Serine deamination by human serine racemase synergizes with antibiotics to curtail the replication of Chlamydia trachomatis.

The obligate intracellular bacterium, Chlamydia trachomatis, has evolved to depend on its human host for many metabolites, including most amino acids and three of the four nucleotides. In view of this, it is not surprising that depletion of a single amino acid in the host cell growth medium blocks chlamydial replication. Paradoxically, supra-normal levels of some amino acids also block productive replication of Chlamydia. Here, we have determined how elevated serine levels, generated by exogenous supplementation, impede chlamydial inclusion development and reduce the generation of infectious progeny. Our findings reveal that human serine racemase, which is broadly expressed in multiple tissues, potentiates the anti-chlamydial effect of elevated serine concentrations. In addition to reversibly converting l-serine to d-serine, serine racemase also deaminates serine via ß-elimination. We have determined that d-serine does not directly impact Chlamydia; rather, ammonia generated by serine deamination limits the productive chlamydial replication. Our findings imply that ammonia produced within host cells can traverse the chlamydial inclusion membrane. Further, this property of serine deaminase can be exploited to sensitize Chlamydia to concentrations of doxycycline that are otherwise not bactericidal. Because exogenously elevated levels of serine can be tolerated over extended periods, the broad expression pattern of serine racemase indicate it to be a host enzyme whose activity can be directed against multiple intracellular bacterial pathogens. From a therapeutic perspective, by demonstrating host metabolism can be skewed to generate an anti-bacterial metabolite that synergizes with antibiotics, we believe our results provide a new approach to target intracellular pathogens.

Folate deficiency modifies the risk of CIN3+ associated with DNA methylation levels: a nested case-control study from the ASCUS-COL trial.

PURPOSE: To our knowledge, there are very few studies evaluating if the levels of folate modify the risk of cervical intraepithelial neoplasia grade 2 and higher (CIN2+ and CIN3+) associated with the levels of HPV genome methylation, two cofactors related to single carbon metabolism and independently associated with cervical cancer in previous studies. We conducted a case-control study nested in a three-arm randomized clinical pragmatic trial (ASCUS-COL trial) to evaluate the risk of CIN3+ associated with methylation levels according to serum folate concentrations. METHODS: Cases (n = 155) were women with histologically confirmed CIN2+ (113 CIN2, 38 CIN3, and 4 SCC) and controls were age and follow-up time at diagnosis-matched women with histologically confirmed ≤ CIN1 (n = 155), selected from the 1122 hrHPV + women of this trial. The concentrations of serum folate were determined by the radioimmunoassay SimulTRAC-SNB-VitaminB12/Folate-RIAKit and the methylation levels by the S5 classifier. Stepwise logistic regression models were used to estimate the association between folate or methylation levels and CIN2+ or CIN3+. The joint effect of folate levels and methylation on the risk of CIN3+ was estimated using combinations of categorical stratifications. RESULTS: Folate levels were significantly lower in women with CIN3+ than in other diagnostic groups (p = 0.019). The risk of CIN3+ was eight times higher (OR 8.9, 95% CI 3.4-24.9) in women with folate deficiency and high methylation levels than in women with normal folate and high methylation levels (OR 1.4, 95% CI 0.4-4.6). CONCLUSION: High methylation and deficient folate independently increased the risk of CIN3+ while deficient folate combined with high methylation was associated with a substantially elevated risk of CIN3+.

Intake Patterns of Specific Alcoholic Beverages by Prostate Cancer Status.

BACKGROUND: Previous studies have shown that different alcoholic beverage types impact prostate cancer (PCa) clinical outcomes differently. However, intake patterns of specific alcoholic beverages for PCa status are understudied. The study's objective is to evaluate intake patterns of total alcohol and the three types of beverage (beer, wine, and spirits) by the PCa risk and aggressiveness status. METHOD: This is a cross-sectional study using 10,029 men (4676 non-PCa men and 5353 PCa patients) with European ancestry from the PCa consortium. Associations between PCa status and alcohol intake patterns (infrequent, light/moderate, and heavy) were tested using multinomial logistic regressions. RESULTS: Intake frequency patterns of total alcohol were similar for non-PCa men and PCa patients after adjusting for demographic and other factors. However, PCa patients were more likely to drink wine (light/moderate, OR = 1.11, p = 0.018) and spirits (light/moderate, OR = 1.14, p = 0.003; and heavy, OR = 1.34, p = 0.04) than non-PCa men. Patients with aggressive PCa drank more beer than patients with non-aggressive PCa (heavy, OR = 1.48, p = 0.013). Interestingly, heavy wine intake was inversely associated with PCa aggressiveness (OR = 0.56, p = 0.009). CONCLUSIONS: The intake patterns of some alcoholic beverage types differed by PCa status. Our findings can provide valuable information for developing custom alcohol interventions for PCa patients.

