Effect of in vitro vitamin E (alpha-tocopherol) supplementation in human spermatozoon submitted to oxidative stress.

The aim of this study was to evaluate the antioxidant effect of in vitro supplementation with vitamin E in human spermatozoon incubated with an oxidative stress inducer. In this study, semen samples from 30 patients were collected and with one aliquot we performed semen analysis according to WHO. The remaining volume was divided into four aliquots: group C: incubated with BWW medium; group I: incubated with 5 mmol 1-1 hydrogen peroxide; group A: incubated with 40 µmol 1-1 vitamin E; and group AI: incubated with both them. After incubations, sperm functional analyses were performed and included: evaluation of oxidative stress, acrosome integrity, mitochondrial activity and DNA fragmentation. Groups were compared using a Friedman test with Bonferroni post hoc (α = 5%). In this study, we observed that in group I there was a decrease in acrosome integrity and mitochondrial activity, and an increase in DNA fragmentation, when compared to group C. Group AI showed an increase in acrosome integrity and mitochondrial activity when compared with group I. Based on our findings, we conclude that the vitamin E supplementation had a positive effect in protecting human spermatozoon from induced oxidative stress.

Measuring spirituality in South Africa: Validation of instruments developed in the USA.

Spirituality is receiving increased attention in the context of the workplace. Research consistently shows that spirituality is significantly correlated with mental health and well-being. Most of the research on spirituality, particularly in the context of the workplace, is conducted with instruments developed in the USA. However, the inter-cultural measurement of constructs remains a concern, because instruments developed in one culture are not necessarily transferable to another culture. In the current study, the transferability of two spiritual measures developed in the USA, namely the Human Spirituality Scale (HSS) and the Organizational Spirituality Values Scale (OSVS) are considered for a sample from South Africa. The results confirm the construct validity of the HSS and the OSVS, but indicate that the factor structures of the HSS and the OSVS should be analysed and reconfirmed when used, particularly in a South African sample. The study provides evidence that the HSS and the OSVS cannot be transferred indiscriminately to a South African sample. This insight contributes to the quality of future research studies in South Africa, not only on the important aspect of spirituality, but also when applying instruments developed elsewhere in the world.

Workplace spirituality and job satisfaction.

In order to obtain an improved understanding of behaviour at work, employees should be studied from physical, psychological, and spiritual dimensions. Although the physical and psychological dimensions of individuals at work have been studied extensively, the spiritual dimension has been neglected for many years. The objective of the current research was to determine the relationship between workplace spirituality and a positive attitude related to work, that is, job satisfaction. A cross-sectional study was conducted with a sample of 600 white-collar workers, chosen from two organizations in different industries in South Africa. The research results indicate that there is a positive relationship between workplace spirituality and job satisfaction. These findings deepen the understanding of personal spirituality, organizational spirituality, and job satisfaction. They bring new insights into the significant role which spirituality plays in the context of the workplace. To survive in the 21st century, organizations need to be spiritually based. This, in turn, will lead to workers being satisfied with their entire work experience.

Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity.

Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.

Effects of six carbohydrate sources on dog diet digestibility and post-prandial glucose and insulin response.

The effects of six extruded diets with different starch sources (cassava flour, brewer's rice, corn, sorghum, peas or lentils) on dog total tract apparent digestibility and glycemic and insulinemic response were investigated. The experiment was carried out on thirty-six dogs with six dogs per diet in a completely randomized design. The diets containing brewer's rice and cassava flour presented the greatest digestibility of dry matter, organic matter and gross energy (p < 0.05), followed by corn and sorghum; pea and lentil diets had the lowest. Starch digestibility was greater than 98% in all diets and was greater for brewer's rice and cassava flour than for lentils and peas diets (p < 0.05). Dogs' immediate post-prandial glucose and insulin responses (AUC < or = 30 min) were greater for brewer's rice, corn, and cassava flour diets (p < 0.05), and later meal responses (AUC > or = 30 min) were greater for sorghum, lentil and pea diets (p < 0.05). Variations in diet digestibility and post-prandial response can be explained by differences in chemical composition of each starch source including fibre content and starch granule structure. The nutritional particularities of each starch ingredient can be explored through diet formulations designed to modulate glycemic response. However, more studies are required to support these.

Individual immunity and susceptibility to female genital tract infection.

