[A multicenter prospective study on early identification of refractory Mycoplasma pneumoniae pneumonia in children].

Objective: To explore potential predictors of refractory Mycoplasma pneumoniae pneumonia (RMPP) in early stage. Methods: The prospective multicenter study was conducted in Zhejiang, China from May 1st, 2019 to January 31st, 2020. A total of 1 428 patients with fever >48 hours to <120 hours were studied. Their clinical data and oral pharyngeal swab samples were collected; Mycoplasma pneumoniae DNA in pharyngeal swab specimens was detected. Patients with positive Mycoplasma pneumoniae DNA results underwent a series of tests, including chest X-ray, complete blood count, C-reactive protein, lactate dehydrogenase (LDH), and procalcitonin. According to the occurrence of RMPP, the patients were divided into two groups, RMPP group and general Mycoplasma pneumoniae pneumonia (GMPP) group. Measurement data between the 2 groups were compared using Mann-Whitney U test. Logistic regression analyses were used to examine the associations between clinical data and RMPP. Receiver operating characteristic (ROC) curves were used to analyse the power of the markers for predicting RMPP. Results: A total of 1 428 patients finished the study, with 801 boys and 627 girls, aged 4.3 (2.7, 6.3) years. Mycoplasma pneumoniae DNA was positive in 534 cases (37.4%), of whom 446 cases (83.5%) were diagnosed with Mycoplasma pneumoniae pneumonia, including 251 boys and 195 girls, aged 5.2 (3.3, 6.9) years. Macrolides-resistant variation was positive in 410 cases (91.9%). Fifty-five cases were with RMPP, 391 cases with GMPP. The peak body temperature before the first visit and LDH levels in RMPP patients were higher than that in GMPP patients (39.6 (39.1, 40.0) vs. 39.2 (38.9, 39.7) ℃, 333 (279, 392) vs. 311 (259, 359) U/L, both P<0.05). Logistic regression showed the prediction probability π=exp (-29.7+0.667×Peak body temperature (℃)+0.004×LDH (U/L))/(1+exp (-29.7+0.667×Peak body temperature (℃)+0.004 × LDH (U/L))), the cut-off value to predict RMPP was 0.12, with a consensus of probability forecast of 0.89, sensitivity of 0.89, and specificity of 0.67; and the area under ROC curve was 0.682 (95%CI 0.593-0.771, P<0.01). Conclusion: In MPP patients with fever over 48 to <120 hours, a prediction probability π of RMPP can be calculated based on the peak body temperature and LDH level before the first visit, which can facilitate early identification of RMPP.

Casp11 Deficiency Alters Subgingival Microbiota and Attenuates Periodontitis.

Periodontitis (PD) is the primary cause of tooth loss in adults. Porphyromonas gingivalis (P.g), a keystone pathogen, has been identified as a crucial contributor to this process. Pyroptosis activation in PD is acknowledged, with accumulating evidence underscoring the crucial role of Caspase-11 (described as Caspase-4/5 in humans)-mediated noncanonical pyroptosis. However, the mechanism behind its impact on PD remains unclear. In this study, we delved into the interplay between the Caspase-11-mediated noncanonical pyroptosis, subgingival microbiota alteration, and macrophage polarization. Clinical samples from PD patients revealed heightened expression of Caspase-4, gasdermin-D, and their active fragments, pointing to the activation of the noncanonical pyroptosis. Single-cell sequencing analysis linked Caspase-4 with gingival macrophages, emphasizing their involvement in PD. In vitro cell experiments confirmed that P.g-induced pyroptosis was activated in macrophages, with Casp11 deficiency attenuating these effects. In an experimental PD mouse model, Casp11 deficiency led to an alteration in subgingival microbiota composition and reduced alveolar bone resorption. Casp11-/- mice cohousing with wild-type mice confirmed the alteration of the subgingival microbiota and aggravated the alveolar bone resorption. Notably, Casp11 deficiency led to decreased M1-polarized macrophages, corresponding with reduced alveolar bone resorption, uncovering a connection between subgingival microbiota alteration, macrophage M1 polarization, and alveolar bone resorption. Taken together, we showed that Caspase-11 fulfilled a crucial role in the noncanonical pyroptosis in PD, potentially influencing the subgingival microbiota and linking to M1 polarization, which was associated with alveolar bone resorption. These findings underscored the pivotal role of the Caspase-11-mediated noncanonical pyroptosis in PD pathogenesis and may provide critical insights into potential therapeutic avenues for mitigating PD.

