Nuclear localization of ING3 is required to suppress melanoma cell migration, invasion and angiogenesis.

Inhibitor of growth family member 3 (ING3), a tumor suppressor, plays crucial roles in cell cycle regulation, apoptosis and transcription. Previous studies suggest important roles of nuclear ING3, however, the nuclear localization sequence (NLS) of ING3 is not defined and its biological functions remain to be elucidated. In this study, various ING3 mutants were generated to identify its NLS. The NLS of ING3 was determined as KKFK between 164 and 167 amino acids. More intriguingly, replacement of Lysine 164 residue of ING3 with alanine (K164A) resulted in retention of ING3 in the cytoplasm. Overexpression of ING3 led to inhibition of melanoma cell migration, invasion, and angiogenesis respectively, however, this inhibition was abrogated in cells with overexpression of ING3-K164A mutant. In conclusion, this study identified the NLS of ING3 and demonstrated the significance of ING3 nuclear localization for tumor suppressive functions of ING3, and future studies await to elucidate the role of ING3 (K164) post-modificaton in its nuclear transportation and cancer development.

Monoclonal Antibodies for the Treatment of Melanoma: Present and Future Strategies.

Metastatic melanoma is a dreadful type of skin cancer arising due to uncontrolled proliferation of melanocytes. It has very poor prognosis, low 5-year survival rates and until recently there were only handful of treatment options for metastatic melanoma patients. The drugs that were approved for the treatment had low response rates and were associated with severe adverse events. With the introduction of monoclonal antibodies against inhibitory immune checkpoints the treatment landscape for metastatic melanoma has changed dramatically. Ipilimumab, the first monoclonal antibody to be approved for the treatment of metastatic melanoma, showed significant improvements in durable response rates in patients and paved the way for next class of monoclonal antibodies. Nivolumab and pembrolizumab, the anti-PD-1 antibodies that were approved 3-years after the approval of ipilimumab, had decent response rates, low relapse rates and showed manageable safety profile. Antibodies against ligands for PD-1 receptors were then developed to overcome the adverse effects of anti-PD-1 antibodies and combination of monoclonal antibodies (ipilimumab plus nivolumab) was tested to increase the response rates. Additional target receptors that regulate T cell activity were identified on T cells and monoclonal antibodies against potential targets such as TIGIT, TIM-3, and LAG-3 were developed. This chapter discusses the details of monoclonal antibodies used for the treatment of melanoma along with the ones that could be introduced in the near future with emphasis on mechanisms by which antibodies stimulate anti-tumor immune response and the specifics of target molecules of the antibodies.

Nobel committee honors tumor immunologists.

This commentary wishes to highlight the 2018 Nobel Prize in Medicine awarded to two cancer immunotherapy scientists, Prof James Allison and Prof Tasuku Honjo, for their discovery on unleashing the body's immune system to attack cancer. Their studies have led to the development of an entire class of drugs that hopefully will bring lasting remissions to many patients who had run out of options.

Prognostic Significance of Nuclear Phospho-ATM Expression in Melanoma.

UV radiation induced genomic instability is one of the leading causes for melanoma. Phosphorylation of Ataxia Telangiectasia Mutated (ATM) is one of the initial events that follow DNA damage. Phospho-ATM (p-ATM) plays a key role in the activation of DNA repair and several oncogenic pathways as well as in the maintenance of genomic integrity. The present study was therefore performed to understand the significance of p-ATM in melanoma progression and to correlate it with patient prognosis. Tissue microarray and immunohistochemical analysis were employed to study the expression of p-ATM in melanoma patients. A total of 366 melanoma patients (230 primary melanoma and 136 metastatic melanoma) were used for the study. Chi-square test, Kaplan-Meier, univariate and multivariate Cox regression analysis were used to elucidate the prognostic significance of p-ATM expression. Results revealed that both loss of, and gain in, p-ATM expression were associated with progression of melanoma from normal nevi to metastatic melanoma. Patients whose samples showed negative or strong p-ATM staining had significantly worse 5-year survival compared to patients who had weak to moderate expression. Loss of p-ATM expression was associated with relatively better 5-year survival, but the corresponding 10-year survival curve almost overlapped with that of strong p-ATM expression. p-ATM expression was found to be an independent prognostic factor for 5-year but not for 10-year patient survival. In conclusion our findings show that loss of p-ATM expression and gain-in p-ATM expression are indicators of worse melanoma patient survival.

