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PubMed2001

Comparison between computerized slow-stage and static liquid nitrogen vapour freezing methods with respect to the deleterious effect on chromatin and morphology of spermatozoa from fertile and subfertile men.

The purpose of this study was to determine the negative effects (cryodamage) on human spermatozoa after freeze-thawing and to determine whether freeze-thawing of spermatozoa with a programmed slow freezer is better than freezing with liquid nitrogen vapour (rapid freezing) with regard to alterations in sperm chromatin and morphology in semen from fertile (donor) and subfertile, IVF/ICSI, patients. Ninety-five semen samples were obtained either from patients attending our IVF unit for treatment (n=34) or from donors (n=25) with proven fertility and normal sperm quality according to WHO guidelines. Each semen sample was divided into two parts after liquefaction and addition of the cryoprotectant. The first part was frozen using a programmed biological freezer and the second part was frozen by means of liquid nitrogen vapour. Smears were made before the freezing and after the thawing procedure to assess morphology (strict criteria) and chromatin condensation (Acridine Orange test). The mean percentage of chromatin condensed spermatozoa in the samples from donors (control group) was 92.4 +/- 8.4% before freezing and this decreased significantly (p < 0.0001) to 88.7 +/- 11.2% after freeze-thawing with the computerized slow-stage freezer and to 87.2 +/- 12.3% after using static liquid nitrogen vapour (p < 0.001). The corresponding values for semen obtained from patients was 78.9 +/- 10.3% before freezing which decreased to 70.7 +/- 10.8 and 68.5 +/- 14.8%, respectively (p < 0.001). On the other hand, the mean percentage of normal sperm morphology in the control group decreased from 26.3 +/- 7.5% before freezing to 22.1 +/- 6.4% (p < 0.0001) after thawing with the computerized slow-stage freezer and to 22.2 +/- 6.6% (p < 0.0001) after the use of static liquid nitrogen vapour. In the patient group, the mean percentage of normal morphology decreased from 11.7 +/- 6.1% after freezing with the biological freezer to 9.3 +/- 5.6% and to 8.0 +/- 4.9% after freezing with static liquid nitrogen vapour. This study demonstrates that chromatin packaging and morphology of human spermatozoa decrease significantly after the freeze-thawing procedure, not only after the use of static liquid nitrogen vapour but also after the use of a computerized slow-stage freezer. However, the chromatin of semen samples with normal semen parameters (donor sperm) withstand the freeze-thaw injury better than those with low quality semen samples. Therefore, the computerized slow stage freezer could be recommended for freezing of human spermatozoa, especially for subnormal semen samples, for example, ICSI and ICSI/TESE candidates and from patients with testicular tumours or Hodgkin's disease, in order to avoid further damage to the sperm chromatin structure.

Chromatin↗
PubMed1996

Hepatitis B nucleotide sequence analysis: linking an outbreak of acute hepatitis B to contamination of a cryopreservation tank.

An epidemiological investigation indicated that six patients treated in a haematology unit who developed acute hepatitis B may have been infected as a result of contamination of a liquid nitrogen bone marrow storage tank. The clinical details are described elsewhere (Tedder et al., 1995); we describe the virological methods used to support the findings. HBV DNA was amplified from sera using a nested PCR with primers for the surface gene, and a region encompassing precore, the 3' end of X, and the 5' end of core. HBV DNA was also extracted from the frozen detritus in the liquid nitrogen storage tank. After equilibration, the aqueous material was filtered, co-precipitated with albumin and polyethylene glycol and the HBV DNA extracted by phenol-chloroform and ethanol precipitation. Direct nucleotide sequence analysis indicated that four patients were infected with HBsAg subtype adw viruses which carried novel amino acid substitutions at codons 145 and 146 of the X gene. HBV DNA extracted from the storage tank detritus contained identical sequences. The samples from two other patients, subtype ayw, did not contain the novel sequence changes in X and had other sequence differences. These findings linked conclusively the four patients as a cluster and the rescue of HBV-DNA sequences from the contaminated storage tank by the method described confirmed this as the common source of infection. Two other HBsAg-positive patients were excluded from the cluster by sequence analysis. Demonstration of infection by this route has implications for the safe storage of bone marrow and other related biological materials.