Insurance status and risk of suicide mortality among patients with cancer: a retrospective study based on the SEER database.

OBJECTIVE: Given that the presence of insurance may affect the risk of suicide mortality in cancer patients, we aimed to examine the association in a population-based study using the Surveillance, Epidemiologic, and End Results (SEER) database. STUDY DESIGN: A retrospective analysis of data from the SEER database. METHODS: We conducted a retrospective study using the SEER database. Hazard ratios (HRs), adjusted HRs (aHRs), and 95% confidence intervals (95% CIs) of suicide death were calculated using Cox proportional hazard models to evaluate the risk of suicide mortality among the cohorts. RESULTS: Multivariable analysis revealed that cancer patients without insurance had an increased risk of suicide death compared with patients with private insurance (aHR, 1.37; 95% CI, 1.01-1.72), whereas no significant result was observed in patients with any Medicaid (aHR, 1.10; 95% CI, 0.93-1.30; P = 0.27). In addition, the stratified analysis indicated that the risk of suicide death in patients in the uninsured and Medicaid groups presented with localized stage of disease (aHR, 1.32; 95% CI, 1.02, 1.69), White (aHR, 1.34; 95% CI, 1.05, 1.71), and American Indian/Alaska Native and Asian/Pacific Islander (aHR, 1.89; 95% CI, 1.08, 3.30) were greater than insured patients. CONCLUSION: Overall, our results indicated that insurance status was a statistically significant predictor of suicide death in patients with cancer. Healthcare providers should identify those patients at high risk of suicide and provide appropriate mental health and psychosocial oncology services in time.

CD28 deficiency attenuates primary blast-induced renal injury in mice via the PI3K/Akt signalling pathway.

INTRODUCTION: Primary blast affects the kidneys due to direct shock wave damage and the production of proinflammatory cytokines without effective treatment. CD28 has been reported to be involved in regulating T cell activation and secretion of inflammatory cytokines. The aim of this study was to investigate the influence of primary blast on the kidney and the effect of CD28 in mice. METHODS: A mouse model of primary blast-induced kidney injury was established using a custom-made explosive device. The severity of kidney injury was investigated by H&E staining. ELISA was applied to study serum inflammation factors' expression. Western blot assays were used to analyse the primary blast-induced inflammatory factors' expression in the kidney. Immunofluorescence analysis was used to examine the PI3K/Akt signalling pathway. RESULTS: Histological examination demonstrated that compared with the primary blast group, CD28 deficiency caused a significant decrease in the severity of the primary blast-induced renal injury. Moreover, ELISA and western blotting revealed that CD28 deficiency significantly reduced the levels of interleukin (IL)-1ß, IL-4 and IL-6, and increased the IL-10 level (p<0.05). Finally, immunofluorescence analysis indicated that PI3K/Akt expression also changed. CONCLUSIONS: CD28 deficiency had protective effects on primary blast-induced kidney injury via the PI3K/Akt signalling pathway. These findings improve the knowledge on primary blast injury and provide theoretical basis for primary blast injury treatment.

Effects of different space allowances on growth performance, blood profile and pork quality in a grow-to-finish production system.

