Atypia of undetermined significance and ThyroSeq v3-positive call rates as quality control metrics for cytology laboratory performance.

BACKGROUND: The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) recommends an upper limit of 10% for atypia of undetermined significance (AUS). Recent data suggest that this category might be overused when the rate of cases with molecular positive results is low. As a quality metric, the AUS and positive call rates for this facility's cytology laboratory and each cytopathologist (CP) were calculated. METHODS: A retrospective analysis of all thyroid cytology cases in a 4.5-year period was performed. Cases were stratified by TBSRTC, and molecular testing results were collected for indeterminate categories. The AUS rate was calculated for each CP and the laboratory. The molecular positive call rate (PCR) was calculated with and without the addition of currently negative to the positive results obtained from the ThyroSeq report. RESULTS: A total of 7535 cases were classified as nondiagnostic, 7.6%; benign, 69%; AUS, 17.5%; follicular neoplasm/suspicious for follicular neoplasm, 1.4%; suspicious for malignancy, 0.7%; and malignant, 3.8%. The AUS rate for each CP ranged from 9.9% to 36.8%. The overall PCR was 24% (range, 13%-35.6% per CP). When including cases with currently negative results, the PCR increased to 35.5% for the cytology laboratory (range, 13%-42.6% per CP). Comparison analysis indicates a combination of overcalling benign cases and, less frequently, undercalling of higher TBSRTC category cases. CONCLUSIONS: The AUS rate in the context of PCR is a useful metric to assess cytology laboratory and cytopathologists' performance. Continuous feedback on this metric could help improve the overall quality of reporting thyroid cytology.

Case report: A case of neuro-Behçet's syndrome presenting as brain stem mass lesions.

Neuro-Behçet's syndrome, a severe and rare manifestation of Behçet's disease (BD), can be misdiagnosed due to its challenging clinical presentation. This article presents the case of a 20-year-old cis-gender male with intermittent fever, bilateral uveitis, and neurological symptoms who was found to have multiple brain stem mass lesions on brain imaging. A careful medical history elicited recurrent painful oral and genital ulcerations which were important in making the correct diagnosis. As there are no validated criteria or definite set of tests available to confirm neuro-Behçet's disease, the diagnosis is often established by exclusion after ruling out other potential etiologies. In our case, after an extensive negative workup for infectious, neuro-degenerative and malignant etiologies combined with the patient's medical history, a diagnosis of Behçet's disease with neurological involvement (neuro-Behçet's syndrome) was made. High doses of steroids were given, and the patient had a favorable outcome. Repeated magnetic resonance imaging of the brain 2 years later showed no new brain lesions. Neuro-Behçet's disease should be included as a differential diagnosis of unexplained brain stem lesions in the right clinical context. In these situations, providers should obtain medical histories related to genital and oral ulcers and eye problems as these may help to narrow down the diagnosis. The clinical presentation and challenges of this uncommon presentation of BD including a brief literature review of neuro-Behçet's disease with brain stem mass lesions are discussed in this case study.

RAGE inhibitor TTP488 (Azeliragon) suppresses metastasis in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic cancer subtype, which is generally untreatable once it metastasizes. We hypothesized that interfering with the Receptor for Advanced Glycation End-products (RAGE) signaling with the small molecule RAGE inhibitors (TTP488/Azeliragon and FPS-ZM1) would impair TNBC metastasis and impair fundamental mechanisms underlying tumor progression and metastasis. Both TTP488 and FPS-ZM1 impaired spontaneous and experimental metastasis of TNBC models, with TTP488 reducing metastasis to a greater degree than FPS-ZM1. Transcriptomic analysis of primary xenograft tumor and metastatic tissue revealed high concordance in gene and protein changes with both drugs, with TTP488 showing greater potency against metastatic driver pathways. Phenotypic validation of transcriptomic analysis by functional cell assays revealed that RAGE inhibition impaired TNBC cell adhesion to multiple extracellular matrix proteins (including collagens, laminins, and fibronectin), migration, and invasion. Neither RAGE inhibitor impaired cellular viability, proliferation, or cell cycle in vitro. Proteomic analysis of serum from tumor-bearing mice revealed RAGE inhibition affected metastatic driver mechanisms, including multiple cytokines and growth factors. Further mechanistic studies by phospho-proteomic analysis of tumors revealed RAGE inhibition led to decreased signaling through critical BC metastatic driver mechanisms, including Pyk2, STAT3, and Akt. These results show that TTP488 impairs metastasis of TNBC and further clarifies the signaling and cellular mechanisms through which RAGE mediates metastasis. Importantly, as TTP488 displays a favorable safety profile in human studies, our study provides the rationale for evaluating TTP488 in clinical trials to treat or prevent metastatic TNBC.