The responses to genital tract infection vary among different women to a far greater extent than has previously been appreciated. All women are not genetically identical and have not been exposed to identical environments; therefore it is naive to expect that a particular microorganism will elicit the identical response and have the identical sequelae for each infected individual. The genes inherited from one's parents, which contain specific polymorphisms in immune response genes, greatly influence the direction and magnitude of the immune response to microorganisms. Similarly, extrinsic variables, such as the type or quantity of a specific infection, whether there is a coinfection with another microorganism, such as an intracellular parasite, and whether an immediate hypersensitivity response is concurrently induced also determine the nature of the host response and thus the consequences of microbial exposure. Finally, factors such as the frequency of sexual intercourse and previous immune sensitization to spermatozoa or other components of a particular ejaculate also influence the outcome. An increased awareness of the uniqueness of each host will lead to the development of more precise individualized treatments and improvements in combating infectious diseases of the female genital tract.

Testing for high-risk human papillomavirus types will become a standard of clinical care.

Testing for high-risk human papillomavirus types should become a standard of care for women in the United States because cervical cancer is an infectious disease. Current care is based on cytologic screening and a pathologic staging of cellular tissue changes. Before these cellular modifications, there is a demonstrable pattern of human papillomavirus infection. Human papillomavirus is the most frequently acquired sexually transmitted disease in women and is usually eliminated without treatment. Persistence of high-risk human papillomavirus types can lead to abnormal cervical cellular changes. When these cervical cellular changes occur, physician interventions hasten human papillomavirus elimination. Currently, adding human papillomavirus screening to the Papanicolaou smear identifies a population for closer follow-up studies. In the future a vaccine should be introduced to prevent infections, and medical treatments to hasten the elimination of high-risk human papillomavirus types should become part of standard medical practice.

Ureaplasma urealyticum colonization in the vaginal introitus and cervix of human immunodeficiency virus-infected women.

Ureaplasma urealyticum colonization was examined in paired cervical and introital specimens from 56 untreated HIV-seropositive women. Specimens were tested for U. urealyticum by polymerase chain reaction (PCR). Peripheral blood was examined for CD4 lymphocyte counts and HIV RNA concentration. U. urealyticum was detected in the cervix of 38 (69.1%) women. Introital U. urealyticum was present in 16 (28.6%) women, all of whom were cervical-positive. Cervical motion pain was present in 40.0% of women with U. urealyticum in the introitus and cervix, 23.8% of those with only cervical U. urealyticum and 5.9% of women negative for this organism (P=0.03). There was no relation between U. urealyticum colonization and CD4 lymphocyte count. However, 64.3% of women with introital U. urealyticum had circulating HIV RNA levels > 10,000 copies per ml as compared with 28.6% of women with only cervical U. urealyticum and 7.1% of women negative for this organism (P < 0.01).

Interleukin 1 receptor antagonist gene polymorphism in women with vulvar vestibulitis.

OBJECTIVE: Vulvar vestibulitis is a chronic inflammatory syndrome of unknown cause and pathogenesis. We examined the relation between vulvar vestibulitis and polymorphisms in the gene coding for the interleukin 1 receptor antagonist, a naturally occurring down-regulator of proinflammatory immune responses. STUDY DESIGN: Cells from the lower genital tract of 68 women with vulvar vestibulitis, 343 women with no history of vulvodynia, and 40 women with human papillomavirus cervical infection were tested by polymerase chain reaction for the different alleles of the gene encoding for interleukin 1 receptor antagonist. The presence of human papillomavirus in the specimens was determined by polymerase chain reaction with the use of degenerate consensus primers to the conserved L1 gene. RESULTS: Allele 2 of the gene encoding the interleukin 1 receptor antagonist was present in homozygous form in 52.9% of women with vulvar vestibulitis. In marked contrast only 8. 5% of the control women and 2.5% of women with human papillomavirus were homozygous for this allele (P

Effect of human seminal fluid on production of messenger ribonucleic acid for metalloproteinase 2 and metalloproteinase 9 in cervical epithelial carcinoma cells.

