Medical Mycology Test 5

What are the two types of Macs?

"Classically" activated macrophages (Th1) Activated by IFN-, IFN-, or by LPS Produce NO and other intermediates to destroy microorganisms Markers - NOS2 (iNOS) "Alternatively" activated macrophages (Th2) Activated by IL-4, IL-10, or IL-13 Produce polyamines and proline, which induce proliferation and collagen production, respectively Markers - ARG-1, YM1, CD206 (MR), FIZZ-1

Is CMI immunity necessary for fungal pathogens?

Yes, as individuals with defects in CMI are more susceptible to several fungal infections. Such as HIV and Solid organ transplant

After activation, what are the three different outcomes for T Cells?

Activation Survival Differentiation

How do DCs influence T Cells?

When an immature Dc becomes mature, the type and cytokines influence T Cell responses.

What are the three types of signals that APCs deliver to T Cells?

1. MHCII presents a peptide antigen to TCR 2. Co-stimulatory molecule interaction 3. Inflammatory cytokines

How are antigens presented?

1. Phagocytosis/Endocytosis/Pinocytosis 2. Phagolysosome forms 3. Degrades into antigens 4. Put on MHC Class II 5. Exocytosis occurs and the molecules can now present to B Cells.

How many TLRs are there? How many are on the endolysosome?

11 TLR 3, 7, 9, 10 are found on the Endolysosome

What is necessary to kill A. fumigatus?

Acidification by the lysosome.

What are some modes of intracellular killing?

Acidification: pH between 3.5-4.5 Antimicrobial Peptides: Defensins and cationic proteins Enzymes: Lysozyme and Acid Hydrolases Competitors: Lactoferin, Vitamin B12 Binding Protein ROS and NOS

What is adjuvant? How does it relate to vaccines?

Adjuvants are substances that enhance the immune response. A vaccine formulation contains antigenic components obtained or derived from the pathogen plus an adjuvant

Explain how TLRs, CLRs and other PRRs determine the functional plasticity of DCs?

All these receptors are found on DCs and allow for the DC to recognize PAMPs on pathogens and soluble PAMPs. This allows them to internalize and present the antigens to T Cells.

What could be responsible for the increased resistance in fluconazole?

Alterations in the drug import, the target (point mutations and over expression, in the efflux pumps (transporters and facilitators) and in the enzymes in the ergosterol biosyn pathway.

What is the molecular basis for immunogenicity on C. neoformans and Fungi?

C. neoformans and Fungi: The mannoproteins Mammals: Sialic Acid, Galactose and N-Acetylglucosamine

What are the two major T Cell populations?

CD4 T Cells: Have CD4 marker, stimulate other T cells. Recognize antigens in the context of MHC class II and multiply and develop into subtype cells that activate cell mediated immune responses: macrophages (Th1- and Th17-type) and humoral responses B cells (Th2-type) CD8 T Cells: Proliferate and differentiate to destroy infected or cancerous "self" cells and in graft rejection. Have CD8 marker and recognize antigens in the context of MHC class I

What are some experimental vaccines?

Candida spp. 1. Live Attenuated: PCA-2 (Kills severely immunocompromised) 2. Subunit: Mannan, Glucans Cryptococcus neoformans 1. Live Attenuated: IFNg 2. Subunit: GXM and Polysaccharide deacetylase Aspergillus fumigatus 1. Subunit: Glucans 2. Dendritic Cell Histoplasma capsulatum 1. Subunit: Hsp60 Blastomyces dermatitidis 1. Live attenuated: Bad1 Mutant 2. Subunit: Recombinant Bad1 Coccidioides spp. 1. Live Attenuated: Deleting two chitinase genes 2. Killed/Inactivated: FKS 3. Subunit: Antigen 2 4. DNA Vaccines: ELI 5. Dendritic Cell: Antigen 2 Paracoccidioides brasiliensis 1. DNA: gp43 Pneumocystis carinii 1. Subunit: gp120 and p55 2. DNA: Kexin

How does Th17 affect the outcome of fungal pathogens?

