HLA polymorphisms as incidence factor in the progression to end-stage renal disease in Brazilian patients awaiting kidney transplant

Chronic renal failure (CRF) leads in the majority of instances to end-stage renal disease (ESRD) requiring renal replacement therapy. Age, gender, genetics, race, hypertension, and smoking among others are factors associated with ESRD. Our interest was to evaluate the possible associations of class I and II HLA antigens with ESRD renal disease independent of other factors, among patients with CRF, having various diagnoses in the Brazilian population of the Sao Paulo state. So 21 HLA-A, 31 HLA-B, and 13 HLA-DR were detected in 105 patients who were compared with 160 healthy controls of both sexes who were not related to the patients evaluated until 2005. We calculated allelic frequencies, haplotypes frequencies, etiological fractions (EF), preventive fractions, and relative risks (RR). We compared demographic data of patients and controls. The antigens positively associated with ESRD were: HLA-A78 (RR = 30.31 and EF = 0.96) and HLA-DR11 (RR = 18.87 and EF = 0.65). The antigens HLAB14 (RR = 29.90 and EF = 0.75) was present at a significantly lower frequency among patients compared with controls. In contrast, no haplotype frequency showed statically significant associations. Further molecular studies may clarify types and subtypes of alleles involved with ESRD progression.

Arteriovenous Fistula Puncture: An Essential Factor for Hemodialysis Efficiency

Objective. To determine the blood recirculation ratio in the vascular access of patients on hemodialysis, and to calculate the Kt/Vs obtained with the different techniques of arteriovenous fistula punctures. Materials and Methods. A total of 174 patients were divided according to the technique used for arteriovenous fistula puncture: group 1, needles in opposite directions and with a distance of 5 cm or more between them; group 2, needles in opposite directions but with a distance of less than 5 cm; group 3, unidirectional needles with both directed to the heart and with a distance of 5 cm or more; group 4, unidirectional needles but separated by a distance of less than 5 cm between needles; and group 5, patients carrying a temporary venous catheter. Blood samples were collected for urea analysis, pre and post-dialysis for Kt/V rate, and other samples for calculation of the access recirculation. Results. Group 1 presented the lowest rate of access recirculation (8.51 +/- 4.90%) and the best Kt/V (1.71 +/- 0.36), while group 4 presented the worst access recirculation (20.68 +/- 4.92%) and Kt/V (1.16 +/- 0.26). All groups differed significantly from group 4 (p < 0.05), except group 5 with regard for Kt/V parameter. Discussion. The technique of arteriovenous fistula puncture is an essential factor to decrease the access recirculation and assure better results of measurement of hemodialysis adequacy. On the basis of the results obtained, insertion of the needles in the same direction and with a distance of less than 5 cm between them should be avoided.

Integration pattern of HIV-1 based lentiviral vector carrying recombinant coagulation factor VIII in Sk-Hep and 293T cells

293T and Sk-Hep-1 cells were transduced with a replication-defective self-inactivating HIV-1 derived vector carrying FVIII cDNA. The genomic DNA was sequenced to reveal LTR/human genome junctions and integration sites. One hundred and thirty-two sequences matched human sequences, with an identity of at least 98%. The integration sites in 293T-FVIIIDB and in Sk-Hep-FVIIIDB cells were preferentially located in gene regions. The integrations in both cell lines were distant from the CpG islands and from the transcription start sites. A comparison between the two cell lines showed that the lentiviral-transduced DNA had the same preferred regions in the two different cell lines.

Human leukocyte antigen (HLA) and single nucleotide polymorphisms (SNPs) tumor necrosis factor (TNF)-alpha-238 and-308 as genetic markers of susceptibility to psoriasis and severity of the disease in a long-term follow-up Brazilian study

Background The strongest genetic marker for psoriasis is Cw*06. Polymorphisms in the tumor necrosis factor (TNF)-alpha promoter region, especially replacement of guanine with adenine in positions -238 and -308 are related to higher TNF-alpha production and higher risk for psoriasis in Caucasoid populations, not found in Asians. We performed a case-control study of 69 patients with psoriasis type I and 70 controls, characterized clinical progression along 10-years of follow-up in mild or severe disease and determined HLA class I, II, and TNF single nucleotide polymorphisms (SNPs) -238 and -308 polymorphisms to demonstrate whether these polymorphisms may be genetic risk for susceptibility to psoriasis or severity of the disease in Brazilians. Methods Polymorphisms were identified using PCR/SSP. Alleles, genotypes, and haplotypes frequencies were compared using Fisher`s test. Results More severe disease was found in male patients. It may be suggested that alleles B*37, Cw*06, Cw*12, and DRB1*07 were associated with severe disease course, while B*57 with mild disease. No statistical difference was found between the patients and controls regarding polymorphisms frequencies in TNF SNPs. This study pointed to a higher TNF-238 G/G genotype frequency (OR: 3.21; CI: 1.06-9.71; P = 0.04) in the group with severe disease. Conclusions Polymorphisms in the TNF-alpha SNPs do not seem to be a more important genetic risk factor for psoriasis than the already known Cw*06 in Brazilian patients, but these markers may be related to clinical manifestations.

