Arnold H. Zea

Background/Objectives: Triple negative breast cancer (TNBC) is an aggressive, molecularly heterogeneous subtype of breast cancer, accounting for approximately 10–15% of all cases. While reproductive and metabolic factors contribute to breast cancer development, growing concerns about environmental exposures, alongside biological and socio-cultural influences, underscore the need for targeted prevention strategies across diverse populations. Despite increasing evidence linking biological, socioeconomic, and environmental factors to TNBC outcomes, the molecular mechanisms underlying these relationships remain poorly understood. Micro-RNAs (miRNAs), which regulate gene expression and play critical roles in cancer development, have emerged as potential mediators between environmental exposures and TNBC progression. The goal of this research is to identify environmental risk factors that directly relate to TNBC stages and enhance understanding of the mechanisms underlying how miRNAs link environmental exposures to TNBC stages. Methods: In this study, we analyzed 434 Formalin-Fixed, Paraffin-Embedded (FFPE) tumor samples from 434 women diagnosed with TNBC between 2009 and 2019, encompassing diverse cancer stages (184 cases from early stage and 250 cases from advanced stage), racial backgrounds, and socioeconomic statuses. The sequencing data were linked with the Louisiana Tumor Registry data and the Environmental Justice index. Results: A total of 348 unique miRNAs were identified as differentially expressed across environmental risk factors statistically associated with TNBC stage, adjusting for plate effects. An UpSet plot revealed 44 miRNAs commonly differentially expressed across TNBC stages and multiple environmental exposures. At least one differentially expressed (DE) miRNA was shared between the TNBC stage and each environmental factor, with many associated with receptor-negative and aggressive breast cancer subtypes. Conclusions: These findings highlight potential biological pathways through which exposures may drive the TNBC progression and contribute to disparities in outcomes.

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.

