Jason Trama

The NFAT-Related Protein NFATL1 (TonEBP/NFAT5) Is Induced Upon T Cell Activation in a Calcineurin-Dependent Manner

NFAT DNA binding complexes regulate programs of cellular activation and differentiation by translating receptor-dependent signaling events into specific transcriptional responses. NFAT proteins, originally defined as calcium/calcineurin-dependent regulators of cytokine gene transcription in T lymphocytes, are expressed in many different cell types and represent critical signaling intermediates that mediate an increasingly wide spectrum of biologic responses. Recent studies have identified a novel protein containing a region of similarity to the NFAT DNA binding domain. Here we demonstrate that this protein, designated NFATL1 (also known as tonicity enhancer binding protein and NFAT5) is expressed at high levels in the thymus but is undetectable in mature lymphocytes. However, NFATL1 can be induced in both primary quiescent T lymphocytes and differentiated Th1 and Th2 cell populations upon mitogen- or Ag receptor-dependent activation. The induction of NFATL1 protein, as well as NFATL1-dependent transcription, is inhibited by cyclosporin A and FK506, and expression of constitutively active calcineurin induces NFATL1-dependent transcription. Overexpression of NFATc1 and inhibition of NFATc activity through the use of a dominant negative NFATc1 protein have no affect on NFATL1-dependent transcription, indicating that NFATc proteins do not play a role in the calcineurin-dependent induction of NFATL1. Interestingly, induction of NFATL1 by a hyperosmotic stimulus is not blocked by the inhibition of calcineurin. Moreover, osmotic stress response genes such as aldose reductase are not induced upon T cell activation. Thus inducible expression of NFATL1 represents a mechanism by which receptor-dependent signals as well as osmotic stress signals are translated into transcriptional responses that regulate cell function.

The Osmoprotective Function of the NFAT5 Transcription Factor in T Cell Development and Activation

The NFAT5/TonEBP transcription factor, a recently identified rel/NF-κB family member, activates transcription of osmocompensatory genes in response to extracellular hyperosmotic stress. However, the function of NFAT5 under isosmotic conditions present in vivo remains unknown. Here we demonstrate that NFAT5 is necessary for optimal T cell development in vivo and allows for optimal cell growth ex vivo under conditions associated with osmotic stress. Transgenic mice expressing an inhibitory form of NFAT5 in developing and mature T cells exhibited a 30% reduction in thymic cellularity evenly distributed among thymic subsets, consistent with the uniform expression and nuclear localization of NFAT5 in each subset. This was associated with a 25% reduction in peripheral CD4+ T cells and a 50% reduction in CD8+ T cells. While transgenic T cells exhibited no impairment in cell growth or cytokine production under normal culture conditions, impaired cell growth was observed under both hyperosmotic conditions and isosmotic conditions associated with osmotic stress. Transgenic thymocytes also demonstrated increased sensitivity to osmotic stress. Consistent with this, the system A amino acid transporter gene ATA2 exhibited NFAT5 dependence under hypertonic conditions but not in response to amino acid deprivation. Expression of the TNF-α gene, a putative NFAT5 target, was not altered in transgenic T cells. These results not only demonstrate an osmoprotective function for NFAT5 in primary cells but also show that NFAT5 is necessary for optimal thymic development in vivo, suggesting that developing thymocytes within the thymic microenvironment are subject to an osmotic stress that is effectively countered by NFAT5-dependent responses.

Survey of vaginal-flora Candida species isolates from women of different age groups by use of species-specific PCR detection.

ABSTRACT A retrospective survey of 93,775 samples testing positive in Candida species-specific PCR tests performed on cervicovaginal swabs over a 4-year period demonstrated consistent yearly distributions of Candida albicans (89%), C. glabrata (7.9%), C. parapsilosis (1.7%), and C. tropicalis (1.4%). However, the species distributions among different age groups revealed increases in the percentages of non-albicans species with increases in age.

Erythromycin and Clindamycin Resistance in Group B Streptococcal Clinical Isolates

ABSTRACT Erythromycin (EM) and clindamycin (CM) susceptibility testing was performed on 222 clinical isolates of group B Streptococcus. A multiplex PCR assay was used to detect the ermB, ermTR, and mefA/E antibiotic resistance genes. These results were compared to the phenotypes as determined by the standard EM/CM double disk diffusion assay.