Association of-308G/A polymorphism in the tumor necrosis factor-alpha gene promoter with susceptibility to development of hantavirus cardiopulmonary syndrome in the Ribeiro Preto region, Brazil

Activation of the immune response in hantavirus cardiopulmonary syndrome (HCPS) leads to a high TNF production, probably contributing to the disease. The polymorphic TNF2 allele (TNF -308G/A) has been associated with increased cytokine production. We investigated the association of the TNF2 allele with the outcome of hantavirus infection in Brazilian patients. A total of 122 hantavirus-exposed individuals (26 presenting HCPS and 96 only hantavirus seroconversion) were studied. The TNF2 allele was more frequently found in HCPS patients than in individuals with positive serology for hantavirus but without a history of HCPS illness, suggesting that the TNF2 allele could represent a risk factor for developing HCPS.

Screening of autosomal gene deletions in patients with hypogonadotropic hypogonadism using multiplex ligation-dependent probe amplification: detection of a hemizygosis for the fibroblast growth factor receptor 1

P>Objective Congenital hypogonadotropic hypogonadism with anosmia (Kallmann syndrome) or with normal sense of smell is a heterogeneous genetic disorder caused by defects in the synthesis, secretion and action of gonadotrophin-releasing hormone (GnRH). Mutations involving autosomal genes have been identified in approximately 30% of all cases of hypogonadotropic hypogonadism. However, most studies that screened patients with hypogonadotropic hypogonadism for gene mutations did not include gene dosage methodologies. Therefore, it remains to be determined whether patients without detected point mutation carried a heterozygous deletion of one or more exons. Measurements We used the multiplex ligation-dependent probe amplification (MLPA) assay to evaluate the potential contribution of heterozygous deletions of FGFR1, GnRH1, GnRHR, GPR54 and NELF genes in the aetiology of GnRH deficiency. Patients We studied a mutation-negative cohort of 135 patients, 80 with Kallmann syndrome and 55 with normosmic hypogonadotropic hypogonadism. Results One large heterozygous deletion involving all FGFR1 exons was identified in a female patient with sporadic normosmic hypogonadotropic hypogonadism and mild dimorphisms as ogival palate and cavus foot. FGFR1 hemizygosity was confirmed by gene dosage with comparative multiplex and real-time PCRs. Conclusions FGFR1 or other autosomal gene deletion is a possible but very rare event and does not account for a significant number of sporadic or inherited cases of isolated GnRH deficiency.

Protective Effect of RC-3095, an Antagonist of the Gastrin-Releasing Peptide Receptor, in Experimental Arthritis

Objective. To evaluate the antiinflammatory effects of RC-3095 in 2 experimental models of arthritis, collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA), and to determine the mechanisms of action involved. Methods. RC-3095 was administered daily to mice with CIA and mice with AIA, after induction of disease with methylated bovine serum albumin. Disease incidence and severity were assessed using a clinical index and evaluation of histologic features, respectively. In mice with CIA, gastrin-releasing peptide receptor (GRPR) was detected by immunohistochemical analysis, while in mice with AIA, migration of neutrophils, presence of glycosaminoglycans, and lymphocyte proliferation, determined using the MTT assay, were assessed. Expression of cytokines interleukin-17 (IL-17), IL-1 beta, and tumor necrosis factor alpha (TNF alpha) was evaluated in all mouse knees using enzyme-linked immunosorbent assay. Treg cell production was assessed by flow cytometry in the joints of mice with AIA. Results. In mice with AIA, administration of RC-3095 reduced neutrophil migration, mechanical hypernociception, and proteoglycan loss. These findings were associated with inhibition of the levels of all 3 proinflammatory cytokines, decreased lymphocyte proliferation, and increased Treg cell numbers. In the CIA model, treatment with RC-3095 led to a significant reduction in arthritis clinical scores and the severity of disease determined histologically. Synovial inflammation, synovial hyperplasia, pannus formation, and extensive erosive changes were all dramatically reduced in the arthritic mice treated with RC-3095. Furthermore, arthritic mice treated with RC-3095 showed a significant reduction in the concentrations of IL-17, IL-1 beta, and TNF alpha, and showed a diminished expression of GRPR. Conclusion. These findings suggest that the GRP pathway has a significant role in chronic arthritis, and its inhibition can be explored as a possible therapeutic strategy in rheumatoid arthritis.

