The Effects of Physical and Chemical Modulating Agents on the Induction of Vaccine-Induced Immune Responses

After the development of the viral-based prostate cancer vaccine, Ad5-PSA, much research has been orientated to help enhance the induced immune response by combining the vaccine with physical and chemical modulating agents, more specifically the polymers polyethylenimine (PEI), chitosan, and chitosan coated with CD3 complex antibodies; all previously shown to stimulate an immune response as isolated gene carriers. To compare the vaccine-induced immune responses between the naked vaccine and the polymer-vaccine combinations, a mouse model using the ovalbumin- specific Ad-OVA vaccine was tested using intracellular cytokine staining (ICS), tetramer staining, and cytotoxic T-cell lymphocyte assays to measure the activation of CD8+ T-cells, interferon gamma proteins (INFƒ×), and the induced cytotoxicity to ovalbumin. The Ad-OVA vaccine combined with both chitosan and chitosan with CD3 complex antibodies, both natural polymers, were found to induce similar immune responses to the naked vaccine while the vaccine combined with the synthetic polymer, PEI, diminished the immune response.

The identification and characterization of the Staphylococcus aureus microbial immunomodulatory molecules (MIMS) Map and Efb

Staphylococcus aureus is an opportunistic pathogen that is a major health threat in the clinical and community settings. An interesting hallmark of patients infected with S. aureus is that they do not usually develop a protective immune response and are susceptible to reinfection, in part because of the ability of S. aureus to modulate host immunity. The ability to evade host immune responses is a key contributor to the infection process and is critical in S. aureus survival and pathogenesis. This study investigates the immunomodulatory effects of two secreted proteins produced by S. aureus, the MHC class II analog protein (Map) and the extracellular fibrinogen-binding protein (Efb). Map has been demonstrated to modulate host immunity by interfering with T cell function. Map has been shown to significantly reduce T cell proliferative responses and significantly reduce delayed-type hypersensitivity responses to challenge antigen. In addition, the effects of Map on the infection process were tested in a mouse model of infection. Mice infected with Map− S. aureus (Map deficient strain) presented with significantly reduced levels of arthritis, osteomyelitis and abscess formation compared to mice infected with the wild-type Map+S. aureus strain suggesting that Map−S. aureus is much less virulent than Map+S. aureus. Furthermore, Map−S. aureus-infected nude mice developed arthritis and osteomyelitis to a severity similar to Map +S. aureus-infected controls, suggesting that T cells can affect disease outcome following S. aureus infection and Map may attenuate cellular immunity against S. aureus. The extracellular fibrinogen-binding protein (Efb) was identified when cultured S. aureus supernatants were probed with the complement component C3. The binding of C3 to Efb resulted in studies investigating the effects of Efb on complement activation. We have demonstrated that Efb can inhibit both the classical and alternative complement pathways. Moreover, we have shown that Efb can inhibit complement mediated opsonophagocytosis. Further studies have characterized the Efb-C3 binding interaction and localized the C3-binding domain to the C-terminal region of Efb. In addition, we demonstrate that Efb binds specifically to a region within the C3d fragment of C3. This study demonstrates that Map and Efb can interfere with both the acquired and innate host immune pathways and that these proteins contribute to the success of S. aureus in evading host immunity and in establishing disease. ^

