HLA class I antigen expression as a measure of response to antiviral therapy of chronic hepatitis B.

HLA class I antigen expression on peripheral blood mononuclear cells was evaluated by flow cytometry in 21 HBeAg-positive patients with chronic hepatitis B. Measurements were made before, during or after treatment with recombinant interferon-alpha-2b, either given alone or after a 6 wk course of prednisone. Immunohistochemical staining for human leukocyte class I antigen was also evaluated in 28 percutaneous liver biopsy specimens either obtained before or after therapy (N = 27) and during therapy in one instance. The amount of HLA class I antigen on peripheral blood mononuclear cells varied markedly among individual patients, but the overall results indicated that the level of inducible antigen did not correlate with increments of ALT during therapy or with a virological response to therapy. Hepatocyte staining for HLA class I antigen was observed in a minority of biopsy specimens (29%) and also did not appear to predict a response or correlate with the severity of histological disease. These data do not support current theories concerning pathogenetic mechanisms in chronic hepatitis B nor do they suggest that spontaneous display of HLA class I antigen on hepatocytes or interferon-induced expression of these antigens on peripheral blood mononuclear cells is a critical determinant for a response to therapy.

Cryopreservation affects endothelial and smooth muscle function of canine autogenous saphenous vein grafts.

Cryopreserved veins used as arterial grafts may be affected by both rejection and the cryopreservation process. Experiments were designed to study changes in endothelial and smooth muscle function after cryopreservation but independent of rejection. One saphenous vein from each of eight dogs was cryopreserved for subsequent use as autografts. After 3 weeks one cryopreserved and one freshly harvested autogenous saphenous vein were implanted as bilateral femoral arterial interposition grafts. Platelet deposition was studied in vivo with indium 111-labeled platelets. At 4 weeks the autografts were removed, and the functional characteristics of the grafts were studied in organ chambers; and the ability of nerve terminals to uptake transmitter was studied with 3H-norepinephrine. Neither patency rates, blood flows, nor platelet deposition were significantly different between freshly harvested and cryopreserved grafts. Uptake of 3H-norepinephrine was significantly reduced in both grafts as compared to unoperated veins. The smooth muscle of the cryopreserved and fresh grafts contracted comparably to alpha-adrenergic agonists and endothelin. In cryopreserved grafts, the maximal tensions that developed to KCl, prostaglandin F2 alpha, and endothelin were greater when the endothelium was present compared to that developed by the smooth muscle alone. Calcium ionophore A23187 caused relaxations only in rings with endothelium; these were not significantly different between graft types. However, relaxations of the smooth muscle to nitric oxide were decreased in the cryopreserved grafts.(ABSTRACT TRUNCATED AT 250 WORDS)

[The role of the initial state of the cells in cold-induced damage]. / Rol' iskhodnogo sostoianiia kletok v kriopovrezhdenii.

The investigations conducted have shown three states of erythrocytes in which they are most resistant to the damaging factors in the cycle of freezing-thawing-removal of cryoprotector. Basing on the results of their own investigations and the literature data on the factors influencing the erythrocyte metabolism and resistance to acid hemolysis, the authors have made a conclusion that the structural regulation in the cell membranes play the main role in cryo-induced damage.

A correlative study of the freezing patterns in rat myocardium using scanning and transmission electron microscopes.

Rat myocardial tissue, both fresh and chemically fixed, was quench frozen in melting Freon 22 at rates that resulted in in the formation of large ice-crystals. The material was studied with both scanning and transmission electron microscopy. Particular attention was paid to the characteristic freezing patterns within the myofibrils and mitochondria, and to features exposed by such freezing that might otherwise remain obscured. The freezing patterns found in tissue subjected to different processes were compared with the ultrastructure of unfrozen, chemically fixed counterparts. Ice-crystal cavities in the contractile material varied considerably along the length of a given myofiber in which the freezing front had progressed in parallel with the long axis of the myofibers. It is suggested that the intercalated disc functioned as a barrier to the freezing process. Ice generally compressed and distorted the contractile material beyond recognition, although the positions of the Z-bands remained evident and in register. Most single-fixed mitochondria were devoid of visible ice-crystal cavities, and the cristae generally remained intact. On the other hand, ice-crystal cavities were commonly seen between cristae of double-fixed mitochondria. No signs of perforations were seen in lipid bilayer membranes. Numerous strand-like connections between mitochondria as well as between mitochondria and the myofibrillar surface, which were believed to be sarcotubules, were made visible by the slow freezing process. Other strands, transverse to the myofibrils, connecting adjacent Z-bands and joining Z-bands to the sarcolemma, were interpreted as cytoskeletal elements. These strands, together with what apparently were SR tubules, exhibited high resistance to the stresses associated with the formation of ice-crystals.

