Renal and antibacterial effects induced by myotoxin I and II isolated from Bothrops jararacussu venom.

Bothrops jararacussu myotoxin I (BthTx-I; Lys 49) and II (BthTX-II; Asp 49) were purified by ion-exchange chromatography and reverse phase HPLC. In this work we used the isolated perfused rat kidney method to evaluate the renal effects of B. jararacussu myotoxins I (Lys49 PLA2) and II (Asp49 PLA2) and their possible blockage by indomethacin. BthTX-I (5 microg/ml) and BthTX-II (5 microg/ml) increased perfusion pressure (PP; ct120=110.28+/-3.70 mmHg; BthTX I=171.28+/-6.30*mmHg; BthTX II=175.50+/-7.20*mmHg), renal vascular resistance (RVR; ct120=5.49+/-0.54 mmHg/ml.g(-1)min(-1); BthTX I=8.62+/-0.37*mmHg/ml g(-1)min(-1); BthTX II=8.9+/-0.36*mmHg/ml g(-1)min(-1)), urinary flow (UF; ct(120)=0.14+/-0.01ml g(-1)min(-1); BthTX I=0.32+/-0.05*ml g(-1)min(-1); BthTX II=0.37+/-0.01*ml g(-1)min(-1)) and glomerular filtration rate (GFR; ct120=0.72+/-0.10 ml g(-1)min(-1); BthTX I=0.85+/-0.13*ml g(-1)min(-1); BthTX II=1.22+/-0.28*ml g(-1)min(-1)). In contrast decreased the percent of sodium tubular transport (%TNa(+); ct(120)=79,76+/-0.56; BthTX I=62.23+/-4.12*; BthTX II=70.96+/-2.93*) and percent of potassium tubular transport (%TK(+);ct120=66.80+/-3.69; BthTX I=55.76+/-5.57*; BthTX II=50.86+/-6.16*). Indomethacin antagonized the vascular, glomerular and tubular effects promoted by BthTX I and it's partially blocked the effects of BthTX II. In this work also evaluated the antibacterial effects of BthTx-I and BthTx-II against Xanthomonas axonopodis. pv. passiflorae (Gram-negative bacteria) and we observed that both PLA2 showed antibacterial activity. Also we observed that proteins Also we observed that proteins chemically modified with 4-bromophenacyl bromide (rho-BPB) decrease significantly the antibacterial effect of both PLA2. In conclusion, BthTx I and BthTX II caused renal alteration and presented activity antimicrobial. The indomethacin was able to antagonize totally the renal effects induced by BthTx I and partially the effects promoted by BthTx II, suggesting involvement of inflammatory mediators in the renal effects caused by myotoxins. In the other hand, other effects could be independently of the enzymatic activity of the BthTX II and the C-terminal domain could be involved in both effects promoted for PLA2.     

Cross-neutralization of the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms by antisera against crotoxin and phospholipase A2 from Crotalus durissus cascavella venom.

We have previously demonstrated that rabbit antisera raised against crotoxin from Crotalus durissus cascavella venom (cdc-crotoxin) and its PLA2 (cdc-PLA2) neutralized the neurotoxicity of this venom and its crotoxin. In this study, we examined the ability of these antisera to neutralize the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms and their major toxins, cdt-crotoxin and bothropstoxin-I (BthTX-I), respectively, in mouse isolated phrenic nerve-diaphragm preparations. Immunoblotting showed that antiserum to cdc-crotoxin recognized cdt-crotoxin and BthTX-I, while antiserum to cdc-PLA2 recognized cdt-PLA2 and BthTX-I. ELISA corroborated this cross-reactivity. Antiserum to cdc-crotoxin prevented the neuromuscular blockade caused by C. d. terrificus venom and its crotoxin at a venom/crotoxin:antiserum ratio of 1:3. Antiserum to cdc-PLA2 also neutralized the neuromuscular blockade caused by C. d. terrificus venom or its crotoxin at venom or toxin:antiserum ratios of 1:3 and 1:1, respectively. The neuromuscular blockade caused by B. jararacussu venom and BthTX-I was also neutralized by the antisera to cdc-crotoxin and cdc-PLA2 at a venom/toxin:antiserum ratio of 1:10 for both. Commercial equine antivenom raised against C. d. terrificus venom was effective in preventing the neuromuscular blockade typical of B. jararacussu venom (venom:antivenom ratio of 1:2), whereas for BthTX-I the ratio was 1:10. These results show that antiserum produced against PLA2, the major toxin in C. durissus cascavella venom, efficiently neutralized the neurotoxicity of C. d. terrificus and B. jararacussu venoms and their PLA2 toxins.     