Candida: Patients with chronic mucocutaneous candidiasis (CMC) have reduced capacity to produce IL-17. Patients with deficiency in Th17 immunity are prone for CMC Vaccine immunity against blastomycosis, histoplasmosis and coccidioidomycosis is dependent on IL-17 production and Th17 response C. albicans and A. fumigatus: Inflammation and infection were exacerbated by a heightened Th17 response Cryptococcus: IL-17 does not appear to be necessary for protection, but may be involved in prevention of dissemination to the brain

What are some adaptor proteins and their receptors.

Card9> Dectin-1 MyD88> TLR2

Explain how resistance is clinical, in vitro and molecular?

Clinical: A patient who fails therapy and succumbs to infection In Vitro: A drug that is not active in the test tube. Molecular: A mechanism by which an organism develops resistance.

What are some examples of macs interaction with a pathogen?

Cryptococcus neoformans - classically activated macrophages associated with protection, alternatively activated macrophages associated with susceptibility Blastomyces and Paracoccidioides - macrophages activated with IFN- showed enhanced killing of the organisms (classically activated macrophages) Pneumocystis - alternatively activated macrophages are associated with protection

What does PRR recognition lead to?

Cytokine production Phagocytosis Killing Antigen presentation and adaptive initiation

Explain the order of activation?

DC > Naive T Cell > Diff > B Cell

Which cells kill by oxidative and non-oxidative means?

DC's and neutrophils (calprotection and defensins)

What bridges the innate and adaptive immunity?

DCs. They recognize fungal cells and present them to T Cells.

What are some of the innate immune cells of the body?

DCs: Phagocytic cells involved in the early immune response. Acquire antigens in the peripheral tissues. They express TLRs and C-Type Lectin. They are the bridge between innate and adaptive as they go to lymph nodes to activate T Cell. It is the only cell that presents to naive T Cells. Macs: Involved in both innate and adaptive response as they are able to phagocytize and kill pathogens. They have TLRs and are able to activate memory T Cells. Are able to polarize to different phenotypes in the adaptive response. Neutrophils: Granulocytes that are involved in phagocytosis and killing. The granules have certain cytotoxic molecules and can cause inflammation.

How do C-Type Lectin receptors play a role in fungal immunity?

Dectin 1: Binds β-1,3 glucans and signals through Syk (laminarin) DC-SIGN (SIGNR1): Binds high mannose carbohydrates (mannan) MR (CD206): Binds terminal mannose, fucose, and N-acetyl glucosamine (mannan and chitin)

What are some fungal PAMPs and the receptor they recognize?

Dectin-1/TLR: B-Glucan/Zymosan Dectin-1: B-Glucan Dectin-2: a-mannan Mincle: a-mannose MR: Mannose TLR2: Phospholipomannan TLR4: O-Linked Mannan

How do pathogens evade phagocytosis?

Depending on the type of Mac, some fungi are able to live and replicate inside of it. This allows for dissemination, H. capsulatum and C. neoformans. Candida and H. capsulatum can grow in Macs but are killed by neutrophils.

How does temperature of the host affect fungal pathogens?

Dimorphic pathogens express virulence factors, while non-pathogen species do not survive. C. albicans - transition to pathogenic hyphal form B. dermatiditis - transition to pathogenic yeast form H. capsulatum - transition to pathogenic yeast form

What are some features of Dectin-1?

Expressed on cells of the monocyte/macrophage and neutrophil lineages, and at lower levels on dendritic cells and a subpopulation of splenic T cells. Mediates host-cell recognition of β-glucan. Important for Saccharomyces, Candida, Coccidioides, Pneumocystis, and Aspergillus. Shown to mediate a variety of both TLR-dependent and TLR-independent antifungal cellular responses. Dectin KO mice are die easier than the WT to Candida infections.

What are some examples of azoles?

Fluconazole: Generally well tolerated, but limited spectrum (good activity against C. albicans, C. neoformans; no activity against moulds) Itraconazole: Against moulds but poor pharmacodynamics/kinetics Voriconazole: An improved fluconazole! Better spectrum (includes A. fumigatus and other moulds) Posaconazole: An improved itraconazole! Increased spectrum to include zygomycetes. Main problem: No IV formulation

How does CD4 provide protection?