INTRODUCTION One third of the world’s population is infected with Mycobaterium tuberculosis (Mtb). There are 9 million new cases of tuberculosis (TB) per year, resulting in approximately 2 million deaths. During the last decade, there has been a significant increase in the number of TB cases that may be the result of increased migration and/or the association of Mtb with HIV. On the other hand, barriers that negatively affect clinical outcomes such as the lengthy multidrug therapy dramatically affects compliance and adherence to medication, patient follow-up, pill burden and toxicity. This results in treatment failures and in the development of drug resistance. Regardless of its origin or negative barriers, the increases in TB burden promote an urgency to better understand host-pathogen and signaling pathways interactions that can lead to increase the susceptibility to TB to control the disease. We have gained new insights into why Mtb has won the battle against its control. Tuberculosis (TB) caused by infection with Mtb, remains as a major worldwide health treat. The standard treatment for TB is often effective, but it is also lengthy, complex, significantly toxic and can be thwarted by antibiotic resistance. Therefore, it is imperative to discover or develop new cost effective treatment strategies, especially in the current climate of drug-resistant tuberculosis. The successful replication, survival and persistence of Mtb in humans depend on its ability to survive inside host macrophages by subverting mechanisms of microbial killing. Upon Mtb infection, intracellular levels of 3’, 5’cyclic adenosine monophosphate (cAMP) increase in macrophages resulting in the induction of the enzyme arginase 2 (ARG2). Then, ARG2 metabolizes L-arginine to generate the production of L-ornithine, a precursor of polyamine synthesis which is necessary for cellular growth, Simultaneously, the increased production of ARG2 blocks the production of inducible nitric oxide synthase (NOS2) and nitric oxide (NO) a molecule in charge to control the growing of intracellular pathogens including Mtb. It has been demonstrated that ARG supports the growth of many parasitic and bacterial pathogens. Therefore, we believe that Mtb has developed the capacity to modulate the L-arginine metabolic pathway as a clever strategy to survive inside the macrophage.Thus, cAMP-induced overproduction of ARG2 may promote growth of Mtb at the expense of an impaired pathogen killing. Hence, the critical interplay between ARG and NOS2 might regulate the outcome of TB. The mechanisms of ARG2 induction in macrophages by Mtb are unknown, however, it appears that in mycobacterial infections, cAMP regulates signal transduction pathways responsible for ARG induction. We hypothesize that Mtb-derived cAMP triggers the induction of ARG and the synthesis of polyamines that are necessary for Mtb to survive. The blockage of cAMP and ARG signaling pathways with specific inhibitors could negatively affect Mtb growth and have a positive impact in the treatment of multi- and extensively-drug resistant tuberculosis. OBJECTIVES With this in mind, we plan to conduct experiments that will test the hypothesis that inhibition of polyamine synthesis suppress intracellular Mtb growth, and restore antimicrobial defenses by increasing NO production. 1. Determine if mycobacterial-cAMP regulates ARG induction within macrophages through a cAMP-PKA dependent mechanism, to promote Mtb growth. 2. Determine if inhibition of cAMP or ARG signaling, negatively affects Mtb growth and survival within the host. METHODS 1. Human monocytes where obtained by negative selection after PBMC isolation. Three million monocytes were plated in six well plates in the presence of 5 ng/ml of hGM-CSF for 7 days to generate macrophages. 2. Macrophages were infected with Mtb-CDC1551 or Mtb-mutant (altered cAMP phenotype) at multiplicity of infection 1:3 for 24, 48 and 72 h were tissue culture supernatants and cell lysates were prepared for determination of arginase activity, arginase and NOS2 protein, cAMP, aminoacid levels (L-arginine, L-ornithine, L-glutamine, citrulline, polyamines), nitrites (NO), cytokines and bacterial culture. These elements were tested by enzymatic assays, western blot, ELISA, HPLC, Greiss reaction, multiplexing and colony forming units (CFU) respectively. 3. Adenylyl cyclases, protein kinase A (PKA), phosphodiesterases (PDE) 3,4,5, ARG, polyamine and NOS2 inhibitors were applied to the cultured macrophages 3 hours before and 3 hours after infection, to test for timing and effectiveness in Mtb control. RESULTS We found that there is a time dependent increase in cAMP levels in macrophages, that parallels with increases in ARG activity when human macrophages were infected with Mtb-CDC1551. PKA inhibitor H89 nearly abolished the induction of cAMP and therefore ARG activity. The inhibition of Mtb growth inside macrophages was markedly reduced in cells treated with H89 inhibitor. Adenylyl cyclase inhibitors were more effective when Mtb was treated before infection. There is a significant induction of ARG2 (p< 0.001) in macrophages infected with Mtb that parallels with increased Mtb growth 4.8, 5.16 and 6.4 log10 CFU at 24, 48 and 72 h respectively. Interestingly when we used the Mtb-mutant (lacking cAMP) we observed a significant decrease in cAMP (p=0.024), ARG (p=0.023) and Mtb growth, After 24 hours of infection there was a significant increase in NO production compared to uninfected cells. However, no increases in NO production were observed after 48 hours. The addition of inhibitors nor-NOHA and BEC to the cultures, significantly inhibited Mtb growth (p= 0.005) associated with a significant decrease in the synthesis of polyamine (p=0.03) as compared to untreated cells. We also observed that ARG inhibition significantly increased the production of NO (p= 0.002). There is a significant decrease in cAMP, ARG and Mtb growth when cells are treated...

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

Renal cell carcinoma (RCC) remains one of the most resistant tumors to systemic chemotherapy, radiotherapy, and immunotherapy. Despite great progress in understanding the basic biology of RCC, the rate of responses in animal models and clinical trials using interferons (IFNs) has not improved significantly. It is likely that the lack of responses can be due to the tumor's ability to develop tumor escape strategies. Currently, the use of targeted therapies has improved the clinical outcomes of patients with RCC and is associated with an increase of Th1-cytokine responses (IFNγ), indicating the importance of IFNγ in inhibiting tumor proliferation. Thus, the present study was designed to investigate a new mechanism by which IFNγ mediates direct anti-proliferative effects against murine renal cell carcinoma cell lines. When cultured RCC cell lines were exposed to murine recombinant IFNγ, a dose dependent growth inhibition in CL-2 and CL-19 cells was observed; this effect was not observed in Renca cells. Growth inhibition in CL-2 and CL-19 cell lines was associated with the intracellular induction of nitric oxide synthase (iNOS) protein, resulting in a sustained elevation of nitric oxide (NO) and citrulline, and a decrease in arginase activity. The inhibition of cell proliferation appears to be due to an arrest in the cell cycle. The results indicate that in certain RCC cell lines, IFNγ modulates L-arginine metabolism by shifting from arginase to iNOS activity, thereby developing a potent inhibitory mechanism to encumber tumor cell proliferation and survival. Elucidating the cellular events triggered by IFNγ in murine RCC cell lines will permit anti-tumor effects to be exploited in the development of new combination therapies that interfere with L-arginine metabolism to effectively combat RCC in patients.