Acute Disease↗
PubMed2006

Anti-HLA antibodies after cryopreserved allograft valve implantation does not predict valve dysfunction at three-year follow up.

BACKGROUND AND AIM OF THE STUDY: As the cause of allograft heart valve degeneration is poorly understood, the study aim was to investigate the host antibody response to allograft valve implantation. METHODS: Sera were obtained from 92 recipients of allograft heart valves (61 pulmonary, 31 aortic). Sera were tested for anti-HLA class I antibodies by ELISA and complement-dependent cytotoxicity (CDC) methods, and anti-HLA class II antibodies by ELISA. Specificities of recipient anti-HLA class I antibodies were defined by standard CDC testing against a panel of T lymphocytes from 80 blood donors. Donor valve HLA typing was performed on stored donor DNA samples using molecular methods. The presence of donor-specific anti-HLA class I antibodies was hence defined in recipient sera. The presence of anti-HLA antibodies and donor-specific anti-HLA class I antibodies were correlated with function of allograft valves at the most recent echocardiographic follow up. RESULTS: At a mean of 3.0 years (range: 0.3-5.4 years) after allograft implantation, 96% (87/92) and 82% (75/92) of patients were positive for anti-HLA class I and II antibodies, respectively, by ELISA testing. Some 68% (61/90) of patients were positive for anti-HLA class I antibody (PRA > 5%) by CDC testing. PRA levels decreased with greater postoperative interval (r = -0.31, p = 0.003). In 68 recipients where donor HLA type was defined, 54% (37/68) of patients had antibodies specific to at least one donor HLA class I antigen. In 87 patients with a recent echocardiographic examination available for analysis (at a mean of 3.5 +/- 1.6 years postoperatively), there was no association between valve dysfunction and antibody status. CONCLUSION: Anti-HLA class I and II antibodies were detected by ELISA methods in most patients after allograft implantation extending to 5.4 years. The clinical significance of these findings is unclear, as no correlation was found between the prevalence of anti-HLA antibody and echocardiographic parameters of valve dysfunction at a mean of 3.5 years follow up.

Adolescent↗
PubMed1985

A new procedure for the cryopreservation of equine embryos.

Early equine blastocysts and blastocysts were collected nonsurgically at six days post-ovulation. Thirty-two embryos were randomly assigned to a 2x2 factorial design. Factors were: 1) 0.5-ml straws or 1-ml glass ampules; and 2) plunging into liquid nitrogen (IN(2)) at -33 C or -38 C. Cryoprotectant, 10% glycerol in PBS plus 5% fetal calf serum (FCS) was added in two steps, 5% then 10%. Embryos were cooled at 4 C/min to -6 C and then seeded, 0.3 C/min to -30 or -35 C and 0.1 C/min to -33 or -38 C. Samples were thawed in 37 C water and glycerol removed in six steps, 10 min per step. Embryo quality and stage of development were evaluated prior to freezing, immediately post-thaw and after 24 h culture in Ham's F10 with 5% FCS. The mean post-thaw quality of embryos plunged at -33 C was superior (P<0.05) to that of embryos plunged at -38 C (2.0 vs 2.9). Embryos frozen in ampules and plunged at -38 C were of poorer quality (P<0.05) than those frozen in ampules and plunged at -33 C or frozen in straws and plunged at -33 C. After 24 h of culture, more embryos developed if frozen in straws compared to ampules, and plunging at -33 C resulted in higher quality embryos than plunging at -38 C. In Experiment 2, 23 embryos were packaged in straws and plunged at -33 C as described in Experiment 1. Six of the 23 surgically transferred frozen embryos were degenerate at thawing and the remaining 17 surgically transferred were via flank incision. Pregnancy rate at 50 days post-ovulation was 53% (nine of 17). Early blastocysts resulted in a higher (P<0.05) pregnancy rate (8 10 , 80%) than expanded blastocysts (1 7 , 14%).

Journal Article↗
PubMed1993

Human neuro-specimen banking 1961-1992. The National Neurological Research Specimen Bank (a donor program of pre- and post-mortem tissues and cerebrospinal fluid/blood; and a collection of cryopreserved human neurological specimens for neuroscientists).