Regulation of Na⁺/H⁺ Exchanger in Dendritic Cells by Akt1.

BACKGROUND/AIMS: Dendritic cells (DCs), antigen-presenting cells critically important for primary immune response and establishment of immunological memory, are activated by bacterial lipopolysaccharides (LPS) resulting in stimulation of Na(+)/H(+) exchanger, ROS formation and migration. The effects are dependent on phosphoinositide 3 (PI3) kinase and paralleled by Akt phosphorylation. The present study explored the contribution of the Akt isoform Akt1. METHODS: Cytosolic pH (pH(i)) (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence), Na(+)/H(+) exchanger activity (Na(+) dependent realkalinization after an ammonium pulse), cell volume (forward scatter in FACS analysis), and ROS production (2',7'-dichlorodihydrofluorescein diacetate [DCFDA] fluorescence) were determined in DCs isolated from bone marrow of mice lacking functional Akt1/PKBα (akt1(-/-)) and their wild type littermates (akt1(+/+)). RESULTS: Forward scatter was lower in akt1(-/-) than in akt1(+/+) DCs, whereas pH(i), Na(+)/H(+) exchanger activity and ROS formation were less in untreated akt1(-/-) and akt1(+/+) DCs. Exposure of DCs to LPS was followed by increase of forward scatter and ROS formation to a similar extent in akt1(-/-) and in akt1(+/+) DCs. A 4 hours treatment with either LPS (1µg/ml) or tert-butylhydroperoxide (tBOOH, 5 µM) significantly stimulated Na(+)/H(+) exchanger activity in both genotypes, effects, however, significantly blunted in akt1(-/-) DCs. CONCLUSION: The present observations demonstrate that Akt1 is required for the full stimulation of Na(+)/H(+) exchanger activity by LPS or oxidative stress in dendritic cells.

The role of the metastasis suppressor gene KAI1 in melanoma angiogenesis.

The tetraspan protein KAI1 (CD82) has been previously shown to have important roles in cell migration, invasion, and melanoma prognosis. In this study, we investigated the role of KAI1 regarding melanoma angiogenesis. KAI1 overexpression strongly suppressed the growth of the human umbilical vein endothelial cells and their tubular structure formation in vitro. Also, KAI1 was able to inhibit both interleukin-6 (IL-6) and VEGF at mRNA and protein levels. Using nude mice in the in vivo study, we showed that KAI1, through the regulation of ING4, inhibited blood vessel formation in matrigel plugs along with the downregulation of IL-6 and VEGF, and the recruitment of CD31-positive cells. Finally, we found that KAI1 was able to suppress the activity of a serine/threonine kinase Akt by suppressing Akt phosphorylation (Ser473). Taken together, our results suggested that KAI1 was able to suppress melanoma angiogenesis by downregulating IL-6 and VEGF expression, and the restoration of KAI1 functionality offered a new approach in human melanoma treatment.

Immunotherapy of melanoma: present options and future promises.

Metastatic melanoma is notorious for its immune evasion and resistance to conventional chemotherapy. The recent success of ipilimumab, a human monoclonal antibody against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), in increasing the median survival time and stabilizing the disease progression renewed, hopes in treatment for melanoma. Currently, ipilimumab and high-dose interleukin-2 (IL-2; Aldesleukin) are approved as monotherapies for the treatment of patients with unresectable advanced melanoma, and pegylated interferon-α2b (p-IFN-α2b) is approved as an adjuvant for the treatment of patients with surgically resected high-risk melanoma. The present review describes the currently approved immune-modulators and the promising immune-based interventions that are currently in clinical trials. We present the four commonly used strategies to boost immune responses against the tumors; monoclonal antibodies, cytokines, cancer vaccines, and adoptive T cell transfer. The corresponding lists of ongoing clinical trials include details of the trial phase, target patients, intervention details, status of the study, and expected date of completion. Further, our review discusses the challenges faced by immunotherapy and the various strategies adopted to overcome them.