Primary Ovarian Mucinous Adenocarcinoma, Expansile Type, Misperceived As Pregnancy by the Patient.

We present the case of a woman in her 20s with an eight-month history of increasing abdominal distention, dyspnea, and night sweats. The patient believed she was pregnant despite being told at another hospital that the pregnancy tests were negative, and no fetus was seen on an abdominal ultrasound. The patient delayed obtaining follow-up because of a distrust of the healthcare system and presented to our hospital at the behest of her mother. On physical examination, the abdomen was distended with a positive fluid wave, and a large mass was palpated in the abdomen. Gynecological examination was limited because of severe abdominal distension but a mass was palpable in the right adnexa. A pregnancy test and fetal ultrasound were performed, and the patient was not pregnant. A CT scan of the abdomen and pelvis revealed a large mass arising from the right adnexa. She underwent right salpingo-oophorectomy, appendectomy, omentectomy, lymph node dissection, and peritoneal implant resection. The biopsy confirmed intestinal-type IIB primary ovarian mucinous adenocarcinoma, expansile type, with peritoneal spread. Chemotherapy was provided for three cycles. A follow-up CT scan of the abdomen showed no evidence of a tumor six months after surgery.

Intestinal parasitosis in children from a rural Caribbean area in Colombia / Parasitosis intestinal en niños de una zona rural del Caribe de Colombia

Abstract In Colombia, the prevalence of intestinal parasitosis varies throughout its regions, social classes, and living conditions. We performed a cohort study (2017-2018) on children from 1-10 years old in El Cedro, Ayapel, Colombia. We tested a convenience sampling of those who accepted and signed the consent form. The National Intestinal Parasite Survey was applied; feces and water source sampling were tested for coprological and microbiology analysis, respectively. Education and pharmacologic treatment to the minor and co-inhabitants were performed. After the recruiting, we followed up at 7 and 12 months. Statistical analysis was performed using IBM® SPSS22. Participants 47, 61,7% male, average age 5,7 years. The caretakers had a low educational background. The monthly income of 72,3% of households was < USD 87. The coprological test showed 61,7% with at least one type of parasite; 32,2% with two or more. Trichuris trichiura was the most frequent. Water sources were positive for Escherichia coli. The population tested showed a high frequency of parasitic infection. We did not find a reduction of intestinal parasitosis with educa tion and pharmacologic treatment at the end of the follow-up. It must be necessary to impact social determinants of public health to achieve intestinal parasitosis control.

Resumen En Colombia, la prevalencia de parasitosis intestinal varía por regiones, clases sociales, condiciones de vida. Realizamos estudio de cohorte (2017-2018) en niños de 1-10 años en El Cedro, Ayapel, Colombia. Muestra por conveniencia, se incluyeron aquellos que aceptaron y firmaron el consentimiento. Se aplicó la Encuesta Nacional de Parásitos Intestinales; se analizaron muestras de heces y fuentes de agua para análisis coprológico y microbiológico, respectivamente. Se realizó educación y tratamiento farmacológico al menor y cohabitantes. Después del reclutamiento, seguimiento a los 7 y 12 meses. El análisis estadístico se realizó con IBM® SPSS22. Participantes 47, 61,7% hombres, promedio de edad 5,7 años. Cuidadores con bajo nivel educativo, ingreso mensual del 72,3% de los hogares fue <USD 87. La población analizada mostró una alta frecuencia de infección parasitaria, un 61,7% con al menos un tipo de parásito; 32,2% con dos o más. Trichuris trichiura fue el más frecuente. Las fuentes de agua fueron positivas para Escherichia coli. Al final del seguimiento, no se redujo la frecuencia de la parasitosis intestinal a pesar de educación y tratamiento farmacológico. Se requiere incidir en los determinantes sociales y de salud pública para lograr el control de las parasitosis intestinales.

