Inhibition of mouse mammary tumor virus-induced T cell responses in vivo by antibodies to an open reading frame protein.

Minor lymphocyte stimulating (Mls) antigens specifically stimulate T cell responses that are restricted to particular T cell receptor (TCR) beta chain variable domains. The Mls phenotype is genetically controlled by an open reading frame (orf) located in the 3' long terminal repeat of mouse mammary tumor virus (MMTV); however, the mechanism of action of the orf gene product is unknown. Whereas predicted orf amino acid sequences show strong overall homology, the 20-30 COOH-terminal residues are strikingly polymorphic. This polymorphic region correlates with TCR V beta specificity. We have generated monoclonal antibodies to a synthetic peptide encompassing the 19 COOH-terminal amino acid residues of Mtv-7 orf, which encodes the Mls-1a determinant. We show here that these antibodies block Mls responses in vitro and can interfere specifically with thymic clonal deletion of Mls-1a reactive V beta 6+ T cells in neonatal mice. Furthermore, the antibodies can inhibit V beta 6+ T cell responses in vivo to an infectious MMTV that shares orf sequence homology and TCR specificity with Mtv-7. These results confirm the predicted extracellular localization of the orf COOH terminus and imply that the orf proteins of both endogenous and exogenous MMTV interact directly with TCR V beta.

Expression pattern of sirtuins in innate immune cells and influence of Sirtuin 1/2 inhibition on innate immune responses to Toll-like receptor ligands and to infection

Purpose/Objective: The family of histone deacetylases comprises 18 members in mammals, among which seven sirtuins (SIRT1-7). Sirtuins are NADP-dependent enzymes that have been involved in the control of cell metabolism, proliferation and survival. The expression pattern of sirtuins and their influence on host response to microbial infection remain largely unknown. The aim of the study was to analyze the expression of SIRT1-7 and to address the effects of SIRT1/2 inhibition on innate immune responses in vitro and in vivo.. Materials and methods: in vitro: Bone marrow (BM), BM-derived macrophages (BMDMs) and dendritic cells (BMDCs) and RAW 264.7 and J774.1 macrophage cell lines were stimulated for 0, 2, 6 and 18 h with LPS, Pam3CSK4 and CpG ODN. SIRT1-7 mRNA was quantified by real time-PCR. TNF was measured by ELISA. In vivo: BALB/c mice were challenged with LPS (350 lg i.p.) with or without a SIRT1/2 inhibitor. Blood and organs were collected after 0, 1, 4, 8 and 24 h to quantify SIRT1-7 and TNF. Mortality was assessed daily. Results: Bone marrow, macrophages and DCs express, in order of abundance, SIRT2 > > SIRT1, SIRT3 and SIRT6 > SIRT4, SIRT5 and SIRT7. Microbial products decrease the expression of all sirtuins except SIRT6 in a time dependent manner in BMDMs (0_24 h). SIRT2 is the most expressed sirtuin also in the liver, kidney (together with SIRT3) and spleen. Upon LPS challenge, SIRT1, SIRT3, SIRT4 and SIRT7 mRNA levels decrease in the liver (from 4 h to 24 h), whereas SIRT1-7 mRNA levels decrease within 1 h in both kidney and spleen. Pharmacological inhibition of SIRT1/2 decreases TNF production by macrophages stimulated with LPS, Pam3CSK4 and CpG ODN (n = 6; P < 0.001). In agreement, prophylactic treatment with a SIRT1/2 inhibitor decreases TNF production (n = 8; P = 0.04) and increases survival (n = 13, P = 0.03) of mice challenged with LPS. Conclusions: Sirtuins are expressed in innate immune cells. Inhibition of SIRT1/2 activity decreases cytokine production by macrophages and protects from endotoxemia, suggesting that sirtuin inhibitors may represent novel adjunctive therapy for treating inflammatory disorders such as sepsis.

Primary motor cortex inhibition in spinal cord injuries.

OBJECTIVES AND METHODS: Excitability changes in the primary motor cortex in 17 spinal-cord injured (SCI) patients and 10 controls were studied with paired-pulse transcranial magnetic stimulation. The paired pulses were applied at inter-stimulus intervals (ISI) of 2 ms and 15 ms while motor evoked potentials (MEP) were recorded in the biceps brachii (Bic), the abductor pollicis brevis (APB) and the tibialis anterior (TA) muscles. RESULTS: The study revealed a significant decrease in cortical motor excitability in the first weeks after SCI concerning the representation of both the affected muscles innervated from spinal segments below the lesion, and the spared muscles rostral to the lesion. In the patients with motor-incomplete injury, but not in those with motor-complete injury, the initial cortical inhibition of affected muscles was temporarily reduced 2-3 months following injury. The degree of inhibition in cortical areas representing the spared muscles was observed to be smaller in patients with no voluntary TA activity compared to patients with some activity remaining in the TA. Surprisingly, motor-cortical inhibition was observed not only at ISI 2 ms but also at ISI 15 ms. The inhibition persisted in patients who returned for a follow-up measurement 2-3 years later. CONCLUSION: The present data showed different evaluation of cortical excitability between patients with complete and incomplete spinal cord lesion. Our results provide more insight into the pathophysiology of SCI and contribute to the ongoing discussion about the recovery process and therapy of SCI patients.