OBJECTIVE: This experiment was conducted to evaluate the optimal space allowance on growth performance, blood profile and pork quality of growing-finishing pigs. METHODS: A total of ninety crossbred pigs [(Yorkshire×Landrace)×Duroc, 30.25±1.13 kg] were allocated into three treatments (0.96: four pigs/pen, 0.96 m2/pig; 0.80: five pigs/pen, 0.80 m2/pig; 0.69: six pigs/pen, 0.69 m2/pig) in a randomized complete block design. Pigs were housed in balanced sex and had free access to feed in all phases for 14 weeks (growing phase I, growing phase II, finishing phase I, and finishing phase II). RESULTS: There was no statistical difference in growing phase, but a linear decrease was observed on average daily gain (ADG, p<0.01), average daily feed intake (ADFI, p<0.01), and body weight (BW, p<0.01) with decreasing space allowance in late finishing phase. On the other hand, a quadratic effect was observed on gain to feed ratio in early finishing phase (p<0.03). Consequently, overall ADG, ADFI, and final BW linearly declined in response to decreased space allowance (p<0.01). The pH of pork had no significant difference in 1 hour after slaughter, whereas there was a linear decrease in 24 h after slaughter with decreasing space allowance. Floor area allowance did not affect pork colors, but shear force linearly increased as floor space decreased (p<0.01). There was a linear increase in serum cortisol concentration on 14 week (p<0.05) with decreased space allocation. Serum IgG was linearly ameliorated as space allowance increased on 10 week (p<0.05) and 14 week (p<0.01). CONCLUSION: Data from current study indicated that stress derived from reduced space allowance deteriorates the immune system as well as growth performance of pigs, resulting in poor pork quality. Recommended adequate space allowance in a grow-to-finish production system is more than 0.80 m2/pig for maximizing growth performance and production efficiency.

Effect of dietary energy levels and phase feeding by protein levels on growth performance, blood profiles and carcass characteristics in growing-finishing pigs.

BACKGROUND: Providing of insufficient nutrients limits the potential growth of pig, while feeding of excessive nutrients increases the economic loss and causes environment pollution. For these reasons, phase feeding had been introduced in swine farm for improving animal production. This experiment was conducted to evaluate the effects of dietary energy levels and phase feeding by protein levels on growth performance, blood profiles and carcass characteristics in growing-finishing pigs. METHODS: A total of 128 growing pigs ([Yorkshire × Landrace] × Duroc), averaging 26.62 ± 3.07 kg body weight, were assigned in a 2 × 4 factorial arrangement with 4 pigs per pen. The first factor was two dietary energy level (3,265 kcal of ME/kg or 3,365 kcal of ME/kg), and the second factor was four different levels of dietary protein by phase feeding (1growing(G)-2finishing(F) phases, 2G-2F phases, 2G-3F phases and 2G-3F phases with low CP requirement). RESULTS: In feeding trial, there was no significant difference in growth performance. The BUN concentration was decreased as dietary protein level decreased in 6 week and blood creatinine was increased in 13 week when pigs were fed diets with different dietary energy level. The digestibility of crude fat was improved as dietary energy levels increased and excretion of urinary nitrogen was reduced when low protein diet was provided. Chemical compositions of longissimus muscle were not affected by dietary treatments. In backfat thickness (P2) at 13 week, pigs fed high energy diet had thicker backfat thickness (P = 0.06) and pigs fed low protein diet showed the trend of backfat thinness reduction (P = 0.09). In addition, water holding capacity was decreased (P = 0.01) and cooking loss was increased (P = 0.07) as dietary protein level reduced. When pigs were fed high energy diet with low subdivision of phase feeding, days to 120 kg market weight was reached earlier compared to other treatments. CONCLUSION: Feeding the low energy diet and subdivision of growing-finishing phase by dietary protein levels had no significant effect on growth performance and carcass characteristics. Also, phase feeding with low energy and low protein diet had no negative effects on growth performance, carcass characteristics but economical profits was improved.

Supplementation of Dried Mealworm (Tenebrio molitor larva) on Growth Performance, Nutrient Digestibility and Blood Profiles in Weaning Pigs.