Paget's disease of the nipple in a Her2-positive breast cancer xenograft model.

PURPOSE: Paget's disease (PD) of the breast is an uncommon disease of the nipple usually accompanied by an underlying carcinoma, often HER2 + , and accounting for 0.5-5% of all breast cancer. To date, histogenesis of PD of the breast remains controversial, as two theories-transformation and epidermotropic-have been proposed to explain this disease. Currently, animal models recapitulating PD of the nipple have not been described. METHODS: HER2-enriched DT13 breast cancer cells were injected into the mammary fat pad of NOD scid gamma null (NSG) female mice. Immunohistochemical staining and pathological studies were performed on tumor samples, and diagnosis of PD of the nipple was confirmed by expression of proteins characteristic of Paget cells (epidermal growth factor 2 (HER2), androgen receptor (AR), cytokeratin 7 (CK7), cytokeratin 8/18 (CK8/18), and mucin 1 (MUC1)). In addition, DT13 cells grown in 2D culture and in soft agar assays were sensitive to in vitro treatment with pharmacological inhibitors targeting Her2, adenylyl cyclase, mTOR, and PI3K signaling pathways. RESULTS: Mice developed tumors and nipple lesions that were detected exclusively on the tumor-bearing mammary fat pad. Tumor cells were positive for proteins characteristic of Paget cells. In vitro, DT13 cells were sensitive to inhibition of Her2, adenylyl cyclase, mTOR, and PI3K signaling pathways. CONCLUSIONS: Our results suggest that injection of HER2 + DT13 cells into the mammary fat pad of NSG mice recapitulates critical aspects of the pathophysiology of PD of the nipple, supporting the epidermotropic theory as the more likely to explain the histogenesis of this disease.

Efecto terapéutico del alprostadil en pacientes con isquemia crítica terminal de los miembros inferiores / Therapeutic effect of alprostadil in patients with critical lower limb ischemia

Resumen Objetivo: El manejo de la isquemia crítica de los miembros inferiores representa un reto para el cirujano vascular debido a la alta tasa de amputaciones y mortalidad. Las opciones de manejo actuales: puente femorodistal, angioplastia con o sin la colocación de Stents y la resección de la placa con láser o de manera mecánica, presentan a largo plazo una tasa de éxito muy baja y un número de amputaciones supracondíleas que continúa siendo elevado. Métodos: Para este estudio prospectivo se reclutaron 173 pacientes con diagnóstico de estadio avanzado, con isquemia crítica de miembro inferior quienes fueron tratados con alprostadil (60- 120 mcgr/día) por vía intravenosa sistémica por 28 días. La respuesta se midió clínicamente por mejoría del llenado capilar y con el uso de la escala análoga visual del dolor. Resultados: Al momento del alta hospitalaria el 94.3% de los pacientes mejoró el puntaje en la escala análoga visual del dolor (p < 0.0001). El seguimiento a más de un año del tratamiento con alprostadil mostró que el 97% de los pacientes mejoró significativamente su estadio de isquemia, evitándose así una amputación mayor. No se observó respuesta al tratamiento en pacientes previamente intervenidos por vía endovascular (5 pacientes). Conclusiones: El tratamiento de pacientes con isquemia crítica de miembro inferior con alpostadil por infusión intravenosa, con bolos diarios de entre 60 y 120 mcg durante 28 días, este medicamento es seguro y presenta mínimos efectos secundarios. Esta terapia mejora sustancialmente el estadio funcional de Rutherford en estos pacientes y evita amputaciones mayores.