OBJECTIVES: The risk for cervical cancer is related to sexual intercourse. One factor associated with tumor invasion and metastases is the production of matrix metalloproteinases, which degrade the extracellular matrix. The ability of human semen to influence production of messenger ribonucleic acid for 2 matrix metalloproteinases associated with cervical cancer, MMP-2 and MMP-9, was examined. STUDY DESIGN: Seminal fluids from 16 men were diluted 1:50 and incubated with the cervical carcinoma cell line HeLa for 18 hours. Cells were harvested, ribonucleic acid was isolated and reverse transcribed into complementary deoxyribonucleic acid, and deoxyribonucleic acid sequences coding for regions of the genes for MMP-2 and MMP-9 were generated by polymerase chain reaction and detected by enzyme-linked immunosorbent assay. RESULTS: All 16 semen samples inhibited production of MMP-2 messenger ribonucleic acid. The MMP-2 messenger ribonucleic acid synthesis in the presence of semen was a mean of 39.5% of the control value. In contrast, MMP-9 messenger ribonucleic acid synthesis was stimulated (mean stimulation, 68.3%) by semen in 10 of the 15 samples tested. Synthesis of beta-actin was comparable in each culture, and cell viability was unaffected by the diluted semen. CONCLUSION: The capacity of semen to influence transcription of the genes for matrix metalloproteinases may be one mechanism whereby sexual activity influences cervical cancer progression.

Relationship between Ureaplasma urealyticum vaginal colonization and polymorphism in the interleukin-1 receptor antagonist gene.

The relationship between polymorphisms in the interleukin-1 receptor antagonist (IL-1ra) gene and microbial vaginal colonization was examined in 88 asymptomatic women of reproductive age. Alleles of the intron 2 region of the IL-1ra gene were identified by polymerase chain reaction (PCR). PCR was also used to detect Ureaplasma urealyticum, Mycoplasma hominis, and Candida albicans; bacterial vaginosis (BV) was identified by clinical criteria. Among the 31 women with vaginal U. urealyticum, only 3 (9.7%) were homozygous for allele 2 of the IL-1ra gene; 21 (36.8%) of the 57 women who were negative for this organism were positive for allele 2 (P=.006). Only 7 women were positive for M. hominis; none were allele 2 homozygotes as opposed to 24 (29.6%) of the 81 women negative for M. hominis. There was no relation between C. albicans or BV and any IL-1ra allele. Reduced susceptibility to vaginal colonization with mycoplasmas may be associated with homozygosity of the 2 allele of the IL-1ra gene.

Heat shock protein expression during gametogenesis and embryogenesis.

When cells are subjected to various stress factors, they increase the production of a group of proteins called heat shock proteins (hsp). Heat shock proteins are highly conserved proteins present in organisms ranging from bacteria to man. Heat shock proteins enable cells to survive adverse environmental conditions by preventing protein denaturation. Thus the physiological and pathological potential of hsps is enormous and has been studied widely over the past two decades. The presence or absence of hsps influences almost every aspect of reproduction. They are among the first proteins produced during mammalian embryo development. In this report, the production of hsps in gametogenesis and early embryo development is described. It has been suggested that prolonged and asymptomatic infections trigger immunity to microbial hsp epitopes that are also expressed in man. This may be relevant for human reproduction, since many couples with fertility problems have had a previous genital tract infection. Antibodies to bacterial and human hsps are present at high titers in sera of many patients undergoing in vitro fertilization. In a mouse embryo culture model, these antibodies impaired the mouse embryo development at unique developmental stages. The gross morphology of these embryos resembled cells undergoing apoptosis. The TUNEL (terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling) staining pattern, which is a common marker of apoptosis, revealed that embryos cultured in the presence of hsp antibodies stained TUNEL-positive more often than unexposed embryos. These data extend preexisting findings showing the detrimental effect of immune sensitization to hsps on embryo development.

Induction of heat shock protein expression in cervical epithelial cells by human semen.