A combination of p300 and Braf expression in the diagnosis and prognosis of melanoma.

BACKGROUND: To date only a handful of drugs are available for the treatment of melanoma. Among them vemurafenib, a BrafV600E specific inhibitor, showed promising results in terms of response rate and increase in median survival time. However, its effectiveness is limited by development of resistance and the search for additional drugs for melanoma treatment is ongoing. The present study was performed to analyze the correlation between Braf expression and the expression of p300, a known down stream target of the mitogen activated protein kinase (MAPK) pathway, which was recently shown by us to be a prognostic marker for melanoma progression and patient survival. METHODS: The expression of Braf and p300 expression were correlated and analyzed by Chi-square test. A total of 327 melanoma patient cases (193 primary melanoma and 134 metastatic melanoma) were used for the study. Classification & regression tree (CRT), Kaplan-Meier, and multivariate Cox regression analysis were used to elucidate the significance of the combination of Braf and p300 expression in the diagnosis and prognosis of melanoma. RESULTS: Our results demonstrate that Braf expression is inversely correlated with nuclear p300 and positively correlated with cytoplasmic p300 expression. Braf and cytoplasmic p300 were found to be associated with melanoma progression, tumor size and ulceration status. CRT analysis revealed that a combination of Braf and p300 expression (nuclear and cytoplasmic), could be used to distinguish between nevi and melanoma, and primary from metastatic melanoma lesions. The combination of Braf and nuclear p300 was significantly associated with patient survival and nuclear p300 was found to be an independent predictor of patient survival. CONCLUSION: Our results indicate a cross-talk between Braf and p300 in melanoma and demonstrate the importance Braf and p300 expression in the diagnosis and prognosis of melanoma.

Loss of XRCC1 confers a metastatic phenotype to melanoma cells and is associated with poor survival in patients with melanoma.

Ultraviolet (UV) radiation-induced DNA damage and genomic instability is one of the leading causes for melanoma. X-ray repair cross-complementary protein 1, XRCC1, plays a critically important role in base excision repair pathway. This study was therefore performed to analyze the correlation between XRCC1 expression, melanoma progression, and patient survival. Using a tissue microarray with a total of 119 patients with melanoma, we demonstrate that loss of XRCC1 expression is associated with the progression of disease from dysplastic nevi to primary melanoma and to metastatic melanoma. We found that the loss of XRCC1 was correlated with the progression of melanoma from AJCC stage II to stage III and with worse overall and disease-specific 5-yr and 10-yr survival of patients with melanoma. Furthermore, we also illustrate the inhibitory effect of XRCC1 on melanoma cell invasion and migration, which are the regulatory events in melanoma metastasis.

Tumor suppressor Ing1b facilitates DNA repair and prevents oxidative stress induced cell death.

Inhibitor of growth (ING) family of proteins are known to coordinate with histone acetyltransferases and regulate the key events of cell cycle and DNA repair. Previous work from our lab showed that Ing1b regulated the nucleotide excision repair by facilitating histone acetylation and subsequent chromatin relaxation. Further, it was also shown that Ing1b protected the cells from genomic instability induced cell death by promoting ubiquitination of proliferating cell nuclear antigen (PCNA). In the present study we explored the role of Ing1b in the repair of oxidized DNA and prevention of oxidative stress induced genotoxic cell death. Using HCT116 cells we show that Ing1b protein expression is induced by treatment with H2O2. Ing1b lacking cells showed decreased ability to repair the oxidized DNA. PCNA monoubiquitination, a critical event of DNA repair was blunted in Ing1b knock down cells and augmented in Ing1b over expressing cells. Moreover, oxidative stress induced cell death was higher in cells lacking Ing1b whereas it was lower in Ing1b over expressing cells. Finally we show that inhibition of histone deacetylases, rescued the Ing1b knock down cells from cytotoxic effects of H2O2 treatment.

