Differential effects of the immunosuppressive calcineurin inhibitors cyclosporine-A and tacrolimus on ovulation in a murine model.

STUDY QUESTION: Do therapeutic levels of cyclosporine-A and tacrolimus affect ovulation in a rat gonadotrophin-induced ovulation model? SUMMARY ANSWER: Cyclosporine-A, but not tacrolimus, decreases ovulation rate when administered for 5 days before induced ovulation. WHAT IS KNOWN ALREADY: The mainstays of immunosuppression in solid organ transplantation, to prevent rejection, are the calcineurin inhibitors cyclosporine-A or tacrolimus. These drugs could potentially affect fertility in transplanted patients. Since ovulation is an inflammation-like process with pivotal roles for several immune cells and modulators, it is possible that the calcineurin inhibitors, with broad effects on the immune system, could interfere with this sensitive, biological process. STUDY DESIGN SIZE DURATION: Experimental design at university-based animal facilities. A total of 45 immature Sprague-Dawley rats were used. The study was carried out over 3 months. PARTICIPANTS/MATERIALS SETTING METHODS: Immature Sprague-Dawley rats (n = 45) were randomly assigned to receive equivalent doses of tacrolimus (0.5 mg/kg/day; TAC), cyclosporine-A (10 mg/kg/day; CyA) or vehicle (Control). Ovarian hyperstimulation was induced with 10 IU of equine chorionic gonadotrophin, and ovulation was triggered with 10 IU of hCG. Oocytes were retrieved from the oviducts and ovulation rates were calculated. Various subpopulations of white blood cells were counted in peripheral blood and ovarian tissue samples. MAIN RESULTS AND THE ROLE OF CHANCE: Animals in the CyA group showed a lower ovulation rate when compared to the TAC and Control groups (CyA: mean 9 oocytes (range 0-22); TAC: 21 oocytes (8-41); Control: 22 oocytes (6-39); P = 0.03). Regarding counts of the white blood cell subpopulations and resident neutrophils in the ovary, no significant differences were observed between the groups. LIMITATIONS REASONS FOR CAUTION: Although the ovulation process is highly conserved within species, the differences between rodents and humans may limit the external translatability of the study. WIDER IMPLICATIONS OF THE FINDINGS: These findings suggest that tacrolimus should be the preferred calcineurin inhibitor of choice in transplanted patients who are aiming for pregnancy. STUDY FUNDING/COMPETING INTERESTS: Swedish Research Council and ALF of Sahlgrenska Academy, Sweden. Rio Hortega Grant from the Instituto de Salud Carlos III, Spain (CM09/00063). There are no conflicts of interest.

Mapping cyclosporine-induced changes in protein secretion by renal cells using stable isotope labeling with amino acids in cell culture (SILAC).

Nephrotoxicity is an adverse event that strongly limits the use of the immunosuppressant cyclosporine in solid organ transplantation and the precise molecular mechanisms underlying this toxicity remain unclear. MS-based proteomic analysis of the secretome of HEK-293 renal cells exposed to cyclosporine was performed to identify changes in protein secretion, as a first step to discover potential biomarkers of such nephrotoxicity. To detect and quantify the perturbed proteins in the culture medium we used SILAC and nano-scale liquid chromatography followed by MALDI-TOF/TOF mass spectrometry. Among 106 proteins identified, 80 were quantified in both forward/reverse SILAC experiments and quantitative proteomic analysis revealed altered levels of expression for 24 secreted proteins. These included the down-regulation of a number of extracellular matrix/cell adhesion components, and the up-regulation of secreted cyclophilins A and B, macrophage inhibition factor and phosphatidylethanolamine-binding protein 1. These changes in protein secretion were not prevented by co-incubation with the antioxidant N-acetylcysteine, suggesting that they were not triggered by cyclosporine-induced oxidative stress. The results from the present study provide important new knowledge to gain insights into the molecular mechanisms of cyclosporine-related toxicity. Some of the proteins identified here should be tested as potential biomarkers of cyclosporine nephrotoxicity in subsequent clinical studies.

