Heterozygous disruption of renal outer medullary potassium channel in rats is associated with reduced blood pressure.

The renal outer medullary potassium channel (ROMK, KCNJ1) mediates potassium recycling and facilitates sodium reabsorption through the Na(+)/K(+)/2Cl(-) cotransporter in the loop of Henle and potassium secretion at the cortical collecting duct. Human genetic studies indicate that ROMK homozygous loss-of-function mutations cause type II Bartter syndrome, featuring polyuria, renal salt wasting, and hypotension; humans heterozygous for ROMK mutations identified in the Framingham Heart Study have reduced blood pressure. ROMK null mice recapitulate many of the features of type II Bartter syndrome. We have generated an ROMK knockout rat model in Dahl salt-sensitive background by using zinc finger nuclease technology and investigated the effects of knocking out ROMK on systemic and renal hemodynamics and kidney histology in the Dahl salt-sensitive rats. The ROMK(-/-) pups recapitulated features identified in the ROMK null mice. The ROMK(+/-) rats, when challenged with a 4% salt diet, exhibited a reduced blood pressure compared with their ROMK(+/+) littermates. More importantly, when challenged with an 8% salt diet, the Dahl salt-sensitive rats with 50% less ROMK expression showed increased protection from salt-induced blood pressure elevation and signs of protection from renal injury. Our findings in ROMK knockout Dahl salt-sensitive rats, together with the previous reports in humans and mice, underscore a critical role of ROMK in blood pressure regulation.

Lack of effects on male fertility from a quadrivalent HPV vaccine in Sprague-Dawley rats.

BACKGROUND: Human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases in both men and women. A recently developed quadrivalent HPV vaccine, Gardasil, has been shown to be highly effective in the prevention of several HPV-mediated diseases. The objective of the present study was to evaluate the potential effects of the vaccine on male fertility including reproductive performance, sperm evaluations, and histology of the testes. In addition, anti-HPV antibodies were measured during the study. METHODS: Group 1 (30 male rats) received the full human dose of vaccine (0.5 mL, ∼200-fold excess based on body weight) by intramuscular injection at 6 weeks, 3 weeks, and 3 days prior to cohabitation. Group 2 males received only 1 dose at 3 days prior to cohabitation. Additional groups (20 male rats each) were administered PBS or Merck Aluminum Adjuvant similarly to Group 1. Ten males in the vaccine-treated groups were bled for immunogenicity assays after each dose. Twenty males per group were mated to untreated female rats. Cesarean sections were performed on Gestation Day 15 or 16. Cohabited males were necropsied and sperm count and motility were evaluated. RESULTS: There were no unscheduled deaths during the study and no evidence of toxicity in vaccine-treated male rats. The vaccine induced a specific antibody response to the 4 HPV types after each injection. There were no effects on the cesarean-section parameters of females or reproductive parameters of the cohabited male rats, including histomorphology of testes and epididymis, sperm count, and sperm motility. CONCLUSIONS: These results demonstrate that this quadrivalent HPV vaccine had no detectable adverse effects on routine measures of male fertility in rats.

Evaluation of organ weights for rodent and non-rodent toxicity studies: a review of regulatory guidelines and a survey of current practices.