Annexin 7 in the regulation of gastric acid secretion.

BACKGROUND/AIMS: Glucocorticoids enhance gastric acid secretion and inhibit gastric cyclooxygenase, thus downregulating formation of PGE2, an inhibitor of gastric acid secretion. In erythrocytes, PGE2 formation is inhibited by annexin 7. The present study thus explored whether annexin 7 participates in the regulation of gastric acid secretion. METHODS: Annexin 7 protein expression was determined by Western blotting, cytosolic pH (pHi) of parietal cells utilizing BCECF-fluorescence, and gastric acid secretion by determination of Na(+)-independent pHi recovery from an ammonium pulse (∆pHi/min). Experiments were performed in isolated glands from gene targeted mice lacking annexin 7 (anx7(-/-)) and in respective wild type animals (anx7(+/+)). RESULTS: Prior to treatment pHi and ∆pHi/min were similar in isolated gastric glands from anx7(-/-) and from anx7(+/+) mice. Aspirin (100 µM added to the glands 1 hr prior to the experiment) significantly increased ∆pHi/min to similar values in both genotypes. The administration of dexamethasone (10 µg/g BW subcutaneously for 4 consecutive days prior to the experiments) significantly increased ∆pH/min in anx7(+/+) mice but not in anx7(-/-) mice. Following dexamethasone treatment, the luminal pH was significantly lower and the acid content significantly higher in anx7(+/+) mice than in anx7(-/-) mice. An increase of extracellular K(+) concentration to 35 mM (replacing Na(+)/NMDG(+)) significantly increased ∆pHi/min in both genotypes. In neither genotype dexamethasone increased ∆pH/min further in the presence of 35 mM K(+) or presence of aspirin. CONCLUSIONS: Annexin 7 is required for the stimulation of gastric acid secretion by glucocorticoids.

Decreased expression of nuclear p300 is associated with disease progression and worse prognosis of melanoma patients.

BACKGROUND: Genomic instability due to UV radiation is one of the leading causes for melanoma. Histone acetyltransferase p300 plays an indispensible role in DNA repair and maintenance of genomic integrity. The present study was performed to analyze the correlation between p300 expression, melanoma progression and patient survival. METHODS: Tissue microarray and immunohistochemical analysis was employed to study the expression of p300 in melanoma patients. A total of 358 melanoma patients (250 primary melanoma and 108 metastatic melanoma) were used for the study. Kaplan-Meier, univariate and multivariate Cox regression analysis, and receiver-operating characteristic curves, were used to elucidate the prognostic significance of p300 expression. RESULTS: Our results demonstrate that p300 is expressed in both nucleus and cytoplasm but the nuclear expression of p300 is predominant. The progression of disease from dysplastic nevi to primary melanoma and to metastatic melanoma was associated with decreased nuclear and increased cytoplasmic p300 expression. Especially, the loss of nuclear and gain in cytoplasmic p300 was correlated with the progression of melanoma from AJCC stage II to stage III, which requires the migration and metastasis of cancer cells from primary sites to lymph nodes. Similarly, decrease in nuclear, and increase in cytoplasmic p300 expression correlated with worse survival of melanoma patients. Nuclear p300 but not cytoplasmic p300 could predict the patient survival independent of AJCC stage, age and gender. CONCLUSION: Loss of nuclear p300 expression is an indicator of worse patient survival and is an independent prognostic marker for melanoma.

PKB/SGK-dependent GSK3-phosphorylation in the regulation of LPS-induced Ca2+ increase in mouse dendritic cells.