Capsule gene regulation in Bacillus anthracis and implications for virulence

The poly-D-glutamic acid capsule of Bacillus anthracis is considered essential for lethal anthrax disease. Yet investigations of capsule function have been limited primarily to attenuated B. anthracis strains lacking certain genetic elements. In work presented in this thesis, I constructed and characterized a genetically complete (pXO1 + pXO2+) B. anthracis strain (UT500) and isogenic mutants deleted for two previously identified capsule gene regulators, atxA and acpA, and a newly-identified regulator, acpB. Results of transcriptional analysis and microscopy revealed that atxA controls expression of the first gene of the capsule biosynthesis operon, capB, via positive transcriptional regulation of acpA and acpB. acpA and acpB appear to be partial functional homologs. Deletion of either gene alone has little effect on capsule synthesis. However, a mutant deleted for both acpA and acpB is noncapsulated. Thus, in contrast to previously published models, my results suggest that atxA is the master regulator of cap gene expression in a genetically complete strain. A detailed transcriptional analysis of capB and the regulatory genes was performed to establish the effects of the regulators and CO2/bicarbonate on specific mRNAs of target genes. CO2/bicarbonate is a well-established signal for B. anthracis capsule synthesis in culture. Taqman RT-PCR results indicated that growth in the presence of elevated CO2 greatly increased expression of acpA, acpB and capB but not atxA. 5′ end mapping of capB and acpA revealed atxA-regulated and atxA-independent transcriptional start sites for both genes. All atxA-regulated start sites were also CO2-regulated. A single atxA-independent start site was identified 5 ′ of acpB. However, RT-PCR analysis indicated that capD and acpB are co-transcribed. Thus, it is likely that atxA-mediated control of acpB expression occurs via transcriptional activation of the atxA-regulated start sites of capB. Finally, I examined the contribution of the B. anthracis capsule to virulence. The virulence of the parent strain, mutants deleted for the capsule biosynthesis genes ( capBCAD), and mutants missing the capsule regulator genes was compared using a mouse model for inhalation anthrax. The data indicate that in this model, capsule is essential for virulence. Mice survived infection with the noncapsulated capBCAD and acpA acpB mutants. These mutants initiated germination in the lung, but did not disseminate to the spleen. The acpA mutant had an LD50 value similar to the parent strain and was able to disseminate and cause lethal infection. Unexpectedly, the acpB mutant had a higher LD 50 and a reduced ability to disseminate. During in vitro culture, the acpB single mutant produces capsule and toxin similar to the parent strain. It is likely that acpB regulates the expression of downstream genes that contribute to the virulence of B. anthracis. ^

B cells in ADA deficiency

Deficiency of the enzyme adenosine deaminase (ADA) results in severe lymphopenia in humans. Mice with an inactivating mutation in the ADA gene also exhibit profound lymphopenia, as well as pulmonary insufficiency and ribcage abnormalities. In fact, the mouse model has a phenotype that is remarkably similar to that of the human disease, making the mice valuable tools for unraveling the mechanism of lymphocyte destruction in absence of this housekeeping gene. T cell deficiency in ADA deficiency has been extensively studied by others, revealing a block in early thymocyte development. In contrast, our studies revealed that early B cell development in the bone marrow is normal. ADA-deficient mice, however, exhibit profound defects in germinal center formation, preventing antigen-dependent B cell maturation in the spleen. ADA-deficient spleen B cells display significant defects in proliferation and activation signaling, and produce more IgM than their normal counterparts, suggesting that extrafollicular plasmablasts are overrepresented. B cells from ADA-deficient mouse spleens undergo apoptosis more readily than those from normal mouse spleens. Levels of ADA's substrates, adenosine and 2′-deoxyadenosine, are elevated in both bone marrow and spleen in ADA-deficient mice. S ′-adenosyihomoeysteine hydrolase (SAH hydrolase) activity is significantly inhibited in both locales, as well. dATP levels, though, are only elevated in spleen, where B cell development is impaired, and not in bone marrow, where B cell ontogeny is normal. This finding points to dATP as the causative agent of lymphocyte death in ADA deficiency. ADA deficiency results in inhibition of the enzyme ribonucleotide reductase, thereby depleting nucleoside pools needed for DNA repair. Another mouse model that lacks a functional gene encoding a protein involved in DNA repair and/or cell cycle checkpoint regulation, p53-binding protein 1, exhibits blocks in T and B cell development that are similar to those seen in ADA-deficient mice. Unraveling the mechanisms of lymphocyte destruction in ADA deficiency may further understanding of lymphocyte biology, facilitate better chemotherapeutic treatment for lymphoproliferative diseases, and improve gene and enzyme therapy regimens attempted for ADA deficiency. ^

Assessment of cone beam computed tomography techniques for imaging lung damage in mice in vivo