Abstract Objetive: Management of critical lower limb ischemia represents a challenge for the vascular surgeon due to the high rate of amputations and mortality. Current management options include femorodistal bypass, angioplasty with or without stent and laser or mechanical resection of the plaque. They present a low success rate in the long run and a number of supracondylar amputations that still remains high. Methods: This prospective study included 173 patients diagnosed with advanced stage critical lower limb ischemia who were treated with systemic intravenous alprostadil (60 - 120 mcg/day) during 8 days. Response was measured clinically with improvement of capillary refill and using the visual analog scale for pain. Results: Upon discharge 94.3% of patients improved their visual analogue scale score for pain (p < 0.0001). Follow-up for more than a year of alprostadil treatment revealed that 97% of patients significantly improved their ischemia status, thus avoiding further amputation. No response to treatment was observed in patients who had previously undergone endovascular surgery (5 patients). Conclusions: Treating patients with critical lower limb ischemia with intravenous alprostadil, administering daily doses of between 60 and 120 mcg during 28 days shows that this drug is safe and causes minimal secondary effects. This therapy significantly improves Rutherford's function state in these patients and avoids further amputations.

Interactions between Adipocytes and Breast Cancer Cells Stimulate Cytokine Production and Drive Src/Sox2/miR-302b-Mediated Malignant Progression.

Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. Obesity is a risk factor for many cancers, but the mechanisms by which it contributes to cancer development and patient outcome have yet to be fully elucidated. Here, we examined the effects of coculturing human-derived adipocytes with established and primary breast cancer cells on tumorigenic potential. We found that the interaction between adipocytes and cancer cells increased the secretion of proinflammatory cytokines. Prolonged culture of cancer cells with adipocytes or cytokines increased the proportion of mammosphere-forming cells and of cells expressing stem-like markers in vitro. Furthermore, contact with immature adipocytes increased the abundance of cancer cells with tumor-forming and metastatic potential in vivo. Mechanistic investigations demonstrated that cancer cells cultured with immature adipocytes or cytokines activated Src, thus promoting Sox2, c-Myc, and Nanog upregulation. Moreover, Sox2-dependent induction of miR-302b further stimulated cMYC and SOX2 expression and potentiated the cytokine-induced cancer stem cell-like properties. Finally, we found that Src inhibitors decreased cytokine production after coculture, indicating that Src is not only activated by adipocyte or cytokine exposures, but is also required to sustain cytokine induction. These data support a model in which cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain proinflammatory cytokine production, and increase tumor-initiating cell abundance and metastatic progression. Collectively, our findings reveal new insights underlying increased breast cancer mortality in obese individuals and provide a novel preclinical rationale to test the efficacy of Src inhibitors for breast cancer treatment.

Infiltrating S100A8+ myeloid cells promote metastatic spread of human breast cancer and predict poor clinical outcome.

The mechanisms by which breast cancer (BrC) can successfully metastasize are complex and not yet fully understood. Our goal was to identify tumor-induced stromal changes that influence metastatic cell behavior, and may serve as better targets for therapy. To identify stromal changes in cancer-bearing tissue, dual-species gene expression analysis was performed for three different metastatic BrC xenograft models. Results were confirmed by immunohistochemistry, flow cytometry, and protein knockdown. These results were validated in human clinical samples at the mRNA and protein level by retrospective analysis of cohorts of human BrC specimens. In pre-clinical models of BrC, systemic recruitment of S100A8+ myeloid cells-including myeloid-derived suppressor cells (MDSCs)-was promoted by tumor-derived factors. Recruitment of S100A8+ myeloid cells was diminished by inhibition of tumor-derived factors or depletion of MDSCs, resulting in fewer metastases and smaller primary tumors. Importantly, these MDSCs retain their ability to suppress T cell proliferation upon co-culture. Secretion of macrophage inhibitory factor (MIF) activated the recruitment of S100A8+ myeloid cells systemically. Inhibition of MIF, or depletion of MDSCs resulted in delayed tumor growth and lower metastatic burden. In human BrC specimens, increased mRNA and protein levels of S100A8+ infiltrating cells are highly associated with poor overall survival and shorter metastasis free survival of BrC patients, respectively. Furthermore, analysis of nine different human gene expression datasets confirms the association of increased levels of S100A8 transcripts with an increased risk of death. Recruitment of S100A8+ myeloid cells to primary tumors and secondary sites in xenograft models of BrC enhances cancer progression independent of their suppressive activity on T cells. In clinical samples, infiltrating S100A8+ cells are associated with poor overall survival. Targeting these molecules or associated pathways in cells of the tumor microenvironment may translate into novel therapeutic interventions and benefit patient outcome.