The National Neurological Research Specimen Bank (The Bank) was established in 1961 to provide a vital service to neuroscientists. Our purpose is to support medical research which seeks the etiopathogenesis for devastating neurological disorders with no known cause, treatment or precise animal model. It serves as a bridge between the neurology clinician who diagnoses and cares for patients with incurable disease and the basic scientist who has need for specimens to find their etiopathogenesis. In the long run this service should advance neurologic diagnoses and serve as the basis for designing treatment. The Bank has grown to be the largest, most diverse neurological specimen bank of its kind in the world. It is a type of "tissue bank" where donor members "will" their central nervous system tissues to science. Then we collect, photograph, seal in air tight bags, quick freeze, cryogenically store and distribute on request the donated tissue to research scientists around the world. All tissue diagnoses are confirmed by clinical records and neuropathologic examination; further histology is conducted on request. In addition to brain and spinal cord tissues, the Bank has samples of other tissues. There are also samples of pre- and post-mortem CSF and sera from normal individuals and patients with various neurological disorders, especially serial specimens on multiple sclerosis patients and HIV-1 seropositive and at risk individuals. This paper outlines the global operations of our human brain bank, based on protocols developed and used by the authors. These operations include donor solicitation, tissue acquisition and documentation, tissue processing and storage, specimen dissemination to users, outcome assessment of banking, quality control, cost of our operation, table of organization and the future.

Blood Donors↗
PubMed2005

Bovine viral diarrhea virus (BVDV) in cell lines used for somatic cell cloning.

Culture of cell lines from fetuses or postnatal animals is an essential part of somatic cell cloning. Fetal bovine serum (FBS) is commonly used in media for propagation of these cells. Unfortunately, bovine fetuses and postnatal animals as well as FBS are all possible sources of non-cytopathic bovine viral diarrhea virus (BVDV) which is widely distributed among cattle. This study was prompted when screening of samples sent to veterinary diagnostic labs revealed that 15 of 39 fetal fibroblast cell lines used in cloning research were positive for BVDV as determined by various assays including reverse transcription-polymerase chain reaction (RT-PCR). Goals of the research were to use both virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR) to confirm which of the cell lines were actually infected with BVDV and to assay samples of media, FBS and the earliest available passages of each cell line in an attempt to determine the source of the viral infections. Sequence analysis of amplified cDNA from all isolates was performed to provide a definitive link between possible sources of virus and infected cell lines. Only 5 of the 39 cell lines were actually infected with BVDV. Three of these five lines were not infected at the earliest cryopreserved passage, leading to the conclusion that they likely became infected after culture in media containing contaminated FBS. In fact, sequence comparison of the amplified cDNA from one lot of FBS confirmed that it was the source of infection for one of these cell lines. Since BVDV was isolated from the remaining two cell lines at the earliest available passage, the fetuses from which they were established could not be ruled out as the source of the virus.

Animals↗
PubMed2004

Obesity and colon cancer: does leptin provide a link?

Obesity, a risk factor for colorectal cancer, is associated with elevated serum levels of leptin, the adipocyte-derived hormone, and insulin. Experimental and epidemiologic studies have indicated a role for insulin in the pathogenesis of colon cancer, and recent experimental studies have suggested a similar role for leptin. In a case-control study nested in the Janus Biobank, Norway, we measured serum levels of leptin and C-peptide (a marker of pancreatic insulin secretion) in cryopreserved prediagnostic sera from men (median age, 45 years) who were diagnosed with cancer of the colon (n = 235) or rectum (n = 143) after blood collection (median time, 17 years), and among 378 controls matched for age and date of blood collection. Conditional logistic regression analyses showed an approximately 3-fold increase in colon cancer risk with increasing concentrations of leptin up to an odds ratio (OR) of 2.72 (95% CI = 1.44-5.12) for top vs. bottom quartile (p(trend) = 0.008). The corresponding OR for C-peptide was 1.81 (95% CI = 0.67-4.86; p(trend) = 0.19). The risk estimates remained unchanged after mutual adjustment. No association of hormone levels with rectal cancer risk was found. Reproducibility of hormone measurements assessed by intraclass coefficients (ICCs) for paired samples taken 1 year apart was high for leptin (ICC = 0.82) but lower for C-peptide (ICC = 0.30). Our results suggest that leptin is a risk factor for colon cancer, and that leptin may provide a link between obesity and colon cancer. Leptin may be directly involved in colon tumorigenesis or it may serve as a sensitive and robust marker of an obesity-induced adverse endocrine environment. Only weak support for an association of insulin with colon cancer was found.