Lung damage is a common side effect of chemotherapeutic drugs such as bleomycin. This study used a bleomycin mouse model which simulates the lung damage observed in humans. Noninvasive, in vivo cone-beam computed tomography (CBCT) was used to visualize and quantify fibrotic and inflammatory damage over the entire lung volume of mice. Bleomycin was used to induce pulmonary damage in vivo and the results from two CBCT systems, a micro-CT and flat panel CT (fpCT), were compared to histologic measurements, the standard method of murine lung damage quantification. Twenty C57BL/6 mice were given either 3 U/kg of bleomycin or saline intratracheally. The mice were scanned at baseline, before the administration of bleomycin, and then 10, 14, and 21 days afterward. At each time point, a subset of mice was sacrificed for histologic analysis. The resulting CT images were used to assess lung volume. Percent lung damage (PLD) was calculated for each mouse on both the fpCT (PLDfpcT) and the micro-CT (PLDμCT). Histologic PLD (PLDH) was calculated for each histologic section at each time point (day 10, n = 4; day 14, n = 4; day 21, n = 5; control group, n = 5). A linear regression was applied to the PLDfpCT vs. PLDH, PLDμCT vs. PLDH and PLDfpCT vs. PLDμCT distributions. This study did not demonstrate strong correlations between PLDCT and PLDH. The coefficient of determination, R, was 0.68 for PLDμCT vs. PLDH and 0.75 for the PLD fpCT vs. PLDH. The experimental issues identified from this study were: (1) inconsistent inflation of the lungs from scan to scan, (2) variable distribution of damage (one histologic section not representative of overall lung damage), (3) control mice not scanned with each group of bleomycin mice, (4) two CT systems caused long anesthesia time for the mice, and (5) respiratory gating did not hold the volume of lung constant throughout the scan. Addressing these issues might allow for further improvement of the correlation between PLDCT and PLDH. ^

Abnormal expression of E2F1 and E2F4 results in hyperproliferation of the ependyma and lethal hydrocephalus

The proliferative role of E2F has been under investigation for several years. However, while it is known that E2F1 and E2F4 play a part in development and differentiation, research has not been centered on determining the exact functions these E2Fs play in brain development, given there high expression levels throughout embryogenesis. A GFAP-E2F1 mouse model directing human E2F1 transgene expression to glial cells, such as ependymal cells, was used in the present study in combination with an E2F4 mutant mouse model. Interestingly, 20% of tgE2F1; E2F4 null mice developed a phenotype consisting of domed head, hunched posture, seizures, tremors, hyperactivity or hypeactivity, dysnea, and low body weight. These mice died during the first three weeks of severe hydrocephalus. Similarly, tgE2F1; E2F4 heterozygous mice also develop severe hydrocephalus, although this occurs at 6 weeks at a 2% frequency. Pathological examination of the brains of those animals uncovered enlarged cerebral ventricles with marked thinning of the cerebral cortices, confirming the diagnosis of three-ventricle hydrocephalus, and the absence of tumors. Careful examination of the aqueduct shows an excess of proliferating cells that may cause a blockage of CSF. Of significance, 44% of ependymal cells in hydrocephalic tgE2F1;E2F4-/- mouse brains were positive for BrdU incorporation. Studies determining the molecular rationale for the hydrocephalic phenotype suggest proliferative ependymal cells may not be exclusively related to dysregulated cell cycle in conjuction with E2F activity. Due in part to the deficiency of E2F4 in this mouse model, we find that differentiation of these ependymal cells is not complete and instead undergoes maturation arrest. This suggestion is confirmed by the expression of genes found in neural stem cells or precursor cell populations, in the ependymal cell region of tgE2F1; E2F4-/-. Therefore, from this study, we conclude that dysregulated E2F1 expression in combination with deficient E2F4 expression results in an undifferentiated ependymal cell population that is hyperproliferative in the ventricular system causing an impediment of CSF circulation. It is further concluded that normal E2F1 and E2F4 expression in brain development is crucial for the proper formation and function of the ventricular system.^

