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PubMed2001

Identification of T-cell clones showing expansion associated with graft-vs-leukemia effect on chronic myelogenous leukemia in vivo and in vitro.

Although the graft-vs-leukemia (GVL) effect induced by donor leukocyte infusion (DLI) is thought to be mediated by T cells, their features, as well as target molecules, remain unknown. To characterize T cells that mediate the GVL effect on chronic myelogenous leukemia (CML), we studied T-cell repertoire in peripheral blood (PB) of two patients treated with DLI for relapsed CML after allogeneic bone marrow transplantation. Peripheral blood mononuclear cells (PBMCs) were obtained at 2-week intervals following DLI and examined for the presence of antigen-driven T-cell proliferation using complementarity-determining region (CDR) 3 size spectratyping of T-cell receptor beta chain subfamilies. Both patients exhibited transient proliferation of a limited number of T cells at a certain point in time (day 132 for patient 1 and day 75 for patient 2) after DLI in association with a decrease in the proportion of Philadelphia chromosome (Ph)-positive cells. In patient 2, who showed expansion of a BV16(+) T cell in PB, expansion of BV16(+) T cells with a similar CDR3 motif containing QDR to that of PB was demonstrated in the bone marrow (BM) sampled on day 33 and in the buccal mucosal tissue, showing chronic graft-vs-host disease (GVHD) on day 138 after DLI. When PBMCs obtained from patient 2 in remission were cultured with cryopreserved CML cells for 2 weeks, proliferation of a BV16(+) T cell with a CDR3 motif of QIR was induced in vitro. These findings indicate that transient proliferation of a limited number of T cells detected in PB 3-5 months after DLI probably reflects the GVL response against CML cells and may serve as a marker for the appearance of the GVL effect induced by DLI.

Adult↗
PubMedDate not supplied

Surface structure of spermatozoa frozen for artificial insemination.

Samples of human semen frozen in liquid nitrogen ( - 196 degrees C) with no glycerol, 5 and 10% glycerol were compared with samples that were untreated, with 10% glycerol but not frozen, and spermatozoa frozen at -20 degrees C. SEM and TEM of the samples indicates that 10% glycerol caused fewer surface changes of the spermatozoa than other treatments. Motility counts after the various freezing treatments were also highest when 10% glycerol was used as the cryoprotectant. Nonetheless, cryopreservation is detrimental to spermatozoa and often causes considerable damage to the acrosome with a leakage of the acrosomal contents.

Cell Count↗
PubMed1997

The effects of cryopreservation on the morphometric dimensions of caprine sperm heads.

Cryopreserved semen has been utilised in the artificial insemination of livestock species for over 40 years, even though the detrimental effects of cryopreservation on sperm function and fertility are well documented. In the present study, computer-automated sperm-head morphometry was used to determine if goat sperm-head morphometry was affected by freezing and thawing. A microscope slide was prepared from single semen samples, collected by artificial vagina, from 10 sexually active Saanen bucks. The remainder of each sample was frozen in a tris-citrate-yolk extender. After thawing, semen smears were prepared on microscope slides. All slides were stained in haematoxylin and mean sperm-head measurements of length, width, width/length, area and perimeter were determined for each slide by computer aided sperm morphometry analysis. The effects of sperm freezing on sperm-head dimensions within and among all bucks were determined. No significant (P > 0.10) freezing effect was found between fresh semen and postthaw samples for length (7.00 microns vs 7.13 microns), width (3.77 microns vs 3.87 microns), width/length (0.54 micron vs 0.54 micron), area (19.67 microns2 vs 20.57 microns2) and perimeter (18.62 microns vs 18.83 microns) when analysed across all bucks. Significant differences (P < 0.05) were however found within three bucks for area, perimeter, length and width, with the percentage increase in measurements being significantly greater than in the remaining bucks. The variability of the morphometric dimensions were not affected by freezing. The results indicate that semen freezing did not affect the overall dimensions of sperm heads across the entire population of bucks sampled. However, since sperm-head dimensions from three bucks were affected, changes in sperm-head morphometry may be indicative of spermatozoa of the semen from individuals to successfully freeze. Because the overall mean sperm-head dimensions acquired from frozen/thawed semen were not different from those of fresh semen, previously reported measurements of goat sperm heads are probably reflective of fresh semen. More importantly, retrospective studies of sperm-head morphometry and fertility may now be performed utilising extensive breeding records from frozen semen.

Animals↗
PubMed2003

Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate.