Adult↗
PubMed2004

Focused microwave irradiation of the brain preserves in vivo protein phosphorylation: comparison with other methods of sacrifice and analysis of multiple phosphoproteins.

At any point in time, net protein phosphorylation represents the contribution of protein kinase and protein phosphatase activities affecting a specific site on a given substrate. Preservation of phosphorylated proteins in neural tissues has traditionally included flash-freezing or fresh tissue processing following tissue isolation. Rapid heat inactivation of protein kinases and phosphatases by focused microwave irradiation sacrifice represents another method to preserve, in vivo, brain protein phosphorylation state. In this study, we compared preservation of the phosphorylation state of a variety of phosphoproteins in the brain following sacrifice of mice by decapitation, decapitation into liquid nitrogen and focused microwave irradiation. We found that microwave irradiation generally provided the highest and most consistent levels of protein phosphorylation, regardless of the substrates examined in striatum and hippocampus. In general, flash-freezing resulted in the least preservation of phospho-state with ERK1/2 and CREB showing almost complete dephosphorylation. When regions of freshly decapitated brains were homogenized and incubated on ice for 30 min, ERK1/2 phosphorylation was completely lost, whereas it was well preserved in microwaved samples left at room temperature for 2 h. Loss of ERK1/2 phosphorylation in the fresh samples could not be attributed to substrate proteolysis. Our results indicate that focused microwave irradiation sacrifice may be required to achieve biologically relevant data for the in vivo protein phosphorylation state of many phosphoproteins.

Animals↗
PubMed2002

Sperm binding to epithelial oviduct explants in bulls with different nonreturn rates investigated with a new in vitro model.

A new in vitro method was developed for analyzing the capacity of sperm to bind to oviductal epithelium to determine whether this binding capacity could be used to predict nonreturn rates (NRR). Sperm binding was evaluated by counting 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1)-labeled spermatozoa attached to oviductal epithelium and by measuring the surface area of the oviduct explants by means of an image analysis program. Hepes + Tyrode albumin lactate pyruvate (TALP) was a more useful medium than in vitro fertilization (IVF)-TALP, TCM-199 medium + 10% fetal calf serum, and TCM-199 medium alone for the investigation of sperm binding to oviductal explants. Oviduct explants with a surface area of < 20 000 micro m(2) provided more consistent results than did explants with a surface area of >100 000 micro m(2). A positive association was found between the log(e) transformed number of spermatozoa bound to 0.1 mm(2) oviductal epithelium and the NRR of the respective sires after 24 h of coincubation, provided that the membrane integrity of the sperm sample was >60%. Determination of the capacity of sperm to bind to oviductal explants could become a reliable in vitro method for predicting the NRR of a given sire.

Animals↗
PubMed2006

Preliminary comparison of quantity, quality, and microarray performance of RNA extracted from formalin-fixed, paraffin-embedded, and unfixed frozen tissue samples.

Microarrays have been used to simultaneously monitor the expression of thousands of genes from biological samples, an approach that can potentially uncover previously unrecognized functions of genes. Microarray analyses can rarely be conducted retrospectively because of the requirement for RNA to be obtained from fresh or unfixed frozen tissues. Archived pathology specimens would need to be used for retrospective analyses, and these are typically preserved as formalin-fixed, paraffin-embedded (FFPE) tissue. Formalin-fixed tissues have been shown to yield compromised RNA compared with that obtained from frozen tissue. To begin to assess the performance of RNA extracted from FFPE samples on a microarray format, we compared RNA from a model system of pelleted lipopolysaccharide-stimulated human bone marrow stromal cells that were snap frozen with RNA from FFPE cells. RNA integrity and Affymetrix quality control parameters were assessed, and differentially regulated genes were analyzed with Ingenuity Pathway Analysis software. Results demonstrate that both snap-frozen and FFPE samples yielded intact RNA suitable for amplification prior to Affymetrix GeneChip analysis. Although some transcriptional information was lost with RNA extracted from the FFPE samples, Ingenuity Pathway Analysis revealed that the major pathways identified as affected by drug treatment were similar. Results show that FFPE samples are amenable to Affymetrix GeneChip analysis, expanding the possibility for expression profiling on archived tissue blocks in pathology laboratories.

