Mechanism of infertility in male guinea pigs immunized with sperm PH-20

PH-20, a testis-specific protein first expressed in haploid germ cells, is present on the posterior head plasma membrane and inner acrosomal membrane of mature guinea pig sperm. PH-20 is bifunctional, having a hyaluronidase activity that allows sperm to penetrate the cumulus layer and a separate activity required for binding of acrosome-reacted sperm to the zona pellucida. The immunization of male guinea pigs with PH-20 reproducibly results in infertility with a duration of 6-12 mo or longer. In this study, we analyzed the immunopathology in the reproductive tract of PH-20-immunized males to probe the mechanism(s) responsible for the induced infertility and found two separate effects. Remarkably, in almost all infertile, PH-20-immunized males, the caudae epididymides were empty (contained no sperm) or contained only abnormal sperm. The complete loss of normal sperm in the epididymis apparently results in infertility. A second effect was the induction of experimental autoimmune orchitis (EAO), representing the first report of EAO induced by a purified testis/sperm molecule of known functions. PH-20-induced EAO differed from EAO induced by crude testis antigens in two respects: 1) an absence of epididymitis with abscess and granuloma and 2) the presence of antibody on germ cells within seminiferous tubules and inside the cauda epididymidis. The former suggests that crude testis antigens other than PH-20 are responsible for epididymitis, and the latter suggests a possible role of antibody in EAO pathogenesis and infertility induction. Return to fertility, after 6-12 mo, was accompanied by regression of EAO and reappearance of spermatozoa in the caudae epididymides.     

Biochemical maturation of Spam1 (PH-20) during epididymal transit of mouse sperm involves modifications of N-linked oligosaccharides

Indirect immunofluorescence of mouse caput and caudal sperm shows distinctly different distributions of Spaml protein, which is associated with structural and functional differences of the molecule. Spam1 is uniformly distributed over the surface of the head of caput sperm while in caudal sperm, light and confocal microscopy demonstrate that it is localized to the anterior and posterior regions. The hyaluronidase activity of Spaml in acrosome-intact caput sperm was significantly lower (4.3-fold; P < 0.0001) than that of caudal sperm. The increase in enzymatic activity in caudal sperm is accompanied by a reduction in the molecular weight (MW): in extracts from caput sperm there was a major band at approximately 74 kDa and a minor band at approximately 67 kDa; while for the cauda there was a major band at approximately 67 kDa and minor bands at approximately 70 and -56 kDa. Additionally, the bands from caput sperm were 4.9 to 7.7-fold less intense than those from caudal sperm. This decreased affinity for the polyclonal anti-Spaml suggests the presence of different surface characteristics of the molecule from the two epididymal regions. Computer analysis of the protein structure from Spam1 cDNA sequence reveals four putative N-linked glycosylation sites, and enzymatic deglycosylation suggests that all sites are functional. After endoglycosidase activity of extracts from caput and caudal sperm, both show a major band with a MW of approximately 56 kDa, the size of the membrane-anchored polypeptide backbone. Based on the difference in size and intensity of the Spaml bands and hyaluronidase activities from caput and caudal sperm, the data suggest that the activation of Spaml during epididymal maturation is regulated by deglycosylation.     

In vitro activation of human herpesviruses 6 and 7 from latency

Human herpesviruses 6 and 7 (HHV-6 and HHV-7) are prevalent lymphotropic viruses that infect more than 80% of children at infancy or during early childhood. Infection ranges from asymptomatic to severe disease. HHV-6B causes exanthem subitum. The virus can be recovered from peripheral blood mononuclear cells during the acute phase of exanthem subitum, but the host remains latently infected throughout life. In immunocompromised patients undergoing kidney, liver, or bone marrow transplantation latent HHV-6B is reactivated, at times causing severe or fatal disease. Here, we describe the establishment of an in vitro system for reactivation of HHV-6B and HHV-7 from latency. HHV-7 is reactivated from latently infected peripheral blood mononuclear cells by T-cell activation. HHV-6B could not be reactivated under similar conditions; however, the latent HHV-6B could be recovered after the cells were infected with HHV-7. Once reactivated, the HHV-6B genomes became prominent and the HHV-7 disappeared. We conclude that HHV-7 can provide a transacting function(s) mediating HHV-6 reactivating from latency. Understanding the activation process is critical for the development of treatments to control the activation of latent viruses so as to avoid these sometimes life threatening infections in transplant recipients.     