Antioxidant supplementation during cooling was assayed to improve the motility of frozen-thawed (FT) boar spermatozoa from two different fractions of the ejaculate, the first component of the sperm-rich fraction (Fraction I) and the rest of the bulk ejaculate (Fraction II). Using a split-sample design, addition of two different concentrations (100 and 200 microMl(-1)) of the water-soluble Vitamin E analogue Trolox (6-hydroxy -2,5,7,8-tetramethylchroman -2-carboxylic acid) was evaluated for an effect on sperm motility (measured both subjectively and by means of a computer assisted motility assessment (CASA)), and on mitochondrial membrane potential using flow cytometry after cell-loading with JC-1. The effect of the Vitamin E analogue was clearly dose-dependent and varied with the fraction of the ejaculate considered. Motility was significantly higher in Trolox-treated spermatozoa (200 microm), from either ejaculate fraction, albeit the effect was more evident in spermatozoa from Fraction II (P<0.05) for any Trolox-concentration. Antioxidant supplementation resulted, also dose-dependent, in a higher number of spermatozoa showing high mitochondrial activity as assessed by the JC-1 staining, in both ejaculate fractions. In the present trial, exogenous Trolox positively affected post-thaw sperm viability (as motility and mitochondrial membrane potential) in both fractions of the ejaculate. The magnitude of the effect appeared, however, to be dependent of the fraction of the ejaculate considered.

Animals↗
PubMed1997

Evaluation of three rapid RNA extraction reagents: relevance for use in RT-PCR's and measurement of low level gene expression in clinical samples.

Although peripheral blood and bone marrow are usually readily available from patients, present techniques of RNA extraction are tedious, require millilitres of starting material and removal of red blood cells before RNA purification. Further, successful reverse transcriptase polymerase chain reaction (RT-PCR) amplification requires the removal of haemoglobin derivatives which interfere with the PCR process. Recently, one step rapid use reagents have become available, claiming to be useful for obtaining high quality RNA from microlitre quantities of whole blood drawn directly from the patient. Their use to date in clinical samples appears limited with little information in the literature documented. In an attempt to overcome this, we tested the Trizol-LS, RNA-STAT-50 and Ultraspec-3 reagents upon a statistically significant number of clinical isolates of fresh and cryopreserved peripheral blood, bone marrow, blood apheresis products and a breast cancer cell line (MCF7) in order to evaluate whether these methods could be applied to routine laboratory use in an RT-PCR method capable of detecting rare gene expression. Our findings showed that there was some variation in the quality of RNA extracted which was indicated by absorbance spectrophotometry at 260 and 280 nm. 1% agarose gel electrophoresis showed that each of these methods could yield total RNA capable of generating the signature 18S and 28S rRNA bands. Using the Kruskal-Wallis non-parametric anova test combined with Dunn's multiple comparison test, the only statistically significant difference (p<0.05) indicated that Trizol-LS was more reliable than RNA-STAT-50-LS and Ultraspec-3 at extracting RNA from fresh peripheral blood. RNA extracted with the Trizol-LS and RNA STAT-50 reagents was successfully amplified in a multiplex RT-PCR reaction for detection of the multi-drug resistance related genes MDR1, the multi-drug resistance related protein (MRP) and topoisomerase IIalpha. Low level MDR1 gene expression could be detected in frozen whole blood. However, PCR products were only seen when the anti-coagulant heparin was removed from all samples prior to cDNA production. RT-PCR amplification was not 100% successful with RNA extracted with Ultraspec-3 reagent. In conclusion, we found that the RNA extracted from whole blood with the Trizol-LS and the RNA-STAT-50 are suitable for use in clinically relevant molecular biology protocols that analyze rare event genes without further purification. Our results indicated that the Trizol-LS reagent was generally more consistent in obtaining a pure and sufficient quantity of RNA from patient material as shown by the mean result of purity and quantity in comparison to either Ultraspec-3 or RNA-STAT-50-LS reagents. Ultraspec-3 is not easily suited for direct use with whole blood products.

Evaluation Studies as Topic↗
PubMed2006

Analysis of the effect of partial vitrification on stress development in cryopreserved blood vessels.

