Immunohistochemical localization of VEGF and its receptors in the corpus luteum of the bitch during diestrus and anestrus

The corpus luteum (CL) is a temporary endocrine gland, whose life span depends on the interaction of luteotrophic and luteolytic factors. Since development and maintenance of CL is based on angiogenesis, angiogenic growth factors may play a role in CL-function of the bitch, as described for other species. The aim of this study was to detect the presence of the vascular endothelial growth factor (VEGF) system in the bitch CL throughout diestrus and early anestrus. For that purpose, blood samples from 24 bitches were collected and analyzed for progesterone to determine ovulation time and the animals were subjected to ovariosalpingohysterectomy 10, 20, 30, 40, 50, 60 or 70 days after ovulation. The corpora lutea were fixed in formalin and embedded in Paraplast resin. Five micrometers sections were submitted to standard immunohistochemistry protocol using three primary antibodies (SC-315, SC-316 and VG76e) for detection of kinase domain region (KDR), fms-like tyrosine kinase 1 (Flt-1) and VEGF, respectively. The VEGF system expression could be detected in all diestrus stages in endothelial as well as luteal cells (responsible for blood vessel formation and progesterone production, respectively), indicating time dependent changes: immunostaining tended to increase from Day 10 to 50 and to decrease until Day 70 post-ovulation. In the CL of the bitch, structure related cells, like pericytes and stroma cells, expressed it in determined time points of diestrus with little intensity variation. We concluded that VEGF might have a modulatory effect in the CL of the dog acting as paracrine and autocrine factor through its receptors, Flt-1 and KDR.     

The control of hyperhomocysteinemia through thiol exchange mechanisms by mesna

In hyperhomocysteinemic patients, after reaction with homocysteine-albumin mixed disulfides (HSS-ALB), mesna (MSH) forms the mixed disulfide with Hcy (HSSM) which can be removed by renal clearance, thus reducing the plasma concentration of total homocysteine (tHcy). In order to assess the HSS-ALB dethiolation via thiol exchange reactions, the distribution of redox species of cysteine, cysteinylglycine, homocysteine and glutathione was investigated in the plasma of healthy subjects: (i) in vitro, after addition of 35 μM reduced homocysteine (HSH) to plasma for 72 h, followed by MSH addition (at the concentration range 10–600 μM) for 25 min; (ii) in vivo, after oral treatment with methionine (methionine, 200 mg/kg body weight, observation time 2–6 h). In both experiments the distribution of redox species, but not the total amount of each thiol, was modified by thiol exchange reactions of albumin and cystine, with changes thermodynamically related to the pKa values of thiols in the corresponding mixed disulfides. MSH provoked a dose–response reversal of the redox state of aged plasma, and the thiol action was confirmed by in vivo experiments. Since it was observed that the dimesna production could be detrimental for the in vivo optimization of HSSM formation, we assume that the best plasma tHcy lowering can be obtained at MSH doses producing the minimum dimesna concentration in each individual.     

Oxidative stress, nitric oxide, endothelial dysfunction and tinnitus

To assess whether pathogenic endothelial dysfunction is involved in acute idiopathic tinnitus we enrolled 44 patients and 25 healthy volunteers. In blood from the internal jugular vein and brachial vein we determined malonaldehyde, 4-hydroxynonenal, myeloperoxidase, glutathione peroxidase, nitric oxide, L-arginine and L-ornitine, thrombomodulin (TM) and von Willebrand factor (vWF) activity during tinnitus and asymptomatic period. Higher plasma concentrations of oxidative markers and L-arginine, and lower nitric oxide and L-ornitine levels were observed in jugular blood of patients with tinnitus, there being a significant difference between brachial and jugular veins. TM and vWF activity were significantly higher in patients' jugular blood than in brachial blood. Our results suggest oxidant, TM, vWF activity production are increased and nitric oxide production reduced in brain circulation reflux blood of patients with acute tinnitus. These conditions are able to cause a general cerebro-vascular endothelial dysfunction, which in turn induce a dysfunction of microcirculation in the inner ear.     

Sulfolobus solfataricus thiol redox puzzle: characterization of an atypical protein disulfide oxidoreductase

Protein disulfide oxidoreductases (PDOs) are proteins involved in disulfide bond formation playing a crucial role in adaptation to extreme environment. This paper reports the functional and structural characterization of Sso1120, a PDO from the hyperthermophilic archaeon Sulfolobus solfataricus. The protein was expressed in Escherichia coli and purified to homogeneity. The functional characterization showed that the enzyme has reductase activity, as tested by insulin assay, but differently from the other PDOs, it does not present isomerase activity. In addition it is able to form a redox couple with the thioredoxin reductase that could be used in undiscovered pathways. The protein revealed a melting point of around 90 °C in CD spectroscopy-monitored thermal denaturation and high denaturant resistance. The X-ray crystallographic structure was solved at 1.80 Å resolution, showing differences with respect to other PDOs and an unexpected similarity with the N-terminal domain of the alkyl hydroperoxide reductase F component from Salmonella typhimurium. On the basis of the reported data and of bioinformatics and phylogenetic analyses, a possible involvement of this atypical PDO in a new antioxidant system of S. solfataricus has been proposed.     