Histoplasmosis Absence of CD4+ T cells converts a nonlethal 1° challenge of yeast into a lethal one Increased risk for progressive, disseminated histoplasmosis if CD4+ cells are <100 cells/μl Depletion of CD4+ T cells during 2° exposure to H. capsulatum reduces rate of clearance (it is cleared however) Pulmonary Pneumocystis Infection Progressive depletion of CD4+ T cells during HIV infection correlates with the risk of developing PCP infection Experimental manipulations resulting in the loss of CD4+ T cell number (KO mice, Ab ablation, ethanol fed mice) render mice susceptible to PCP Reconstitution of CD4+ T cells (clinically due to HAART or experimentally in animal models) results in a decrease in PCP Pulmonary B. dermatitidis infection CD4+ T cells are chiefly responsible for vaccine resistance in immune competent host Some compensatory mechanisms in immune deficient host (CD8+ T cell help) Pulmonary C. neoformans infection CD4+ T cell deficiency is associated with reduced clearance from lungs, increased dissemination to the CNS, and increased lethality CD4+ T cell depleted mice have increased susceptibility to experimental C. neoformans infection Pulmonary Coccidioides infection Severity of coccidioidomycosis directly correlates with depressed cell-mediated immune responses, Patients with a CD4 count under 250 are at highest risk for becoming infected Oral C. albicans infection Oropharyngeal candidiasis occurs in HIV+ patients with CD4 cell counts <200 Over 90% of patients will experience OPC during the progression to AIDS Symptomatic infection is associated with T cell immunosuppression HIV, transplants, corticosteroid therapy, chemotherapy

Where do DCs acquire the antigens from?

In the peripheral tissue, and then they take them to the lymphoid tissue.

How does 5-fly-Cytosine work?

Inhibits nucleic acid syn, but it is not used as a monotherapy and used mostly against crypto.

When does innate immunity activate? Adaptive?

Innate: 0-4hrs Adaptive: Greater than 96hrs Early induced innate response occurs in the meantime.

What is the difference between intrinsic resistance and acquired resistance?

Intrinsic is when the organism is naturally resistant. Can be species and strains. Acquired is when the organism develop resistance after exposure to the drug.

What organism is intrinsically resistant to Echinocandins? Acquired?

Intrinsic: C. neo Acquired. C. albicans/globrata

How do azoles work?

It binds to 14-a-demethylase to inhibit the production of ergosterol from lanosterol. Can have some cross reactivity for cytochrome p450 enzymes.

Is Th-17 important to fungal protection?

It is currently under study, but it can cause the recruitment of neutrophil granulocytes. Th17 cells are also able to recruit neutrophil granulocytes via the induction of CXCL8 production by macrophages through the combined activity of IL-17A, IL-17F and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as the direct production of CXCL8 and GM-CSF. IL-17A, IL-17F, IL-22 and IL-26 also stimulate epithelial cells to produce chemokine CC ligand (CCL)20 that results in the recruitment of Th17 cells themselves. IL-21 activates natural killer (NK) cells and CD8+ cytotoxic T cells, is a powerful B cell stimulatory factor, and also provides an autocrine amplification loop for Th17 cells themselves.

What is the advantage of lipid formulations of amphotericin B?

It is more expensive but it lessens nephrotoxicity.

Mac activation can be influenced by?

Macrophages can be differentially activated depending on recognition of the pathogen and the local environment

What are some features of Mannose Receptor?

MR (CD206) is a receptor for terminal mannose residues, found on macrophages and DCs Fungal cell walls contain long chains of terminal mannose residues This distinguishes fungal glycosylation from mammalian glycosylation

How does normal flora act as a first line of defense?

Microorganisms provide protection by: Cover binding sites Compete for nutrients Stimulate immune response

Is there good correlation between in vitro antifungal drug susceptibility testing and clinical outcome?

No, since most of the studies done have been on Candida in immunocompromised patients.

How do granules form?