Bone Marrow Cells↗
PubMed2001

Single-cell dissection and microdroplet chemistry.

The unique roles of individual cells may be critical to the physiology of an organism. In such cases, micromethods are essential to elucidating the molecular biology, biochemistry and biophysics of the specialized cells or even subcellular compartments of the important cells. The great proliferation of micromethods testifies to their value and no single review can be comprehensive. This review therefore provides only a generalized overview of one approach, namely dissection that provides a pure sample for subsequent extraction and analysis by microdroplet chemistry. As a means of illustrating the utility of this approach, an application-study of the interaction of cytosolic malate concentration and guard-cell phosphoenolpyruvate carboxylase-is provided.

Cell Compartmentation↗
PubMed1993

Scanning electron microscopy of high-pressure-frozen sea urchin embryos.

High-pressure-freezing permits direct cryo-fixation of sea urchin embryos having a defined developmental state without the formation of large ice crystals. We have investigated preparation protocols for observing high-pressure-frozen and freeze-fractured samples in the scanning electron microscope. High-pressure-freezing was superior to other freezing protocols, because the whole bulk sample was reasonably well frozen and the overall three-dimensional shape of the embryos was well preserved. The samples were either dehydrated by freeze-substitution and critical-point-drying, or imaged in the partially hydrated state, using a cold stage in the SEM. During freeze-substitution the samples were stabilized by fixatives. The disadvantage of this method was that shrinking and extraction effects, caused by the removal of the water, could not be avoided. These disadvantages were avoided when the sample was imaged in the frozen-hydrated state using a cold-stage in the SEM. This would be the method of choice for morphometric studies. Frozen-hydrated samples, however, were very beam sensitive and many structures remained covered by the ice and were not visible. Frozen-hydrated samples were partially freeze-dried to make visible additional structures that had been covered by ice. However, this method also caused drying artifacts when too much water was removed.

Animals↗
PubMed1993

Intrauterine insemination. Effect of wash media on pregnancy rates and sperm motility.

Testyolk Buffer has been shown to enhance sperm penetration in hamster penetration assays and increase fertilization of oocytes in in vitro fertilization. Based on these findings, we compared pregnancy rates and sperm motility in intrauterine inseminations done with sperm samples washed and resuspended in Ham's F10 as compared with Testyolk buffer. Charts were reviewed retrospectively from 1,098 husband and donor intrauterine inseminations performed at the University of Florida. Data were analyzed using life table analysis and the curves compared with the Mantel-Haenszel statistical test. In addition, sperm motility in fresh sperm was observed in samples incubated in Testyolk or Ham's F10, with motility counts performed at 0, 6 and 24 hours. Four hundred ninety-two Testyolk cycles and 579 Ham's F10 cycles were compared, with cumulative pregnancy rates at one year of 53% and 44%, respectively (P = .58). With donor sperm, 229 cycles with Testyolk and 314 cycles with Ham's F10 had cumulative pregnancy rates of 68% and 48%, respectively (P = .52). With husband insemination, 264 Testyolk and 253 Ham's F10 cycles had pregnancy rates of 37% and 35%, respectively (P = .23). Fresh sperm motility in 22 samples compared at 0, 6 and 24 hours in Ham's F10 (76%, 67.8%, 56.6%) versus Testyolk (76%, 67.7%, 58.8%) revealed no significant differences. There was also no difference in total motile sperm inseminated and postwash motility in 1,098 samples with Testyolk versus Ham's F10. This study demonstrates that there is no enhanced pregnancy rate or increased sperm motility when sperm are treated with Testyolk Buffer instead of Ham's F10.

Adult↗
PubMedDate not supplied

Concentration of glutathione and expression of glutathione peroxidases 1 and 4 in fresh sperm provide a forecast of the outcome of cryopreservation of human spermatozoa.