Cleavage of L1 in exosomes and apoptotic membrane vesicles released from ovarian carcinoma cells.

PURPOSE: The L1 adhesion molecule (CD171) is overexpressed in human ovarian and endometrial carcinomas and is associated with bad prognosis. Although expressed as a transmembrane molecule, L1 is released from carcinoma cells in a soluble form. Soluble L1 is present in serum and ascites of ovarian carcinoma patients. We investigated the mode of L1 cleavage and the function of soluble L1. EXPERIMENTAL DESIGN: We used ovarian carcinoma cell lines and ascites from ovarian carcinoma patients to analyze soluble L1 and L1 cleavage by Western blot analysis and ELISA. RESULTS: We find that in ovarian carcinoma cells the constitutive cleavage of L1 proceeds in secretory vesicles. We show that apoptotic stimuli like C2-ceramide, staurosporine, UV irradiation, and hypoxic conditions enhance L1-vesicle release resulting in elevated levels of soluble L1. Constitutive cleavage of L1 is mediated by a disintegrin and metalloproteinase 10, but under apoptotic conditions multiple metalloproteinases are involved. L1 cleavage occurs in two types of vesicles with distinct density features: constitutively released vesicles with similarity to exosomes and apoptotic vesicles. Both types of L1-containing vesicles are present in the ascites fluids of ovarian carcinoma patients. Soluble L1 from ascites is a potent inducer of cell migration and can trigger extracellular signal-regulated kinase phosphorylation. CONCLUSIONS: We suggest that tumor-derived vesicles may be an important source for soluble L1 that could regulate tumor cell function in an autocrine/paracrine fashion.     

ABL mutations in late chronic phase chronic myeloid leukemia patients with up-front cytogenetic resistance to imatinib are associated with a greater likelihood of progression to blast crisis and shorter survival: a study by the GIMEMA Working Party on Chronic Myeloid Leukemia.

PURPOSE: Point mutations within the ABL kinase domain of the BCR-ABL gene have been associated with clinical resistance to imatinib mesylate in chronic myeloid leukemia (CML) patients. To shed further light on the frequency, distribution, and prognostic significance of ABL mutations, we retrospectively analyzed a homogeneous cohort of late chronic phase CML patients who showed primary cytogenetic resistance to imatinib. PATIENTS AND METHODS: Using denaturing high-performance liquid chromatography (D-HPLC) and sequencing, we screened for ABL mutations in a total of 178 bone marrow and/or peripheral blood samples from 40 late chronic phase CML patients homogeneously treated with imatinib 400 mg/d, who did not reach a major cytogenetic response at 12 months. RESULTS: Mutations were found in 19 of 40 patients (48%). Mutations were already detectable by D-HPLC at a median of 3 months from the onset of therapy. The presence of a missense mutation was significantly associated with a greater likelihood of subsequent progression to accelerated phase/blast crisis (P = .0002) and shorter survival (P = .001). Patients carrying mutations falling within the P-loop seemed to have a particularly poor outcome in terms of time to progression (P = .032) and survival (P = .045). CONCLUSION: Our results show that, irrespective of the hematologic response, monitoring for emerging mutations in the first months of therapy may play a role in detecting patients with worse prognosis, for whom a revision of the therapeutic strategy should be considered.     

Detection of spontaneous CD4+ T-cell responses in melanoma patients against a tyrosinase-related protein-2-derived epitope identified in HLA-DRB1*0301 transgenic mice.

PURPOSE: The frequently expressed differentiation antigen tyrosinase-related protein-2 (TRP-2) has repeatedly been described as a target of spontaneous cytotoxic T-cell responses in melanoma patients, suggesting that it might be an ideal candidate antigen for T cell-based immunotherapy. As a prerequisite for immunization, T-cell epitopes have to be identified. Whereas a number of HLA class I-presented TRP-2-derived epitopes are known, information about HLA class II-presented antigenic ligands recognized by CD4+ T helper (Th) cells is limited. EXPERIMENTAL DESIGN: The search for TRP-2-derived Th epitopes was carried out by competitive in vitro peptide binding studies with predicted HLA-DRB1*0301 ligands in combination with peptide and protein immunizations of HLA-DRB1*0301 transgenic mice. In vivo selected candidate epitopes were subsequently verified for their immunogenicity in human T-cell cultures. RESULTS: This strategy led to the characterization of TRP-2(60-74) as an HLA-DRB1*0301-restricted Th epitope. Importantly, TRP-2(60-74)-reactive human CD4+ Th cell lines, specifically recognizing target cells loaded with recombinant TRP-2 protein, could be established by repeated peptide stimulation of peripheral blood lymphocytes from several HLA-DRB1*03+ melanoma patients. Even short-term peptide stimulation of patients' peripheral blood lymphocytes showed the presence of TRP-2(60-74)-reactive T cells, suggesting that these T cells were already activated in vivo. CONCLUSION: Peptide TRP-2(60-74) might be a useful tool for the improvement of immunotherapy and immune monitoring of melanoma patients.     