The function of dendritic cells (DCs) is modified by glycogen synthase kinase GSK3 and GSK3 inhibitors have been shown to protect against inflammatory disease. Regulators of GSK3 include the phosphoinositide 3 kinase (PI3K) pathway leading to activation of protein kinase B (PKB/Akt) and serum and glucocorticoid inducible kinase (SGK) isoforms, which in turn phosphorylate and thus inhibit GSK3. The present study explored, whether PKB/SGK-dependent inhibition of GSK3 contributes to the regulation of cytosolic Ca(2+) concentration following stimulation with bacterial lipopolysaccharides (LPS). To this end DCs from mutant mice, in which PKB/SGK-dependent GSK3α,ß regulation was disrupted by replacement of the serine residues in the respective SGK/PKB-phosphorylation consensus sequence by alanine (gsk3(KI)), were compared to DCs from respective wild type mice (gsk3(WT)). According to Western blotting, GSK3 phosphorylation was indeed absent in gsk3(KI) DCs. According to flow cytometry, expression of antigen-presenting molecule major histocompatibility complex II (MHCII) and costimulatory molecule CD86, was similar in unstimulated and LPS (1µg/ml, 24h)-stimulated gsk3(WT) and gsk3(KI) DCs. Moreover, production of cytokines IL-6, IL-10, IL-12 and TNFα was not significantly different in gsk3(KI) and gsk3(WT) DCs. In gsk3(WT) DCs, stimulation with LPS (1µg/ml) within 10min led to transient phosphorylation of GSK3. According to Fura2 fluorescence, LPS (1µg/ml) increased cytosolic Ca(2+) concentration, an effect significantly more pronounced in gsk3(KI) DCs than in gsk3(WT) DCs. Conversely, GSK3 inhibitor SB216763 (3-[2,4-Dichlorophenyl]-4-[1-methyl-1H-indol-3-yl]-1H-pyrrole-2,5-dione, 10µM, 30min) significantly blunted the increase of cytosolic Ca(2+) concentration following LPS exposure. In conclusion, PKB/SGK-dependent GSK3α,ß activity participates in the regulation of Ca(2+) signaling in dendritic cells.

Upregulation of intestinal NHE3 following saline ingestion.

BACKGROUND: Little is known about the effect of salt content of ingested fluid on intestinal transport processes. Osmosensitive genes include the serum- and glucocorticoid-inducible kinase SGK1, which is up-regulated by hyperosmolarity and cell shrinkage. SGK1 is in turn a powerful stimulator of the intestinal Na(+)/H(+) exchanger NHE3. The present study was thus performed to elucidate, whether the NaCl content of beverages influences NHE3 activity. METHODS: Mice were offered access to either plain water or isotonic saline ad libitum. NHE3 transcript levels and protein abundance in intestinal tissue were determined by confocal immunofluorescent microscopy, RT-PCR and western blotting, cytosolic pH (pHi) in intestinal cells from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence and Na(+)/H(+) exchanger activity from the Na(+) dependent realkalinization following an ammonium pulse. RESULTS: Saline drinking significantly enhanced fluid intake and increased NHE3 transcript levels, NHE3 protein and Na(+)/H(+) exchanger activity. CONCLUSIONS: Salt content of ingested fluid has a profound effect on intestinal Na(+)/H(+) exchanger expression and activity.

SGK3 regulates Ca(2+) entry and migration of dendritic cells.