Anti-aversive effects of the atypical antipsychotic, aripiprazole, in animal models of anxiety

Aripiprazole is a unique antipsychotic that seems to act as a partial agonist at dopamine D2-receptors, contrasting with other drugs in this class, which are silent antagonists. Aripiprazole may also bind to serotonin receptors. Both neurotransmitters may play major roles in aversion-, anxiety-and panic-related behaviours. Thus, the present work tested the hypothesis that this antipsychotic could also have anti-aversive properties. Male Wistar rats received injections of aripiprazole (0.1-10 mg/kg) and were tested in the open field, in the elevated plus and T mazes (EPM and ETM, respectively) and in a contextual fear conditioning paradigm. Aripiprazole (1mg/kg) increased the percentage of entries onto the open arms of the EPM and attenuated escape responses in the ETM. In the latter model, the dose of 0.1 mg/kg also decreased the latency to leave the enclosed arm, suggesting anxiolytic- and panicolytic-like properties. This dose also decreased the time spent in freezing in a contextual fear conditioning. No significant motor effects were observed at these doses. The present data support the hypothesis that aripiprazole could inhibit anxiety-related responses. Acting as a partial agonist at dopamine receptors, this drug could effectively treat schizophrenia and, in contrast with most antipsychotic drugs, alleviate aversive states.

Granulocyte-Colony Stimulating Factor (G-CSF) induces mechanical hyperalgesia via spinal activation of MAP kinases and PI(3)K in mice

Granulocyte-colony stimulating factor (G-CSF) is a current pharmacological approach to increase peripheral neutrophil counts after anti-tumor therapies. Pain is most relevant side effect of G-CSF in healthy volunteers and cancer patients. Therefore, the mechanisms of G-CSF-induced hyperalgesia were investigated focusing on the role of spinal mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated kinase). JNK (Jun N-terminal Kinase) and p38, and PI(3)K (phosphatidylinositol 3-kinase). G-CSF induced dose (30-300 ng/paw)-dependent mechanical hyperalgesia, which was inhibited by local post-treatment with morphine. This effect of morphine was reversed by naloxone (opioid receptor antagonist). Furthermore, G-CSF-induced hyperalgesia was inhibited in a dose-dependent manner by intrathecal pre-treatment with ERK (PD98059), JNK (SB600125), p38 (SB202190) or PI(3)K (wortmanin) inhibitors. The co-treatment with MAP kinase and PI(3)K inhibitors, at doses that were ineffective as single treatment, significantly inhibited G-CSF-induced hyperalgesia. Concluding, in addition to systemic opioids, peripheral opioids as well as spinal treatment with MAP kinases and PI(3)K inhibitors also reduce G-CSF-induced pain. (C) 2011 Elsevier Inc. All rights reserved.

Nitric oxide-mediated anxiolytic-like and antidepressant-like effects in animal models of anxiety and depression

The effects of microinjection of the nitric oxide (NO) precursor L-arginine (L-Arg), the NO synthase (NOS) inhibitors N-methyl-L-arginine (L-NAME) and 7-nitroindazole (7-NI), and the cyclic guanosine 3`,5`-monophosphate (cGMP) analog 8-Br-cGMP into the dorsal raphe nucleus (DRN) were assessed in rats using the elevated plus maze (EPM) and the forced swim test (FST). L-Arg (100 and 200 nmol) produced an anxiolytic-like effect in the EPM. 8-Br-cGMP (25 and 50 nmol) dose-dependently increased locomotor activity. In the FST, antidepressant-like effects were produced by L-Arg (50 and 100 nmol) and 8-Br-cGMP (12.5 and 25 nmol). Dual effects were observed with NOS inhibitors L-NAME and 7-NI in both the EPM and FST. While low doses of L-NAME (25 nmol) or 7-NI (1 nmol) induced a selective increase in EPM open arm exploration and a decrease in immobility time in the FST, high doses (L-NAME 400 nmol, 7-NI 10 nmol) decreased locomotor activity. These results show that interference with NO-mediated neurotransmission in the DRN induced significant and complex motor and emotional effects. Further studies are needed to elucidate the mechanisms involved in these effects. (C) 2007 Elsevier Inc. All rights reserved.