A Framework for the Virtual Medical Interview Process: Considerations for the Applicant and the Interviewer.

Background: Videoconferencing platforms are being used for the purposes of interviewing in academic medicine because of the coronavirus disease 2019 pandemic. We present considerations applicable to interviewers and interviewees in the virtual space, with a focus on medical school and residency applicants. Methods: We reviewed the literature regarding the virtual interview process for medical school and residency by searching PubMed using the following keywords and terms: "interview," "academic medicine," "medical school application," "residency application," "virtual interviews," and "videoconferencing." Our search identified 701 results, from which we selected 36 articles for review. Results: The garnered information focuses on strategies for optimizing the virtual interview process from the standpoint of both the interviewer and the interviewee. We discuss the advantages and disadvantages of the virtual interview process and present recommendations. Conclusion: While the future of the interview process for medical school and residency is uncertain, virtual interviewing is a common and growing practice that will continue to be at least part of the medical interview process for years to come. Interviewers and interviewees should prepare to adapt to the evolving changes in the process.

ISGylation is increased in the peripheral blood mononuclear cells derived from symptomatic COVID-19 patients.

Cytokine-driven hyper inflammation has been identified as a critical factor behind poor outcomes in patients severely infected with SARS-CoV-2 virus. Notably, protein ISGylation, a protein conjugated form of Type 1 IFN-inducible ubiquitin-like protein ISG15 (Interferon-Stimulated Gene 15), induces cytokine storm (CS) and augments colonic inflammation in colitis-associated colon cancers in mouse models. However, whether ISGylation is increased and causally responsible for CS and hyper inflammation in symptomatic COVID-19 patients is unknown. Here, we measured ISGylation levels in peripheral blood mononuclear cells (PBMCs) from 10 symptomatic (SARS-CoV-2-positive with symptoms) and asymptomatic (SARS-CoV-2-positive with no symptoms) COVID-19 patients, and 4 uninfected individuals (SARS-CoV-2-negative), using WesTm assay. Strikingly, we note significant increases in protein ISGylation and MX-1 (myxovirus-resistance protein-1) protein levels, both induced by type-I IFN, in symptomatic but not in asymptomatic patients and uninfected individuals. Knowing that ISGylation augments CS and intestinal inflammation in colon cancers, we propose that increased ISGylation may be an underlying cause of CS and inflammation in symptomatic patients.

Improving Multi-site Interaction Through Remote Learning Technology: Report from a Training Program to Increase Underrepresented Undergraduate and Medical Students in Health Disparities Research.

Since 2018, we have evaluated the effectiveness of various teaching technologies for training young investigators on translational research in cancer health disparities. The Southeast Partnership for Improving Research and Training in Cancer Health Disparities (SPIRIT-CHD) unites Moffitt Cancer Center and the Louisiana State University Health Sciences Center. One of the main components of the SPIRIT-CHD is the Cancer Research Education Program (CREP) for training undergraduate and medical students from underrepresented backgrounds. The CREP utilizes a web-based didactic curriculum to engage students at both institutions in biobanking, precision medicine, and cancer health disparities topics. We report experiences from our cross-institutional cancer education program, specifically evaluating the cohorts' satisfaction and learning gains using various communication technologies and instructional approaches. Trainees completed a survey with questions evaluating the curriculum and technology. Trainees reported satisfaction with the flipped classroom model (FCM) content and overall program (mean score = 3.2, SD = 0.79), and would recommend the program to peers. Yet, despite improved program delivery, trainees felt interaction between the two sites (mean score = 1.5, SD = 0.85) and engagement with faculty (mean score = 2.80, SD = 1.14) could be improved. The technology with the highest reported use was e-mail, with a mean score of 4.6 (SD = 0.52). LinkedIn and Twitter had the lowest frequency of use with mean scores at 1.90 (SD = 0.99) and 1.30 (SD = 1.34). Our study highlights the successes and challenges of remote learning using technology to increase interaction and engagement among trainees and faculty in a multi-site cancer research training program.