A novel role for neural crest cells in thymus organogenesis

Classical ablation studies have shown that neural crest cells (NCC) are critical for thymus organogenesis, though their role in this process has never been determined. We have used a mouse model deficient in NCC near the thymus rudiment to investigate the role of NCC in thymus organogenesis. Splotch mice exhibit a lack of NCC migration due to mutation in the gene encoding the transcription factor Pax 3. Homozygous mutants, designated Pax3Sp/Sp, display a range of phenotypes including spina bifida, cardiac outflow tract deformities, and craniofacial deformities. Pax3Sp/Sp, mice have also been reported to have hypoplastic and abnormal thymi, which is consistent with the expected result based on the classical ablation studies. However, in contrast to the dogma, we find that the thymus lobes in Pax3Sp/Sp, mice are even larger in size than those of littermate controls, although they fail to migrate and are therefore ectopic. Differentiation of the thymic epithelial compartments occurs normally, including the ability to import hematopoietic precursors, until the embryos die at embryonic day E13.0. We also investigated the patterning of the third pharyngeal pouch which gives rise to both the thymus and the parathyroid. Using RNA probes to detect expression of transcription factors exclusively expressed in the ventral, thymus- or dorsal, parathyroidfated domains of the E11.5 third pouch, we show that the parathyroid domain is restricted and the thymus-fated domain is expanded in Pax3Sp/Sp, embryos. Furthermore, mixing of the boundary between these domains occurs at E12.0. These results necessitate reconsideration of the previously accepted role for NCC in thymus organogenesis. NCC are not required for outgrowth of the thymus up to E13.0, and most strikingly, we have discovered a novel role for NCC in establishing parathyroid versus thymus fate boundaries in the third pharyngeal pouch. ^

Regulation of T cell function by complement C5A in mice during mycobacterial infection

Tuberculosis is the leading cause of death in the world due to a single infectious agent, making it critical to investigate all aspects of the immune response mounted against the causative agent, Mycobacterium tuberculosis , in order to better treat and prevent disease. Previous observations show a disparity in the ability to control mycobacterial growth between mouse strains sufficient in C5, such as C57BL/6 and B10.D2/nSnJ, and those naturally deficient in C5, such as A/J and B10.D2/nSnJ, with C5 deficient mice being more susceptible. It has been shown that during M. tuberculosis infection, C5 deficient macrophages have a defect in production of interleukin (IL)-12, a cytokine involved in the cyclical activation between infected macrophages and effector T cells. T cells stimulated by IL-12 produce interferon (IFN)-γ, the signature cytokine of T helper type 1 (Th1) cells. It is known that a cell-mediated Th1 response is crucial for control of M. tuberculosis in the lungs of humans and mice. This study demonstrates that murine T cells express detectable levels of CD88, a receptor for C5a (C5aR), following antigen presentation by macrophages infected with mycobacteria. T cells from C5 deficient mice infected with M. tuberculosis were found to secrete less IFN-γ and had a reduced Th1 phenotype associated with fewer cells expressing the transcription factor, T-box expressed in T cells (T-bet). The altered Th1 phenotype in M. tuberculosis infected C5 deficient mice coincided with a rise in IL-4 and IL-10 secretion from Th2 cells and inducible regulatory T cells, respectively. It was found that the ineffective T cell response to mycobacteria in C5 deficient mice was due indirectly to a lack of C5a via poor priming by infected macrophages and possibly by a direct interaction between T cells and C5a peptide. Therefore, these studies show a link between the cells of the innate and adaptive arms of the immune system, macrophages and T cells respectively, that was mediated by C5a using a mouse model of M. tuberculosis infection. ^

An innovative approach to impacting student academic achievement and attitudes: Pilot study of the HEADS UP Virtual Molecular Biology Lab

Objective. To evaluate the HEADS UP Virtual Molecular Biology Lab, a computer-based simulated laboratory designed to teach advanced high school biology students how to create a mouse model. ^ Design. A randomized clinical control design of forty-four students from two science magnet high schools in Mercedes, Texas was utilized to assess knowledge and skills of molecular laboratory procedures, attitudes towards science and computers as a learning tool, and usability of the program. ^ Measurements. Data was collected using five paper-and-pencil formatted questionnaires and an internal "lab notebook." ^ Results. The Virtual Lab was found to significantly increase student knowledge over time (p<0.005) and with each use (p<0.001) as well as positively increase attitudes towards computers (p<0.001) and skills (p<0.005). No significant differences were seen in science attitude scores.^ Conclusion. These results provide evidence that the HEADS UP Virtual Molecular Biology Lab is a potentially effective educational tool for high school molecular biology education.^