Plasma angiotensin-(1-8) octapeptide measurement to assess acute angiotensin-converting enzyme inhibition with captopril administered parenterally to normal subjects.

The blood pressure (BP), heart rate (HR), and humoral effects of single intravenous (i.v.) doses of the angiotensin-converting enzyme (ACE) inhibitor captopril was investigated in five normotensive healthy volunteers. Each subject received at 1-week intervals a bolus dose of either captopril (1, 5, and 25 mg) or its vehicle. The study was conducted in a single-blind fashion, and the order of treatment phases was randomized. The different doses of captopril had no acute effect on BP and HR. They induced a dose-dependent decrease in plasma ACE activity and plasma angiotensin II levels. The angiotensin-(1-8) octapeptide was isolated by solid-phase extraction and high-performance liquid chromatography (HPLC) prior to radioimmunoassay (RIA). All three doses of captopril reduced circulating angiotensin II levels within 15 min of drug administration. Only with the 25-mg dose was the angiotensin II concentration below the detection limit at 15 min and still significantly reduced 90 min after drug administration. Simultaneous and progressive decreases in plasma aldosterone levels were observed both with ACE inhibition and during vehicle injection, but the relative fall was more pronounced after captopril administration. No adverse reaction was noticed. These results demonstrate that captopril given parenterally blocks the renin-angiotensin system in a dose-dependent manner. Only with the dose of 25 mg was the inhibition of plasma-converting enzyme activity and the reduction of plasma angiotensin II sustained for at least 1 1/2 h.

Importance of TNF-alpha in the course of acute infection with Trypanosoma cruzi: influence of its inhibition by pentoxifylline treatment

Infection of C3H/He mice with the Peruvian strain of Trypanosoma cruzi (Biodeme type I, Z2b), a macrophagotropic strain, determined severe parasitism of macrophages, necrosis of the spleen, and high host mortality. In the present study, pentoxifylline (PTX), an inhibitor of TNF-alpha was investigated on its action upon splenic necrosis, parasitemia and host survival. Immunohistochemical data suggested the importance of this cytokine in parasite destruction and decreasing of parasitemia, although paradoxically contributing to the high mortality of infected mice. Necrotic lesions involving several organs, specially the heart, in acute Chagas disease, are important aggravating factors, increasing cardiac morbidity. Advantage of inhibiting TNF-alpha action was herein investigated. Infected mice were divided into two groups: untreated (n = 24), and PTX treated mice (n = 25). PTX was administered in two daily doses of 30 mg/kg/bw, by intraperitoneal route. Normal controls either treated with PTX or saline were also included. Histopathology of the spleen and in situ immunolabeling of TNF-alpha, using anti-TNF-alpha monoclonal antibody, were performed. Necrotic areas were evaluated by morphometry. Mice treated with PTX showed a significant decrease of necrotic areas and diminution of TNF-alpha expression in spleen tissue, suggesting that PTX treatment could control TNF-alpha effects, and thus be used as an adjuvant in the treatment of acute Chagas' disease.

The utility of the polymerase chain reaction assay for aetiologic definition of unspecified bacterial meningitis cases

Most patients with acute suppurative meningitis are otherwise healthy individuals with regard to immune mechanisms against invasive bacterial disease. This medical emergency is among the most dramatic and potentially ravaging diseases that affect humans, particularly young children. The illness often strikes suddenly, and can either result in death or leave the survivors with significant neurological dysfunctions. The demonstration of a bacterial aetiology is necessary for decisions regarding treatment and prophylaxis. Conventional bacteriological methods frequently fail to identify an agent, as a result of administration of antibiotics or delayed lumbar punctures. We investigated the major aetiologic sources of unspecified bacterial meningitis cases (G00.9, ISCD-10) by polymerase chain reaction (PCR)-based identification of Neisseria meningitidis (crgA), Streptococcus pneumoniae (ply) and Haemophilus influenzae (bexA) in cerebrospinal fluid samples. The multiplex PCR detected N. meningitidis in 92%, S. pneumoniae in 4% and H. influenzae in 1% of the 192 clinical samples assayed; 3% were negative for all three DNA targets. Bacterial DNA detection was found to be a valuable adjunct to enhance bacterial meningitis surveillance when the yield of specimens by culture is reduced. The implementation of PCR assays as a diagnostic procedure in Public Health Laboratories is perceived to be a significant advance in the investigation of bacterial meningitis.