The effects of anti-rheumatoid drugs on the in vitro activity of human serum hyaluronidase

Fourteen antirheumatoid drugs were tested for their effects on the in vitro hyaluronidase activity of normal human serum. Four drugs produced significant changes in enzyme activity. Different results were obtained with ovine testicular hyaluronidase when diluted with either saline or inactivated human serum. No increase in serum hyaluronidase activity was found in patients with rheumatoid arthritis. There was no evidence for the existence of tissue specific isoenzymes of hyaluronidase in the serum of either normal subjects or patients with rheumatoid arthritis.     

Evaluation of methods of sperm preparation for human in vitro fertilization

Congenital absence of useful ilio-caval venous segment is a very infrequent congenital anomaly and makes unfit grafting of a kidney transplant in iliac fossa. We report the case of a 18 years old male affected by this abnormality who was transplanted in intraabdominal situation. We review technical alternatives offered by the literature.     

In vitro fertilization treatment for severe male factor: a comparative study of intracytoplasmic sperm injection with testicular sperm extraction and with spermatozoa from ejaculate

PURPOSE: Our purpose was to evaluate whether the source of spermatozoa influences the results of intracytoplasmic sperm injection (ICSI) treatment in couples with severe male-factor infertility. METHODS: A retrospective analysis of 40 cases of ICSI with testicular-retrieved spermatozoa, matched with 40 cases of ICSI with ejaculated spermatozoa, was performed. We included only couples with normoovulatory females younger than 37 years who were matched according to the day of ovum pickup with the patients in the study group. RESULTS: Eighty cycles were analyzed: 40 cycles using testicular spermatozoa and 40 cycles using ejaculated spermatozoa. In 32 (80%) of the 40 ICSI transcutaneous needle aspiration cycles, we obtained enough spermatozoa to inject all the mature oocytes retrieved. In eight (20%) cases there were not enough spermatozoa to inject all the oocytes. Only 76 (54%) of 141 available oocytes were injected in these eight patients. The oocyte fertilization rates were 42% for the study group and 55.5% for the controls (P < 0.005). Thirty-six (90%) patients in the group with nonobstructive a zoospermia (NOA) and 37 (92.5%) patients in the oligoteratoasthenospermia (OTA) group had embryos for replacement. The mean cleavage rates per cycle (96% with testicular and 93% with ejaculated spermatozoa), the mean number of embryos per transfer (3.72 +/- 1.6 in the NOA group and 4.24 +/- 1.5 in the OTA group), the embryo quality (cumulative embryo scoring = 34.03 +/- 22.62 in the testicular sperm group and 36.08 +/- 19.28 in the ejaculated sperm group), and the clinical pregnancy rates (22.5% in the NOA patients and 20% in the ejaculate group) were not significantly different between groups. CONCLUSIONS: High fertilization, cleavage, and pregnancy rates can be achieved with intracytoplasmic testicular sperm injection from patients with NOA, reaching levels comparable with those of ICSI using ejaculated spermatozoa.     

In vitro stimulation of lymphocytes by neutral proteinases from human polymorphonuclear leukocyte granules

Two neutral proteinases from human polymorphonuclear leukocytes (PMN), an elastase and the chymotrypsin-like cathepsin G, were purified, and their actions on lymphocytes in culture were studied. Both PMN proteinases stimulate lymphocytes from human peripheral blood and from mouse spleen in vitro, but do not affect thymic cells from either normal or hydrocortisone-treated mice. In stimulated mouse spleen cell cultures, most of the developing blast cells bear surface immunoglobulins, and subsequently appear to engage in antibody synthesis. In their stimulatory action, the two PMN proteinases thus resemble the classic B-cell mitogen LPS and neutral pancreatic proteinases such as trypsin, chymotrypsin, and elastase. The effects of proteinase inhibitors indicate that lymphocyte stimulation is dependent on the proteolytic activity of the enzymes. This work suggests that PMN proteinases, which are released at sites of inflammation, may modulate the function of lymphocytes.     