This experiment was conducted to investigate the effects of dried mealworm (Tenebrio molitor larva) on growth performance, nutrient digestibility and blood profiles in weaning pigs. A total of 120 weaning pigs (28±3 days and 8.04±0.08 kg of body weight) were allotted to one of five treatments, based on sex and body weight, in 6 replicates with 4 pigs per pen by a randomized complete block design. Supplementation level of dried mealworm was 0%, 1.5%, 3.0%, 4.5%, or 6.0% in experimental diet as treatment. Two phase feeding programs (phase I from 0 day to 14 day, phase II from 14 day to 35 day) were used in this experiment. All animals were allowed to access diet and water ad libitum. During phase I, increasing level of dried mealworm in diet linearly improved the body weight (p<0.01), average daily gain (ADG) (p<0.01) and average daily feed intake (ADFI) (p<0.01). During phase II, ADG also tended to increase linearly when pigs were fed higher level of dried mealworm (p = 0.08). In addition, increasing level of dried mealworm improved the ADG (p<0.01), ADFI (p<0.05) and tended to increase gain to feed ratio (p = 0.07) during the whole experimental period. As dried mealworm level was increased, nitrogen retention and digestibility of dry matter as well as crude protein were linearly increased (p = 0.05). In the results of blood profiles, decrease of blood urea nitrogen (linear, p = 0.05) and increase of insulin-like growth factor (linear, p = 0.03) were observed as dried mealworm was increased in diet during phase II. However, there were no significant differences in immunoglobulin A (IgA) and IgG concentration by addition of dried mealworm in the growth trial. Consequently, supplementation of dried mealworm up to 6% in weaning pigs' diet improves growth performance and nutrient digestibility without any detrimental effect on immune responses.

Overexpression of maelstrom promotes bladder urothelial carcinoma cell aggressiveness by epigenetically downregulating MTSS1 through DNMT3B.

We have recently identified and characterized a novel oncogene, maelstrom (MAEL) from 1q24, in the pathogenesis of hepatocellular carcinoma. In this study, MAEL was investigated for its oncogenic role in urothelial carcinoma of the bladder (UCB) tumorigenesis/aggressiveness and underlying molecular mechanisms. Here, we report that overexpression of MAEL in UCB is important in the acquisition of an aggressive and/or poor prognostic phenotype. In UCB cell lines, knockdown of MAEL by short hairpin RNA is sufficient to inhibit cell growth, invasiveness/metastasis and suppressed epithelial-mesenchymal transition (EMT), whereas ectopic overexpression of MAEL promoted cell growth, invasive and/or metastatic capacity and enhanced EMT both in vitro and in vivo. We further demonstrate that MAEL could induce UCB cell EMT by downregulating a critical downstream target, the metastasis suppressor 1 (MTSS1) gene, ultimately leading to an increased invasiveness of cancer cells. Notably, overexpression of MAEL in UCB cells substantially enhanced the enrichment of DNA methyltrans-ferase (DNMT)3B and histone deacetylase (HDAC)1/2 on the promoter of the MTSS1, and thereby epigenetically suppressing the MTSS1 transcription. Downregulation of MTSS1 by MAEL in UCB cells is partially dependent on DNMT3B. Furthermore, we identify that beside the gene amplification of MAEL, miR-186 is a key negative regulator of MAEL and downregulation of miR-186 is another important mechanism for MAEL overexpression in UCBs. These data suggest that overexpression of MAEL, caused by gene amplification and/or decreased miR-186, has a critical oncogenic role in UCB pathogenesis by downregulation of MTSS1, and MAEL could be used as a novel prognostic marker and/or effective therapeutic target for human UCB.

Preoperative evaluation improves the outcome in heart transplant recipients with pulmonary hypertension--retrospective analysis of 106 cases.

OBJECTIVES: The objectives of this study were to retrospectively analyze the value of preoperative evaluation and to analyze the risk factors of long-term mechanical ventilation in heart transplant recipients. METHODS: We analyzed the data of 106 patients prepared to receive heart transplants. Before the operation, according to the pulmonary vascular resistance (PVR) and transpulmonary gradient (TPG), the patients were divided into 2 groups: a pulmonary hypertension (PH) group and a pulmonary artery pressure normal (N) group. The vasodilator conditioning test as a predictive factor was performed in the PH group. Univariate analysis and logistic regression were used to examine the relationship between risk factors and long-term mechanical ventilation. The 30-day and long-term survival rates were followed. RESULTS: PVR and TPG significantly decreased among the PH group after intravenous infusion epoprostenol and inhalation (nitric oxide). After preoperative evaluation, 96 patients underwent heart transplantation. There were no short-term deaths related to PH. There was no significant difference in mortality between the PH group and the N group. PH, hypotension after cardiopulmonary bypass, renal dysfunction, donor heart ischemia time, and ejection fraction <25% were risk factors for long-term mechanical ventilation. PH was not related to long-term deaths. CONCLUSION: A vasodilator conditioning test was reliable for PH patients undergoing heart transplantation for preoperative evaluation and preparation.