Erectile Dysfunction in Young Non-Obese Type II Diabetic Goto-Kakizaki Rats is Associated with Decreased eNOS Phosphorylation at Ser1177

Introduction. Diabetes mellitus (DM) is a risk factor for erectile dysfunction (ED). Although type 2 DM is responsible for 90-95% diabetes cases, type 1 DM experimental models are commonly used to study diabetes-associated ED. Aim. Goto-Kakizaki (GK) rat model is relevant to ED studies since the great majority of patients with type 2 diabetes display mild deficits in glucose-stimulated insulin secretion, insulin resistance, and hyperglycemia. We hypothesized that GK rats display ED which is associated with decreased nitric oxide (NO) bioavailability. Methods. Wistar and GK rats were used at 10 and 18 weeks of age. Changes in the ratio of intracavernosal pressure/mean arterial pressure (ICP/MAP) after electrical stimulation of cavernosal nerve were determined in vivo. Cavernosal contractility was induced by electrical field stimulation (EFS) and phenylephrine (PE). In addition, nonadrenergic-noncholinergic (NANC)- and sodium nitroprusside (SNP)-induced relaxation were determined. Cavernosal neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) mRNA and protein expression were also measured. Main Outcome Measure. GK diabetic rats display ED associated with decreased cavernosal expression of eNOS protein. Results. GK rats at 10 and 18 weeks demonstrated impaired erectile function represented by decreased ICP/MAP responses. Ten-week-old GK animals displayed increased PE responses and no changes in EFS-induced contraction. Conversely, contractile responses to EFS and PE were decreased in cavernosal tissue from GK rats at 18 weeks of age. Moreover, GK rats at 18 weeks of age displayed increased NANC-mediated relaxation, but not to SNP. In addition, ED was associated with decreased eNOS protein expression at both ages. Conclusion. Although GK rats display ED, they exhibit changes in cavernosal reactivity that would facilitate erectile responses. These results are in contrast to those described in other experimental diabetes models. This may be due to compensatory mechanisms in cavernosal tissue to overcome restricted pre-penile arterial blood supply or impaired veno-occlusive mechanisms. Carneiro FS, Giachini FRC, Carneiro ZN, Lima VV, Ergul A, Webb RC, and Tostes RC. Erectile dysfunction in young non-obese type II diabetic Goto-Kakizaki rats is associated with decreased eNOS phosphorylation at Ser1177. J Sex Med 2010;7:3620-3634.

Role of platelet-activating factor in the pathogenesis of 5-fluorouracil-induced intestinal mucositis in mice

Gastrointestinal mucositis is a common side effect of cancer chemotherapy. Platelet-activating factor (PAF) is produced during gut inflammation. There is no evidence that PAF participates in antineoplastic-induced intestinal mucositis. This study evaluated the role of PAF in 5-fluorouracil (5-FU)-induced intestinal mucositis using a pharmacological approach and PAF receptor knockout mice (PAFR(-/-)). Wild-type mice or PAFR(-/-) mice were treated with 5-FU (450 mg/kg, i.p.). Other mice were treated with saline or BN52021 (20 mg/kg, s.c.), an antagonist of the PAF receptor, once daily followed by 5-FU administration. After the third day of treatment, animals were sacrificed and tissue samples from the duodenum were removed for morphologic evaluation. In addition, myeloperoxidase activity and the cytokine concentration were measured. 5-FU treatment decreased the duodenal villus height/crypt depth ratio, increased MPO activity, and increased the concentration of TNF-alpha, IL-1 beta and KC in comparison with saline-treated animals. In PAFR(-/-) mice and PAFR antagonist-treated mice, 5-FU-dependent intestinal damage was reduced and a decrease in duodenal villus height/crypt depth ratio was attenuated. However, the 5-FU-dependent increase in duodenum MPO activity was not affected. Without PAFR activation, 5-FU treatment did not increase the TNF-alpha, IL-1 beta and KC concentration. In conclusion, our study establishes the role of PAFR activation in 5-FU-induced intestinal mucositis. This study implicates treatment with PAFR antagonists as novel therapeutic strategy for this condition.