In vitro culture of human peritoneal fluid cells

The author has studied the behaviour of cells from human ascitic fluid in long-term culture (5-6 months). Three cellular types are described with different morphological features, namely the cellular shape, the fashion in which the cell spreads on the glass, the nucleocytoplasmic ratio, the chromatin appearance and the abundance of mitochondria. The three cellular types can phagocytose, but each one in a different way. The first one phagocytoses exclusively erythrocytes 'by contact' without emission of pseudopods; the second one phagocytoses degenerating nucleated cells in the same way as the first; the third type phagocytoses degenerating nucleated cells by emission of long pseudopods. The origin of these three cellular types is discussed; it is felt that they are transformed mesothelial cells. According to this study, it cannot be excluded, especially for the second and the third type, that they are histiocytes coming from serous membranes. The life in vitro of the three cellular types is depending upon the composition of nourishing medium. Cells can divide by mitosis only during the first 10 - 15 days of culture (mitotic index 0.1-3.0(0/00). Nuclear amitosis, nucleolus expulsion into cytoplasm and cytoplasmatic DNA synthesis can be observed in healthy cells.     

In vitro percutaneous penetration of methyl-parathion from a commercial formulation through the human skin

OBJECTIVE: To compare in vitro percutaneous absorption of methyl-parathion dissolved in an acetone vehicle and in the form of a commercial formulation. METHODS: Penetration through the human skin was measured in Franz diffusion cells with full thickness skin from a human cadaver as the membrane. The two tailed non-parametric Mann-Whitney U test was used to compare the cumulative diffusion of methyl-parathion in the receptor fluid of the cells at various time intervals. RESULTS: In vitro skin penetration of methyl-parathion was significantly higher with the commercial formulation. The percentage of the applied dose absorbed after 24 hours was 5.20% v 1.35%. The mean lag time was < 8 hours. CONCLUSION: Assessments of uptake and internal dose after exposure to pesticides should be based on the commercial products rather than active ingredients, because of the crucial role of the vehicle, as shown in this study.     

In vitro growth and drug sensitivity of tumor colony-forming units from human tumor xenografts

To investigate the feasibility of using tissue obtained from human tumor xenografts for in vitro screening of antineoplastic agents, we grew human tumor colony-forming units (CFU) in semisold agar from xenografts serially passaged in nude mice. Growth of human tumor CFU was accomplished from nine xenografts representing five different histological tumor types (ovarian carcinoma, adenocarcinoma of the colon, malignant melanoma, epidermoid carcinoma of the lung, and malignant astrocytoma). Cloning efficiency ranged from 0.04 to 0.1% and showed significant variability both between tumor types and between individual animals bearing the same type of xenograft. A high percentage of tumor CFU was in S phase [47 +/- 20% (S.D.)] as determined by the thymidine "suicide" technique. The number of tumor CFU observed increased linearly with increasing numbers of cells plated. In vitro drug sensitivity of the tumor CFU was assessed to Adriamycin, cis-platinum, and melphalan. The patterns of drug sensitivity were found to be reproducible and stable over a period of 9 months. Drug sensitivity curves to Adriamycin for five xenografts representing four tumor types showed complex patterns with plateau portions similar to those described for tumor CFU from primary tumors. The rank order of sensitivity of the tumors was compared to that of normal granulocyte-macrophage progenitors and, with the exception of the melanomas, was found to correlate well with clinical experience (order of sensitivity = colon less than ovary less than bone marrow). Growth of human tumor CFU from xenografts represents a reproducible and stable means for the study of the biology of tumor CFU and has potential applications as a means for screening new anticancer agents.     