Enhanced susceptibility of adriamycin-treated human renal cell carcinoma cells to lysis by peripheral blood lymphocytes and tumor infiltrating lymphocytes.

Previous studies indicated that the anticancer agent adriamycin (ADR) could induce activation of cytotoxic T lymphocytes (CTL) and natural killer cells. In this study, we investigated the effect of ADR on the susceptibility of human renal cell carcinoma (RCC) cells to lysis by peripheral blood lymphocytes (PBL) and tumor infiltrating lymphocytes (TIL). Treatment of human RCC cell line ACHN and freshly derived RCC cells with ADR at 1 microg/ml or more for 3 h significantly enhanced their susceptibility to lysis by PBL (P<0.05). This ADR-induced enhancement of susceptibility of RCC cells to lysis by PBL was also observed when freshly derived TIL were used as effector cells (P<0.05). ADR up-regulated the expression of leukocyte function-associated antigen-3 (LFA-3) and intercellular adhesion molecule-1 (ICAM-1), which are critical in the binding and killing of CTL against cancer cells. Of the five fresh RCC cell cultures treated with ADR, LFA-3 was increased in all and ICAM-1 was increased in three of them, respectively (P<0.05). Up-regulation of LFA-3 and ICAM-1 was also observed in ACHN cells treated with two derivatives of ADR, epirubicin and pirarubicin. ADR further significantly increased the bindings of PBL to RCC cells (P<0.05). These findings suggest that treatment of RCC patients with low doses of ADR may sensitize the RCC cells to killing by PBL and TIL and may be a novel immunotherapeutic modality for the treatment of drug-resistant and/or immune-resistant RCC. The inducing of LFA-3 and ICAM-1 by ADR may be involved in the enhancement of susceptibility of PBL and TIL-mediated cytolysis in human RCC cells.

Myosin light chain kinase-independent inhibition by ML-9 of murine TRPC6 channels expressed in HEK293 cells.

BACKGROUND AND PURPOSE: Myosin light chain kinase (MLCK) plays a pivotal role in regulation of cellular functions, the evidence often relying on the effects of extracelluarly administered drugs such as ML-9. Here we report that this compound exerts non-specific inhibitory actions on the TRPC6 channel, a transient receptor potential (TRP) protein. EXPERIMENTAL APPROACH: Macroscopic and single channel currents were recorded from transfected HEK293 cells by patch-clamp techniques. KEY RESULTS: Cationic currents elicited by carbachol (CCh; 100 microM) in HEK293 cells overexpressing murine TRPC6 (I(TRPC6)) were dose-dependently inhibited by externally applied ML-9 (IC(50)=7.8 microM). This inhibition was voltage-dependent and occurred as fast as external Na(+) removal. Another MLCK inhibitor, wortmannin (3 microM), and MLCK inhibitory peptides MLCK-IP(11-19) (10 microM) and -IP(480-501) (1 microM) showed little effects on I(TRPC6) density and the inhibitory efficacy of ML-9. The extent of the inhibition also unchanged with co-expression of wild-type or a dominant negative mutant of MLCK. Inhibitory effects of ML-9 on I(TRPC6) remained unaffected whether TRPC6 was activated constitutively or by a diacylglycerol analogue OAG (100 microM). Similar rapid inhibition was also observed with a ML-9 relative, ML-7. Intracellular perfusion of ML-9 via patch pipette, dose-dependently suppressed I(TRPC6). In inside-out patch configuration, bath application of ML-9 (and ML-7) rapidly diminished approximately 35pS single TRPC6 channel activities. Contrarily, currents due to TRPC7 expression were rapidly enhanced by externally applied ML-9 and ML-7, which was not prevented by MLCK inhibitory peptides. CONCLUSION AND IMPLICATIONS: These results strongly suggest that ML compounds inhibit TRPC6 channels via a mechanism independent of inhibition of MLCK activity.

Angiotensin type 2 receptor-mediated hypotension in angiotensin type-1 receptor-blocked rats.