Role of Inflammasome Activation in Systemic Lupus Erythematosus: Are Innate Immune Cells Activated?

BACKGROUND: Systemic lupus erythematosus (SLE) is characterized by a wide spectrum of clinical and immunological abnormalities. New data have emerged about the role of inflammasomes in autoimmune diseases. We aimed to investigate whether basal inflammasome activation occurs in SLE patients, and whether a relationship between inflammasome-related-cytokines and disease activity exists. METHODS: Fourteen (14) consecutive SLE patients and 13 healthy individuals, matched by sex, age and ethnicity, were included. Demographics, laboratory and clinical data were recorded. Peripheral blood mononuclear cells (PBMCs) from patients and controls were obtained and monocytes were isolated by negative selection. Purified monocytes were stimulated with LPS in the presence or absence of Caspase-1 inhibitor. CD14 and Caspase-1 expression were analyzed by flow cytometry. Cytokine levels were determined in plasma and culture supernatants by ELISA. Student's t test and Mann-Whitney tests were used for statistical analysis. RESULTS: The percentage of CD14+/Caspase-1+ was significantly higher in monocytes from SLE patients compared to normal controls (p<0.01). These findings paralleled with higher plasma levels of IL-1ß (p<0.05) and IL-18 (p<0.01) in those patients. Purified monocytes from SLE patients displayed a robust inflammatory response after LPS stimulation where Caspase-1, IL-1ß and IL-18 were highly expressed. Plasma levels of IL-18 were also significantly higher in SLE patients with active disease (p<0.05). In addition, the production of IL-18 was reduced by 3 fold when Caspase-1 inhibitor was added to the cultures. CONCLUSIONS: Monocytes from SLE patients exhibited increased inflammasome activation, characterized by high expression of Caspase-1, IL-1ß and IL-18. Caspase-1 specific inhibitor decreased inflammasome activation (in vitro) by suppressing the production of IL-18.

Patient-Derived Xenografts as an Innovative Surrogate Tumor Model for the Investigation of Health Disparities in Triple Negative Breast Cancer.

Despite a decline in overall incidence rates for cancer in the past decade, due in part to impressive advancements in both diagnosis and treatment, breast cancer (BC) remains the leading cause of cancer-related deaths in women. BC alone accounts for ∼30% of all new cancer diagnoses in women worldwide. Triple-negative BC (TNBC), defined as having no expression of the estrogen or progesterone receptors and no amplification of the HER2 receptor, is a subtype of BC that does not benefit from the use of estrogen receptor-targeting or HER2-targeting therapies. Differences in socioeconomic factors and cell intrinsic and extrinsic characteristics have been demonstrated in Black and White TNBC patient tumors. The emergence of patient-derived xenograft (PDX) models as a surrogate, translational, and functional representation of the patient with TNBC has led to the advances in drug discovery and testing of novel targeted approaches and combination therapies. However, current established TNBC PDX models fail to represent the diverse patient population and, most importantly, the specific ethnic patient populations that have higher rates of incidence and mortality. The primary aim of this review is to emphasize the importance of using clinically relevant translatable tumor models that reflect TNBC human tumor biology and heterogeneity in high-risk patient populations. The focus is to highlight the complexity of BC as it specifically relates to the management of TNBC in Black women. We discuss the importance of utilizing PDX models to study the extracellular matrix (ECM), and the distinct differences in ECM composition and biophysical properties in Black and White women. Finally, we demonstrate the crucial importance of PDX models toward novel drug discovery in this patient population.

A novel patient-derived xenograft model for claudin-low triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) subtypes are clinically aggressive and cannot be treated with targeted therapeutics commonly used in other breast cancer subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research are limited. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by cell-line derived xenograft methods. METHODS: We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new CL-TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0. RESULTS: Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including the whole and intact breast tumor, specific cell populations within the tumor, and the extracellular matrix. CONCLUSIONS: Here, we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.