BACKGROUND/AIMS: Dendritic cells (DCs) are antigen-presenting cells linking innate and adaptive immunity. DC maturation and migration are governed by alterations of cytosolic Ca(2+) concentrations ([Ca(2+)](i)). Ca(2+) entry is in part accomplished by store-operated Ca(2+) (SOC) channels consisting of the membrane pore-forming subunit Orai and the ER Ca(2+) sensing subunit STIM. Moreover, DC functions are under powerful regulation of the phosphatidylinositol-3-kinase (PI3K) pathway, which suppresses proinflammatory cytokine production but supports DC migration. Downstream targets of PI3K include serum- and glucocorticoid-inducible kinase isoform SGK3. The present study explored, whether SGK3 participates in the regulation of [Ca(2+)](i) and Ca(2+)-dependent functions of DCs, such as maturation and migration. METHODS/RESULTS: Experiments were performed with bone marrow derived DCs from gene targeted mice lacking SGK3 (sgk3(-/-)) and DCs from their wild type littermates (sgk3(+/+)). Maturation, phagocytosis and cytokine production were similar in sgk3(-/-) and sgk3(+/+) DCs. However, SOC entry triggered by intracellular Ca(2+) store depletion with the endosomal Ca(2+) ATPase inhibitor thapsigargin (1 µM) was significantly reduced in sgk3(-/-) compared to sgk3(+/+) DCs. Similarly, bacterial lipopolysaccharide (LPS, 1 µg/ml)- and chemokine CXCL12 (300 ng/ml)- induced increase in [Ca(2+)](i) was impaired in sgk3(-/-) DCs. Moreover, currents through SOC channels were reduced in sgk3(-/-) DCs. STIM2 transcript levels and protein abundance were significantly lower in sgk3(-/-) DCs than in sgk3(+/+) DCs, whereas Orai1, Orai2, STIM1 and TRPC1 transcript levels and/or protein abundance were similar in sgk3-/- and sgk3(+/+) DCs. Migration of both, immature DCs towards CXCL12 and LPS-matured DCs towards CCL21 was reduced in sgk3(-/-) as compared to sgk3(+/+) DCs. Migration of sgk3(+/+) DCs was further sensitive to SOC channel inhibitor 2-APB (50 µM) and to STIM1/STIM2 knock-down. CONCLUSION: SGK3 contributes to the regulation of store-operated Ca(2+) entry into and migration of dendritic cells, effects at least partially mediated through SGK3-dependent upregulation of STIM2 expression.

OSR1-sensitive regulation of Na+/H+ exchanger activity in dendritic cells.

The oxidative stress-responsive kinase 1 (OSR1) is activated by WNK (with no K kinases) and in turn stimulates the thiazide-sensitive Na-Cl cotransporter (NCC) and the furosemide-sensitive Na-K-2Cl cotransporter (NKCC), thus contributing to transport and cell volume regulation. Little is known about extrarenal functions of OSR1. The present study analyzed the impact of decreased OSR1 activity on the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs were cultured from bone marrow of heterozygous WNK-resistant OSR1 knockin mice (osr(KI)) and wild-type mice (osr(WT)). Cell volume was estimated from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescence, and Na(+)/H(+) exchanger activity from Na(+)-dependent realkalinization following ammonium pulse and migration utilizing transwell chambers. DCs expressed WNK1, WNK3, NCC, NKCC1, and OSR1. Phosphorylated NKCC1 was reduced in osr(KI) DCs. Cell volume and pH(i) were similar in osr(KI) and osr(WT) DCs, but Na(+)/H(+) exchanger activity and ROS production were higher in osr(KI) than in osr(WT) DCs. Before LPS treatment, migration was similar in osr(KI) and osr(WT) DCs. LPS (1 µg/ml), however, increased migration of osr(WT) DCs but not of osr(KI) DCs. Na(+)/H(+) exchanger 1 inhibitor cariporide (10 µM) decreased cell volume, intracellular reactive oxygen species (ROS) formation, Na(+)/H(+) exchanger activity, and pH(i) to a greater extent in osr(KI) than in osr(WT) DCs. LPS increased cell volume, Na(+)/H(+) exchanger activity, and ROS formation in osr(WT) DCs but not in osr(KI) DCs and blunted the difference between osr(KI) and osr(WT) DCs. Na(+)/H(+) exchanger activity in osr(WT) DCs was increased by the NKCC1 inhibitor furosemide (100 nM) to values similar to those in osr(KI) DCs. Oxidative stress (10 µM tert-butyl-hydroperoxide) increased Na(+)/H(+) exchanger activity in osr(WT) DCs but not in osr(KI) DCs and reversed the difference between genotypes. Cariporide virtually abrogated Na(+)/H(+) exchanger activity in both genotypes and blunted LPS-induced cell swelling and ROS formation in osr(WT) mice. In conclusion, partial OSR1 deficiency influences Na(+)/H(+) exchanger activity, ROS formation, and migration of dendritic cells.