An association between chromosomal abnormalities in rapidly frozen 2-cell mouse embryos and the ice-forming properties of the cryoprotective solution.

This paper investigates the effect of straw handling on the viability of 2-cell mouse embryos rapidly frozen in dimethyl sulphoxide (DMSO) solutions. During the brief (3 min) equilibration step, straws were either rotated periodically to keep the embryos in suspension, or kept still to allow the embryos to settle onto the the inner surface of the straw. The effects of these straw movements were tested with cryoprotectant solutions containing 1.5, 3.0 or 4.5 M-DMSO. Rapidly cooled straws containing 4.5 M-DMSO vitrify throughout on cooling, but ice forms on warming. The survival and normality of embryos frozen in 4.5 M-DMSO was not influenced by straw handling as 91-92% formed blastocysts in vitro, 77-78% formed normal fetuses, and no chromosomal rearrangements were observed. In solutions containing less than 4.5 M-DMSO ice formation occurred throughout (1.5 M-DMSO), or in parts (3.0 M-DMSO) of the cryoprotectant during cooling. The viability of embryos frozen in 3.0 or 1.5 M-DMSO solutions was reduced both in vitro and in vivo and structural chromosome aberrations, predominantly tri- and quadri-radial rearrangements, were observed. The reduction in embryo viability, and the chromosomal damage was particularly pronounced in embryos frozen in 3.0 M-DMSO in straws which were rotated during the equilibration step (47% blastocysts, 15% fetuses, 77% chromosome rearrangements). The results indicate that rapid freezing of 2-cell mouse embryos in 4.5 M-DMSO is safe and efficient, whereas freezing at lower DMSO concentrations is associated with severe chromosome damage, and reduced viability in vitro and in vivo.

The influence of in-vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts.

Blastocysts from 198 patients were frozen using glycerol as cryoprotectant. No difference in the post-thaw survival of blastocysts or implantation rates was found between 177 patients (122 transfers) with all surplus embryos cultured to blastocysts before freezing and 20 patients (12 transfers) whose embryos were considered unsuitable for freezing during cleavage and were then frozen as blastocysts. Nineteen pregnancies were achieved, of which six aborted. Pre-freezing morphology was similar in blastocysts of patients in groups 1 and 2 and did not relate to their survival after cryopreservation. A significantly lower proportion with suspected damage after thawing was present among patients becoming pregnant after transfers of single blastocysts (P less than 0.01) and implanting embryos were in general more expanded at the time of transfer. No differences were detected between blastocysts resulting in normal development and those leading to abortion. The developmental consequences of damage to human blastocysts are discussed.

Human preimplantation embryo cryopreservation: selected aspects.