The High-Risk Human Papillomavirus E6 Oncogene Exacerbates the Negative Effect of Tryptophan Starvation on the Development of Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular pathogen that requires specific essential nutrients from the host cell, one of which is the amino acid tryptophan. In this context interferon gamma (IFNγ) is the major host protective cytokine against chlamydial infections because it induces the expression of the host enzyme, indoleamine 2,3-dioxygenase 1, that degrades tryptophan, thereby restricting bacterial replication. The mechanism by which IFNγ acts has been dissected in vitro using epithelial cell-lines such as HeLa, HEp-2, or the primary-like endocervical cell-line A2EN. All these cell-lines express the high-risk human papillomavirus oncogenes E6 & E7. While screening cell-lines to identify those suitable for C. trachomatis co-infections with other genital pathogens, we unexpectedly found that tryptophan starvation did not completely block chlamydial development in cell-lines that were HR-HPV negative, such as C33A and 293. Therefore, we tested the hypothesis that HR-HPV oncogenes modulate the effect of tryptophan starvation on chlamydial development by comparing chlamydial development in HeLa and C33A cell-lines that were both derived from cervical carcinomas. Our results indicate that during tryptophan depletion, unlike HeLa, C33A cells generate sufficient intracellular tryptophan via proteasomal activity to permit C. trachomatis replication. By generating stable derivatives of C33A that expressed HPV16 E6, E7 or E6 & E7, we found that E6 expression alone was sufficient to convert C33A cells to behave like HeLa during tryptophan starvation. The reduced tryptophan levels in HeLa cells have a biological consequence; akin to the previously described effect of IFNγ, tryptophan starvation protects C. trachomatis from clearance by doxycycline in HeLa but not C33A cells. Curiously, when compared to the known Homo sapiens proteome, the representation of tryptophan in the HR-HPV E6 & E6AP degradome is substantially lower, possibly providing a mechanism that underlies the lowered intracellular free tryptophan levels in E6-expressing cells during starvation.

Activation of the IL-2 Receptor in Podocytes: A Potential Mechanism for Podocyte Injury in Idiopathic Nephrotic Syndrome?

The renal podocyte plays an important role in maintaining the structural integrity of the glomerular basement membrane. We have previously reported that patients with idiopathic nephrotic syndrome (INS) have increased IL-2 production. We hypothesized that podocytes express an IL-2 receptor (IL-2R) and signaling through this receptor can result in podocyte injury. To confirm the presence of the IL-2R, we tested a conditionally immortalized murine podocyte cell line by flow cytometry, qPCR, and Western blot. To test for the presence of the IL-2R in vivo, immunohistochemical staining was performed on human renal biopsies in children with FSGS and control. Podocytes were stimulated with IL-2 in vitro, to study signaling events via the JAK/STAT pathway. The results showed that stimulation with IL-2 resulted in increased mRNA and protein expression of STAT 5a, phosphorylated STAT 5, JAK 3, and phosphorylated JAK 3. We then investigated for signs of cellular injury and the data showed that pro-apoptotic markers Bax and cFLIP were significantly increased following IL-2 exposure, whereas LC3 II was decreased. Furthermore, mitochondrial depolarization and apoptosis were both significantly increased following activation of the IL-2R. We used a paracellular permeability assay to monitor the structural integrity of a podocyte monolayer following IL-2 exposure. The results showed that podocytes exposed to IL-2 have increased albumin leakage across the monolayer. We conclude that murine podocytes express the IL-2R, and that activation through the IL-2R results in podocyte injury.

"Omics" of Selenium Biology: A Prospective Study of Plasma Proteome Network Before and After Selenized-Yeast Supplementation in Healthy Men.

