Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa

The objective was to test the hypothesis that the optimal cryoprotective agent for cryopreservation of human spermatozoa would be a solute for which cells have the highest plasma membrane permeability, resulting in the least amount of volume excursion during its addition and removal. To test this hypothesis, theoretical simulations were performed using membrane permeability coefficients to predict optimal procedures for the addition and removal of a cryoprotectant. Simulations were performed using data from four different cryoprotectants: (i) glycerol, (ii) dimethyl sulphoxide, (iii) propylene glycol and (iv) ethylene glycol. Thermodynamic formulations were applied to determine approaches for the addition and removal of 1 M and 2 M final concentrations of cryoprotectant, allowing the spermatozoa to maintain a cell volume within their osmotic tolerance limits. Based on these data, ethylene glycol was predicted to be optimal for minimizing volume excursions among the solutes evaluated. These predictions were then experimentally tested using glycerol as the control cryoprotectant and ethylene glycol as the experimental cryoprotectant. The results indicate that there was a higher (P < 0.05) recovery of motile spermatozoa after cryopreservation when using 1 M ethylene glycol than with 1 M glycerol, supporting the hypothesis that use of the cryoprotectant for which the cell has the highest permeability will result in higher cell survival.      

Comparison of isolation of Haemophilus vaginalis (Corynebacterium vaginale) from peptone-starch-dextrose agar and Columbia colistin-nalidoxic acid agar.

A total of 447 cervical or vaginal specimens were inoculated in parallel onto peptone-starch-dextrose (PSD) and Columbia colistin (10 mg/ml)-nalidixic acid (15 mug/ml) (CNA) agar and were incubated for 48 h at 35 degrees C in an atmosphere with 2 to 10% CO2. One hundred (22.4%) of the cultures were positive for Haemophilus vaginalis. Forty-eight of the isolates were recovered from both PSD and Columbia CNA agar, five from PSD only, and 47 from Columbia CNA agar only (P less than 0.001). On Columbia CNA agar, 76 of the isolates were detected after 24 h of incubation, and the remainder were detected within 4 days of incubation.     

Identifying secondary active solute transport in epithelia

This Editorial Review analyzes the adequacy of the strategies that are commonly used to establish whether the active transport of a solute does or does not occur by secondary active transport. First, the energetics of noncoupled (passive) diffusion, primary active transport, secondary active transport (coupled diffusion), and solvent drag are briefly described. Second, the kinetics of noncoupled and coupled diffusion processes are discussed. Third, the special difficulties of identifying secondary active solute transport in epithelia are outline. Several factors are described that may obscure or mimic true coupled diffusion. Last, the use of transport inhibitors for identifying coupled diffusion processes is briefly illustrated. The adequacy of the strategies that have been employed to evaluate the role of luminal membrane Na+-H+ exchange in mediating proximal tubular acidification is particularly emphasized. It is concluded that many of the findings apparently supporting a major contribution of Na+-independent, primary active H+ secretion to the proximal tubular acidification process are not necessarily inconsistent with secondary active H+ secretion via Na+-H+ exchange.     

Pathological and virological features of arenavirus disease in guinea pigs. Comparison of two Pichinde virus strains.

A guinea pig passage-adapted strain of the arena-virus Pichinde (adPIC) is highly virulent in inbred guinea pigs, whereas the related strain PIC3739 is attenuated. Both viruses were macrophage tropic and infected peritoneal, splenic, liver, and alveolar macrophages during experimental Pichinde virus infection. Infection with the virulent strain was associated with unlimited viral replication in the face of exaggerated delayed-type hypersensitivity response, manifested by the macrophage disappearance reaction. Histopathological lesions unique to adPIC-infected guinea pigs included intestinal villus blunting with mucosal infiltration by pyknotic debris-laden macrophages and apoptosis of crypt epithelial cells. Splenic red pulp necrosis was also significantly associated with adPIC infection but not PIC3739 infection. These findings may provide clues to the pathogenesis of a group of poorly understood human viral hemorrhagic fevers.     

C1-esterase inhibitor blocks T lymphocyte proliferation and cytotoxic T lymphocyte generation in vitro

We have previously shown that activated C1s complement and activated T cells cleave beta2-microglobulin (beta2m) in vitro leading to the formation of desLys58 beta2m. This process can specifically be inhibited by C1-esterase inhibitor (C1-inh). Furthermore we showed that exogenously added desLys58 beta2m in nanomolar amounts to a one-way allogenic mixed lymphocyte culture (MLC) increased the endogenous production of IL-2 and the generation of allo-specific cytotoxic T lymphocytes. C1-inh was purified from fresh human plasma and added to human or murine MLC and mitogen-stimulated lymphocyte cultures grown in the presence of complement-inactivated serum. Read-outs were cell proliferation, lymphokine production and development of T cell-mediated cytotoxicity. We found that addition of C1-inh to MLC and mitogen- exposed murine and human lymphocyte cultures inhibited proliferation, the development of allospecific cytotoxic activity, and changed the endogenous production of IL-2, IL-4, IL-10, IL-12 and IFN-gamma. These data clearly demonstrate a regulatory function of C1-inh on T cell- mediated immune functions.