This report describes the results of cryopreserving human preimplantation zygotes and cleaved embryos (2-4 cells) in our in-vitro fertilization programme. Cryopreserved zygotes and cleaved embryos resulted in similar post-thaw survival rates (74.8 versus 70.9%). Pregnancy rates per retrieval cycle (RC) and embryos transferred per pregnancy for frozen-thawed zygotes versus frozen-thawed cleaved embryos were 21.8 versus 11.5% (P less than 0.2) and 12.6 versus 17.5 (P less than 0.2), respectively. Pregnancy rates increased significantly for both fresh (P less than 0.0005) and frozen-thawed (P less than 0.05) embryos as the number of embryos replaced per transfer increased from one to three or more. Frozen-thawed embryos resulted in multiple implantation rates per transfer of 25 compared to 6.4% (P less than 0.1) for fresh embryos when two embryos were replaced. Pregnancy rates were reduced for fresh (P less than 0.05) and frozen-thawed (P less than 0.1) embryos obtained from patient retrieval cycle numbers greater than 3. The method of follicular stimulation during the retrieval cycle did not affect frozen-thawed embryo survival rates. There was no difference in pregnancy rates from frozen-thawed embryos replaced during natural or clomiphene citrate transfer cycles. Patients with cryopreserved embryos had cumulative pregnancy rates of 37.1% (66/178) compared to 23.5% (110/468) (P less than 0.01) for patients with no embryos cryopreserved; cryopreservation of preimplantation embryos is a reliable therapeutic procedure that enhances achievement of pregnancy through in-vitro fertilization.

Enumeration of IgE secreting B cells. A filter spot-ELISA.

To assess the frequency of IgE producing cells in humans a filter immunoplaque assay has been developed to detect IgE secretion from individual B lymphocytes in unfractionated peripheral blood mononuclear cells (PBMC). PBMC were incubated in microfilter plates containing nitrocellulose membranes coated with polyclonal anti-human IgE antibody, and the IgE production by a single cell was detected using a specific anti-human IgE monoclonal antibody followed by enzymatic development. The products of the enzymatic reaction were visualized as blue plaques on the membranes. The assay was both sensitive and specific as determined by: (1) a near 1:1 correlation between direct cell counts of an IgE producing myeloma cell line (U266) and the number of plaques in the filter immunoassay; and (2) the absence of detectable plaques generated by human B cells transformed by Epstein-Barr Virus (EBV) and producing only IgG or IgM. The presence of other cell types in PBMC did not affect the ability to detect IgE secreting cells. Replicate cultures of highly purified B lymphocytes, first transformed with EBV and then stimulated with recombinant human interleukin-4, produced IgE levels in culture supernatants that correlated closely with the number of IgE producing cells (r = 0.93; P less than 0.001). Furthermore, using the same transformed cells, the number of IgE secreting cells assessed by the immunoplaque assay was significantly correlated (r = 0.94; P = 0.002) with the number of IgE producing cells assessed by immunofluorescence staining of intracytoplasmic IgE. This assay provides a simple and direct method to assess the frequency of IgE producing lymphocytes in humans.

Bovine 1-2-cell embryo development using a simple medium in three oviduct epithelial cell coculture systems.

These studies were designed to develop a coculture system using a simple medium to promote development of 1-cell bovine embryos through the 8-16-cell stage to morula and blastocyst stages. Monolayers for coculture were prepared from bovine oviduct epithelial cells (BOEC). In vivo-fertilized 1-2-cell embryos and ova (384) were surgically collected from superovulated cows. In Experiment 1, embryos cocultured in a simple glucose-free and serum-free medium (CZB) developed with superior scores of embryo quality than embryos cocultured in Ham's F-10 with serum, and a greater percentage developed past 8-16 cells than embryos cocultured in CMRL-1066 with serum (p less than 0.05). In Experiment 2, embryos cocultured with fresh BOEC monolayers averaged more (p less than 0.05) cells than did embryos in coculture with frozen-thawed BOEC monolayers or in BOEC-conditioned medium. Without glucose in the simple medium for the first 48 h of culture, more embryos blastulated (p less than 0.01) by Day 5.5 of culture (Day 6.5 of donor's estrous cycle) than embryos in the same medium with glucose present throughout. In Experiment 3, more embryos tended to hatch in BOEC coculture (p less than 0.10) than in conditioned medium. These results show that a chemically simple medium with fresh BOEC monolayers can provide a significant benefit for coculture of early bovine embryos.

Computerized slow-staged freezing of semen from men with testicular tumors or Hodgkin's disease preserves sperm better than standard vapor freezing.