Low selenium levels have been linked to a higher incidence of cancer and other diseases, including Keshan, Chagas, and Kashin-Beck, and insulin resistance. Additionally, muscle and cardiovascular disorders, immune dysfunction, cancer, neurological disorders, and endocrine function have been associated with mutations in genes encoding for selenoproteins. Selenium biology is complex, and a systems biology approach to study global metabolomics, genomics, and/or proteomics may provide important clues to examining selenium-responsive markers in circulation. In the current investigation, we applied a global proteomics approach on plasma samples collected from a previously conducted, double-blinded placebo controlled clinical study, where men were supplemented with selenized-yeast (Se-Yeast; 300 µg/day, 3.8 µmol/day) or placebo-yeast for 48 weeks. Proteomic analysis was performed by iTRAQ on 8 plasma samples from each arm at baseline and 48 weeks. A total of 161 plasma proteins were identified in both arms. Twenty-two proteins were significantly altered following Se-Yeast supplementation and thirteen proteins were significantly changed after placebo-yeast supplementation in healthy men. The differentially expressed proteins were involved in complement and coagulation pathways, immune functions, lipid metabolism, and insulin resistance. Reconstruction and analysis of protein-protein interaction network around selected proteins revealed several hub proteins. One of the interactions suggested by our analysis, PHLD-APOA4, which is involved in insulin resistance, was subsequently validated by Western blot analysis. Our systems approach illustrates a viable platform for investigating responsive proteomic profile in 'before and after' condition following Se-Yeast supplementation. The nature of proteins identified suggests that selenium may play an important role in complement and coagulation pathways, and insulin resistance.

Defining Plasma MicroRNAs Associated With Cognitive Impairment In HIV-Infected Patients.

Human Immunodeficiency Virus (HIV)-infected individuals are at increased risk for developing neurocognitive disorders and depression. These conditions collectively affect more than 50% of people living with HIV/AIDS and adversely impact adherence to HIV therapy. Thus, identification of early markers of neurocognitive impairment could lead to interventions that improve psychosocial functioning and slow or reverse disease progression through improved treatment adherence. Evidence has accumulated for the role and function of microRNAs in normal and pathological conditions. We have optimized a protocol to profile microRNAs in body fluids. Using this methodology, we have profiled plasma microRNA expression for 30 age-matched, HIV-infected (HIV(+) ) patients and identified highly sensitive and specific microRNA signatures distinguishing HIV(+) patients with cognitive impairment from those without cognitive impairment. These results justify follow-on studies to determine whether plasma microRNA signatures can be used as a screening or prognostic tool for HIV(+) patients with neurocognitive impairment. J. Cell. Physiol. 231: 829-836, 2016. © 2015 Wiley Periodicals, Inc.

Chronic alcohol increases CD8+ T-cell immunosenescence in simian immunodeficiency virus-infected rhesus macaques.

Activated CD8+ T-cells correlate with viral load and may foretell antiretroviral therapy (ART) failure. HIV infection has been suggested to accelerate immunosenescence through chronic persistent inflammation. Alcohol-use disorders (AUD) are prevalent in persons living with HIV/AIDS (PLWHA). We tested the hypothesis that hazardous alcohol consumption accelerates immune activation and immunosenescence. Immune activation and immunosenescence were examined in CD8+ T lymphocytes (CD3+CD4-CD8+) isolated from intestinal biopsies, axillary lymph nodes, and peripheral blood mononuclear cells (PBMCs) of chronic binge alcohol (CBA)-consuming simian immunodeficiency virus (SIV)-infected male rhesus macaques with and without antiretroviral therapy (ART; CBA/ART+, CBA/ART-) and in PBMCs isolated from a cohort of PLWHA. Polychromatic flow cytometry was used to phenotype cells isolated from intestinal biopsies, lymph nodes, and peripheral blood from rhesus macaques and PLWHA. The Alcohol Use Disorders Identification Test (AUDIT) identified hazardous alcohol drinking in PLWHA. Viral load was determined by RT-qPCR and telomere length was measured using qPCR. PBMC CD8+ T-cell activation (CD38+HLA-DR+) and immunosenescence (CD28-) were increased over baseline levels (857% ± 334, p < 0.05; 398% ± 80, p < 0.05, respectively) only in CBA animals not receiving ART. Viral load correlated with CD8+ T-cell immunosenescence in macaque PBMCs (r(s) = 0.49, p = 0.02). Activated immunosenescent T-cell (CD8+CD38+CD28-) frequencies in PBMCs from PLWHA significantly correlated with AUDIT scores (r(s) = 0.75, p = 0.001), while no correlation was observed with CD4+ T-cell and AUDIT scores (r(s) = -0.24, p = 0.38). Activated immunosenescent T-cells had shorter telomeres than CD8+ T-cells (CD8+CD28+) from PLWHA. Our results suggest that CBA and AUD augment immune activation and immunosenescence in SIV-infected macaques and PLWHA.

