Everolimus, cyclosporine, and thrombotic microangiopathy: clinical role and preventive tools in renal transplantation.

INTRODUCTION: Thrombotic microangiopathy (TMA) is characterized by endothelial cell injury and formation of fibrin thrombi within capillary and arterioles. In renal allograft recipients, TMA mainly presents as hemolytic uremic syndrome. Its occurrence is rare, and diagnosis requires a high degree of suspicion. Drug toxicity, in particular from calcineurin inhibitors (CNIs) and mTOR inhibitors (mTORi), is the most common cause posttransplant and has recently been emphasized in the setting of lung transplantation. OBJECTIVE: The goal of this study was to investigate the role of mTORi as an added risk factor in the development of TMA to propose strategies for modulation of immunosuppressive (IS) therapy. PATIENTS AND METHODS: From a database of 496 renal graft recipients, we analyzed 350 renal graft biopsy specimens gathered at our center from 1998 to 2012. In patients undergoing combined therapy with mTORi and CNI, we compared drugs levels in TMA-affected and TMA-free groups, using mTORi and CNI TLC and the summation of [everolimus TLC+(cyclosporine C2/100)] (Σ) as a surrogate marker of combined exposition to 2 drugs. Receiver-operating characteristic analysis of association of EVL TLC+(C2/100) was performed for patients exposed to mTORi. RESULTS: Histologic features of TMA were found in 36 patients (prevalence of 7.3%). The caseload was divided into 2 groups: not drug-related TMA (n=19) and drug-related TMA (n=17). Despite the prevalence of TMA in patients exposed to mTORi being greater (8 of 153; prevalence, 5.3%) compared with therapies without mTORi (9 of 324; prevalence, 2.8%), statistical difference was not reached. Patients treated with mTORi who developed de novo drug-related TMA had higher blood levels of IS drugs compared with those who did not develop TMA. Receiver-operating characteristic analysis found a significant threshold of 12.5 ng/mL (area under the curve, 0.803; P=.006). CONCLUSIONS: Results confirm the pivotal role of IS drugs in the onset of de novo TMA. On the basis of literature, we could speculate a sequence of endothelial damage by CNI, on which everolimus fits hindering the repair of endothelial injury. Therefore, high blood levels of CNI and mTORi seem to predispose patients to posttransplant TMA. Combined monitoring of these 2 drugs might be used to prevent the complication. Σ [everolimus TLC + (cyclosporine C2/100)]>12.5 ng/mL should be avoided as a surrogate risk factor for adverse effects.

Identification of a sperm penetration factor in the oviduct of the golden hamster.

Previously, we found oviductal eggs to be significantly more penetrable and fertilizable in vitro than ovulated eggs collected from the ovarian bursa, while bursal eggs were comparable to mature (unovulated) follicular eggs. Incubation of follicular eggs with a soluble eluate of oviductal egg cumulus complexes (COF) increased sperm penetration: the activity was macromolecular, was destroyed at 56 degrees C, and was produced in the oviduct. We now report purification of this oviductal factor that enhances penetration of follicular eggs and have identified it as oviductin (OVN). Oviducts, 1-1.5 h post-LH from eCG-primed females, were homogenized and the cytosolic fraction was chromatographed on a Helix pomatia lectin affinity column; specific proteins were eluted with 0.2 M N-acetyl-D-galactosamine. Fractions were monitored by dot-blot assay using as the primary antibody monoclonal antibody (mAb) 1C4 against OVN. Proteins were resolved by one-dimensional SDS-gel electrophoresis, followed by electrotransfer and immunostaining of Western blots. OVN fractions were indexed to COF by quantitative dot-blot assay, and activity was bioassayed by penetration of follicular eggs within 1 h of coincubation with precapacitated sperm +/- factors: COF and BSA (high and low controls, respectively) and fractions from the lectin-isolated peak. The mean penetration rates for three isolations were 17 +/- 4.0a, 51.7 +/- 5.0b, and 49 +/- 2.7b% for BSA, COF, and column fractions, respectively (p < or = 0.05). Purified OVN bound to follicular zonae during culture. Acrosome-intact sperm heads bound OVN during 30 min of incubation both before (t = 0 h) and after capacitation (t = 5.5 h) (visualized by indirect immunofluorescence).(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of spermatozoa and zona pellucida properties by the soluble acrosome reaction-inducing factor of the ovulated egg-cumulus complex.

Three sources of hamster periovulatory fluids (+/- heat inactivation at 56 degrees C), with bovine serum albumin (BSA) as control, were tested for effects on penetration of three classes of eggs by hamster sperm precapacitated in BSA. These fluids were a soluble extract of cumulus oophorus fluid (COF) from the ovulated hamster egg-cumulus complex, serum, and follicular fluid. Egg types were ovulated, salt-stored (ovulated), and follicular. In both COF and serum, there were significant differences among egg types in mean penetration, and significant effects of fluid addition. In contrast, there was no effect of follicular fluid and no differences between follicular and stored eggs. For the follicular eggs (combined data, normalized, ranked), patterns of response to the three factors (+/- heating) were different: only unheated COF and heated serum increased penetration significantly above BSA control levels (average rank 20.2, 41.4, 38, for BSA, COF (unheated), serum (heated), respectively). This indicated that the active component in COF was heat labile, not present in either serum or follicular fluid, and, therefore, of oviductal origin. Oviduct and/or COF exposure of eggs and sperm was tested for effects as an acrosome reaction inducing factor (ARIF) for acrosome reactions (AR; zona-bound and free-swimming sperm) and on sperm:zona binding and penetration. The COF ARIF for free-swimming sperm AR was heat stable. Penetration of follicular eggs increased after incubation in COF prior to sperm addition, but a greater response occurred when COF was added to eggs with sperm. In kinetic experiments, 25 min following sperm attachment, follicular eggs had lost 41% of initially bound sperm, vs. 23% for ovulated eggs, and had only 16 AR sperm/egg, vs. 26 for ovulated. Follicular eggs incubated in COF (then washed three times) had the same number of bound AR sperm as ovulated eggs. Acid solubilized zona pellucida (ASZP) from ovulated eggs was more effective as an ARIF per zona than ASZP from follicular eggs. Zonae of follicular eggs, as evidenced by dissolution times in beta-mercaptoethanol (beta-MEOH), were not "harder" than those of ovulated eggs. There were differences in lectin binding antigens on zonae of both fresh and stored, follicular and ovulated, eggs. We conclude that multiple biological factors orchestrate sperm:egg interactions in the ampulla. Our data are consistent with the presence of at least three effective components: 1) the oviductal lectin-binding antigen (ZPO or oviductin), 2) an additional heat-labile component, and 3) the heat-stable ARIF for free-swimming sperm.

SAG: a Schwann cell membrane glycoprotein.

We report on characterization of a 170,000 Da glycoprotein found exclusively in the PNS. We refer to this protein as the Schwann cell membrane glycoprotein (SAG). SAG contains the HNK-1 carbohydrate, which is considered by some to be a marker of adhesion molecules. Its N-terminal sequence is not similar to previously known polypeptide sequences. SAG is found exclusively in the PNS, is present in rat sciatic nerve prior to myelination, and is in both myelinating and nonmyelinating Schwann cells. Tumors of Schwann cell lineage express SAG where axons are present (neurofibromas) but do not in the absence of axons (schwannomas). Schwannoma cells in culture do not express SAG even when exposed to forskolin, an activator of adenylate cyclase. However, schwannoma cells grown in the presence of a neuronal cell line (PC12) express SAG.