In vitro effects of diazepam on human ciliary function

This study demonstrated the in vitro effect of diazepam on human ciliary beat frequency (CBF) from fifteen normal subjects using brush cytology. Tube A contained culture medium, tube B the diluent that diazepam intravenous injectate was carried in and culture medium (controls). Tube C, D and E contained, 0.4 mg/l, 4.0 mg/l and 40.0 mg/l of diazepam in culture medium respectively. The mean effective diazepam concentration in plasma is 0.4 mg/l. CBF was measured photometrically. The most vigorous cilia were measured in 5 areas taking 10 readings on each sample, 30 minutes and 1 hour after mixing. Standard deviation (SD) and confidence limits were calculated along with significance testing (p < 0.05) using the paired t-test and ANOVA. The mean of the CBF of tubes A and B were 13.44 (SD 2.65) and 13.67 (SD 2.48). There was a reduction of the CBF with increasing concentrations of diazepam at 30 minutes, 11.32 (SD 2.14), 10.29 (SD 1.58) and 4.14 (SD 1.57) tubes C. D and E respectively. There was a significant lowering in CBF of 17% (p < 0.01) of diazepam at the mean effective plasma level (tube C) when compared against the controls. CBF decreased over time and at 1 hour was 10.57 (SD, 1.36), 9.02 (SD, 1.39) and 3.58 (SD, 1.31) tubes C, D and E respectively. A proposed mechanism of altered intracellular calcium flux via the action diazepam on GABA receptors is described.     

Genetics of human sperm

The mechanism by which low doses of methotrexate act in psoriasis to restore a clinically normal skin is poorly understood. Apoptosis is a programmed cell death activated when cell removal is needed. The purpose of the present work was to examine using an organotypical model of keratinocyte culture, the possibility that low doses of methotrexate can induce apoptosis of keratinocytes. Epidermal explants were cultivated on dead deepidermized dermis under air-exposed conditions. After 10 days, methotrexate (10(-7) M) was added. After a further 5 days, one part of each culture was fixed and submitted to routine histology, DNA nick end labelling (TUNEL) to detect DNA fragmentation (a molecular marker of apoptotic cell death) and immunohistochemical detection of p53 (a protein involved in apoptosis induced by DNA-damaging agents). The other part of each culture was processed for electron microscopy. A significant proportion of keratinocytes (1%) were damaged and exhibited the morphological features of apoptotic cell death. Immunohistochemical overexpression of p53 was detected in the basal layer of the cultures treated with methotrexate. Low doses of methotrexate induce apoptosis. This mode of action could explain the reduction in epidermal hyperplasia during treatment of psoriasis with methotrexate.     

Decondensation of sperm nuclei in vitro

The urine of five patients with three distinct diseases ("I Cell disease" and two new types of mucolipidosis) contains sialic acid-rich oligosaccharides in a high amount: 50- to 500-fold the normal. The structure of the major components are as follows: alphaAcNeu(2 leads to 6)betaGal(1 leads to 4)betaGlcNac(1 leads to 2)alphaMan(1 leads to 3)betaMan(1 leads to 4)GlcNac,[alphaAcNeu(2 leads to 6)]betaGal(1 leads to 4)betaGlcNAc(1 leads to 2)alphaMan(1 leads to 3)[betaGal(1 leads to 4)betaGlcNac(1 leads to 2)alphaMan(1 leads to 6)]betaMan(1 leads to 4)GlcNAc and alphaAcNeu(2 leads to 6)betaGal(1 leads to 4)betaGlcNAc(1 leads to 2)alphaMan(1 leads to 3)[alphaAcNeu(2 leads to 6)betaGal(1 leads to 4)betaGlcNAc(1 leads to 2)alphaMan(1 leads to 6)]betaMan(1 leads to 4)GlcNAc. These results suggest that a deficit in alpha-neuraminidase is associated to these three different disorders and that an endo-beta-D-N-acetylglucosaminidase is able to release sialyoligosaccharides by splitting the sialylglycans of glycoproteins.     

