Non-invasive measurement of adrenocortical and gonadal activity in male and female guinea pigs (Cavia aperea f. porcellus)

Taking blood samples is a common method in biomedical and biological research using guinea pigs. However, most blood sampling techniques are complicated and highly invasive and may therefore not be appropriate for certain research topics concerning stress and reproduction. Thus, a non-invasive method to measure steroid hormones is critically needed. The aim of this study was the biological validation of corresponding enzyme immunoassays for the measurement of fecal cortisol, progesterone, estrogen, and testosterone metabolites in guinea pigs. We examined the effect of subcutaneous injections of ACTH or saline on fecal cortisol metabolites to investigate the suitability of fecal samples to monitor adrenocortical activity. Furthermore, we investigated whether fecal sex steroid metabolites accurately reflected endocrine changes observed in plasma samples during female estrous cycles and male puberty, respectively. In addition, we compared fecal testosterone metabolites of intact males, castrated males, and females to investigate the reliability of fecal samples in discriminating gonadal status of males. Concentrations of fecal cortisol metabolites were significantly increased following ACTH challenge, indicating that adrenocortical activity can be monitored via fecal samples. Secondly, in females, plasma and fecal gonadal steroids were significantly correlated in most subjects. The assay for testosterone metabolites, on the other hand, could not clearly discriminate between test groups. From these findings we conclude that fecal samples can be used for the non-invasive assessment of adrenocortical and female reproductive status in guinea pigs. Testosterone metabolism seems to be more complex and further investigations are needed to establish a more suitable assay.     

Analysis of the PTPN11 gene in idiopathic short stature children and Noonan syndrome patients

Background  Mutations in the PTPN11 gene are the main cause of Noonan syndrome (NS). The presence of some NS features is a frequent finding in children with idiopathic short stature (ISS). These children can represent the milder end of the NS clinical spectrum and PTPN11 is a good candidate for involvement in the pathogenesis of ISS.
Objective  To evaluate the presence of mutations in PTPN11 in ISS children who presented NS-related signs and in well-characterized NS patients.
Patients and methods  We studied 50 ISS children who presented at least two NS-associated signs but did not fulfil the criteria for NS diagnosis. Forty-nine NS patients diagnosed by the criteria of van der Burgt et al . ³ were used to assess the adequacy of these criteria to select patients for PTPN11 mutation screening. The coding region of PTPN11 was amplified by polymerase chain reaction (PCR), followed by direct sequencing.
Results  No mutations or polymorphisms were found in the coding region of the PTPN11 gene in ISS children. Nineteen of the 49 NS patients (39%) presented mutations in PTPN11 . No single characteristic enabled us to distinguish between NS patients with or without PTPN11 mutations.
Conclusion  Considering that no mutations were found in the present cohort with NS-related signs, it is unlikely that mutations would be found in unselected ISS children. The van der Burgt et al . criteria are adequate in attaining NS diagnosis and selecting patients for molecular studies. Mutations in the PTPN11 gene are commonly involved in the pathogenesis of NS but are not a common cause of ISS.     

Essential role for Galpha13 in endothelial cells during embryonic development

Toward identifying the roles of protease-activated receptor-1 (PAR1) and other G protein-coupled receptors important for vascular development, we investigated the role of Galpha13 in endothelial cells in the mouse embryo. LacZ inserted into Galpha13 exon 1 was highly expressed in endothelial cells at midgestation. Endothelial-specific Galpha13 knockout embryos died at embryonic days 9.5-11.5 and resembled the PAR1 knockout. Restoration of Galpha13 expression in endothelial cells by use of a Tie2 promoter-driven Galpha13 transgene rescued development of endothelial-specific Galpha13 knockout embryos as well the embryonic day 9.5 vascular phenotype in Galpha13 conventional knockouts; transgene-positive Galpha13-/- embryos developed for several days beyond their transgene-negative Galpha13-/- littermates and then manifested a previously uncharacterized phenotype that included intracranial bleeding and exencephaly. Taken together, our results suggest a critical role for Galpha13 in endothelial cells during vascular development, place Galpha13 as a candidate mediator of PAR1 signaling in this process, and reveal roles for Galpha13 in other cell types in the mammalian embryo.     

Endurance exercise training in orthostatic intolerance: a randomized, controlled trial

Orthostatic intolerance is a syndrome characterized by chronic orthostatic symptoms of light-headedness, fatigue, nausea, orthostatic tachycardia, and aggravated norepinephrine levels while standing. The aim of this study was to assess the protective effect of exercise endurance training on orthostatic symptoms and to examine its usefulness in the treatment of orthostatic intolerance. 2768 military recruits were screened for orthostatic intolerance by questionnaire. Tilt-table testing identified 36 cases of orthostatic intolerance out of the 2768 soldiers. Subsequently, 31 of these subjects with orthostatic intolerance entered a randomized, controlled trial. The patients were allocated randomly to either a "training" (3 months jogging) or a "control" group. The influence of exercise training on orthostatic intolerance was assessed by determination of questionnaire scores and tilt-table testing before and after intervention. After training, only 6 individuals of 16 still had orthostatic intolerance compared with 10 of 11 in the control group. The Fisher exact test showed a highly significant difference in diagnosis between the 2 groups (P=0.008) at the end of the study. Analysis of the questionnaire-score showed significant interaction between time and group (P=0.001). The trained subjects showed an improvement in the average symptom score from 1.79+/-0.4 to 1.04+/-0.4, whereas the control subjects showed no significant change in average symptom score (2.09+/-0.6 and 2.14+/-0.5, respectively). Our data demonstrate that endurance exercise training leads to an improvement of symptoms in the majority of patients with orthostatic intolerance. Therefore, we suggest that endurance training should be considered in the treatment of orthostatic intolerance patients.     