The Society of Toxicologic Pathology convened a working group to evaluate current practices regarding organ weights in toxicology studies. A survey was distributed to pharmaceutical, veterinary, chemical, food/nutritional and consumer product companies in Europe, North America, and Japan. Responses were compiled to identify organs routinely weighed for various study types in rodent and non-rodent species, compare methods of organ weighing, provide perspectives on the value of organ weights and identify the scientist(s) responsible for organ weight data interpretation. Data were evaluated as a whole as well as by industry type and geographic location. Regulatory guidance documents describing organ weighing practices are generally available, however, they differ somewhat dependent on industry type and regulatory agency. While questionnaire respondents unanimously stated that organ weights were a good screening tool to identify treatment-related effects, opinions varied as to which organ weights are most valuable. The liver, kidneys, and testes were commonly weighed and most often considered useful by most respondents. Other organs that break were commonly weighed included brain, adrenal glands, ovaries, thyroid glands, uterus, heart, and spleen. Lungs, lymph nodes, and other sex organs were weighed infrequently in routine studies, but were often weighed in specialized studies such as inhalation, immunotoxicity, and reproduction studies. Organ-to-body weight ratios were commonly calculated and were considered more useful when body weights were affected. Organ to brain weight ratios were calculated by most North American companies, but rarely according to respondents representing veterinary product or European companies. Statistical analyses were generally performed by most respondents. Pathologists performed interpretation of organ weight data for the majority of the industries.

Society of Toxicologic Pathology position paper: organ weight recommendations for toxicology studies.

The evaluation of organ weights in toxicology studies is an integral component in the assessment of pharmaceuticals, chemicals, and medical devices. The Society of Toxicologic Pathology (STP) has created recommendations for weighing organs in GLP general toxicology studies lasting from 7 days to 1 year. The STP recommends that liver, heart, kidneys, brain, testes, and adrenal glands be weighed in all multidose general toxicology studies. Thyroid gland and pituitary gland weights are recommended for all species except mice. Spleen and thymus should be weighed in rodent studies and may be weighed in non-rodent studies. Weighing of reproductive organs is most valuable in sexually mature animals. Variability in age, sexual maturity, and stage of cycle in non-rodents and reproductive senescence in female rodents may complicate or limit interpretation of reproductive organ weights. The STP recommends that testes of all species be weighed in multidose general toxicology studies. Epididymides and prostate should be weighed in rat studies and may be weighed on a case-by-case basis in non-rodent and mouse studies. Weighing of other organs including female reproductive organs should be considered on a case-by-case basis. Organ weights are not recommended for any carcinogenicity studies including the alternative mouse bioassays. Regardless of the study type or organs evaluated, organ weight changes must be evaluated within the context of the compound class, mechanism of action, and the entire data set for that study.

Histone acetyltransferase (HAT) activity of p300 modulates human T lymphotropic virus type 1 p30II-mediated repression of LTR transcriptional activity.

Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T cell leukemia/lymphoma, and is implicated in a variety of lymphocyte-mediated inflammatory disorders. HTLV-1 provirus has regulatory and accessory genes in four pX open reading frames. HTLV-1 pX ORF-II encodes two proteins, p13II and p30II, which are incompletely defined in virus replication or pathogenesis. We have demonstrated that pX ORF-II mutations block virus replication in vivo and that ORF-II encoded p30II, a nuclear-localizing protein that binds with CREB-binding protein (CBP)/p300, represses CREB and Tax responsive element (TRE)-mediated transcription. Herein, we have identified p30II motifs important for p300 binding and in regulating TRE-mediated transcription in the absence and presence of HTLV-1 provirus. Within amino acids 100-179 of p30II, a region important for repression of LTR-mediated transcription, we identified a single lysine residue at amino acid 106 (K3) that significantly modulates the ability of p30II to repress TRE-mediated transcription. Exogenous p300, in a dose-responsive manner, reverses p30II-dependent repression of TRE-mediated transcription, in the absence or presence of the provirus, In contrast to wild type p300, p300 HAT mutants (defective in histone acetyltransferase activity) only partially rescued p30(II)-mediated LTR repression. Deacetylation by histone deacetylase-1 (HDAC-1) enhanced p30II-mediated LTR repression, while inhibition of deacetylation by trichostatin A decreases p30(II)-mediated LTR repression. Collectively, our data indicate that HTLV-1 p30II modulates viral gene expression in a cooperative manner with p300-mediated acetylation.

Calcium-dependent enhancement of transcription of p300 by human T-lymphotropic type 1 p12I.