PARP is activated in human asthma and its inhibition by olaparib blocks house dust mite-induced disease in mice.

Our laboratory established a role for poly(ADP-ribose)polymerase (PARP) in asthma. To increase the clinical significance of our studies, it is imperative to demonstrate that PARP is actually activated in human asthma, to examine whether a PARP inhibitor approved for human testing such as olaparib blocks already-established chronic asthma traits in response to house dust mite (HDM), a true human allergen, in mice and to examine whether the drug modulates human cluster of differentiation type 4 (CD4(+)) T-cell function. To conduct the study, human lung specimens and peripheral blood mononuclear cells (PBMCs) and a HDM-based mouse asthma model were used. Our results show that PARP is activated in PBMCs and lung tissues of asthmatics. PARP inhibition by olaparib or gene knockout blocked established asthma-like traits in mice chronically exposed to HDM including airway eosinophilia and hyper-responsiveness. These effects were linked to a marked reduction in T helper 2 (Th2) cytokine production without a prominent effect on interferon (IFN)-γ or interleukin (IL)-10. PARP inhibition prevented HDM-induced increase in overall cellularity, weight and CD4(+) T-cell population in spleens of treated mice whereas it increased the T-regulatory cell population. In CD3/CD28-stimulated human CD4 (+)T-cells, olaparib treatment reduced Th2 cytokine production potentially by modulating GATA binding protein-3 (gata-3)/IL-4 expression while moderately affecting T-cell proliferation. PARP inhibition inconsistently increased IL-17 in HDM-exposed mice and CD3/CD28-stimulated CD4(+) T cells without a concomitant increase in factors that can be influenced by IL-17. In the present study, we provide evidence for the first time that PARP-1 is activated in human asthma and that its inhibition is effective in blocking established asthma in mice.

Distinct genetic profile in peripheral blood mononuclear cells of psoriatic arthritis patients treated with methotrexate and TNF-inhibitors.

Psoriatic arthritis (PsA) is a systemic inflammatory condition associated with psoriasis. Despite considerable heterogeneity in clinical presentation, genetic studies and animal models support the notion that PsA is a distinct disease. We aimed to characterize the PsA genotype by gene expression profile and to research the effect in gene modulation of methotrexate (MTX) and TNF-inhibitors (TNF-I) in PsA-treated patients. Nine PsA patients, according to CASPAR criteria, and three healthy controls were recruited from an outpatient rheumatology clinic. Three out of nine PsA patients were naïve to treatment, three received TNF-I, and the remaining three were on MTX-monotherapy. Blood samples were collected and analyzed by human genome U95 Array-Affymetrix (GeneChip® instrument system). Identification of statistically significant differences between differentially expressed genes was determined by Mann-Whitney and t test (p < 0.05). The microarray profile identified a predominance of differentially expressed genes with an increased expression in baseline PsA patients: 115/12,000 genes were up-regulated and 13/12,000 down-regulated, as compared to healthy controls. The great majority were involved in inflammatory cells and pathways. In the biologic-treated patients, a higher number of down-regulated genes were expressed vs. the MTX patients, 161 vs. 33, respectively. This study shows that in PsA patients, TNF-I and MTX are able to modulate the gene expression in a synergistic and additive manner.

Dual effect of interferon (IFNγ)-induced nitric oxide on tumorigenesis and intracellular bacteria.

Nitric oxide (NO) is a key messenger involved in numerous physiological functions including inflammatory and immune responses. The functions of NO and their underlying mechanisms have been elucidated by extensive studies over the past 10 years. However, the complexity of the interactions between different levels of NO and multiple aspects of tumor development/progression as well as bacterial pathogenesis has led to apparently conflicting findings. The precise role of NO in bacterial and tumor pathogenesis involves a multitude of inter- and intracellular signaling pathways in which interferon gamma signaling and L-arginine metabolism are the major pathways involved in NO synthesis and regulation. The availability of the amino acid L-Arg can be a key factor to control the expression of inducible nitric oxide synthase (NOS2) and cellular NO levels. The role played by the NOS2/NO system both in bacterial pathogenesis and in tumor development is complex due to the dual role these molecules can play promoting or inhibiting infections and cancer. This duality brings to the table a double challenge to determine the net impact of NO on cancer or bacterial behavior and to define the therapeutic role of NO-centered anticancer or antibacterial strategies. We believe that a comprehensive and dynamic understanding of the cascade of molecular and cellular events underlying tumor biology and bacterial pathogenesis that are affected by NO will allow researchers to exploit the potential antitumor and antibacterial properties of drugs interfering with NO metabolism. The contrasting roles of NO/NOS2 in these processes are clarified in this chapter.