Effects of gum arabic (Acacia senegal) on renal function in diabetic mice.

BACKGROUND/AIMS: Gum arabic (GA) is a Ca(2+)-, Mg(2+)- and K(+)-rich dietary fiber used for the treatment of patients with chronic kidney disease in Middle Eastern countries. In healthy mice, GA treatment increases creatinine clearance, renal ADH excretion, as well as intestinal and renal excretion of Mg(2+) and Ca(2+). GA decreases plasma Pi concentration, urinary Pi and Na(+) excretion. The present study explored the effects of GA on renal function in diabetic mice. METHODS: Metabolic cage experiments were performed on Akita mice (akita(+/-)), which spontaneously develop insulin deficiency and thus hyperglycemia. Plasma and urinary concentrations of Na(+), K(+) and Ca(2+) were measured by flame photometry (AFM 5051, Eppendorf, Germany), creatinine by the Jaffé method, phosphate photometrically, urea by an enzymatic method, glucose utilizing a glucometer and an enzymatic kit, aldosterone using an RIA, urinary albumin fluorometrically, and blood pressure by the tail-cuff method. RESULTS: GA (10% in drinking water) significantly increased urinary excretion of Ca(2+) and significantly decreased plasma phosphate and urea concentrations, urinary flow rate, urinary Na(+), phosphate and glucose excretion, blood pressure and proteinuria. CONCLUSIONS: GA treatment decreases blood pressure and proteinuria in diabetic mice and may thus prove beneficial in diabetic nephropathy.

Effect of azathioprine on Na(+)/H(+) exchanger activity in dendritic cells.

Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na(+)/H(+) exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na(+)/H(+) exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) was estimated utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 µg/ml) led to a transient increase of Na(+)/H(+) exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 µM) did not significantly alter the Na(+)/H(+) exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na(+)/H(+) exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na(+)/H(+) exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug.

Rapamycin sensitive ROS formation and Na(+)/H(+) exchanger activity in dendritic cells.

Rapamycin, a widely used immunosuppressive drug, has been shown to interfere with the function of dendritic cells (DCs), antigen-presenting cells contributing to the initiation of primary immune responses and the establishment of immunological memory. DC function is governed by the Na(+)/H(+) exchanger (NHE), which is activated by bacterial lipopolysaccharides (LPS) and is required for LPS-induced cell swelling, reactive oxygen species (ROS) production and TNF-α release. The present study explored, whether rapamycin influences NHE activity and/or ROS formation in DCs. Mouse DCs were treated with LPS in the absence and presence of rapamycin (100 nM). ROS production was determined from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, and TNF-α production utilizing ELISA. In the absence of LPS, rapamycin did not significantly modify cytosolic pH, NHE activity or cell volume but significantly decreased ROS formation. LPS stimulated NHE activity, enhanced forward scatter, increased ROS formation, and triggered TNF-α release, effects all blunted in the presence of rapamycin. NADPH oxidase inhibitor Vas-2870 (10 µM) mimicked the effect of rapamycin on LPS induced stimulation of NHE activity and TNF-α release. The effect of rapamycin on TNF-α release was also mimicked by the antioxidant ROS scavenger Tempol (30 µM) and partially reversed by additional application of tert-butylhydroperoxide (10 µM). In conclusion, in DCs rapamycin disrupts LPS induced ROS formation with subsequent inhibition of NHE activity, cell swelling and TNF-α release.