Human T-lymphotropic virus type 1 (HTLV-1) p12I localizes to the endoplasmic reticulum and Golgi causing sustained release of calcium, T cell activation, and enhanced expression of several calcium-regulated genes. In recent microarray studies, p300 mRNA was increased in T cells expressing p12I. The co-activator p300 is a key regulator of cellular and viral transcription; however, factors that influence its transcriptional regulation are less well studied. We hypothesized that the transcription of p300 is calcium dependent and that sustained low magnitude increases in intracellular calcium may enhance the transcription of p300. Herein, we report enhanced expression of p300 in T cells by p12I in a calcium-dependent, but calcineurin-independent manner. Sustained low magnitude calcium release induced by ionomycin in T cells was sufficient to increased mRNA and protein levels of p300 resulting in enhanced transcription from a p300-dependent promoter. Promoter analysis of the p300 gene was used to predict calcium-responsive transcription factor binding sites. Using mutant forms of p12I, we demonstrate that ER localization of the viral protein is required to increase p300. In addition, p12I reversed the repression of HTLV-1 LTR-driven transcription by HTLV-1 p30II, a p300-binding protein. HTLV-1 p12I-mediated enhancement of p300 expression represents a novel mechanism of regulation of cellular gene expression by viral proteins. By targeting a ubiquitous second messenger such as calcium, HTLV-1 p12I may regulate the expression of the cellular transcriptional co-activator p300 to modulate viral gene expression and promote lymphocyte survival.

Human T-lymphotropic virus type 1 mitochondrion-localizing protein p13II sensitizes Jurkat T cells to Ras-mediated apoptosis.

Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia. In addition to typical retroviral structural and enzymatic gene products, HTLV-1 encodes unique regulatory and accessory proteins, including a singly spliced pX open reading frame II (ORF II) product, p13(II). We have demonstrated that proviral clones of HTLV-1 which are mutated in pX ORF II fail to obtain typical proviral loads and antibody responses in a rabbit animal model. p13(II) localizes to mitochondria and reduces cell growth and tumorigenicity in mice, but its function in human lymphocytes remains undetermined. For this study, we analyzed the functional properties of Jurkat T cells expressing p13(II), using both transient and stable expression vectors. Our data indicate that p13(II)-expressing Jurkat T cells are sensitive to caspase-dependent, ceramide- and FasL-induced apoptosis. p13(II)-expressing Jurkat T cells also exhibited reduced proliferation when cultured at a high density. Furthermore, preincubation of the p13(II)-expressing cells with a farnesyl transferase inhibitor, which blocks the posttranslational modification of Ras, markedly reduced FasL-induced apoptosis, indicating the participation of the Ras pathway in p13(II)'s influence on lymphocyte survival. Our data are the first to demonstrate that p13(II) alters Ras-mediated apoptosis in T lymphocytes, and they reveal a potential mechanism by which HTLV-1 alters lymphocyte proliferation.

Human T lymphotropic virus type 1 accessory protein p12I modulates calcium-mediated cellular gene expression and enhances p300 expression in T lymphocytes.

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia/lymphoma (ATLL), an aggressive CD4+ T lymphocyte malignancy. Activation of T lymphocytes is required for effective retroviral integration into the host cell genome and subsequent viral replication, but the molecular mechanisms involved in HTLV-1-mediated T cell activation remain unclear. HTLV-1 encodes various accessory proteins such as p12I, which has been demonstrated to be critical for HTLV-1 infectivity in vivo in rabbits and in vitro in quiescent primary human T lymphocytes. This hydrophobic protein localizes in the endoplasmic reticulum, increases intracellular calcium, and activates nuclear factor of activated T cell-mediated transcription. To further elucidate the role of p12I in regulation of cellular gene expression, we performed gene array analysis on stable p12I-expressing Jurkat T cells, using Affymetrix U133A arrays. Our data indicate that p12I altered the expression of genes associated with a network of interrelated pathways including T cell signaling, cell proliferation, and apoptosis. Expression of several calcium-regulated genes was found to be altered by p12I, consistent with known properties of the viral protein. Gene array findings were confirmed by semiquantitative RT-PCR in Jurkat T cells and primary CD4+ T lymphocytes. Furthermore, dose-dependent expression of p12I in Jurkat T cells resulted in significant increases in p300 and p300-dependent transcription. This is the first report of a viral protein influencing the transcription of p300, a rate-limiting coadapter critical in HTLV-1-mediated T cell activation. Collectively, our data strongly indicate that HTLV-1 p12I modulates cellular gene expression patterns to hasten the activation of T lymphocytes and thereby promote efficient viral infection.