The role of CD1d in adipogenesis

Atherosclerosis-associated coronary heart disease is the number one cause of death in the western society, and often triggered by metabolic disorders, such as hyperlipidemia, hypertension, obesity, and diabetes. The CD1 molecules are a group of evolutionarily conservative transmembrane proteins that have recently emerged as novel lipid-binding and transporting molecules. The current study was aimed at illustrating the role of CD1d in regulation of lipid metabolism and adipogenesis. In stably transfected smooth muscle cells where CD1d is overexpressed via a pCMV promoter, high levels of binding of the oxysterol 7-ketocholesterol was found. Adipogenic treatment induces the cells to transdifferentiate into an adipocyte-like morphology. This adipocyte morphology of CD1d transfected SMCs strongly resembles that of the pre-adipocytes 3T3-L1 cells grown in the same adipogenic media. Adipogenic treatment of CD1d transfected 3T3-L1 cells led to an increased accumulation of lipids compared to mock transfected cells. Induction of adipogenic gene expression and activation, such as PPARγ and lipoprotein lipase (LPL), was achieved in adipogenically treated smooth muscle cells as well as 3T3-L1 cells with overexpression of CD1d. For determination of the role of CD1d in regulation of adipogenesis, a CD1d transgenic mouse strain was created using the CD1d-smooth muscle promoter construct. Compared to wild type control mice matched in age and sex, the transgenic mice show an age-dependent increase in abdominal and visceral fat tissue. Histopathological examination demonstrated marked enlargement of adipocytes in the transgenic fat tissue which otherwise remained a normal fat tissue structure. Immunohistochemical analysis of CD1d expression in the fat tissue revealed much stronger membrane CD1d immunostaining in the transgenic tissue than the wild type fat tissue. Under normal chow diets, CD1d-transgenic mice also developed fatty livers. In conclusion, CD1d serves as a regulator of lipid metabolism, which may transducer signals from oxysterols to induce expression of genes important in lipogenesis. These experimental results point to a novel mechanism by which CD1d mediates lipid metabolism in adipose tissue and contributes to the development of obesity. ^

Role of telomere dysfunction, DNA damage response and p53 mutations in tumorigenesis and aging

The ends of eukaryotic chromosomes are protected by specialized ribonucleoprotein structures termed telomeres. Telomeres protect chromosomes from end-to-end fusions, inappropriate repair and degradation. Disruption of this complex activates an ATM/ATR DNA damage response (DDR) pathway. One component of the complex is the Protection Of Telomeres 1 (POT1) protein, an evolutionarily conserved protein which binds single-stranded 3' overhang and is required for both chromosomal end protection and telomere length regulation. The mouse contains two POT1 orthologs, Pot1a and Pot1b. Here we show that both proteins colocalize with telomeres through interaction with the adapter protein TPP1. In addition, compared to Pot1a, the OB-folds of Pot1b possess less sequence specificity for telomeres. Disruption of POT1 proteins result in telomere dysfunction and activation of an ATR-dependent DDR at telomeres, suggesting that this response is normally suppressed by POT1 binding to the single-stranded G-overhang. ^ Telomeres are maintained by telomerase, and its absence in somatic cells results in telomere progressive loss that triggers the activation of p53. Telomere dysfunction initiates genomic instability and induces both p53-dependent replicative senescence and apoptosis to suppress tumorigenesis. In the absence of functional p53, this genomic instability promotes cancer. It was previously not known which aspect of the p53 dependent DNA damage response is important to suppress tumorigenesis initiated by dysfunctional telomeres. The p53R172P knock-in mouse, which is unable to induce apoptosis but retains intact cell cycle arrest/cellular senescence pathways, allowed us to examine whether p53-dependent apoptosis is a major tumor suppression pathway initiated in the setting of telomere dysfunction. Spontaneous tumorigenesis remains potently suppressed in late generation telomerase null mice possessing the p53P/P mutation. These results suggest that suppression of spontaneous tumorigenesis initiated by dysfunctional telomeres requires activation of a p53-dependent senescence pathway. In addition, we used another knock-in mouse model with a p53R172H (p53H) point mutation to test the hypothesis that telomere dysfunction promotes chromosomal instability and accelerates the onset of tumorigenesis in vivo in the setting of this most common gain-of-function mutation in the human Li Fraumeni cancer syndrome. We unexpectedly observed that telomerase null mice possessing dysfunctional telomeres in the setting of the p53H/+ mutation develop significantly fewer tumors, die prematurely and exhibit higher level of cellular senescence, apoptosis and elevated genomic instability compared to telomerase intact p53H/+ and telomerase null p53+/+ mice. These contrasting results thus link cancer and aging to the functional status of telomeres and the integrity of the p53 pathway. ^