Thermal stress development in blood vessels, during processes associated with vitrification (vitreous means glassy in Latin), is studied. This paper addresses the limiting case where the specimen completely crystallizes, while the cryoprotectant medium (CPA) completely vitrifies. This case is expected to provide upper boundary estimates for stresses for the more common problem of a partially vitrified sample. The CPA is modeled as a linear viscoelastic medium, with viscosity increasing exponentially with decreasing temperature; given the assumption of complete crystallization, the blood vessel is modeled as linear elastic below the freezing temperature. Consistent with previous observations, the CPA is found to behave linear elastically below a set-temperature, at which point the viscosity rises sufficiently quickly with further cooling. This observation reduces computational efforts and allows for parametric studies based on suitably chosen wholly elastic models. Both 2D concentric cylinder models of the blood vessel in a straight configuration and a 3D model of the vessel curled in a vial of CPA are studied; 2D models are shown to bound the results of the more general 3D problem. It is found that stress in the CPA decreases with increase in CPA volume, at least under conditions where the temperature can be viewed as uniform. Planar cracks are predicted to form transverse to the vessel axis, and to propagate right up to the blood vessel wall. Should such cracks propagate into the vessel, even over only a few mum, the mechanical damage to the lumen, or to endothelial cells, may cause the blood vessel to completely loose its functionality at the end of the cryopreservation protocol.

Animals↗
PubMed1994

Ultrastructural localization of endogenous hormones and receptors in bone tissue: an immunocytological approach in frozen samples.

In addition to their well-known mechanical role, bones are essential for the regulation of calcium phosphate metabolism. Most of the peptide and steroid hormones acting on bone tissue exert their cellular effects directly on bone cells after binding to cell receptors. A combined method using immunocytology on ultrathin frozen sections has been applied to bone tissue and made possible the ultrastructural localization of endogenous hormones and hormone receptors. There is immunocytological evidence, at the osteoblast level, for: (1) the presence of calcitonin, parathyroid hormone, growth hormone, 1,25 dihydroxyvitamin D3, aldosterone, corticosterone and progesterone; (2) the existence of 1,25 dihydroxyvitamin D3 and progesterone receptors; (3) the absence of estradiol and receptors, testosterone and gonadoliberin. Ultimate goals of future studies are to extend the ultrastructural localization of hormones at the osteoclast level, to assess the distribution of adhesive proteins and of their receptors in bone cells and matrix, and to use well-calcified human bone samples in order to evaluate better the role of therapeutic agents.

Animals↗
PubMed2005

Fertility evaluation of frozen/thawed semen.

In vitro semen analyses have been used for more than half a century to estimate the fertilizing potential of a semen sample. Unfortunately, none of the assays developed provide results that consistently correlate well with fertility. The reasons for this lack of consistency, due in part to the complexity of the spermatozoon itself, the collection of fertility data, and factors beyond control of the semen analyses themselves, are discussed. Different spermatozoal attributes that are necessary for a spermatozoon to fertilize an oocyte are presented and assays used to evaluate each attribute described. Although laboratory assay results do not correlate well with semen fertility, the importance of conducting laboratory assays on every semen sample used for artificial insemination or to attempt to determine causes for infertility, is discussed.

Animals↗
PubMed1999

[Missense mutations of the butyrylcholinesterase gene in six Japanese patients with low cholinesterasemia: genetic analysis using sera stored in a freezer].

Six serum samples with no detectable butyrylcholinesterase (BCHE) activity had been stored at -70 degrees C for more than 10 years. These sera were used for amplification of BCHE gene using polymerase chain reaction (PCR) and for nucleotide sequence analysis. Five of them demonstrated a C-->T transition at codon 100 (CCA-->TCA), resulting in a Pro-->Ser substitution. The other one was a compound heterozygote as revealed a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and G-->C transversion at codon 365 from GGA (Gly) to CGA (Arg). These results showed sera stored in a freezer could be used as a starting material for amplification of genomic DNA when it is not possible to obtain fresh blood samples.

Asian People↗
PubMed2004

Should cryopreserved epididymal or testicular sperm be recovered from obstructive azoospermic men for ICSI?

OBJECTIVE: To determine the effect of the anatomical site of sperm recovery on intracytoplasmic sperm injection (ICSI) embryo implantation, pregnancy and live birth rates in couples with isolated obstructive azoospermia as the sole cause of infertility. DESIGN: Controlled, single centre, retrospective clinical study. SETTING: University Hospital, Centre for Reproductive Medicine. SAMPLE: One hundred and fifty-one cycles of ICSI were performed, using surgically recovered sperm, between August 1996 and March 2002. METHODS: The outcome of ICSI, with surgically recovered sperm, was compared between epididymal (Group E) and testicular (Group T) derived sperm. Inclusion was limited to couples undergoing their first treatment cycle, where female age was < or =39 years and a minimum of five oocytes were available for injection. Women with a history of ovarian surgery, ultrasonic evidence of polycystic ovaries, uterine anomalies or hydrosalpinx were excluded. MAIN OUTCOME MEASURES: Clinical pregnancy, implantation and live birth rate. RESULTS: Forty-two of 151 cycles met the strict inclusion criteria. Groups E and T were comparable with respect to age, basal serum FSH, ovarian response; number of oocytes injected and number of embryos available and transferred. No difference existed between Groups E and T in implantation, clinical pregnancy or live birth rate (28.8% vs 25.8%, 42.9% vs 42.9% and 39.3% vs 42.9%, respectively). CONCLUSIONS: Cryopreserved epididymal and testicular sperm, from men with obstructive azoospermia, appear equally effective in ICSI. Epididymal recovery should remain the method of first choice for obstructive azoospermic men but further study of sperm DNA damage rates in different testicular sites is required.