Oxidative stress imbalance potentially leads to damage of the structure of the cell and macromolecules such as plasma membrane components, proteins, and DNA. The plasma membrane of the sperm cell, which has high levels of polyunsaturated fatty acids, renders it particularly sensitive to free radical-mediated attacks. The freezing and subsequent thawing of sperm is a physically stressful process carried out during routine procedures in assisted reproduction techniques, which results in a highly variable and unpredictable reduction in the number of motile sperm cells. Subsequently, oxidative status can positively or negatively affect the motility, viability, and fertilizing capacity of thawed sperm. These effects are counteracted by various oxidative defense enzymes and anti-oxidants such as glutathione peroxidase isoforms GPx1 and GPx4, glutathione reductase (GR), and cellular glutathione (reduced) (GSH). In this way, oxidative status could represent a predictive marker of sperm quality following the freeze-thaw process. This study was based on 56 human sperm samples. We observed direct positive and negative relationships between the postthaw motile sperm recovery rate and GPx1 and GPx4 expression and activity, on the one hand, and GSH concentrations, on the other. No correlation was found between this recovery rate and GR or basic semen parameters. Predictive values clearly demonstrate that, among the molecules analyzed, the most accurate diagnoses result when analyses are conducted for GPx1 and GPx1 messenger RNA expression, GPx1 and GPx4 enzymatic activity, and GSH concentration. In conclusion, a reserve of glutathione, together with GPx expression, is necessary to eliminate free radicals using GSH or a like structural protein and seems to be essential for a good postthaw recovery. These molecules can be employed as indicators of postthaw sperm quality.

Cryopreservation↗
PubMed2005

Nitric oxide-induced capacitation of cryopreserved bull spermatozoa and assessment of participating regulatory pathways.

The effect of nitric oxide (NO*) on the capacitation rates of cryopreserved bull spermatozoa and the participation of protein kinases in the capacitation process were evaluated. A pool of spermatozoa from four bulls were incubated in TALP medium in the presence of heparin (10 IU/ml) or sodium nitroprusside (SNP, 0.05-100 microM), a NO* donor. The participation of NO* was confirmed by the use of scavengers, i.e. methylene blue (50,100 microM) and hemoglobin (20-40 microg/ml). The role of nitric oxide synthase in heparin-induced capacitation was evaluated using enzyme inhibitors Nomega-nitro-L-arginine methyl ester (L-NAME) and Nomega-nitro-L-arginine (L-NA) in concentrations ranging from 1 to 500 microM. The effects of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK), on NO*-induced capacitation were evaluated by incubation with specific inhibitors of these enzymes (H-89, 50 microM; bisindolylmaleimide I, 0.1 microM and genistein, 3 microM). The role of hydrogen peroxide or superoxide anion in NO*-induced capacitation was evaluated by incubation with catalase (20-100 microg/ml) or superoxide dismutase (SOD, 0.05-0.5 mg/ml), respectively. Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC). SNP concentrations employed had no effect on progressive motility or sperm viability. Capacitation values of the 0.05 microM SNP treatment (31 +/- 5.15%) were similar to those of heparin treated samples (33 +/- 4.27%). Inhibitors of nitric oxide synthase (NOS) diminished capacitation percentages in a dose-dependent manner as did the addition of NO*- scavengers (P <0.05). The presence of PKA, PKC and PTK inhibitors likewise decreased capacitation percentages (6.25 +/- 0.71, 12.75 +/- 1.41, 9.00 +/- 1.41%, respectively). The presence of catalase or SOD in the incubation medium had no effect on capacitation percentages. These results indicate that NO* may be generated by a sperm NOS during heparin-induced capacitation and that exogenous NO* acts as a capacitation inducer and involves the participation of PKA, PKC and PTK as part of the intracellular mechanisms that lead to capacitation in cryopreserved bull spermatozoa.

Animals↗
PubMed2000

Flow cytometric analysis of enzymes in live spermatozoa before and after cryostorage.

