated with Autoimmune Diseases:

‐ Gender; numerous autoimmune diseases occur more frequently (3‐10x) in women than men. ‐ Target tissue products (immune activators/inhibitors) may influence the course of autoimmune responses and the severity of the pathology that they cause.

Immunosuppressive treatments to prevent graft rejection

‐ RADIATION ‐ CYTOTOXIC CHEMOTHERAPY Side‐effects that accompany immunosuppressive treatments often involve increased susceptibility to infection, and general or selective tissue toxicities.