Adult↗
PubMed2004

Antioxidant supplementation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture.

Previous studies have shown sperm quality after cryopreservation differs depending on the fraction of seminal plasma the boar spermatozoa are contained in. Thus, spermatozoa contained in the first 10 ml of the sperm-rich fraction (portion I) withstand handling procedures (extension, handling and freezing/thawing) better than those contained in the latter part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction; portion II). The present study evaluated whether an exogenous antioxidant, the water-soluble vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), could, when added to the freezing extender in a split-sample design trial, improve the post-thaw viability and membrane quality of this particular portion of the ejaculate, with particular attention to the status of the plasma membrane. Using a split-sample design, the initial changes in the fluidity status of the sperm plasmalemma after thawing were measured by flow cytometry (FC) after loading with Merocyanine-540 and YO-PRO-1. The FC-derived data revealed a clear ejaculate portion-dependent effect of the antioxidant supplementation. While no beneficial effect of the antioxidant supplementation was visible in spermatozoa from portion I, more spermatozoa with intact membranes were observed in the supplemented samples of portion II, suggesting the protective effect of vitamin E is dependent of the portion of the boar ejaculate considered.

Animals↗
PubMedDate not supplied

A chemically 'defined' diluent for cryopreservation of bovine spermatozoa.

A chemically 'defined' extender for the cryopreservation of bovine spermatozoa was developed using commercially available lecithin as an additive to protect cells against freeze damage. The effectiveness of different media in preventing cryoinjury was evaluated by assessing motion activity immediately post-thaw and after 2 h incubation at 37 degrees C by means of automatic computer image analysis. The extender best suited to maintains swimming activity consisted of the zwitter ion buffer system HEPES-Tris/Citrate (HTC) supplemented with 0.9% lecithin. The effectiveness of this diluent was compared with that of an extender containing egg yolk used routinely in breeding stations. While immediately post-thaw sperm samples frozen in HTC showed a lower motion activity than those preserved in egg yolk diluent, the opposite result was obtained after 2 h incubation at 37 degrees C.

Animals↗
PubMedDate not supplied

Desiccation and freezing tolerance of embryonic axes from Citrus sinensis [L.] osb. pretreated with sucrose.

Embryonic axes of Citrus sinensis L. were successfully cryopreserved. While fully hydrated unfrozen axes germinated 100%, survival decreased as axes water content dropped, and total loss of viability was observed when the water content dropped to 0.04 and 0.10 mg H2O/mg dry mass, for axes without and with sucrose preculture, respectively. Fully hydrated axes did not survive exposure to liquid nitrogen. Highest seedling recovery (93-100%) for untreated axes was observed at 0.26 to 0.15 mg H2O/mg dry mass. Differential scanning calorimetry revealed the presence of broad melting peaks in fully hydrated embryonic axes. The size of the melting peak diminished as water was removed by desiccation. Minimum melting of water was observed at the point axes survived cryopreservation. Occurrence of a glass transition upon warming was not a condition for axes to survive liquid nitrogen exposure. In untreated axes, glucose, increased with desiccation to 0.2 mg H2O/mg dry mass, and decreased as the axes were desiccated to lower water contents. Fructose and sucrose levels did not increase when untreated samples were desiccated for the same periods of time. Raffinose and stachyose levels decreased as untreated and precultured embryonic axes were desiccated. In sucrose precultured axes, sucrose and fructose levels increased when they were dehydrated, reaching maximum levels at 0.2 mg H2O/mg dry mass. Tissue glucose did not change significantly with desiccation. Raffinose and stachyose levels dropped as precultured embryonic axes were dried.

Citrus sinensis↗
PubMed1983

Penetration of zona-free hamster ova and bovine cervical mucus by fresh and frozen human spermatozoa.