Primary breast tumor-derived cellular models: characterization of tumorigenic, metastatic, and cancer-associated fibroblasts in dissociated tumor (DT) cultures.

Our goal was to establish primary cultures from dissociation of breast tumors in order to provide cellular models that may better recapitulate breast cancer pathogenesis and the metastatic process. Here, we report the characterization of six cellular models derived from the dissociation of primary breast tumor specimens, referred to as "dissociated tumor (DT) cells." In vitro, DT cells were characterized by proliferation assays, colony formation assays, protein, and gene expression profiling, including PAM50 predictor analysis. In vivo, tumorigenic and metastatic potential of DT cultures was assessed in NOD/SCID and NSG mice. These cellular models differ from recently developed patient-derived xenograft models in that they can be used for both in vitro and in vivo studies. PAM50 predictor analysis showed DT cultures similar to their paired primary tumor and as belonging to the basal and Her2-enriched subtypes. In vivo, three DT cultures are tumorigenic in NOD/SCID and NSG mice, and one of these is metastatic to lymph nodes and lung after orthotopic inoculation into the mammary fat pad, without excision of the primary tumor. Three DT cultures comprised of cancer-associated fibroblasts (CAFs) were isolated from luminal A, Her2-enriched, and basal primary tumors. Among the DT cells are those that are tumorigenic and metastatic in immunosuppressed mice, offering novel cellular models of ER-negative breast cancer subtypes. A group of CAFs provide tumor subtype-specific components of the tumor microenvironment (TME). Altogether, these DT cultures provide closer-to-primary cellular models for the study of breast cancer pathogenesis, metastasis, and TME.

Triple negative breast cancer initiating cell subsets differ in functional and molecular characteristics and in γ-secretase inhibitor drug responses.

Increasing evidence suggests that stem-like cells mediate cancer therapy resistance and metastasis. Breast tumour-initiating stem cells (T-ISC) are known to be enriched in CD44(+) CD24(neg/low) cells. Here, we identify two T-ISC subsets within this population in triple negative breast cancer (TNBC) lines and dissociated primary breast cancer cultures: CD44(+) CD24(low+) subpopulation generates CD44(+) CD24(neg) progeny with reduced sphere formation and tumourigenicity. CD44(+) CD24(low+) populations contain subsets of ALDH1(+) and ESA(+) cells, yield more frequent spheres and/or T-ISC in limiting dilution assays, preferentially express metastatic gene signatures and show greater motility, invasion and, in the MDA-MB-231 model, metastatic potential. CD44(+) CD24(low+) but not CD44(+) CD24(neg) express activated Notch1 intracellular domain (N1-ICD) and Notch target genes. We show N1-ICD transactivates SOX2 to increase sphere formation, ALDH1+ and CD44(+) CD24(low+) cells. Gamma secretase inhibitors (GSI) reduced sphere formation and xenograft growth from CD44(+) CD24(low+) cells, but CD44(+) CD24(neg) were resistant. While GSI hold promise for targeting T-ISC, stem cell heterogeneity as observed herein, could limit GSI efficacy. These data suggest a breast T-ISC hierarchy in which distinct pathways drive developmentally related subpopulations with different anti-cancer drug responsiveness.

Paget's disease of the nipple.

Paget's disease of the breast is a disorder of the nipple-areola complex that, while rare, is often associated with an underlying carcinoma. It is characterized by eczematoid changes of the nipple. Two theories have been proposed to explain the pathogenesis of Paget's disease. The Epidermotropic, which is the most accepted theory, suggests that Paget's cells originate from ductal cancer cells that had migrated from the underlying breast parenchyma. It is supported by the predominance of breast cancer markers found in Paget's disease. This article provides an overview of Paget's disease of the breast with special attention to immunohistochemistry and raises the question of new therapeutic approaches.

A new mouse model for the study of human breast cancer metastasis.