The type-2 (AT(2)) angiotensin (Ang) II receptor has been characterized as potentially counterregulatory to the actions of Ang II at its type-1 (AT(1)) receptor. We investigated the effects of Ang II and CGP-42112A (CGP), a selective peptide AT(2) receptor agonist, on blood pressure (BP) in rats with or without pharmacological blockade of the AT(1) receptor with losartan (LOS) or valsartan (VAL). In anesthetized rats (n=5 per group) receiving normal sodium intake, Ang II (200 pmol/kg per minute IV) alone increased BP from a control of 112+/-3 to 168+/-7 mm Hg (P<0.001) and LOS (30 mg/kg) alone decreased BP to 89+/-7 mm Hg (P<0.0001 from control). Ang II administered together with LOS decreased BP further to 71+/-4 mm Hg (P<0.00001 from control and LOS alone). AT(2) receptor antagonist PD 123,319 (PD) completely blocked the hypotensive response to LOS combined with Ang II (P=NS from control). In conscious rats (n=5 per group) receiving normal sodium intake, VAL (10 mg/kg) alone decreased BP from a control of 98+/-5 to 86+/-3 mm Hg (P<0.00001). Ang II combined with VAL induced a consistent, highly significant decline in BP for 6 days to a nadir of 69+/-3 mm Hg (P<0.01 versus daily VAL alone). PD completely blocked the chronic hypotensive response to the combination of Ang II and VAL to control levels before VAL administration. In another study in conscious rats (n=5 per group), CGP (70 microg/kg per minute) also decreased BP in VAL-treated conscious rats. BP was 119+/-3 mm Hg during the control period, decreased to 86+/-6 mm Hg during 3 days of VAL alone, (P<0.00001) and decreased further to 65+/-7 mm Hg (P<0.001 from daily VAL alone) with 7 days of CGP in the presence of VAL. In the absence of VAL, CGP decreased BP for 4 consecutive days, and this response was blocked by PD. Also, the CGP-induced decrease in BP over a 7-day period was blocked by N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase. The results strongly suggest that the AT(2) receptor induces a systemic vasodilator response mediated by NO that counterbalances the vasoconstrictor action of Ang II at the AT(1) receptor.

Renal interstitial cGMP mediates natriuresis by direct tubule mechanism.

The objective of this study was to test the hypothesis that renal interstitial (RI) cGMP is natriuretic in vivo. In conscious rats (n=8), urinary sodium excretion (U(Na)V) was significantly greater on days 3 and 4 of RI infusion of cGMP (1.17+/-0.14 and 1.61+/-0.11 mmol/24 h, respectively) than during vehicle infusion (0.56+/-0.15 and 0.70+/-0.17 mmol/24 h, respectively) (P<0.01). Similarly, U(Na)V was greater on days 3 and 4 of RI infusion of 8-bromo-cGMP (2.15+/-0.42 and 2.16+/-0.1 mmol/24 h, respectively). Protein kinase G inhibitor Rp-8-pCPT-cGMPS reduced cGMP-induced and 8-bromo-cGMP-induced U(Na)V to control levels. Acute RI infusion of L-arginine (L-Arg, 40 mg. kg(-1). min(-1)), but not D-arginine, caused an increase in U(Na)V from 1.65+/-0.11 to 4.07+/-0.1 micromol/30 min (P<0.01). This increase was blocked by RI infusion of N(G)-nitro-L-arginine methyl ester (100 ng. kg(-1). min(-1)) by the phosphodiesterase (PDE II) activator 5,6DMcBIMP (0.01 micromol/microL), by PDE II (0.03 U. kg(-1). min(-1)) itself, or by the soluble guanylyl cyclase inhibitor 1-H-[1,2,4]oxadiazolo-[4,2-alpha]quinoxalin-1-one (ODQ, 0.12 mg. kg(-1). min(-1)). The PDE II activator also blocked L-Arg-stimulated cGMP levels. The NO donor S-nitroso-N-acetylpenicillamine (SNAP, 0.12 micromol. L(-1). kg(-1). min(-1)) increased U(Na)V from 1.65+/-0.11 to 2.93+/-0.08 micromol/30 min (P<0.01), and this response was blocked completely by ODQ. Renal arterial but not RI administration of the heat-stable enterotoxin of Escherichia coli induced natriuresis. RA infusion of cGMP (3 microg/min) increased U(Na)V, renal blood flow (RBF), and glomerular filtration rate (GFR). Renal cortical interstitial cGMP infusion increased U(Na)V with no effect on total RBF, renal cortical blood flow, or GFR. Similarly, the natriuretic actions of renal interstitial L-Arg or SNAP were not accompanied by any change in RBF or GFR. Medullary cGMP infusion had no effect on U(Na)V, total RBF, or medullary blood flow. Texas red-labeled cGMP infused via the RI space was distributed exclusively to cortical renal tubular cells. The results demonstrate that RI cGMP inhibits renal tubular sodium absorption via protein kinase G independently of hemodynamic changes. These observations indicate that the cortical interstitial compartment provides a potentially important domain for cell-to-cell signaling within the kidney.