One Th1 cells activate, it attracts and retains inflammatory cells in the lungs and organizes into a structure to contain the pathogen. Walling off preventing spread of organisms Failure to form granulomas may lead to dissemination. Granulomas may serve as reservoir for future infections/relapses. Granulomas are found in infections by Cryptococcus, Blastomyces, Histoplasma, and Coccidioides

What is needed to uptake C. neoformans?

Opsonization with either complement (C3b) or with anticapsular antibodies?

What is the importance of ROS?

Patients with Chronic Granulomatous Disease resulting from a deficiency in oxidant formation have increased susceptibility to aspergillosis, systemic candidiasis, and cryptococcosis. Myeloperoxidase (a lysosomal hemoprotein found in granules of neutrophils and monocytes but not macrophages) deficiency predisposes individuals to pulmonary candidiasis

What are some fungal PAMPs?

Plasma Membrane Mannoproteins GPI Anchor Proteins Chitin B-(1,3)-Glucans B-(1,6)-Glucans

What are some of the drug targets for specific antifungals?

Polyenes: Membrane function Echinocandins: Cell Wall Synthesis 5-Flucytosine: DNA Syn Azoles: Ergosterol Biosyn Terbinafine: Ergosterol

What are some of the main drug problems?

Polyenes: kidney toxicity, solubility Azoles: static drugs, resistance 5FC: resistance Echinocandins: recent, solubility, only IV

What are some of the functions of CD8 T Cells?

Production of IFN-γ cytokine Release of antimicrobial peptides (perforin, granzyme, etc.) Lysis of fungal-containing phagocytes Mediate protection against some fungi in the absence of CD4+ T cells

What is the difference between a prophylactic and therapeutic vaccine?

Prophylactic prevents disease, while the therapeutic treats disease.

CD4 T Cells are responsible for protection against what infections?

Pulmonary Blastomyces dermatiditis infection Oral Candida albicans infection Pulmonary Coccidioides spp. infection Pulmonary Cryptococcus neoformans infection Histoplasma capsulatum infection Pulmonary Pneumocystis jiroveci (PJP or PCP)

How common is resistance to Amphotericin B?

Rare but can occur is C. lusitaniae, C. Krusei and A. terreus .

What is the antifungal activity of CD8 T Cells?

Required for optimal elimination of fungal pathogens Assist in clearance of fungal infections Depletion of CD4+ and CD8+ T cells results in increased fungal burden compared to CD4+ T cell depletion alone Oropharyngeal candidiasis Vaccine immunity to pulmonary histoplasmosis Clearance of pulmonary cryptococcosis in CD4+ T cell-deficient mice

What are some differences between innate and adaptive?

Response time Specificity Diversity: Adaptive is highly diverse. Memory Self Discrimination Soluble Components: Antimicrobials/Complement v. Antibodies

How do fungal pathogens evade the host immune system?

Shielding of PAMPS Modulation of Inflammatory signals Shedding of decoy components (GXM and gpA) Persistence of Intracellular environments Complement evasion

What are some cytokines and TF that T Cells secrete?

TH1: Il-2, IFNg, Il-2 TH2: IL-4, Il-5 TH17: IL-6, IL-17

Which TLRs are important to fungal infections?

TLR 2/4: In C. neoformans infections, there is a major role for MyD88 and minor roles for TLR4 and TLR2 in the response. Decreased survival of MyD88(-/-) mice correlated with increased lung CFU and serum and lung GXM levels. Macrophage response to C. posadasii spherules is dependent on TLR2. TLR 9: A. fumigatus DNA potently stimulates the production of pro-inflammatory cytokines in DCs through TLR9. TLR9-/- mice survive longer than WT mice following challenge with Candida albicans hyphae and A. fumigatus conidia. DNA from C. neoformans activates myeloid DCs via a TLR9-dependent pathway. MyD88 TLR Dependent Pathway: Required for resistance to C. albicans, A. fumigatus, C. neoformans, and C. posadii. MyD88 adaptor molecule signals through members of the IL-1R/TLR superfamily - IL-1R, TLR2, TLR4, and TLR9 Role of the individual receptors varies depending on fungal species, fungal morphotypes, and route of infection. Individual TLRs and IL-1R activate specialized antifungal effector functions on neutrophils, which correlates with susceptibility to infection. MyD88-dependent signaling on dendritic cells is crucial for priming antifungal Th1 responses

Which CD4 T Cell is important for antifungal immunity?