Sanguinarine and chelerythrine: assessment of safety on pigs in ninety days feeding experiment.

Sanguinaria canadesis, Chelidonium majus and Macleya cordata have been used for centuries as alternative medicines. Currently the extracts from these medicinal plants are components of veterinary and human phytopreparations, and of oral-hygiene agents. Sanguinarine and chelerythrine (SA/CHE) are biologically active components of these extracts. They display distinct antibacterial and anti-inflammatory properties, but, on the other hand, they have been reported as having adverse effects - genotoxicity and hepatotoxicity. This paper is aimed at evaluation of the effects of daily administration of the extract from Macleya cordata (2 mg and 100 mg in 1 kg feed, sanguinarine:chelerythrine 3:1) in the diet on the health status of swine. After 90-day administration, alkaloids were retained to a different extent in tissues. The highest SA/CHE retention was detected in the gingiva (0.55 microg/g) and liver (0.15 microg/g), no SA/CHE were detected in muscles. Plasma SA levels attained 0.11 microg/ml. Treated animals did not display any results of hematological, biochemical or histological assay different from controls. A (32)P-postlabeling assay proved that no DNA-adducts with SA/CHE were detected in pig livers. We did not observe any symptom linked to epidemic dropsy syndrome often attributed to sanguinarine. In conclusion, an average daily oral dose of alkaloids up to 5 mg per 1 kg animal body weight proved to be safe.     

Cerebrospinal anandamide levels are elevated in acute schizophrenia and are inversely correlated with psychotic symptoms.

The endocannabinoids are a family of bioactive lipids that activate CB1 cannabinoid receptors in the brain and exert intense emotional and cognitive effects. Here, we have examined the role of endocannabinoid signaling in psychotic states by measuring levels of the endocannabinoid anandamide in cerebrospinal fluid (CSF) of acute paranoid-type schizophrenic patients. We found that CSF anandamide levels are eight-fold higher in antipsychotic-naive first-episode paranoid schizophrenics (n = 47) than healthy controls (n = 84), dementia patients (n = 13) or affective disorder patients (n = 22). Such an alteration is absent in schizophrenics treated with 'typical' antipsychotics (n = 37), which antagonize dopamine D2-like receptors, but not in those treated with 'atypical' antipsychotics (n = 34), which preferentially antagonize 5HT(2A) receptors. Furthermore, we found that, in nonmedicated acute schizophrenics, CSF anandamide is negatively correlated with psychotic symptoms (rS = -0.452, P = 0.001). The results suggest that anandamide elevation in acute paranoid schizophrenia may reflect a compensatory adaptation to the disease state.     

In vivo biocompatibility of the PLGA microparticles in parotid gland

Poly(lactic-co-glycolic acid) (PLGA) microparticles are used in various disorders for the controlled or sustained release of drugs, with the management of salivary gland pathologies possible using this technology. There is no record of the response to such microparticles in the glandular parenchyma. The purpose of this study was to assess the morphological changes in the parotid gland when injected with a single dose of PLGA microparticles. We used 12 adult female Sprague Dawley rats (Rattus norvegicus) that were injected into their right parotid gland with sterile vehicle solution (G1, n=4), 0.5 mg PLGA microparticles (G2, n=4), and 0.75 mg PLGA microparticles (G3, n=4); the microparticles were dissolved in a sterile vehicle solution. The intercalar and striated ducts lumen, the thickness of the acini and the histology aspect in terms of the parenchyma organization, cell morphology of acini and duct system, the presence of polymeric residues, and inflammatory response were determined at 14 days post-injection. The administration of the compound in a single dose modified some of the morphometric parameters of parenchyma (intercalar duct lumen and thickness of the glandular acini) but did not induce tissue inflammatory response, despite the visible presence of polymer waste. This suggests that PLGA microparticles are biocompatible with the parotid tissue, making it possible to use intraglandular controlled drug administration.