Human T lymphotropic virus type-1 p30II alters cellular gene expression to selectively enhance signaling pathways that activate T lymphocytes.

BACKGROUND: Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T-cell leukemia/lymphoma and is implicated in a variety of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13II and p30II, which are incompletely defined in the virus life cycle or HTLV-1 pathogenesis. Proviral clones of the virus with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. Exogenous expression of p30II differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and represses tax/rex RNA nuclear export. RESULTS: Herein, we further characterized the role of p30II in regulation of cellular gene expression, using stable p30II expression system employing lentiviral vectors to test cellular gene expression with Affymetrix U133A arrays, representing approximately 33,000 human genes. Reporter assays in Jurkat T cells and RT-PCR in Jurkat and primary CD4+ T-lymphocytes were used to confirm selected gene expression patterns. Our data reveals alterations of interrelated pathways of cell proliferation, T-cell signaling, apoptosis and cell cycle in p30II expressing Jurkat T cells. In all categories, p30II appeared to be an overall repressor of cellular gene expression, while selectively increasing the expression of certain key regulatory genes. CONCLUSIONS: We are the first to demonstrate that p30II, while repressing the expression of many genes, selectively activates key gene pathways involved in T-cell signaling/activation. Collectively, our data suggests that this complex retrovirus, associated with lymphoproliferative diseases, relies upon accessory gene products to modify cellular environment to promote clonal expansion of the virus genome and thus maintain proviral loads in vivo.

Human T-cell lymphotropic virus type 1 p12I enhances interleukin-2 production during T-cell activation.

Human T-cell lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATLL) and a variety of lymphoproliferative disorders. The early virus-cell interactions that determine a productive infection remain unclear. However, it is well recognized that T-cell activation is required for effective retroviral integration into the host cell genome and subsequent viral replication. The HTLV-1 pX open reading frame I encoding protein, p12(I), is critical for the virus to establish persistent infection in vivo and for infection in quiescent primary lymphocytes in vitro. p12(I) localizes in the endoplasmic reticulum (ER) and cis-Golgi apparatus, increases intracellular calcium and activates nuclear factor of activated T cells (NFAT)-mediated transcription. To clarify the function of p12(I), we tested the production of IL-2 from Jurkat T cells and peripheral blood mononuclear cells (PBMC) expressing p12(I). Lentiviral vector expressed p12(I) in Jurkat T cells enhanced interleukin-2 (IL-2) production in a calcium pathway-dependent manner during T-cell receptor (TCR) stimulation. Expression of p12(I) also induced higher NFAT-mediated reporter gene activities during TCR stimulation in Jurkat T cells. In contrast, p12 expression in PBMC elicited increased IL-2 production in the presence of phorbal ester stimulation, but not during TCR stimulation. Finally, the requirement of ER localization for p12(I)-mediated NFAT activation was demonstrated and two positive regions and two negative regions in p12(I) were identified for the activation of this transcription factor by using p12(I) truncation mutants. These results are the first to indicate that HTLV-1, an etiologic agent associated with lymphoproliferative diseases, uses a conserved accessory protein to induce T-cell activation, an antecedent to efficient viral infection.