Acetate metabolism and the control of environmental pH in Candida albicans and Saccharomyces cerevisiae

Candida albicans is the most common opportunistic fungal pathogen of humans. The balance between commensal and pathogenic C. albicans is maintained largely by phagocytes of the innate immune system. Analysis of transcriptional changes after macrophage phagocytosis indicates the C. albicans response is broadly similar to starvation, including up-regulation of alternate carbon metabolism. Systems known and suspected to be part of acetate/acetyl-CoA metabolism were also up-regulated, importantly the ACH and ACS genes, which manage acetate/acetyl-CoA interconversion, and the nine-member ATO gene family, thought to participate in transmembrane acetate transport and also linked to the process of environmental alkalinization. ^ Studies into the roles of Ach, Acs1 and Acs2 function in alternate carbon metabolism revealed a substantial role for Acs2 and lesser, but distinct roles, for Ach and Acs1. Deletion mutants were made in C. albicans and were phenotypically evaluated both in vitro and in vivo. Loss of Ach function resulted in mild growth defects on ethanol and acetate and no significant attenuation in virulence in a disseminated mouse model of infection. While loss of Acs1 did not produce any significant phenotypes, loss of Acs2 greatly impaired growth on multiple carbon sources, including glucose, ethanol and acetate. We also concluded that ACS1 and ACS2 likely comprise an essential gene pair. Expression analyses indicated that ACS2 is the predominant form under most growth conditions. ^ ATO gene function had been linked to the process of environmental alkalinization, an ammonium-mediated phenomenon described here first in C. albicans. During growth in glucose-poor, amino acid-rich conditions C. albicans can rapidly change its extracellular pH. This process was glucose-repressible and was accompanied by hyphal formation and changes in colony morphology. We showed that introduction of the ATO1G53D point mutant to C. albicans blocked alkalinization, as did over-expression of C. albicans ATO2, the only C. albicans ATO gene to lack the conserved N-terminal domain. A screen for alkalinization-deficient mutants revealed that ACH1 is essential for alkalinization. However, addition of acetate to the media restored alkalinization to the ach1 mutant. We proposed a model of ATO function in which Atos regulated the cellular co-export of ammonium and acetate. ^

Evaluation of the C5a anaphylatoxin and its receptor (CD88) in allergic lung disease

Allergic asthma is characterized by airflow obstruction, airway hyperresponsiveness (AHR) and chronic airway inflammation. We and others have reported that complement component C3 and the anaphylatoxin C3a receptor promote while C5 protects against the development of the biological and physiological hallmarks of allergic lung disease in mice. In this study, we assessed if the protective responses could be mediated by C5a, an activation-induced C5 cleavage product. Mice with ablation of the C5a receptor (C5aR) either by genetic deletion or by pharmacological blockade exhibited significantly exacerbated AHR compared to allergen-challenged wild-type (WT) mice. However, there were no significant differences in many of the other hallmarks of asthma such as airway infiltration by eosinophils or lymphocytes, pulmonary IL-4-producing cell numbers, goblet cell metaplasia, mucus secretion or total serum IgE levels. In contrast to elevated AHR, numbers of IL-5 and IL-13 producing pulmonary cells, and IL-5 and IL-13 protein levels, were significantly reduced in allergen-challenged C5aR-/- mice compared to allergen-challenged WT mice. Administration of a specific cysteinyl leukotriene receptor 1 (cysLT1R) antagonist before each allergen-challenge abolished AHR in C5aR-/- as well as in WT mice. Pretreatment with a C3aR antagonist dose-dependently reduced AHR in allergen-challenged WT and C5aR-/- mice. Additionally, allergen-induced upregulation of pulmonary C3aR expression was exaggerated in C5aR-/- mice compared to WT mice. In summary, deficiency or antagonism of C5aR in a mouse model of pulmonary allergy increased AHR, which was reversed or reduced by blockade of the cysLT1R and C3aR, respectively. In conclusion, this study suggests that C5a and C5aR mediate protection against AHR by suppressing cysLT and C3aR signaling pathways, which are known to promote AHR. This also supports important and opposing roles of complement components C3a/C3aR and C5a/C5aR in AHR. ^