Tadalafil analgesia in experimental arthritis involves suppression of intra-articular TNF release

BACKGROUND AND PURPOSE We investigated the effect of the phosphodiesterase-5 inhibitor, tadalafil, on the acute hypernociception in rat models of arthritis. EXPERIMENTAL APPROACH Rats were treated with either an intra-articular injection of zymosan (1 mg) or surgical transection of the anterior cruciate ligament (as an osteoarthritis model). Controls received saline intra-articular or sham operation respectively. Joint pain was evaluated using the articular incapacitation test measured over 6 h following zymosan or between 4 and 7 days after anterior cruciate ligament transection. Cell counts, tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and the chemokine, cytokine-induced neutrophil chemoattractant-1 (CINC-1) were measured in joint exudates 6 h after zymosan. Groups received tadalafil (0.02-0.5 mg.kg(-1) per os) or saline 2 h after intra-articular zymosan. Other groups received the mu-opioid receptor antagonist naloxone or the cGMP inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) before tadalafil. KEY RESULTS Tadalafil dose-dependently inhibited hypernociception in zymosan and osteoarthritis models. In zymosan-induced arthritis, tadalafil significantly decreased cell influx and TNF-alpha release but did not alter IL-1 or CINC-1 levels. Pretreatment with ODQ but not with naloxone prevented the anti-inflammatory effects of tadalafil. CONCLUSIONS AND IMPLICATIONS Therapeutic oral administration of tadalafil provided analgesia mediated by guanylyl cyclase and was independent of the release of endogenous opioids. This effect of tadalafil was associated with a decrease in neutrophil influx and TNF-alpha release in inflamed joints.

An anti-inflammatory lectin from Luetzelburgia auriculata seeds inhibits adhesion and rolling of leukocytes and modulates histamine and PGE2 action in acute inflammation models

To study and characterize the in vivo effect of the lectin from Luetzelburgia auriculata seed on acute inflammation models. The lectin was purified from the crude saline extract by affinity chromatography on a guar-gum matrix. Native, heat-treated, and digested lectin was evaluated for anti-inflammatory activity by using peritonitis and paw edema models. The anti-inflammatory activity was characterized by intravital microscopy, nitric oxide production, and myeloperoxidase activity. The lectin exhibited anti-inflammatory activity (2 mg/kg) on both models, reducing local myeloperoxidase activity. Galactose or heat treatment (100A degrees C, 10 min) reduced anti-inflammatory action. Anti-inflammation involves the inhibition of adhesion and rolling of leukocytes along with augmentation of nitric oxide in serum. The lectin inhibited the edematogenic effect of histamine and prostaglandins (PGE2) but did not alter the chemoattractant effect of IL-8. The results indicate that this lectin is a potent anti-inflammatory molecule. Its effects engage diverse modulatory events.

Antidepressant-like effects of cannabidiol in mice: possible involvement of 5-HT(1A) receptors

Background and purpose: Cannabidiol (CBD) is a non-psychotomimetic compound from Cannabis sativa that induces anxiolytic- and antipsychotic-like effects in animal models. Effects of CBD may be mediated by the activation of 5-HT(1A) receptors. As 5-HT(1A) receptor activation may induce antidepressant-like effects, the aim of this work was to test the hypothesis that CBD would have antidepressant-like activity in mice as assessed by the forced swimming test. We also investigated if these responses depended on the activation of 5-HT(1A) receptors and on hippocampal expression of brain-derived neurotrophic factor (BDNF). Experimental approach: Male Swiss mice were given (i.p.) CBD (3, 10, 30, 100 mg.kg(-1)), imipramine (30 mg.kg(-1)) or vehicle and were submitted to the forced swimming test or to an open field arena, 30 min later. An additional group received WAY100635 (0.1 mg.kg(-1), i.p.), a 5-HT(1A) receptor antagonist, before CBD (30 mg.kg(-1)) and assessment by the forced swimming test. BDNF protein levels were measured in the hippocampus of another group of mice treated with CBD (30 mg.kg(-1)) and submitted to the forced swimming test. Key results: CBD (30 mg.kg(-1)) treatment reduced immobility time in the forced swimming test, as did the prototype antidepressant imipramine, without changing exploratory behaviour in the open field arena. WAY100635 pretreatment blocked CBD-induced effect in the forced swimming test. CBD (30 mg.kg(-1)) treatment did not change hippocampal BDNF levels. Conclusion and implications: CBD induces antidepressant-like effects comparable to those of imipramine. These effects of CBD were probably mediated by activation of 5-HT(1A) receptors. British Journal of Pharmacology (2010) 159, 122-128; doi:10.1111/j.1476-5381.2009.00521.x; published online 4 December 2009