Role of the angiotensin AT2 receptor in blood pressure regulation and therapeutic implications.

The angiotensin (ANG) Type 2 (AT2) receptor is one of two major ANG II receptors that have been identified, cloned, and sequenced. Most of the biologic actions of ANG II are thought to be mediated by the AT1 receptor, but evidence is beginning to emerge that the AT2 receptor has a significant role in the regulation of blood pressure. In the adult rat, the AT2 receptor is expressed, albeit in low concentrations in kidney, mesenteric blood vessels, and heart. Most of the evidence suggests that the AT2 receptor stimulates a vasodilator signaling cascade that includes bradykinin, nitric oxide, and guanosine cyclic 3',5'-monophosphate. At lease some of the beneficial actions of AT1 receptor blockade are mediated by the AT2 receptor through this pathway. Several recent studies suggest that AT2 receptors may mediate vasodilation and hypotension. The AT2 receptor represents a potential therapeutic target for agonist action and a candidate molecule in the pathophysiology of hypertension.

Green tea protects against psoralen plus ultraviolet A-induced photochemical damage to skin.

The use of psoralens combined with exposure to ultraviolet A radiation is a major form of treatment for psoriasis and a number of other common skin diseases. Although psoralen plus ultraviolet A treatment is highly effective, careful follow-up cohort studies have shown that it greatly increases risk for the development of cutaneous squamous cell carcinoma and melanoma. Strategies to reduce the risk of cancer development in psoralen plus ultraviolet A-treated populations are highly desirable. In prior studies, we demonstrated that green tea and constituent polyphenols protect against ultraviolet B-induced carcinogenesis and reduce the growth rate of established tumors in skin. In this study, we show that pre- and post-treatment with standardized green tea extract in psoralen plus ultraviolet A treatment populations abrogates the psoralen plus ultraviolet A-induced photochemical damage to skin. Intact mouse and human skin and reconstituted human skin were employed to assess the effect of both topical and oral administration of standardized green tea extract against psoralen plus ultraviolet A-induced photodamage. Oral administration of standardized green tea extract prior to and during multiple psoralen plus ultraviolet A treatments reduced hyperplasia and hyperkeratosis in murine skin. Standardized green tea extract treatment also inhibited accumulation of c-fos and p53 protein induction following a single exposure to psoralen plus ultraviolet A. c-fos and p53 positive cells in psoralen plus ultraviolet A-treated skin were found to be increased by 55.4 +/- 13. 6% and 62.3 +/- 10.5%, respectively, compared with saline-treated unexposed control skin. Oral administration of 0.4 or 0.8% standardized green tea extract inhibited c-fos protein accumulation by 18.5% and 46.2% (p < 0.05), respectively, and p53 protein accumulation by 26.1% and 54.3% (p < 0.05), respectively. Similarly proliferating cell nuclear antigen staining, a marker of cell proliferation was induced (73.7%) in psoralen plus ultraviolet A-treated skin. Oral administration of 0.4% or 0.8% standardized green tea extract 1 d after psoralen plus ultraviolet A treatment was effective in reducing psoralen plus ultraviolet A-induced inflammatory responses including erythema and edema formation. When standardized green tea extract was applied to EpiDerm, a reconstituted human skin equivalent, psoralen plus ultraviolet A-induced 8-methoxypsoralen-DNA adduct formation and p53 protein accumulation were inhibited. Topical application of 0.2 mg 8-methoxypsoralen per cm2 followed by exposure to ultraviolet A (2.5 J per cm2) resulted in delayed erythema formation in human subjects. Pretreatment of human skin with topical application of 0.2 mg standardized green tea extract per cm2 30 min prior to psoralen plus ultraviolet A treatment resulted in an almost complete abrogation of psoralen plus ultraviolet A-induced erythema. In summary, these data demonstrate that standardized green tea extract protects against psoralen plus ultraviolet A-induced phototoxicity by inhibiting DNA damage and diminishing the inflammatory effects of this modality.

