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 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.

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.Group B Streptococcus (GBS) is one of the leading causes of neonatal bacterial infection. This type of infection commonly leads to pneumonia, septicemia, or meningitis. Because of the serious nature of neonatal GBS infections, the suggested standard protocol for the obstetrician/gynecologist is that pregnant women should be tested for the presence of GBS at 35 to 37 weeks of gestation (7,15). Once GBS colonization is diagnosed, the typical treatment for these patients is penicillin, to which there is no known resistance. However, there is a significant population of penicillin-allergic patients, a reported 12% of pregnant women (12), for whom the macrolide (erythromycin [EM]) or lincosamide (clindamycin [CM]) class of drugs needs to be administered, in particular, for those patients who are at high risk for anaphylactic shock. Previous reports have cited resistance of GBS to EM and CM of up to 37% and 17%, respectively (7). The resistance is commonly caused by three genes: ermB, ermTR, and mefA/E (1, 9, 10). The ermB and ermTR genes encode 23S rRNA methylases, which alter the binding of the antibiotic target site. The expression of these genes leads to the constitutively expressed and the erythromycin-induced macrolide, lincosamide, and streptogramin B (cMLS and iMLS, respectively) resistance phenotypes (9). The mefA and mefE genes, which are 90% identical, encode 14-and 15-member macrolide efflux pumps and lead to the macrolide only (M) resistance phenotype (1). Because of the presence of ermB, ermTR, mefA/E, and other antibiotic resistance genes on plasmids and/or transposons, these genes can pass from organism to organism, and the monitoring of the antibiotic resistance of GBS should occur regularly (13). We used a multiplex PCR assay to screen for the prevalence of the ermB, ermTR, and mefA/E genes in GBS clinical isolates from 222 patients for whom physicians ordered GBS testing. The samples, representing 20 states in the United States and 60% of which were from Florida, New Jersey, and Texas, were chosen at random. Patient ages ranged from 15 to 82 years, with an average of 31.3 Ϯ 11.8 years. These results were compared to the antibiotic resistance phenotypes as determined by the standard EM/CM double disk diffusion assay (3,11,15)

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.Vulvovaginal candidiasis (VVC) is a common fungal infection that affects healthy women of all ages. At least 75% of women will develop one or more infections once during their lifetime, with 5 to 8% of those individuals developing recurrent infections (5, 7). Current literature examining the species distribution of Candida isolates involved in VVC is limited; however, several important observations have been made. For example, one study shows that Candida albicans accounts for 70 to 90% of all VVC cases, with a recent emergence of nonalbicans species (10). The rise in VVC infections, more specifically in those caused by non-albicans species, could be due to several factors, ranging from an increase in over-the-counter antifungal use to an increase in high-risk patient populations (i.e., diabetics and menopausal women). Candida glabrata is the primary non-albicans species emerging in VVC, accounting for up to 14% of infections in immune-competent women (9, 10).In addition to an increase in non-albicans species overall, it is becoming clear that certain patient populations may experience higher risks of infection from these non-albicans species, often leading to limited treatment options. Interestingly, in a few small studies, C. glabrata was found to be the primary species isolated from diabetic (61.3%) and elderly (51.2%) patients with VVC (2, 4, 6, 11). Often, these non-albicans species are associated with elevated MIC levels for the azoles, the most commonly prescribed class of antifungal drugs. It has been well documented that C. glabrata demonstrates both intrinsically low susceptibility to the azoles and the ability to develop frank resistance (8,12,13,14,15,16). Moreover, a recent increase in the trailing phenotype, with low-level resistance to the azoles, has been observed for the Candida tropicalis isolates (1, 3). This highlights the importance of identifying Candida species within clinical samples in order to provide physicians with information concerning the proper treatment for their patients.