Th1 since it can: IFN-γ, GM-CSF, TNF-α, and IL-12 IL-12, TNF-α drive IFN-γ production Th1-cytokine production key to activation of phagocytes at site of infection Stimulates leukocyte migration, adherence, phagocytosis and phagocyte oxidative killing, MHC Class I & II expression, enhances antigen presentation

Which CD4 T Cell are not associated with protection?

Th2 Th2 Type cytokines: IL-4, IL-5, IL-13 Th2-dominant cytokine profile in the saliva of those with HIV or OPC

How is antifungal susceptibility tested for?

The CLSO made a standardized testing called MIC (Min Inhibitory Concentration). This is based on: Inoculum Temp Media time (48hrs) Standards Drug Resuspension Reproducibility

What is the first line of defense?

The skin

What are some polymorphisms in PRRs?

There are two different Dectin 1 receptors: 1. Y2235: Reduced zymosan binding and IFNg production which leads to resistance to oropharyngeal candidiasis. 2. Y23X: Decreased surface expression of the molecule and b-glucan binding capacity along with impaired cytokine production. This leads to chronic mucocutaneous candidiasis and invasive aspergillosis leading to more susceptibility.

What are PRRs?

These are receptors on the host cell that recognize PAMPs, which can be on the pathogen itself or of tissue damage.

What happens when Toll was knocked out in Drosophila?

They became infected with Aspergillus fumigatus. TLRs use MyD88

How do polyenes and nystatin work?

They bind to ergosterol in the cell membrane which destabilizes the membrane. This causes leakage of intracellular cations.

What are other sterol biosyn inhibitors?

They inhibit ergosterol biosyn at other steps. EGR1: Terbinafine is Lamisil and Tolnaftate is Tinactin. EGR2/EGR24: Amorolfine and Fenpropimorph.

What do TLRs do when the detect a PAMP?

They signal to the nucleus to start gene expression to make chemokines and cytokines.

How do Echinocandins work?

They taget the b-glucan synthesis to inhibit cell wall formation. They target the b-(1,3) glucan synthase. This leads to osmotic fragility. Crypto is immune. Usually well tolerated with few side effects.

Why are boosters necessary?

To maximize the efficiency.

What is the best way to treat fungal infections?

To restore the host immune system

What are the different types of vaccines?

Traditional: 1. Live Attenuated- The person is immunized with live pathogen that has had its virulence reduced by chemicals, temp, or serial passaging. This is a low cost method that elicits a strong immune response but it has a limited shelf life and can revert to its virulent form. 2. Killed/Inactivated: The pathogen has been killed or inactivated by a chemical, such as formaldehyde, temperature, irradiation pr lysis. There is no chance of virulence returning and has a long shelf life. It is more expensive to make. New Vaccines: 1. Subunit- Only part of the pathogen is used, usually the immunogenic surface molecules. These epitopes are collected and put in a vaccine. 2. DNA- Insert the DNA of a pathogen into the host cell. The host's own cells take up these genes and express the encoded proteins which then trigger an immune response. When the pathogen is present the immune system launches an attack against the pathogen. 3. Vector- Viruses are used as vehicles to carry the vaccine and trigger an immune response. 4. Peptide- Mimic epitopes of pathogens that have been chemically syn. They molecules were relatively small and it is of utmost importance that their 3D structure be similar to the native epitopes of the pathogen. You can find the epitopes via epitope mapping (B/T Cell epitopes).

What are vaccines?

Vaccines are antigenic preparations that administered in small quantities stimulate our immune system to subsequently evoke a "memory" response to activate defense mechanisms and confer protection against an invading pathogen

What happens when the normal flora is disrupted?

When the balance is upset some fungi can replicate without restraint and cause an inflammatory response. For instance Vulvovaginal candidiasis.

What is the problem with amphotericin B?

While it is able to make a colloidal solution, and is effective against most human fungal pathogens, it is very toxic.

Are there any antifungal vaccines?

there is no commercially available vaccine