Arteriogenesis proceeds via ICAM-1/Mac-1- mediated mechanisms

Monocyte adhesion to shear stress-activated endothelium stands as an important initial step during arteriogenesis (collateral artery growth). Using multiple approaches, we tested the hypothesis that monocyte adhesion via intercellular adhesion molecule-1 (ICAM-1) and selectin interactions is essential for adaptive arteriogenesis. Forty-eight New Zealand White rabbits received either solvent, monocyte chemoattractant protein-1 (MCP-1) alone, MCP-1 plus ICAM-mab, or MCP-1 plus an IgG2a isotype control via osmotic minipumps. After 7 days, collateral conductance was evaluated: solvent 4.01 (mL/min per 100 mm Hg), MCP-1 plus ICAM-mab 8.04 (versus solvent P=NS), and MCP-1 alone 33.11 (versus solvent P<0.05). Furthermore, the right femoral arteries of ICAM-1-/-, Mac-1-/- and mice having defective selectin interactions (FT4/7-/-) as well as their corresponding controls were ligated. One week later, perfusion ratios were determined by the use of fluorescent microspheres. FT4/7-/- mice did not show any significant difference in perfusion restoration whereas ICAM-1-/- and Mac-1-/- mice had a significant reduction in arteriogenesis as compared with matching controls (FT4/7-WT 37+/-9%, FT4/7-/- 32+/-3%, P=0.31; C57BL/6J 59+/-9%, ICAM-1-/- 36+/-8%, P<0.05; Mac-1-/- 42+/-3%, P<0.05). ICAM-1/Mac-1-mediated monocyte adhesion to the endothelium of collateral arteries is an essential step for arteriogenesis, whereas this process can proceed via selectin interaction independent mechanisms. Furthermore, in vivo treatment with monoclonal antibodies against ICAM-1 totally abolishes the stimulatory effect of MCP-1 on collateral artery growth, suggesting that the mechanism of the MCP-1-induced arteriogenesis proceeds via the localization of monocytes rather than the action of the MCP-1 molecule itself.     

Effects of perfluorocarbon emulsions on microvascular blood flow and oxygen transport in a model of severe arterial gas embolism

Background

Arterial gas embolism (AGE) is a clinical problem that occurs directly in cardiopulmonary bypass machines in open-heart surgeries, or indirectly (through cardiac or pulmonary right to left shunts) in dive accidents, resulting in serious morbidity and even death. Perfluorocarbon (PFC) emulsions have been used for the treatment of AGE in an animal model. We hypothesized that PFC emulsions enhance microvascular blood flow, speed bubble resolution, and oxygenation in AGE compared with saline in a model of cremaster muscle from anesthetized rats.

Materials and methods

AGE was induced by direct air injection into the femoral artery ipsilateral to the studied cremaster muscle. Microhemodynamics, microvascular, and tissue oxygenation were determined before and after treatment with two different commercial PFC emulsions (C₁₀F₂₀, Oxycyte; Oxygen Biotherapeutics, Inc and C₁₀F₁₈, PHER-O₂; Sanguine Corporation, Inc) compared with saline in real time using brightfield and phosphorescence microscopy.

Results

Blood pressure and heart rate remained unchanged. Systemic PO₂, oxygen (O₂) content, and glucose were higher in PFC groups, whereas hematocrit dropped in all groups. Arteriolar blood flow went up 85% and 80% of baseline after C₁₀F₂₀ and C₁₀F₁₈ treatments, respectively, versus 11% after saline treatment. Arteriolar and tissue PO₂, and O₂ delivery were higher in PFC groups compared with the control group. There was an increase in arteriolar blood flow, reduction in diffusional resistance of O₂ in the plasma, and improved tissue oxygenation.

Conclusions

Administration of PFC emulsions in AGE is superior to saline primarily because of surfactant properties along with air bubble reabsorption.     

Direct-acting antiviral agents for hepatitis C virus-mixed cryoglobulinaemia: dissociated virological and haematological responses

The hepatitis C virus-positive (HCV+) mixed cryoglobulinaemia (MC) is associated with haematological alterations such as monoclonal B-cell lymphocytosis or non-Hodgkin lymphomas (NHLs). Antiviral therapy for MC, based on interferon and ribavirin, has been shown to be able to eliminate the viral replication as well as the B-cell monoclonal alterations. Many studies have reported the efficacy of direct-acting antivirals (DAAs) in the treatment of HCV+ MC. However, some authors noticed the persistence of haematological diseases despite HCV eradication. To verify the effects of DAAs on B-cell proliferation, we evaluated 67 patients with HCV+ MC. Six patients had an overt NHL and 30% had monoclonal B-lymphocytosis. In 20% of the patients, the mutation L265P of the myeloid differentiation factor 88 (MYD88) gene was detected in peripheral blood. All patients had negative HCV viraemia at week 12; one had a breakthrough, while two cases relapsed. A complete clinical response of vasculitis was seen in 60% of the patients. Among the six patients with NHL, one showed a complete response, whereas in the others there were no changes in the number and size of the nodes. Among the patients carrying a clonal population in peripheral blood, only 22% became negative. These data indicate that DAAs are not able to eliminate the clonal alterations induced by HCV in a large proportion of cases.