Quantitative proteomic analysis of cyclosporine-induced toxicity in a human kidney cell line and comparison with tacrolimus.

The calcineurin-inhibitors (CNIs) cyclosporine (CsA) and tacrolimus (TAC) remain the pillars of modern immunosuppression regimens used in solid organ transplantation. Nephrotoxicity is an adverse effect that limits their successful use. The precise molecular mechanisms underlying this nephrotoxicity remain unclear. Using SILAC together with LC-MALDI-TOF/TOF, we investigated the CNIs-induced proteomic perturbations in renal cells. Among the 495 proteins quantifiable in both forward and reverse SILAC, 69 displayed CsA-induced perturbations: proteins involved in ER-stress/protein folding, apoptosis, metabolism/transport or cytoskeleton pathways were up-regulated, while cyclophilin B as well as nuclear and RNA-processing proteins were down-regulated. Co-administration of CsA with the antioxidant N-acetylcysteine significantly decreased lipid peroxidation and also partially corrected the CsA-induced unfolded protein response. TAC toxicity profile was apparently different from that of CsA, especially without perturbation of cyclophilins A and B, up-regulation of ER-chaperones nor down-regulation of a number of nuclear proteins. These results provide a new insight and are consistent with recent data regarding the molecular mechanisms of CNIs-induced nephrotoxicity. Our findings offer new directions for future research aiming to identify specific biomarkers of CsA nephrotoxicity.

Safety and immunogenicity of a combined hepatitis B virus-Haemophilus influenzae type B vaccine comprising a synthetic antigen in healthy adults.

The combined HB-Hib vaccine candidate Hebervac HB-Hib (CIGB, La Habana), comprising recombinant HBsAg and tetanus toxoid conjugate synthetic PRP antigens has shown to be highly immunogenic in animal models. A phase I open, controlled, randomized clinical trial was carried out to assess the safety and immunogenicity profile of this bivalent vaccine in 25 healthy adults who were positive for antibody to HBsAg (anti-HBs). The trial was performed according to Good Clinical Practices and Guidelines. Volunteers were randomly allocated to receive the combined vaccine or simultaneous administration of HB vaccine Heberbiovac-HB and Hib vaccine QuimiHib (CIGB, La Habana). All individuals were intramuscularly immunized with a unique dose of 10 microg HBsAg plus 10 microg conjugated synthetic PRP. Adverse events were actively recorded after vaccine administration. Total anti-HBs and IgG anti-PRP antibody titers were evaluated using commercial ELISA kits at baseline and 30 days post-vaccination. The combined vaccine candidate was safe and well tolerated. The most common adverse reactions were local pain, febricula, fever and local erythema. These reactions were all mild in intensity and resolved without medical treatment. Adverse events were mostly reported during the first 6-72 hours post-vaccination. There were no serious adverse events during the study. No severe or unexpected events were either recorded during the trial. The combined vaccine elicited an anti-HBs and anti-PRP booster response in 100% of subjects at day 30 of the immunization schedule. Anti-HBs and anti-PRP antibody levels had at least a two-fold increase compared to baseline sera. Even more, anti-HBs antibody titer showed a four-fold increase in 100% of volunteers in the study group. The results indicate that the combined HB-Hib vaccine produces increased antibody levels in healthy adults who have previously been exposed to these two antigens. To our knowledge, this is the first demonstration of safety and immunogenicity for a combined vaccine comprising recombinant HBV and synthetic Hib antigens. The present results support phase I-II clinical trial in the target population, two months old healthy infants.

Endocardial carbon-braid electrodes. A new concept for lower defibrillation thresholds.