Compartmentalization of extracellular cGMP determines absorptive or secretory responses in the rat jejunum.

We examined potential mechanisms by which angiotensin subtype-2 (AT2) receptor stimulation induces net fluid absorption and serosal guanosine cyclic 3',5'-monophosphate (cGMP) formation in the rat jejunum. L-arginine (L-ARG) given intravenously or interstitially enhanced net fluid absorption and cGMP formation, which were completely blocked by the nitric oxide (NO) synthase inhibitor, N-nitro-L-arginine methylester (L-NAME), but not by the specific AT2 receptor antagonist, PD-123319 (PD). Dietary sodium restriction also increased jejunal interstitial fluid cGMP and fluid absorption. Both could be blocked by PD or L-NAME, suggesting that the effects of sodium restriction occur via ANG II at the AT2 receptor. L-ARG-stimulated fluid absorption was blocked by the soluble guanylyl cyclase inhibitor 1-H-[1,2,4]oxadiazolo[4, 2-alpha]quinoxalin-1-one (ODQ). Cyclic GMP-specific phosphodiesterase in the interstitial space decreased extracellular cGMP content and prevented the absorptive effects of L-ARG. Angiotensin II (ANG II) caused an increase in net Na+ and Cl- ion absorption and 22Na+ unidirectional efflux (absorption) from the jejunal loop. In contrast, intraluminal heat-stable enterotoxin of Escherichia coli (STa) increased loop cGMP and fluid secretion that were not blocked by either L-NAME or ODQ. These findings suggest that ANG II acts at the serosal side via AT2 receptors to stimulate cGMP production via soluble guanylyl cyclase activation and absorption through the generation of NO, but that mucosal STa activation of particulate guanylyl cyclase causes secretion independently of NO, thus demonstrating the opposite effects of cGMP in the mucosal and serosal compartments of the jejunum.

Reconstituted 3-dimensional human skin as a novel in vitro model for studies of carcinogenesis.

EpiDerm (MatTek Co., MA) is a reconstituted human skin equivalent which exhibits morphological and growth characteristics similar to human skin. This model has previously been utilized to evaluate the cytotoxicity and irritant potential of various cosmetic and household products. In this study, we show for the first time that EpiDerm can be used successfully to evaluate the genotoxicity of different types of known carcinogenic agents such as benzo[a]pyrene (BaP), ultraviolet B radiation (UVB), ultraviolet A radiation (UVA), and psoralen-ultraviolet A radiation (PUVA) at the molecular level. The topical application of 50 microg/cm2 BaP to EpiDerm resulted in the accumulation of BaP-DNA adducts and c-fos and p53 proteins as evidenced by immunohistochemical localization. Similarly, exposure to UVB (50 mJ/cm2) and UVA (2.5 J/cm2) enhanced the epidermal expression of c-fos and p53 proteins in the human skin equivalent. PUVA treatment of EpiDerm, however, resulted in the formation of both DNA-8-MOP adducts and augmented expression of c-fos and p53 proteins. Most of these changes reached a peak 8 h after the treatments except in the case of UVA where maximum changes in the expression of c-fos and p53 proteins were observed 24 h after treatment. These results are similar to those previously reported in human and murine skin following exposure to BaP, UVB, UVA, or PUVA indicating that human skin equivalents can be used as a convenient and cost-effective alternative to animal testing for assessing the genotoxicity and mechanism of action of mutagens/carcinogens in human skin.