Role of T cell response during vaccine immunity to tuberculosis

Tuberculosis remains one of the leading causes of death in man due to a single infectious agent. An estimated one-third of the world's population is infected with the causative agent, Mycobacterium tuberculosis (Mtb), despite the availability of the widely used vaccine, BCG. BCG has significantly varying protection rates with the lowest level of protection seen with the most common form of TB, adult pulmonary TB. Thus, numerous studies are being conducted to develop a more efficient vaccine. The ideal candidate vaccine would possess the ability to induce a solid and strong Th1 response, as this is the subset of T cells primarily involved in clearance of the infection. A novel vaccine should also induce such a response that may be recalled and expanded upon subsequent infection. Our group has introduced a mutant of a virulent strain of Mtb which lacks a component of the immunogenic antigen 85 complex (Ag85). Our vaccine, ΔfbpA, does not secrete the fibronectin binding protein Ag85A, and this has shown to lead to its attenuation in both murine macrophages and mice. Previous studies have also proven that ΔfbpA is more protective in mice than BCG against virulent aerosol challenge with Mtb. This study addresses the mechanisms of protection observed with ΔfbpA by phenotyping responding T cells. We first evaluated the ability of dendritic cells to present the mycobacteria to naïve T cells, an in vitro mock of primary immunization. We also measured the response of primed T cells to macrophage-presented mycobacteria to interpret the possible response of a vaccinated host to a boost. We concluded that ΔfbpA can elicit a stronger Th1 response compared to BCG in vitro, and further observed that this enhanced response is at least partly due to the presence of proteins encoded by a region of the genome absent in all strains of BCG. Finally, we observed this heightened Th1 response in the mouse model after primary vaccination and a virulent aerosol challenge. The cytolytic T cell response was also measured after virulent challenge and was found to be superior in the ΔfbpA-treated group when compared to the BCG group. ^

Alternative carbon metabolism: Virulence and regulation in Candida albicans

The interaction between C. albicans and innate immune cells is a key determinant to disease progression. Transcriptional profiling showed that C. albicans responds to macrophage phagocytosis by inducing pathways required for alternative carbon metabolism (beta-oxidation, the glyoxylate cycle, and gluconeogenesis), suggesting these pathways are important for virulence of C. albicans. ^ We have shown that deleting key genes (FOX2, FBP1) in these pathways results in virulence defects in an in vivo mouse model for systemic infection. Like icl1Δ/Δ mutants, fbp1Δ/Δ mutants are severely attenuated and fox2Δ/Δ mutants are mildly but significantly attenuated, indicating that carbon starvation is a relevant stress in vivo. ^ However, fox2Δ/Δ mutants also had unexpected phenotypes on certain carbon sources, unlike the case in Saccharomyces cerevisiae, suggesting these pathways are regulated differently in C. albicans. To test this, we identified the C. albicans regulators of these pathways based on those from S. cerevisiae and Aspergillus nidulans. ^ C. albicans has a partly conserved framework, but lacks two regulators (Oaf1p, Pip2p) controlling peroxisome biogenesis and beta-oxidation genes in yeast. Instead, C. albicans has a homolog, CTF1, of the A. nidulans fatty acid catabolism regulators FarA and FarB. We have shown that CTF1 is needed for growth on oleate (like FarA and FarB), expression of beta-oxidation and glyoxylate cycle genes, and full virulence. No function for CTF1 has previously been identified in C. albicans. Our data demonstrate a role for alternative carbon metabolism in the virulence of C. albicans and suggest that the regulation of these pathways is a mixture of the filamentous fungi and budding yeast systems. ^