Breast cancer is the most common cancer in women, and this prevalence has a major impact on health worldwide. Localized breast cancer has an excellent prognosis, with a 5-year relative survival rate of 85%. However, the survival rate drops to only 23% for women with distant metastases. To date, the study of breast cancer metastasis has been hampered by a lack of reliable metastatic models. Here we describe a novel in vivo model using human breast cancer xenografts in NOD scid gamma (NSG) mice; in this model human breast cancer cells reliably metastasize to distant organs from primary tumors grown within the mammary fat pad. This model enables the study of the entire metastatic process from the proper anatomical site, providing an important new approach to examine the mechanisms underlying breast cancer metastasis. We used this model to identify gene expression changes that occur at metastatic sites relative to the primary mammary fat pad tumor. By comparing multiple metastatic sites and independent cell lines, we have identified several gene expression changes that may be important for tumor growth at distant sites.

Transcriptional regulation of gene expression during osmotic stress responses by the mammalian target of rapamycin.

Although stress can suppress growth and proliferation, cells can induce adaptive responses that allow them to maintain these functions under stress. While numerous studies have focused on the inhibitory effects of stress on cell growth, less is known on how growth-promoting pathways influence stress responses. We have approached this question by analyzing the effect of mammalian target of rapamycin (mTOR), a central growth controller, on the osmotic stress response. Our results showed that mammalian cells exposed to moderate hypertonicity maintained active mTOR, which was required to sustain their cell size and proliferative capacity. Moreover, mTOR regulated the induction of diverse osmostress response genes, including targets of the tonicity-responsive transcription factor NFAT5 as well as NFAT5-independent genes. Genes sensitive to mTOR-included regulators of stress responses, growth and proliferation. Among them, we identified REDD1 and REDD2, which had been previously characterized as mTOR inhibitors in other stress contexts. We observed that mTOR facilitated transcription-permissive conditions for several osmoresponsive genes by enhancing histone H4 acetylation and the recruitment of RNA polymerase II. Altogether, these results reveal a previously unappreciated role of mTOR in regulating transcriptional mechanisms that control gene expression during cellular stress responses.

Simultaneous measurement of ERα, HER2, and phosphoERK1/2 in breast cancer cell lines by flow cytometry.

The activation of human epidermal growth factor receptor-2 (HER2) results in the activation of the mitogen-activated protein kinase (MAPK) cascade that may lead to the resistance to anti-estrogen therapy in estrogen receptor (ERα) expressing breast cancer by means of phosphorylation of ERα in the N-terminal region by phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) and by means of decreasing ERα expression. Immunohistochemistry is the most widely used technique for the detection of ERα and HER2 in breast cancer specimens, however, is inadequate in its ability to assess the relationship between ERα, HER2, and MAPK cascade at the single cell level. To clear this major hurdle, we devised a novel flow cytometric method to quantify the expression of ERα, HER2, and the activation of MAPK cascade simultaneously in single cells. The method was validated by concurrent Western blotting in established cell lines: MDA-231 (ERα and HER2-negative), MCF-7 (ERα-positive, HER2-negative), MCF-7 cells overexpressing ERα after long-term incubation in estrogen-free medium, and HER2 transfected MCF7 cells. Using the flow cytometry method, we confirmed the previous finding that ERα expression is down-regulated upon epidermal growth factor mediated ERK1/2 phosphorylation in EGFR/MCF-7 cells. To our knowledge, this is the first such assay to incorporate simultaneous single cell measurement for all of these pathways, which may prove useful to determine the intratumoral heterogeneity in breast tumors or the receptor status in circulating tumor cells.

NFAT5 regulates T lymphocyte homeostasis and CD24-dependent T cell expansion under pathologic hypernatremia.