Potential value of biliary CEA assay in early detection of colorectal adenocarcinoma liver metastases.

Elevation of the biliary CEA level in patients with liver metastases from colorectal carcinoma has been reported. The aim of this study is to determine the potential value of biliary CEA assay in the early detection of liver metastases. Biliary and serum CEA levels were determined in patients operated on for a colorectal cancer and in control groups. Among 13 patients with liver metastases from colorectal carcinomas, biliary CEA levels were markedly elevated (> 40 ng/ml) in nine, moderately elevated (5-40 ng/ml) in two and normal (arbitrarily defined as < 5 ng/ml) in two. Of 28 patients with primary colorectal carcinoma without detectable hepatic secondaries, three had marked CEA elevation in the bile, 10 had moderate CEA elevation and 15 had normal levels. Among nine patients with non-malignant hepatobiliary pathology, there was one marked biliary CEA elevation, one moderate elevation and seven normal levels. None of the 13 individuals with no identified hepatobiliary pathology had elevated biliary CEA levels. The follow-up of patients with a primary colorectal tumour, no evidence of hepatic secondaries and a biliary CEA elevation is of particular interest. If subsequent appearance of liver metastases is found in such cases, intra-operative biliary CEA assay could be considered a valuable diagnostic test. Further studies will then have to prove the possible benefit of a specific treatment for this group of patients.

Performance of indirect immunofluorescence assay, immunochromatography assay and reverse transcription-polymerase chain reaction for detecting human respiratory syncytial virus in nasopharyngeal aspirate samples

Comparison of the use of indirect immunofluorescence assay (IFA), immunochromatography assay (ICA-BD) and reverse transcription-polymerase chain reaction (RT-PCR) for detecting human respiratory syncytial virus (HRSV) in 306 nasopharyngeal aspirates samples (NPA) was performed in order to assess their analytical performance. By comparing the results obtained using ICA-BD with those using IFA, we found relative indices of 85.0% for sensitivity and 91.2% for specificity, and the positive (PPV) and negative (NPV) predictive values were 85.0% and 91.2%, respectively. The relative indices for sensitivity and specificity as well as the PPV and NPV for RT-PCR were 98.0%, 89.0%, 84.0% and 99.0%, respectively, when compared to the results of IFA. In addition, comparison of the results of ICA-BD and those of RT-PCR yielded relative indices of 79.5% for sensitivity and 95.4% for specificity, as well as PPV and NPV of 92.9% and 86.0%, respectively. Although RT-PCR has shown the best performance, the substantial agreement between the ICA-BD and IFA results suggests that ICA-BD, also in addition to being a rapid and facile assay, could be suitable as an alternative diagnostic screening for HRSV infection in children.

Duplex-PCR assay for the detection of adenovirus and respiratory syncytial virus in nasopharyngeal samples

Human adenovirus (HAdV) and human respiratory syncytial virus (HRSV) are important etiologic agents of acute respiratory infections. In this study, a duplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of HAdV and HRSV in clinical samples. Sixty previously screened nasopharyngeal aspirates were used: 20 HAdV-positive, 20 HRSV-positive and 20 double-negative controls. Eight samples were positive for both viruses. The duplex PCR assay proved to be as sensitive and specific as single-target assays and also detected the mixed infections with certainty. The identification of both viruses in a single reaction offers a reduction in both cost and laboratory diagnostic time.

Regulation of Placental Leptin Expression by Cyclic Adenosine 5 '-Monophosphate Involves Cross Talk between Protein Kinase A and Mitogen-Activated Protein Kinase Signaling Pathways