BACKGROUND: In the treatment of patients with life-threatening ventricular arrhythmia, transvenous implantable cardioverter/defibrillators provide significant advantages over devices requiring a thoracotomy. This study tested the hypothesis that a new carbon-fiber electrode, designed at the Technische Universität in Munich, Germany, has a lower defibrillation threshold (DFT) than standard transvenous defibrillation electrodes. METHODS AND RESULTS: In 8 mongrel dogs (weight, 25.2 +/- 0.8 kg; heart weight, 192 +/- 19 g), we examined the efficacy and electrical characteristics of a right ventricular endocardial carbon prototype defibrillation electrode (9.5F, 4.4-cm2 surface) compared with a standard CPI 0062 Endotak electrode and a Medtronic 6966 Transvene endocardial right ventricular defibrillation electrode. The new electrode consists of 24 braided, tubular carbon filaments, each containing 1000 highly isotropic carbon fibers of 7-microns diameter, yielding a theoretical electrical surface of 480 cm2. The DFTs were determined in random order between each of the three right ventricular electrodes and a subcutaneous wire array anode placed on the left thorax. A standard step-down/up DFT protocol of 20-V shock steps was applied. Two different biphasic waveforms with a 1-ms delay between phases were tested: 3.2-ms first phase/2.0-ms second phase, and 6.0-ms first phase/6.0-ms second phase. For the 3.2/2.0-ms waveform, we found a significantly lower DFT for the carbon lead (4.96 +/- 1.58 J) compared with the CPI 0062 (6.93 +/- 1.67 J) and the Medtronic 6966 (7.49 +/- 0.99 J) leads. For the 6.0/6.0-ms waveform, the DFT for the carbon electrode (5.97 +/- 2.09 J) was significantly lower than for the Medtronic 6966 lead (8.55 +/- 1.93 J) but not for the CPI 0062 lead (6.30 +/- 1.41 J). The impedance with carbon was lower than with the other two leads for the 6.0/6.0-ms waveform but not for the 3.2/2.0-ms waveform. For the carbon electrode, the 3.2/2.0-ms waveform had a lower DFT than the 6.0/6.0-ms waveform. CONCLUSIONS: The present canine study found a lower DFT for a new carbon electrode compared with DFTs for endocardial defibrillation electrodes made of standard metal. Further long-term animal studies and clinical studies are needed to determine whether carbon materials and braided-lead technology are practical and beneficial in patients.

Improved defibrillation threshold with a new epicardial carbon electrode compared with a standard epicardial titanium patch.

BACKGROUND: Recent studies show that depending on the type of shock morphology used, 5% to 15% of patients requiring implantable defibrillators cannot be treated with a nonthoracotomy system. In these cases, an epicardial patch-based system becomes necessary. In this study, we investigated a newly developed epicardial carbon electrode as an alternative to a standard epicardial titanium patch. METHODS AND RESULTS: A tubular epicardial braided carbon electrode of 7F diameter and 14-cm length applied in a U-shape to the epicardium was compared with a standard left ventricular epicardial 15-cm2 titanium mesh patch (CPI Inc). As cathode, a CPI endocardial lead, a Medtronic lead, or a carbon-platinum-iridium prototype electrode was used. Ventricular fibrillation was induced with a 60-Hz generator and allowed to continue for 10 seconds before a shock was given. Two different biphasic shock waveforms (3.2/2- and 6/6-millisecond) were delivered by the six electrode configurations. Eight dogs (weight, 24.5 +/- 1.3 kg) underwent an up-down defibrillation protocol. The order of testing the epicardial electrodes, the endocardial cathodes, and the waveform was randomized. With the epicardial carbon electrode, the mean defibrillation threshold (DFT) energy decreased 39% to 56% and the voltage decreased 24% to 35% compared with the titanium patch: from 8.3 +/- 2.5 to 4.9 +/- 3.6 J with the CPI lead and the 3.2/2-millisecond waveform, from 6.2 +/- 2.5 to 2.9 +/- 2.1 J with the carbon-platinum-iridium prototype, and from 6.4 +/- 3.4 J to 3.5 +/- 2.6 J with the Medtronic lead (P < or = .05). The DFT determinations with the 6/6-millisecond biphasic waveform showed a similar trend with slightly higher values. CONCLUSIONS: Compared with a titanium patch, the new braided epicardial electrode significantly decreases the defibrillation energy requirements. This effect can be maximized by using an endocardial carbon-platinum-iridium prototype as cathode and a short duration biphasic waveform.