Semen samples from 25 fertile donors and 34 infertility patients were analyzed and evaluated both fresh and frozen with the zona-free hamster egg penetration test (EPT) and the bovine cervical mucus penetration test (MPT). Cryopreserved sperm penetrated 47.2% (average) of the hamster eggs, and fresh sperm penetrated 55.9%. Frozen sperm retained 86% of the fresh EPT values (P, not significant), but only 38% of the fresh MPT values (P less than 0.001). Cryopreserved sperm yielded the same fertility classification as fresh sperm in 94% of the cases with the EPT but only 43% with the MPT. The use of cryopreserved sperm simplifies the procedure and should enable more laboratories to utilize the EPT, but the poor results with the MPT preclude utilization of cryopreserved sperm for routine fertility evaluation with the MPT.

Animals↗
PubMed1997

Effects of cryopreserved semen quality and timed intrauterine insemination on pregnancy rate and gender of offspring in a donor insemination program.

PURPOSE: Our purpose was to study the relationship among cryopreserved donor semen quality, pregnancy rates, and preconception sex selection after intrauterine insemination. METHODS: We reviewed the records of the 203 women in our donor insemination program from 1987 to 1994 who became pregnant after more than one insemination cycle and had no female-factor infertility. They were categorized according to the number of cycles required for pregnancy. Semen samples from 54 donors were analyzed before freezing and after thawing. Specimens resulting in pregnancy were compared to specimens from the same donor that did not. Semen characteristics were compared to gender of the child. RESULTS: Two hundred fifty two-women became pregnant of the 422 who were enrolled. The pregnancy rate per cycle was 13%. Semen quality was not related to pregnancy outcome or offspring gender. However, more male children (101 vs 83) were born. CONCLUSIONS: Semen characteristics in good-quality cryopreserved donor semen do not affect pregnancy rate or offspring gender.

Cryopreservation↗
PubMed1997

Donor helper T-cell frequencies as predictors of acute graft-versus-host disease in bone marrow transplantation between HLA-identical siblings.

BACKGROUND: Despite the current level of sophistication of molecular typing for class I and class II alleles, a significant proportion (20-40%) of recipients of HLA-identical sibling marrow develop severe, acute graft-versus-host disease (GVHD) after bone marrow transplantation. It has been suggested that the frequency of patient-specific helper T lymphocyte precursors (HTLp) detected in the HLA-identical sibling donor correlates with the incidence and severity of acute GVHD after transplantation. METHODS: This study group consisted of 42 patients who all received bone marrow from HLA-identical sibling donors from January 1990 to December 1996. Using a limiting dilution analysis, donor HTLp frequencies were determined on samples collected before transplantation. The HTLp assay used the cytotoxic T-cell line, CTLL-2, which proliferates in the presence of interleukin-2. The reliability and reproducibility of this assay was established by using cryopreserved batches of CTLL-2 cells of known sensitivity. RESULTS: The recipient-directed HTLp frequencies detected in the donor before transplantation were correlated with the incidence and severity of acute GVHD experienced by the recipient after transplantation. Statistical analysis revealed an extremely significant correlation between donor precursor frequencies and the development of acute GVHD in the patient after transplantation (P<0.0001). CONCLUSIONS: This study suggests that together with molecular typing the HTLp frequency should be considered when selecting the most suitable sibling donor for bone marrow transplantation.

Adolescent↗
PubMed2002

Use of blood outgrowth endothelial cells for gene therapy for hemophilia A.

A culture of human blood outgrowth endothelial cells (BOECs) was established from a sample of peripheral blood and was transfected using a nonviral plasmid carrying complementary DNA for modified human coagulation factor VIII (B domain deleted and replaced with green fluorescence protein). BOECs were then chemically selected, expanded, cryopreserved, and re-expanded in culture. Stably transfected BOECs were administered intravenously daily for 3 days to NOD/SCID mice at 4 cell dose levels (from 5 x 10(4) to 40 x 10(4) cells per injection). In 156 days of observation, mice showed levels of human FVIII that increased with cell dose and time. Mice in all cell dose groups achieved therapeutic levels (more than 10 ng/mL) of human FVIII, and mice in the 3 highest dose groups acquired levels that were normal (100-200 ng/mL) or even above the normal range (highest observed value, 1174 ng/mL). These levels indicate that the BOECs expanded in vivo after administration. When the mice were killed, it was found that BOEC accumulated only in bone marrow and spleen and that these cells retained endothelial phenotype and transgene expression. Cell doses used here would make scale-up to humans feasible. Thus, the use of engineered autologous BOECs, which here resulted in sustained and therapeutic levels of FVIII, may comprise an effective therapeutic strategy for use in gene therapy for hemophilia A.

Animals↗