Two methods of freezing semen taken from patients with testicular tumors or Hodgkin's disease before treatment were compared. Many patients already have semen abnormalities, so an optimal method is extremely important. Ejaculates from 8 patients with testicular tumors and 20 with Hodgkin's disease were frozen by fast-freezing or by slow-staged freezing. Effects of motility, viability, and swelling after thawing were significantly impaired with both methods. However, cryosurvival was better after slow- than fast-freezing: motility 24% +/- 12.4% versus 15% +/- 11.2%; viability 24.1% +/- 11.4% versus 17.3% +/- 10.4%, swelling 33.3% +/- 11% versus 27.6% +/- 12.8%. The effects were equal for normal and abnormal sperm. Sperm from tumor patients should be frozen by slow-staged freezing method in spite of the higher cost and longer time.

Cold-induced ultrastructural changes in bull and boar sperm plasma membranes.

The effect of low temperatures on the ultrastructure of the plasma membrane of bull and boar spermatozoa was investigated. Cold-induced changes in the organization of sperm plasma membrane components were demonstrated by the use of fast-freezing combined with freeze-fracture electron microscopy. This preparation technique ensures fixation without artifacts. At 38 degrees C bull and boar spermatozoa exhibited a random distribution of intramembranous particles over the plasma membrane of both head and tail. Exposure to 0 degree C resulted in redistribution of the intramembranous particles: on the head and principal piece of bull spermatozoa and on the principal piece of boar spermatozoa, particle-free areas were observed, whereas on the boar sperm head, particle aggregates were present. The original particle distribution was restored upon rewarming of bull and boar spermatozoa to 38 degrees C, as well as after freezing and thawing of bull spermatozoa. Dilution of bull and boar semen into Tris-dilution buffer and Beltsville Thaw Solution-dilution buffer, respectively, could not prevent cold-induced redistribution of intramembranous particles. The observed particle reorganization upon cooling was interpreted as the result of lateral phase separation in the plasma membrane. Species-dependent differences in cold-induced ultrastructural changes were considered to be determined by lipid composition and asymmetry of the plasma membrane, and might be related to differences in cold resistance between species.

Examination by fluorescence and EPR spectroscopy of the state of mitochondrial and lysosomal membranes upon freeze-thawing.

The results of spectroscopic examination of mitochondria and lysosomes indicate that freeze-thawing leads to alterations of different character and extent in membrane structural organization which manifest as changes in the molecular packing of the organelle membrane lipid bilayer, lateral separation of lipids into individual domains, and impairment of membrane permeability. Supercooling of organelle suspensions without crystallization of external water has been found not to affect membrane barrier function markedly; however, such a decrease in the temperature results in a slight loosening of the membrane with an increase in the volume of subcellular structures. The crystallization of external water causes dehydration of organelles, which favors a decrease in their volume, increasing the viscosity of the liquid phase inside subcellular structures and packing the lipid bilayer. Changes in the permeability of mitochondrial and lysosomal membranes manifest during thawing after the formation of an external liquid phase and might be due to the sharp rehydration of these membranes through latent membrane defects formed upon freezing.

Propane-1,2-diol as a potential component of a vitrification solution for corneas.

Any method of cryopreservation of the cornea must maintain integrity of the corneal endothelium, a monolayer of cells on the inner surface of the cornea that controls corneal hydration and keeps the cornea thin and transparent. During freezing, the formation of ice damages the endothelium, and vitrification has been suggested as a means of achieving ice-free cryopreservation of the cornea. To achieve vitrification at practicable cooling rates, tissues must be equilibrated with high concentrations of cryoprotectants. In this study, the effects of propane-1,2-diol on the structure and function of rabbit corneal endothelium were studied. Corneas were exposed to concentrations of propane-1,2-diol ranging from 10 to 30% v/v in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, and 6% w/v bovine serum albumin. Endothelial function was assessed by monitoring corneal thickness during perfusion of the endothelial surface at 34 degrees C for 6 hr. Exposure to 10-15% v/v propane-1,2-diol was well tolerated for 20 min at 4 degrees C when the cryoprotectant was removed in steps or by sucrose dilution. However, exposure to 25% v/v propane-1,2-diol for 20 min at 0 or -5 degrees C was consistently tolerated only when 2.5% w/v chondroitin sulfate was included in the vehicle solution. Exposure to 30% v/v propane-1,2-diol was harmful at -5 and -10 degrees C. The endothelial damage following exposure to 30% v/v propane-1,2-diol was probably the result of a toxic effect rather than osmotic stress. Although 25% v/v propane-1,2-diol does not vitrify at cooling rates that are practicable for corneas, it could at this concentration form a major component of a vitrification solution comprising a mixture of cryoprotectants.