Atopobium vaginae triggers an innate immune response in an in vitro model of bacterial vaginosis

Bacterial vaginosis is the most common vaginal disorder among women of reproductive age. The pathogenesis of bacterial vaginosis is poorly understood, but is defined by a transition in the vaginal flora from the predominant Lactobacillus species to other bacterial species such as Atopobium vaginae and Gardnerella vaginalis. This change is associated with an increase in vaginal cytokine secretion. We hypothesize that vaginal epithelial cells respond to bacterial vaginosis-associated bacteria by triggering an innate immune response. We observed that vaginal epithelial cells secreted interleukin-6 and interleukin-8 in response to Atopobium vaginae and Gardnerella vaginalis, but not to Lactobacillus crispatus. Atopobium vaginae induced increased levels of interleukin-6 and interleukin-8 transcripts, as well as increased transcripts for the antimicrobial peptide beta-defensin 4. This innate immune response required live bacteria capable of protein synthesis in direct contact with vaginal epithelial cells. The response of vaginal epithelial cells was mediated by Toll-like receptor 2, required the adaptor protein MyD88, and involved activation of the NFkappaB signaling pathway. These results suggest that Atopobium vaginae stimulates an innate immune response from vaginal epithelial cells, leading to localized cytokine and defensin production, and possibly contributes to the pathogenesis of bacterial vaginosis.

Oncoprotein DEK as a tissue and urinary biomarker for bladder cancer

BackgroundBladder cancer is a significant healthcare problem in the United States of America with a high recurrence rate. Early detection of bladder cancer is essential for removing the tumor with preservation of the bladder, avoiding metastasis and hence improving prognosis and long-term survival. The objective of this study was to analyze the presence of DEK protein in voided urine of bladder cancer patients as a urine-based bladder cancer diagnostic test.MethodsWe examined the expression of DEK protein by western blot in 38 paired transitional cell carcinoma (TCC) bladder tumor tissues and adjacent normal tissue. The presence of DEK protein in voided urine was analyzed by western blot in 42 urine samples collected from patients with active TCC, other malignant urogenital disease and healthy individuals.ResultsThe DEK protein is expressed in 33 of 38 bladder tumor tissues with no expression in adjacent normal tissue. Based on our sample size, DEK protein is expressed in 100% of tumors of low malignant potential, 92% of tumors of low grade and in 71% of tumors of high grade. Next, we analyzed 42 urine samples from patients with active TCC, other malignant urogenital disease, non-malignant urogenital disease and healthy individuals for DEK protein expression by western blot analysis. We are the first to show that the DEK protein is present in the urine of bladder cancer patients. Approximately 84% of TCC patient urine specimens were positive for urine DEK.ConclusionBased on our pilot study of 38 bladder tumor tissue and 42 urine samples from patients with active TCC, other malignant urogenital disease, non-malignant urogenital disease and healthy individuals; DEK protein is expressed in bladder tumor tissue and voided urine of bladder cancer patients. The presence of DEK protein in voided urine is potentially a suitable biomarker for bladder cancer and that the screening for the presence of DEK protein in urine can be explored as a noninvasive diagnostic test for bladder cancer.

Uropathogenic Escherichia coli dominantly suppress the innate immune response of bladder epithelial cells by a lipopolysaccharide- and Toll-like receptor 4-independent pathway.