Background: L-arginine is the common substrate for the two isoforms of arginase. Arginase I, highly expressed in the liver and arginase II mainly expressed in the kidney. Arginase I-producing myeloid derived suppressor cells have been shown to inhibit T-cell function by the depletion of L-arginine. On the other hand, arginase II has been detected in patients with cancer and is thought to metabolize L-arginine to L-ornithine needed to sustain rapid tumor growth; however its role in L-arginine depletion is unclear. Thus, in tumor biology, L-arginine metabolism may play a dual role in tumor growth and in the induction of T cell dysfunction. Therefore, we studied in murine renal cell carcinoma (RCC) cell lines, the effect of arginase II on tumor cell proliferation and L-arginine depletion. The effect of arginase inhibitors on cell proliferation was also tested. Methods: Three murine renal cell carcinoma (mRCC) cell lines were tested for the presence of arginase. nor-NOHA, an arginase inhibitor was used to substantiate the effect of arginase on cell growth and L-arginine depletion. Amino acid levels were tested by HPLC. Results: Our results show that mRCC cell lines express only arginase II and were able to deplete L-arginine from the medium. Cell growth was independent of the amount of arginase activity expressed by the cells. nor-NOHA significantly (P = 0.01) reduced arginase II activity and suppressed cell growth in cells exhibiting high arginase activity. The depletion of L-arginine by mRCC induced the decrease expression of CD3ζ a key element for T-cell function. Conclusion: The results of this study show for the first time that arginase II produced by RCC cell lines depletes L-arginine resulting in decreased expression of CD3ζ. These results indicate that RCC cell lines expressing arginase II can modulate the L-arginine metabolic pathway to regulate both cell growth and T-cell function. Blocking arginase may lead to a decrease in RCC cell growth and aid in restoring immune function by increasing L-arginine availability for T-cell use. Understanding the interplay between arginase II and its interaction with the immune system may provide future therapeutic benefits to treat patients with RCC.

To study Toll-like receptor-2 (TLR-2) and TLR-4 expression in antigen-presenting cells from patients with psoriatic arthritis (PsA). We measured expression of TLR-2 and TLR-4 in monocyte-derived dendritic cells from patients with PsA and with rheumatoid arthritis (RA), and in healthy controls. Dendritic cells were obtained from freshly isolated monocytes, stimulated with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) after 6 days in culture. To obtain mature dendritic cells, lipopolysaccharide stimulation and 2 additional days in culture were necessary. The expression of TLR-2, TLR-4, HLA-DR, and CD86 was studied at baseline, at 6 days, and at 8 days by flow cytometry. To establish the functional properties of TLR expression we studied the following cytokines in cell supernatants: tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, and GM-CSF. TLR-2 expression was confirmed by Western blot analysis. Ten PsA patients with active disease and 8 healthy controls were studied, along with 4 patients with RA. TLR-2 expression was increased in immature dendritic cells from patients with PsA. Monocytes and mature dendritic cells did not show statistically significant differences. No difference was observed in the expression of TLR-4 in any cell type. The supernatant expression of cytokines showed a Th1 pattern, mostly with increased expression of TNF-alpha, IFN-gamma, and IL-2. Western blot analysis confirmed the increased expression of TLR-2. Upregulation of TLR-2 expression provides support for a role of the innate immune system in the pathogenesis of PsA.

BackgroundThe protective immune response against Mycobacterium tuberculosis relies both on antigen-presenting cells and on T lymphocytes. In patients