OBJECTIVE: The 70kD heat shock protein (Hsp70), induced when cells are subjected to environmental stress, prevents the denaturation and incorrect folding of polypeptides and may expedite replication and transmission of DNA and RNA viruses. We analyzed whether messenger RNA (mRNA) for Hsp70 was expressed following exposure of a cultured human cervical cell line (HeLa cells) to human semen or in cervical cells from sexually active women. STUDY DESIGN: HeLa cells were co-cultured with a 1:50 dilution of semen from four men or with purified spermatozoa or cell-free seminal fluid. Endocervical swabs were acquired at mid-cycle from 53 women. Heat shock protein 70 mRNA was detected by a reverse transcriptase-polymerase chain reaction utilizing specific primer pairs and analysis on agarose gels. In cervical cells Hsp70 mRNA was measured identically followed by hybridization with an Hsp70-specific internal probe and detection by enzyme-linked immunosorbent assay (ELISA). Cervical immunoglobulin A (IgA) antibodies to the human Hsp70 were determined by ELISA. RESULTS: HeLa cell-semen co-culture resulted in the induction of Hsp70 mRNA. In addition, cell-free seminal plasma and motile sperm incubated individually with HeLa cells also induced this mRNA. Heat shock protein 70 mRNA was detected in 28 (52.8%) of 53 endocervical samples obtained from women at various time points following intercourse. The percentage of samples expressing this mRNA was 37.5% at less than 10 hours, 64.3% at 10 hours, 70% at 11 hours, and between 36% and 50% at later times after semen exposure. The detection of cervical IgA antibodies to the Hsp70 was highly associated with Hsp70 gene transcription. CONCLUSION: Human semen induces transcription of Hsp70 in cervical epithelial cells.

Human semen induces interleukin 10 and 70 kDa heat shock protein gene transcription and inhibits interferon-gamma messenger RNA production in peripheral blood mononuclear cells.

The influence of semen on immunity in sexually active women has been scarcely studied. The effect of human semen on production of messenger RNA (mRNA) for the anti-inflammatory TH2-related cytokine, interleukin-10 (IL-10), the 70 kDa heat shock protein (HSP70) and the pro-inflammatory TH1-related cytokine, interferon-gamma (IFN-gamma) was examined. Co-incubation of peripheral blood mononuclear cells (PBMC) from 10 women with a non-cytotoxic 1:50 dilution of semen lead to induction of IL-10 mRNA. Semen from each of seven different men tested induced IL-10 mRNA in PBMC. IL-10 protein was also released into the culture supernatant after PBMC-semen co-culture. Similarly, semen induced transcription of the HSP70 gene in PBMC obtained from 10 women. In contrast, semen did not induce IFN-gamma mRNA in any of the female PBMC donors. Furthermore, semen markedly inhibited IFN-gamma mRNA production without affecting cell viability in PBMC that were cocultured with phytohaemagglutinin, a potent IFN-gamma-inducing T-cell mitogen. Thus, human semen is both an inducer of an anti-inflammatory (TH2) immune response and an inhibitor of pro-inflammatory (TH1) cell-mediated immunity.

Cell-mediated immunity to human and Escherichia coli 60-kDa heat shock protein in women: association with a history of spontaneous abortion and endometriosis.

PROBLEM: Heat shock proteins are expressed during early pregnancy and in peritoneal fluids from women with endometriosis. The relationship between a cell-mediated immune response to human 60-kDa heat shock protein (hsp60), spontaneous abortion, and endometriosis was examined. METHOD OF STUDY: Peripheral blood mononuclear cells (PBMCs) from 110 female partners of infertile couples undergoing in vitro fertilization and 41 fertile control subjects were incubated with human hsp60 or Escherichia coli hsp60. PBMC proliferation was measured by [3H]thymidine incorporation, and a stimulation index was calculated. RESULTS: Lymphocytes from 21.8% of the infertile women, as opposed to 7.3% of the fertile women, proliferated in response to human hsp60 (P = 0.05). In contrast, proliferation in response to the E. coli hsp60 was equivalent in both groups. Within the infertile group, the response to human hsp60 was 40.7% among women with a history of spontaneous abortion and only 12% in those with no history of spontaneous abortion (P = 0.003). There was no association between immunity to E. coli hsp60 and spontaneous abortion or between immunity to human hsp60 and therapeutic abortion or the cause of infertility. Immunity to the E. coli hsp60 was associated with endometriosis. CONCLUSIONS: A cell-mediated autoimmune response to human hsp60 is associated with a history of spontaneous abortion, whereas immunity to E. coli hsp60 was most prevalent in women with endometriosis.

Induction of messenger RNA for the 70 kDa heat shock protein in HeLa cells and the human endocervix following exposure to semen: implications for antisperm antibody production and susceptibility to sexually transmitted infections.