Urinary tract infections are a major source of morbidity among women, with the majority caused by uropathogenic Escherichia coli. Our objective was to test if uropathogenic E. coli suppress the innate immune response of bladder epithelial cells. We found that bladder epithelial cells secrete interleukin-6 and interleukin-8 in response to non-pathogenic E. coli, whereas they failed to do so in response to uropathogenic E. coli. Uropathogenic E. coli prevented interleukin-6 secretion in response to non-pathogenic E. coli and a panel of Toll-like receptor agonists, as well as to interleukin-1beta, but not to tumor necrosis factor alpha. These results indicate that receptors with a Toll/interleukin-1 receptor domain are specifically targeted, and that suppression is not a consequence of toxicity. One candidate for mediating immune suppression is bacterial lipopolysaccharide. However, lipopolysaccharide isolated from either uropathogenic or non-pathogenic E. coli stimulated interleukin-6 secretion to similar levels. In addition, uropathogenic E. coli did not stimulate interleukin-6 secretion from cells expressing a dominant negative Toll-like receptor 4, and prevented cells lacking Toll-like receptor 4 from secreting interleukin-6 in response to synthetic lipoprotein. We conclude that uropathogenic E. coli suppress the innate immune response through a pathway partially independent of lipopolysaccharide and Toll-like receptor 4.

Detecting DNA methylation of the BCL2, CDKN2A and NID2 genes in urine using a nested methylation specific polymerase chain reaction assay to predict bladder cancer.

PURPOSE Detection of methylated DNA has been shown to be a good biomarker for bladder cancer. Bladder cancer has the highest recurrence rate of any cancer and, as such, patients are regularly monitored using invasive diagnostic techniques. As urine is easily attainable, bladder cancer is an optimal cancer to detect using DNA methylation. DNA methylation is highly specific in cancer detection. However, it is difficult to detect because of the limited amount of DNA present in the urine of patients with bladder cancer. Therefore, an improved, sensitive and noninvasive diagnostic test is needed. MATERIALS AND METHODS We developed a highly specific and sensitive nested methylation specific polymerase chain reaction assay to detect the presence of bladder cancer in small volumes of patient urine. The genes assayed for DNA methylation are BCL2, CDKN2A and NID2. The regions surrounding the DNA methylation sites were amplified in a methylation independent first round polymerase chain reaction and the amplification product from the first polymerase chain reaction was used in a real-time methylation specific polymerase chain reaction. Urine samples were collected from patients receiving treatment at Wolfson Medical Center in Holon, Israel. RESULTS In a pilot clinical study using patient urine samples we were able to differentiate bladder cancer from other urogenital malignancies and nonmalignant conditions with a sensitivity of 80.9% and a specificity of 86.4%. CONCLUSIONS We developed a novel methylation specific polymerase chain reaction assay for the detection and monitoring of bladder cancer using DNA extracted from patient urine. The assay may also be combined with other diagnostic tests to improve accuracy.

Detection of Aspergillus fumigatus and a Mutation That Confers Reduced Susceptibility to Itraconazole and Posaconazole by Real-Time PCR and Pyrosequencing

ABSTRACT A real-time PCR and pyrosequencing method was developed to detect Aspergillus fumigatus in whole blood by amplifying the cyp51A gene and sequencing the codon for glycine 54. Mutations in this codon can result in amino acid substitutions that confer reduced susceptibility to itraconazole and posaconazole.

CIP2A expression is elevated in cervical cancer

Early detection of cervical cancer is critical for a favorable prognosis. Standard cytological detection methods, such as Pap smear, are highly subjective and HPV detection is not a reliable marker for predicting the malignancy potential of cervical lesions. As a result, there is a demand for a diagnostic assay capable of sensitive and specific detection of cervical cancer. In this preclinical exploratory study, qRT-PCR and western blotting were used to assess expression levels of CIP2A and p16INK4a in cervical tissue samples (n(normal adjacent) = 23, n(tumor) = 29). CIP2A was abundantly expressed in cervical cancer cell lines and was not expressed in normal epithelial cells. CIP2A mRNA levels were higher in cervical tumor tissues in comparison to the level of CIP2A mRNA in normal adjacent tissue from cervical cancer patients. CIP2A protein was specifically expressed in cervical tumor tissues at different cancer grades and stages, and was not observed in normal adjacent tissue. Elevated CIP2A mRNA levels in cervical tissues had a sensitivity of 80% and specificity of 91% and CIP2A protein expression detection had a sensitivity of 83% and specificity of 100%, similar to that of p16INK4a, with no correlation of CIP2A expression with HPV infection, age, race, or other patient characteristics. However the number of samples analyzed in this preliminary study is limited and a large prospective cohort study is necessary to further evaluate CIP2A as a biomarker for cervical cancer.