Synthetic fluorogenic substrates, like the CellProbe reagents, can determine enzymes in vital human spermatozoa. These substrates will enter the cells without previous cell permeabilization and exhibit fluorescence after cleavage depending on enzyme activity. They consist of different peptide sequences, specific for the enzymes, and a fluorescein- or rhodamine 110-dye moiety. The number of positive cells and the intensity of the fluorescence can be determined by flow cytometric analysis. We investigated several enzymes (peptidases, proteinases, esterases, elastases and collagenases) in intact spermatozoa before and after cryoprotection. Semen samples with normal spermiogram parameters were cryoprotected using the freezing medium TEST yolk buffer (TYB). Fresh spermatozoa showed a marked fluorescence after incubation with the synthetic substrates for the aminopeptidase M, butyryl esterase, fluorescein diacetate (FDA)-and FDA/sodium fluoride (NAF)-esterase, ala-ala-pro-val (AAPV)-elastase, gly pro-leu-gly pro-(GPLGP)-collagenase, gly gly leu-(GGL)-subtilisin as well as lys-ala-(LA)-dipeptidyl peptidase (DPP) II. After cryopreservation the spermatozoal fluorescence increased applying substrates for butyryl esterase (P<0.05), prolyl-aminopeptidase (P<0.001) and val-lys-(VK)-cathepsin (P<0.001) most probably due to elevated enzyme activities. The activities of FDA-esterase (P<0.05) and FDA/NAF-esterase (P<0.05), AAPV-elastase (P<0.01), GPLGP-collagenase (P<0.05) and GGL-subtilisin (P<0.001) decreased after cryopreservation. The substrates for arg-gly glut-ser-(RGES)-elastase, gly phenyl-gly ala-(GFGA)-collagenase and threo-pro-(TP)-cathepsin were not cleaved before as well as after cryostorage. The substrates for subtilisin an

Cryopreservation↗
PubMed1997

High-dose therapy with iodine-131-labeled monoclonal antibody CC49 in patients with gastrointestinal cancers: a phase I trial.

PURPOSE: A phase I trial that evaluated for extrahematopoietic toxicity was conducted with iodine-131 (131I) labeled monoclonal antibody (MAb) CC49. Correlative studies included pharmacokinetic and biodistribution analyses, estimates of absorbed radiation dose, and measurement of human antimonoclonal antibodies (HAMA). PATIENTS AND METHODS: After collection and cryopreservation of hematopoietic stem cells, 15 patients with gastrointestinal cancers were administered a tracer dose of 131I-MAb CC49. Within 5 to 6 days, 14 patients (two to three per activity level) underwent a single treatment with 131I-MAb CC49 (50, 100, 150, 200, 250, and 300 mCi/m2). Biodistribution was determined using planar and single photon emission computer tomographic (SPECT) imaging. Pharmacokinetic studies were performed by measuring radioactivity in serial blood samples. In some patients, biopsies of metastases and related normal tissues were obtained for radioactivity measurements. Radiation dosimetry estimates were calculated using available biodistribution, pharmacokinetic, and tissue biopsy data. Toxicity was evaluated using the National Cancer Institute (NCI) Common Toxicity Criteria. RESULTS: No dose-limiting extrahematopoietic toxicity was identified. Twelve patients experienced grade IV myelosuppression and met criteria for infusion of hematopoietic stem cells. Radioimmunolocalization was excellent. The T1/2 for 131I-MAb CC49 after diagnostic and therapeutic administration was 39.7 +/- 10.4 and 46.1 +/- 10.6 hours, respectively. The percent injected dose per killigram of tumor ranged from 0.2 to 2.1. Absorbed radiation dose in metastatic tumor sites ranged from 630 to 3300 cGy. CONCLUSION: Although extrahematopoietic dose-limiting toxicity was neither observed or predicted, suboptimal absorbed dose estimates suggested that further escalation of 131I-MAb CC49 would not be useful. Future studies should focus on the use of radionuclides with high energy beta emissions, such as yttrium 90, and on strategies to optimize access of antibody to target antigens.

Adenocarcinoma↗
PubMed2006

SELDI-TOF MS of quadruplicate urine and serum samples to evaluate changes related to storage conditions.

Proteomic profiling with SELDI-TOF MS has facilitated the discovery of disease-specific protein profiles. However, multicenter studies are often hindered by the logistics required for prompt deep-freezing of samples in liquid nitrogen or dry ice within the clinic setting prior to shipping. We report high concordance between MS profiles within sets of quadruplicate split urine and serum samples deep-frozen at 0, 2, 6, and 24 h after sample collection. Gage R&R results confirm that deep-freezing times are not a statistically significant source of SELDI-TOF MS variability for either blood or urine.

Albumins↗