Cryogenic preservation of low-quality human semen.

The article describes the technical strategies for clinical cryogenic preservation of low-quality human semen. To compensate for sperm dilution resulting from the use of a cryogenic medium, ejaculated semen was concentrated before freezing by means of continuous-step density gradient centrifugation. Freezing was simplified by employing the carbon dioxide pellet method with KS-II cryogenic medium, which contains Pluronic F-68, a nonionic detergent, to solubilize egg yolk (a major cryogenic protectant). Semen of less than 50% motility (n = 23) was processed and then cryogenically preserved. Sperm concentration was increased by a factor of 1.8 +/- 0.96 (n = 23). Sperm motility was improved from 35.9% +/- 13.9% to 69.4% +/- 10.8%. Even after thawing 59.4% +/- 17.5% motility remained, with a mean survival rate of 85% +/- 14%. The concentration of sperm and improved sperm motility by the use of the continuous-step density gradient and the high survival rate ensured by the carbon dioxide pellet method with KS-II cryogenic medium compensated for the lowering of sperm quality during cryogenic preservation.

Cryopreservation of mammalian sperm: what we ask them to survive.

Techniques for freezing bull sperm developed over the past 40 years have not yielded protocols for preserving sperm from other species. Recent advances in our understanding of cell membrane structure function and metabolism now permit alternative modes of investigation. These data will allow development of unique studies which should have a higher probability of yielding successful protocols for sperm from other species. In this review the authors will: (1) provide a general overview of cryopreservation; (2) review emerging concepts of membrane structure and the relationship of membrane composition to water and cryoprotectant movement; (3) emphasize how these parameters affect cell volume and surface areas; (4) focus attention on the concept that cryoprotectants will alter membrane structure and function in addition to their well-recognized effects on bulk solvent; and (5) emphasize the effect of the processing protocol on metabolic balance. These concepts are reintroduced in the context of the established and successful protocol for freezing bull sperm to illustrate the molecular responses that may be necessary to survive a freeze-thaw cycle.

Fertilization in vitro of human oocytes by spermatozoa collected in different stressful situations.

It has been shown that semen quality is impaired in couples undergoing in-vitro fertilization (IVF), probably due to stress. A possible effect of stress on the ability of spermatozoa to fertilize human oocytes in vitro was analysed in the present study composed of 26 couples with normozoospermic men undergoing IVF. A semen sample was obtained during the infertility work-up and was cryopreserved (sample 1). A second sample (sample 2) was provided after oocyte retrieval during the IVF cycle. Sample 1 was thawed and both samples were washed and preincubated for oocyte insemination. One-hundred-and-five oocytes were inseminated using thawed sample 1, and 120 with sample 2. Semen parameters such as density, progressive motility and percentage of abnormal forms were compared between sample 1, before and after freezing, and sample 2. Only motility was significantly (P less than 0.01) decreased by cryopreservation in sample 1, but no parameter was significantly different when fresh sample 1 was compared to sample 2. The fertilization rate was 78.6% using sample 1 in comparison to 87.5% when sample 2 was employed (not significant, NS). Cleavage rates were 77.7 and 89.7%, respectively (NS). A group of five patients undergoing IVF who needed donor semen served as a control for the effect of sperm cryopreservation on IVF. In these cases, the donor was asked to provide a fresh sample. Half of this sample was frozen and thawed. Subsequently, fresh and thawed samples were prepared for insemination and oocytes inseminated either with the fresh preparation (n = 24) or the frozen and thawed spermatozoa (n = 22).(ABSTRACT TRUNCATED AT 250 WORDS)