Immune cells rely on the transcription factor NFAT5 to adapt to hypertonic stress. The hypertonicity-dependent role of NFAT5 in T cells in vivo remains unclear because mouse models of NFAT5 deficiency have produced substantially different T cell phenotypes. In this study, we analyzed the T cell compartment in NFAT5-null and T cell-specific NFAT5 knockout mice. We found that NFAT5-null mice had constitutive, pronounced hypernatremia and suffered a severe immunodeficiency, with T cell lymphopenia, altered CD8 naive/memory homeostasis, and inability to reject allogeneic tumors. By contrast, T cell-specific NFAT5 knockout mice had normal plasma tonicity, rejected allogeneic tumors, and exhibited only a mild, low-penetrance memory bias in CD8 cells. Notably, when T cells from these mice were cultured ex vivo in hypernatremic media, they exhibited features found in NFAT5-null mice, with pronounced naive/memory imbalance and impaired homeostatic survival in response to IL-7, as well as a severe inhibition of their mitogen-induced proliferation. By analyzing surface receptors whose expression might be affected in NFAT5-deficient cells, we identified CD24 as a novel NFAT5 target induced by hypertonicity both in vitro and in vivo, and required to sustain T cell expansion under osmostress. NFAT5 bound to the Cd24 promoter in response to hypertonicity facilitated the local derepression of chromatin and enhanced the expression of CD24 mRNA and protein. Altogether, our results indicate that the systemic hypernatremia of NFAT5-null mice is a major contributor to their immunodeficiency, and highlight the role of NFAT5 and CD24 in the homeostasis of T cells under osmostress in vivo.

The transcription factor NFAT5 is required for cyclin expression and cell cycle progression in cells exposed to hypertonic stress.

BACKGROUND: Hypertonicity can perturb cellular functions, induce DNA damage-like responses and inhibit proliferation. The transcription factor NFAT5 induces osmoprotective gene products that allow cells to adapt to sustained hypertonic conditions. Although it is known that NFAT5-deficient lymphocytes and renal medullary cells have reduced proliferative capacity and viability under hypertonic stress, less is understood about the contribution of this factor to DNA damage responses and cell cycle regulation. METHODOLOGY/PRINCIPAL FINDINGS: We have generated conditional knockout mice to obtain NFAT5(-/-) T lymphocytes, which we used as a model of proliferating cells to study NFAT5-dependent responses. We show that hypertonicity triggered an early, NFAT5-independent, genotoxic stress-like response with induction of p53, p21 and GADD45, downregulation of cyclins, and cell cycle arrest. This was followed by an NFAT5-dependent adaptive phase in wild-type cells, which induced an osmoprotective gene expression program, downregulated stress markers, resumed cyclin expression and proliferation, and displayed enhanced NFAT5 transcriptional activity in S and G2/M. In contrast, NFAT5(-/-) cells failed to induce osmoprotective genes and exhibited poorer viability. Although surviving NFAT5(-/-) cells downregulated genotoxic stress markers, they underwent cell cycle arrest in G1/S and G2/M, which was associated with reduced expression of cyclins E1, A2 and B1. We also show that pathologic hypertonicity levels, as occurring in plasma of patients and animal models of osmoregulatory disorders, inhibited the induction of cyclins and aurora B kinase in response to T cell receptor stimulation in fresh NFAT5(-/-) lymphocytes. CONCLUSIONS/SIGNIFICANCE: We conclude that NFAT5 facilitates cell proliferation under hypertonic conditions by inducing an osmoadaptive response that enables cells to express fundamental regulators needed for cell cycle progression.

Regulation of the hypertonic stress response and other cellular functions by the Rel-like transcription factor NFAT5.

Stress, be it from environmental factors or intrinsic to the cell as result of growth and metabolism, can be harmful to cells. Mammalian cells have developed numerous mechanisms to respond to diverse forms of stress. These mechanisms combine signaling cascades and activation of gene expression programs to orchestrate an adaptive response that will allow the cell to survive and resume its normal functioning. In this review we will focus on the transcription factor NFAT5, a fundamental regulator of the response to osmotic stress in mammalian cells. Identified in 1999, NFAT5 is the latest addition to the Rel family, which comprises the NF-kappaB and NFATc proteins. Though in some of its structural and functional features NFAT5 is a hybrid between these two major groups of Rel proteins, it has unique characteristics that make it stand on its own as a third type of Rel transcription factor. Since its discovery, NFAT5 has been studied mostly in the context of the hypertonicity stress response. The advent of mouse models deficient in NFAT5 and other recent advances have confirmed a fundamental osmoprotective role for this factor in mammals, but also revealed features that suggest it may have a wider range of functions.