The 70 kDa heat shock protein (HSP70) is induced in cells exposed to chemical or physical stress. HSP70 facilitates cell survival by preventing protein denaturation and incorrect assembly of polypeptides. Induction of HSP70 messenger RNA (mRNA) synthesis also inhibits transcription of genes coding for pro-inflammatory cytokines. We analyzed whether HSP70 mRNA was expressed in a cultured human cervical cell line (HeLa cells) following exposure to human semen, or in cells obtained from the endocervices of sexually active women. HeLa cells were co-cultured with a 1:50 dilution of semen from four men, with purified spermatozoa, or with cell-free seminal fluid. Endocervical swabs were obtained at mid-cycle from 53 women. HSP70 mRNA was detected in HeLa cells by a reverse transcriptase-polymerase chain reaction (RT-PCR) and analysis on agarose gels. HSP70 mRNA in cervical cells was measured by RT-PCR followed by hybridization with an HSP70-specific internal probe and detection by ELISA. Cervical IgA antibodies to HSP70 were measured by ELISA. HeLa cell-semen co-culture led in each case to induction of HSP70 mRNA. Cell-free seminal fluid and isolated motile spermatozoa also induced HSP70 mRNA when incubated individually with HeLa cells. HSP70 mRNA was detected in 28 (52.8%) of 53 endocervical cell samples obtained from women at varying times after sexual intercourse. The percentage of samples expressing HSP70 mRNA was 37.5% at <10 h, 64.3% at 10 h, 70.0% at 11 h and between 36 and 50% at later times after semen exposure. Cervical IgA antibodies to HSP70 were also detected in some women and their occurrence was highly correlated with HSP70 gene transcription (P < 0.0001). The data demonstrate that exposure to semen induces HSP70 mRNA in endocervical cells.

Chlamydia trachomatis Infection, Immunity, and Pregnancy Outcome.

Chlamydia trachomatis can ascend from the cervix to the fallopian tubes and survive for long periods of time without causing symptoms. The immune response to infection clears the extracellular organisms but leads to development of a persistent intracellular infection. Repeated cycles of productive infection and persistence eventually induce tubal occlusion and infertility. Persistently infected cells continue to synthesize the chlamydial 60 kD heat shock protein (hsp60). Immunity to conserved regions of hsp60 may result in autoimmunity to human hsp60. Expression of hsp60 by the embryo and decidua during early pregnancy may reactivate hsp60-sensitized lymphocytes, disturb pregnancy-induced immune regulatory mechanisms, and lead to immune rejection of the embryo. Due to this mechanism women with tubal infertility who are sensitized to the human hsp60 may have a decreased probability of successful outcome after undergoing in vitro fertilization and embryo transfer.

The 60 kDa heat shock protein in human semen: relationship with antibodies to spermatozoa and Chlamydia trachomatis.

The presence of the 60 kDa heat shock protein (hsp60) in seminal fluid and its relationship to sperm autoimmunity or a localized immune response to Chlamydia trachomatis were examined. Semen from 64 male partners of infertile couples with no history of a chlamydial infection were investigated. Hsp60 was identified by an enzyme-linked immunosorbent assay (ELISA) using a monoclonal anti-hsp60 antibody bound to wells of a microtitre plate and a polyclonal anti-hsp60 antibody for detection. Antisperm antibodies on motile spermatozoa were detected by immunobead binding, while antichlamydial immuno-globulin (Ig) A and IgG in seminal fluid were identified by a commercial ELISA (SeroELISA: Savyon Diagnostics, Beer-Sheva, Israel). RNA was purified from isolated seminal round mononuclear cells and tested for hsp60-specific mRNA by a reverse transcription polymerase chain reaction ELISA. Hsp60 was present in semen from nine (14.1%) men, 12 (18.8%) men had antisperm autoantibodies. 16 (25.0%) were positive for antichlamydial IgA and 17 (26.6%) had detectable hsp60-specific mRNA. The presence of hsp60 in semen correlated with the occurrence of antichlamydial IgA (P = 0.0005), hsp60 mRNA (P = 0.04) and antisperm antibodies (P = 0.05). Thus, hsp60 was present in a soluble form in semen primarily in men with evidence of immune system activation within their genital tract. The role of hsp60 in promoting or inhibiting immune responses within the genital tract remains to be determined.