Leptin, a 16-kDa protein mainly produced by adipose tissue, has been involved in the control of energy balance through its hypothalamic receptor. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it was found to be expressed. In the current study, we examined the effect of cAMP in the regulation of leptin expression in trophoblastic cells. We found that dibutyryl cAMP [(Bu)(2)cAMP], a cAMP analog, showed an inducing effect on endogenous leptin expression in BeWo and JEG-3 cell lines when analyzed by Western blot analysis and quantitative RT-PCR. Maximal effect was achieved at 100 microM. Leptin promoter activity was also stimulated, evaluated by transient transfection with a reporter plasmid construction. Similar results were obtained with human term placental explants, thus indicating physiological relevance. Because cAMP usually exerts its actions through activation of protein kinase A (PKA) signaling, this pathway was analyzed. We found that cAMP response element-binding protein (CREB) phosphorylation was significantly increased with (Bu)(2)cAMP treatment. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor CREB caused a significant stimulation on leptin promoter activity. On the other hand, the cotransfection with a dominant negative mutant of the regulatory subunit of PKA inhibited leptin promoter activity. We determined that cAMP effect could be blocked by pharmacologic inhibition of PKA or adenylyl ciclase in BeWo cells and in human placental explants. Thereafter, we decided to investigate the involvement of the MAPK/ERK signaling pathway in the cAMP effect on leptin induction. We found that 50 microm PD98059, a MAPK kinase inhibitor, partially blocked leptin induction by cAMP, measured both by Western blot analysis and reporter transient transfection assay. Moreover, ERK 1/2 phosphorylation was significantly increased with (Bu)(2)cAMP treatment, and this effect was dose dependent. Finally, we observed that 50 microm PD98059 inhibited cAMP-dependent phosphorylation of CREB in placental explants. In summary, we provide some evidence suggesting that cAMP induces leptin expression in placental cells and that this effect seems to be mediated by a cross talk between PKA and MAPK signaling pathways.

Resistance of melanized yeast cells of Paracoccidioides brasiliensis to antimicrobial oxidants and inhibition of phagocytosis using carbohydrates and monoclonal antibody to CD18

Paracoccidioides brasiliensis, a thermal dimorphic fungal pathogen, produces a melanin-like pigment in vitro and in vivo. We investigated the involvement of carbohydrates and monoclonal antibody to CD18, on phagocytosis inhibition, involving macrophage receptors and the resistance of melanized fungal cells to chemically generated nitric oxide (NO), reactive oxygen species (ROS), hypochlorite and H2O2. Our results demonstrate that melanized yeast cells were more resistant than nonmelanized yeast cells to chemically generated NO, ROS, hypochlorite and H2O2, in vitro. Phagocytosis of melanized yeast cells was virtually abolished when mannan, N-acetyl glucosamine and anti-CD18 antibody were added together in this system. Intratracheal infection of BALB/c mice, with melanized yeast cells, resulted in higher lung colony forming units, when compared to nonmelanized yeast cells. Therefore, melanin is a virulence factor of P. brasiliensis.

In house colorimetric reverse hybridisation assay for detection of the mutation most frequently associated with resistance to isoniazid in Mycobacterium tuberculosis

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC→ACC (Ser→Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100% of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.

Inhibition of Trypanosoma cruzi proline racemase affects host-parasite interactions and the outcome of in vitro infection

Proline racemase is an important enzyme of Trypanosoma cruzi and has been shown to be an effective mitogen for B cells, thus contributing to the parasite's immune evasion and persistence in the human host. Recombinant epimastigote parasites overexpressing TcPRAC genes coding for proline racemase present an augmented ability to differentiate into metacyclic infective forms and subsequently penetrate host-cells in vitro. Here we demonstrate that both anti T. cruzi proline racemase antibodies and the specific proline racemase inhibitor pyrrole-2-carboxylic acid significantly affect parasite infection of Vero cells in vitro. This inhibitor also hampers T. cruzi intracellular differentiation.

Development and Preliminary Evaluation of a Rapid Oligochromatographic Assay for Specific Detection of New Human Influenza A H1N1 Virus

A new oligochromatographic assay, Speed-Oligo Novel Influenza A H1N1, was designed and optimized for the specific detection of the 2009 influenza A H1N1 virus. The assay is based on a PCR method coupled to detection of PCR products by means of a dipstick device. The target sequence is a 103-bp fragment within the hemagglutinin gene. The analytical sensitivity of the new assay was measured with serial dilutions of a plasmid that contained the target sequence, and we determined that down to one copy per reaction of the plasmid was reliably detected. Diagnostic performance was assessed with 103 RNAs from suspected cases (40 positive and 63 negative results) previously analyzed with a reference real-time PCR technique. All positive cases were confirmed, and no false-positive results were detected with the new assay. No cross-reactions were observed when other viral strains or clinical samples with other respiratory viruses were tested. According to these results, this new assay has 100% sensitivity and specificity. The turnaround time for the whole procedure was 140 min. The assay may be especially useful for the specific detection of 2009 H1N1 virus in laboratories not equipped with real-time PCR instruments

Further evaluation of an updated PCR assay for the detection of Schistosoma mansoni DNA in human stool samples

A previously reported sensitive PCR assay for the detection of Schistosoma mansoni DNA was updated and evaluated. Changes in the DNA extraction method, including the use of a worldwide available commercial kit and the inclusion of additional quality control measures, increased the robustness of the test, as confirmed by the analysis of 67 faecal samples from an endemic area in Brazil. The PCR assay is at hand as a proven, reliable diagnostic test for the control of schistosomiasis in specific settings.