Expression of adhesion molecule T-cadherin is increased during neointima formation in experimental restenosis

Phenotypic modulation, migration and proliferation of vascular smooth muscle cells (SMCs) are major events in restenosis after percutaneous transluminal angioplasty. Surface cell adhesion molecules, essential to morphogenesis and maintenance of adult tissue architecture, are likely to be involved, but little is known about cell adhesion molecules expressed on SMCs. T-cadherin is a glycosyl phosphatidylinositol-anchored member of the cadherin superfamily of adhesion molecules. Although highly expressed in vascular and cardiac tissues, its function in these tissues is unknown. We previously reported increased expression of T-cadherin in intimal SMCs in atherosclerotic lesions and proposed a role for T-cadherin in phenotype control. Here we performed immunohistochemical analysis of spatial and temporal changes in vascular T-cadherin expression following balloon catheterisation of the rat carotid artery. T-cadherin expression in SMCs markedly increases in the media early (1-4 days) after injury, and later (day 7-28) in forming neointima, especially in its preluminal area. Staining for monocyte/macrophage antigen ED-1, proliferating cell nuclear antigen and smooth muscle alpha-actin revealed that spatial and temporal changes in T-cadherin level coincided with the peak in cell migration and proliferation activity during neointima formation. In colchicine-treated cultures of rat aortic SMCs T-cadherin expression is increased in dividing M-phase cells but decreased in non-dividing cells. Together the data support an association between T-cadherin expression and SMC phenotype.     

Characterization of a multifunctional protein disulfide oxidoreductase from Sulfolobus solfataricus

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been ascribed to a new family of protein disulfide oxidoreductases (PDOs). We report on the characterization of SsPDO, isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. SsPDO was cloned and expressed in Escherichia coli. We revealed that SsPDO is the substrate of a thioredoxin reductase in S. solfataricus (K(M) 0.3 microm) and not thioredoxins (TrxA1 and TrxA2). SsPDO/S. solfataricus thioredoxin reductase constitute a new thioredoxin system in aerobic thermophilic archaea. While redox (reductase, oxidative and isomerase) activities of SsPDO point to its central role in the biochemistry of cytoplasmic disulfide bonds, chaperone activities also on an endogenous substrate suggest a potential role in the stabilization of intracellular proteins. Northern and western analysis have been performed in order to analyze the response to the oxidative stress.     

Final antigenic Melan-A peptides produced directly by the proteasomes are preferentially selected for presentation by HLA-A*0201 in melanoma cells

The melanoma-associated protein Melan-A contains the immunodominant CTL epitope Melan-A(26/27-35)/HLA-A*0201 against which a high frequency of T lymphocytes has been detected in many melanoma patients. In this study we show that the in vitro degradation of a polypeptide encompassing Melan-A(26/27-35) by proteasomes produces both the final antigenic peptide and N-terminally extended intermediates. When human melanoma cells expressing the corresponding fragments were exposed to specific CTL, those expressing the minimal antigenic sequence were recognized more efficiently than those expressing the N-terminally extended intermediates. Using a tumor-reactive CTL clone, we confirmed that the recognition of melanoma cells expressing an N-terminally extended intermediate of Melan-A is inefficient. We demonstrated that the inefficient cytosolic trimming of N-terminally extended intermediates could offer a selective advantage for the preferred presentation of Melan-A peptides directly produced by the proteasomes. These results imply that both the proteasomes and postproteasomal peptidases limit the availability of antigenic peptides and that the efficiency of presentation may be affected by conditions that alter the ratio between fully and partially processed proteasomal products.     

Immune Responses to the Cancer Testis Antigen XAGE-1b in Non Small Cell Lung Cancer Caucasian Patients

Immunotherapy approaches using checkpoint blockade, alone, or in combination with tumor antigen vaccination, or adoptive cell transfer, are emerging as promising approaches for the treatment of non-small cell lung cancer (NSCLC). In preparation for upcoming combined immunotherapy approaches in NSCLC, here, we have assessed spontaneous immune responses to XAGE-1b, a tumor specific antigen of the Cancer Testis Antigen group that has been previously reported to be spontaneously immunogenic in the Japanese population, in a cohort of Caucasian patients with NSCLC. We found spontaneous serological responses to XAGE-1b in 9% of the patients. Importantly, these responses were limited to, and represented 13% of, patients with adenocarcinoma tumors, the most frequent histological subtype, for which immunotherapy approaches are under development. Using a set of overlapping peptides spanning the entire XAGE-1b protein, and in support of the serological data, we detected significant XAGE-1b specific CD4⁺ T cell responses in all XAGE-1b seropositive patients and identified several CD4⁺ T cell epitopes. Altogether, our results support the relevance of the XAGE-1b antigen in Caucasians NSCLC patients with adenocarcinoma, and the implementation of future immunotherapies exploiting the high immunogenicity of the antigen in this patient population.