Immunosuppression in cervical cancer with special reference to arginase activity.

INTRODUCTION: Cervical cancer is characterized by an immunosuppressive microenvironment and a Th2-type cytokine profile. Expression of arginase (ASE), the enzyme that converts L-arginine into L-ornithine and urea, is stimulated by Th2-type cytokines. OBJECTIVE: To assess the association of ASE activity and L-Arg metabolism products with cervical cancer. METHODS: Sera of 87 and 41 women with histologically confirmed by colposcopy-directed biopsy SCC and CIN3 respectively and 79 with normal cytology or Low-Grade Squamous Intraepithelial Lesion (LSIL), were evaluated. Cytokines were measured using Milliplex Human cytokine/chemokine kit. Arginase (ASE) activity was determined using an enzymatic assay. Levels of L-arginine, L-ornithine, putrescine and spermine were determined by HPLC. RESULTS: Significantly higher levels of ASE activity were observed in women with CIN3 (age-adjusted OR: 24.3; 95%CI: 3.82-155) and SCC (AOR: 9.8; 95%CI: 2.34-40.8). As expected, possibly due to high levels of ASE activity, higher levels of l-Arg were negatively associated with CIN3 (AOR: 0.03; 95%CI: 0.004-0.19) and SSC (AOR: 0.06; 95%CI: 0.02-0.24). Consistent with the role of ASE in the conversion of L-arginine to L-ornithine and polyamine production therefrom, women with cervical cancer had higher levels of spermine and putrescine. A correlation analysis revealed a significant albeit weak relationship between high levels of IL-10 and high levels of ASE (Pearson r=0.32, p-value=0.003) in women with cervical cancer. CONCLUSION: This study indicates that ASE activity and L-Arg degradation mechanisms of immunosuppression are present in cervical cancer. The results foster research in the design of possible strategies to inhibit ASE activity for therapy of cervical cancer.

Influence of the tryptophan-indole-IFNγ axis on human genital Chlamydia trachomatis infection: role of vaginal co-infections.

The natural history of genital Chlamydia trachomatis infections can vary widely; infections can spontaneously resolve but can also last from months to years, potentially progressing to cause significant pathology. The host and bacterial factors underlying this wide variation are not completely understood, but emphasize the bacterium's capacity to evade/adapt to the genital immune response, and/or exploit local environmental conditions to survive this immune response. IFNγ is considered to be a primary host protective cytokine against endocervical C. trachomatis infections. IFNγ acts by inducing the host enzyme indoleamine 2,3-dioxgenase, which catabolizes tryptophan, thereby depriving the bacterium of this essential amino acid. In vitro studies have revealed that tryptophan deprivation causes Chlamydia to enter a viable but non-infectious growth pattern that is termed a persistent growth form, characterized by a unique morphology and gene expression pattern. Provision of tryptophan can reactivate the bacterium to the normal developmental cycle. There is a significant difference in the capacity of ocular and genital C. trachomatis serovars to counter tryptophan deprivation. The latter uniquely encode a functional tryptophan synthase to synthesize tryptophan via indole salvage, should indole be available in the infection microenvironment. In vitro studies have confirmed the capacity of indole to mitigate the effects of IFNγ; it has been suggested that a perturbed vaginal microbiome may provide a source of indole in vivo. Consistent with this hypothesis, the microbiome associated with bacterial vaginosis includes species that encode a tryptophanase to produce indole. In this review, we discuss the natural history of genital chlamydial infections, morphological and molecular changes imposed by IFNγ on Chlamydia, and finally, the microenvironmental conditions associated with vaginal co-infections that can ameliorate the effects of IFNγ on C. trachomatis.