Selection of antigens for use in a virus-vectored immunocontraceptive vaccine: PH-20 as a case study

In this work we had assumed to clarify the role of the water structure in the process of the radiation damage of DNA. It is known that the aliphatic alcohols stabilize the water structure until the critical concentration. In this connection we have analyzed the changes of the long- and short-distance interactions in the DNA irradiated in the water-ethanol solutions with various concentration of the ethanol and ions of metal. It was shown that as the water structure becomes more stable the conformational damages in the DNA are decreasing and finally at the some concentration of the alcohol in the irradiated solution the damages disappear. By the achieving of the alcohol concentration which lead to the destruction of the water structure the radiation results in the same changes of the considered parameters as in the case of the DNA irradiated in the water-salt solution with ethanol. The analyses of the experimental data allow us to conclude that the radiation destroys the structure of the water and thus helps the positive ions from the solution to come nearer to the DNA, to say, the radiation reduce intramolecular electrostatic interactions. This concept allows us to explain the observed changes of intrinsic viscosity and the difference in the polarizabilities of the DNA in the process of the radiation damage.     

In vitro infection of human macrophages with human T-cell leukemia virus type 1

The tropism of the human T-cell leukemia virus type 1 (HTLV-1) for the cells of monocyte-macrophage lineage was evaluated by the coculture of blood monocyte-derived macrophages, with irradiated cells of HTLV-1 producing cell lines MT2 or C91/PL. The susceptibility to HTLV-1 was assessed by the detection of viral DNA using the polymerase chain reaction method. HTLV-1 gene expression in the cells was detected using in situ hybridization and by immunofluorescent staining of viral antigen. The presence of type C virus-like particles detected by electron microscopy and the ability to infect normal cord blood lymphocytes demonstrated that the infected macrophages produced infectious virus. These results indicate that human macrophages are susceptible in vitro to productive HTLV-1 infection, and thus might be involved in the pathogenesis of HTLV-1-related diseases.     

In vitro generation of human dendritic cells and cell therapy

HHV-7 growth on Sup-T1, an immature T-cell line, was studied using different HHV-7 isolates obtained in our laboratory. Titration of viral yields showed that all the virus isolates propagate on this cell line more efficiently than in cord blood lymphocytes, the cells usually recommended for HHV-7 growth. The permissivity of Sup-T1 to HHV-6, whose ability to replicate in these cells was still unknown, was also investigated using two virus isolates representative of variants A and B respectively. Both isolates were able to propagate on Sup-T1 and viral titres were similar to those obtained in cord blood lymphocytes. As the efficient propagation of both HHV-7 and HHV-6 isolates in Sup-T1 cultures, these cells may replace more time consuming and expensive cord blood lymphocyte preparations for the propagation of both the viruses.     

In vitro receptor imaging for characterization of human solid tumors

Artificial neural networks have been applied to a variety of pattern recognition tasks in medical imaging and have been shown to be a powerful classification tool. The potential usefulness to discriminate normal from abnormal cerebral perfusion patterns was investigated. METHODS: Cerebral perfusion imaging with 99mTc-labeled hexamethylpropyleneimine oxime was performed on 52 normal control subjects, 29 patients with clinically diagnosed Alzheimer's disease (AD) and 25 patients with chronic cocaine polydrug abuse. Each study was registered and scaled to a common anatomic coordinate system, yielding 120 standardized cortical regions. A back-propagation neural network classifier based on regional perfusion was used to classify normal and abnormal perfusion patterns. The neural network was trained to discriminate patients with AD from age-matched normal controls and cocaine polydrug abuse patients from normal controls. The performance of the neural network in these two tasks was evaluated quantitatively by receiver operating characteristic (ROC) analysis using cross-validation. RESULTS: For patients with AD, the area under the ROC curve was 0.93 +/- 0.04. When testing with the cocaine polydrug abuser data, the area under the ROC curve was 0.89 +/- 0.04. CONCLUSION: Neural networks provide a potentially useful tool in the decision-making task to discriminate patients with AD and cocaine abuse from normal controls.     

In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines.