Effect of the metabolic syndrome on male reproductive function: a case-controlled pilot study.

The metabolic syndrome (MetS) is a constellation of various risk factors. This study aimed to investigate the effect of MetS on testosterone and progesterone, and semen parameters, in a case-controlled pilot study. Male patients (n = 54) had body mass index, waist-to-hip ratio (WHR) and blood pressure recorded. Blood was analysed for HDL cholesterol, triglycerides and glucose. Saliva was assayed for free testosterone and free progesterone. Ejaculates were analysed for volume, sperm concentration, total sperm count, motility, vitality, mitochondrial membrane potential (MMP), DNA fragmentation and leucocyte concentration. Participants were divided into the control group (n = 28) and the MetS group (n = 26). Differences were found between the groups for body mass index, WHR, blood pressure, high-density lipoprotein (HDL), triglycerides and glucose. The MetS group showed significant reductions in sperm concentration (P = 0.0026), total sperm count (P = 0.0034), total motility (P = 0.0291), sperm vitality (P = 0.002), MMP (P = 0.0039), free testosterone (P = 0.0093) and free progesterone (P = 0.0130), while values for DNA fragmentation increased (P = 0.0287). Results indicate that patients with MetS have compromised sperm parameters in the absence of leucocytospermia. A reduction in free progesterone suggests that steroidogenesis cascades may be compromised. It is hypothesised that a systemic pro-inflammatory state with oxidative stress associated with MetS may provide a novel explanation.

Plasma sarcosine does not distinguish early and advanced stages of prostate cancer.

INTRODUCTION: Diagnosis of prostate cancer by prostate specific antigen (PSA) is error-prone and cannot distinguish benign prostatic hyperplasia (BPH) from malignant disease, nor identify aggressive and indolent types. METHODS: We determined serum sarcosine (N-methylglycine) in 328 cancer patients by gas chromatography (GC)/mass spectroscopy (MS) and searched for correlations with early (stage T1/T2) and advanced (stage T3/T4) disease. RESULTS: Serum sarcosine of male control patients ranged from 1.7 µmol/l to 4.8 µmol/l. In prostate cancer patients, sarcosine ranged from 2.8 µmol/l to 20.1 µmol/l. Expressed as the sarcosine/alanine ratio, serum control values were 9.4 ± 5.5 x 10(-3) (mean ± SD) compared with 21.6 ± 9.0; 28.5 ± 16.6; 22.7 ± 7.7 and 22.2 ± 11.0 for patients diagnosed with T1, T2, T3 and T4 prostate tumours, respectively. The small differences between T1, T2, T3 and T4 patients were not statistically significant (p=0.51). However, the conventional PSA marker significantly correlated with T stage in these patients (r=0.63; p<0.009). CONCLUSIONS: The median sarcosine/alanine ratios among patients with early and advanced prostatic cancer ranged from 21.6 ± 9.0 to 28.5 ± 16.6 and were fairly constant, showing no statistically significant differences between T-stages. The results are consistent with published data in urine and serum which find differences between controls and patients with metastatic prostate cancer to be small and sarcosine to be uninformative regarding prostate cancer progression. By multi-comparison of PSA with T-stages in the same group of patients, we found significant correlations confirming the well-known merits and limitations of this marker.

TUNEL assay and SCSA determine different aspects of sperm DNA damage.

For the determination of sperm DNA damage, different assays are used. However, no further distinction is made and the literature generally speaks about DNA damage. Thus, this study aimed at comparing the sperm chromatin structure assay (SCSA) and the TUNEL assay. In 79 patients, sperm DNA damage was determined flow cytometrically using the SCSA and the TUNEL assay. Moreover, normal sperm morphology was evaluated according to strict criteria. A statistical comparison of the two methods was performed using standard correlations, Bland and Altman plots, Passing-Bablok regressions and concordance correlation. Results show a significant difference between P- and G-pattern morphology only for the mean channel fluorescence of the SCSA. Spearman's rank correlations between the different parameters of both assays, SCSA and TUNEL, revealed significant associations between the parameters of the assays. However, when applying Bland and Altman plots, Passing-Bablok regression and concordance correlation results showed that these methods are not comparable. These different techniques determine different aspects of sperm DNA damage, i.e. 'real' DNA damage for the TUNEL assay and 'potential' DNA damage in terms of susceptibility to DNA denaturation for the SCSA. Thus, one should clearly distinguish between the different assays, not only practically and methodologically but also linguistically.

The role of phytosterols and phytosterolins in immune modulation: a review of the past 10 years.

Although plant sterols (phytosterols) were chemically described in 1922, their biological role in human and animal health has been underestimated. Their ability to control cholesterol plasma levels in hypercholesterolimic patients was first described in 1983 when the structure of phytosterols implied that they could, by steric hindrance, inhibit the absorption of cholesterol from our diets. This has led to the development of functional foods containing high contents of these plant molecules or their esters as cholesterol controlling foods. Over the last 15 years, however, several reports have appeared in the literature indicating that phytosterols have some immunological activity as highlighted in animal models of inflammation or even in in-vitro and in-vivo models of cancer (colorectal and breast cancer). These findings were paralleled by epidemiological studies correlating the reduced risk of numerous diseases and the dietary intake of phytosterols. It is only in the last 10 years, however, that their direct immune modulatory activity on human lymphocytes has been proven and the mechanism of action in cancer cells has been elucidated. The use of phytosterols as supportive therapies in certain chronic conditions has been tested under clinical trial conditions. This review presents a summary of the in-vitro and in-vivo studies published to date.

Major histocompatibility complex class II invariant chain expression in non-antigen-presenting cells.

In contrast to the generally accepted belief, the major histocompatibility complex (MHC) class II invariant chain (Ii) is commonly expressed intracellularly in cells that do not present exogenous antigens. Such cells include resting peripheral blood T cells and natural killer (NK) cells. In T cells, the Ii is associated with a 77 000 molecular-weight molecule (p77) that has yet to be identified. This molecule is co-precipitated with the anti-Ii monoclonal antibody (mAb) VCD-1, but not with mAb BU-45. This suggests that in the p77-Ii complex, the extracellular epitope of Ii recognized by BU-45 is hidden, whereas the Ii epitope for VCD-1 remains exposed. In antigen-presenting cells (APCs), p77 association with the Ii was minimal, if detectable. The p77-Ii association in non-professional APCs suggests that the Ii may have another, more general, function other than the one accepted in antigen presentation.

The effects of B-sitosterol (BSS) and B-sitosterol glucoside (BSSG) mixture on selected immune parameters of marathon runners: inhibition of post marathon immune suppression and inflammation.

A pilot study was undertaken to investigate the effects of the intake of capsules containing the plant sterols and sterolins (BSS:BSSG mixture) on selected immune parameters of volunteers participating in an ultra-marathon in Cape Town, South Africa. Those runners having received active capsules (n=9) showed less neutrophilia, lymphopenia and leukocytosis when compared to their counterparts having received placebo capsules (n=8): the placebo treated individuals showed significant increases in their total white blood cell numbers as well as in their neutrophils (p=0.03 and 0.03 respectively). Furthermore, statistically significant increases within lymphocyte subsets were observed in the runners having received the active capsules: CD3+ cells increased (p=0.02) as did CD4+ cells (p=0.03). In parallel, the BSS:BSSG capsules decreased the plasma level of IL6 in the runners using the active capsules (p=0.08) and significantly decreased the cortisol: DHEAs ratio (p=0.03), suggesting that these volunteers had less of an inflammatory response and were less immune suppressed during the post-marathon recovery period. These findings justify further investigations into the use of the phytosterols to prevent the subtle immunosuppression associated with excessive physical stress.

Plant sterols and sterolins: a review of their immune-modulating properties.

Beta-sitosterol (BSS) and its glycoside (BSSG) are sterol molecules which are synthesized by plants. When humans eat plant foods phytosterols are ingested, and are found in the serum and tissues of healthy individuals, but at concentrations orders of magnitude lower than endogenous cholesterol. Epidemiological studies have correlated a reduced risk of numerous diseases with a diet high in fruits and vegetables, and have concluded that specific molecules, including b-carotene, tocopherols, vitamin C, and flavonoids, confer some of this protective benefit. However, these epidemiologic studies have not examined the potential effect that phytosterols ingested with fruits and vegetables might have on disease risk reduction. In animals, BSS and BSSG have been shown to exhibit anti-inflammatory, anti-neoplastic, anti-pyretic, and immune-modulating activity. A proprietary BSS:BSSG mixture has demonstrated promising results in a number of studies, including in vitro studies, animal models, and human clinical trials. This phytosterol complex seems to target specific T-helper lymphocytes, the Th1 and Th2 cells, helping normalize their functioning and resulting in improved T-lymphocyte and natural killer cell activity. A dampening effect on overactive antibody responses has also been seen, as well as normalization of the DHEA:cortisol ratio. The re-establishment of these immune parameters may be of help in numerous disease processes relating to chronic immune-mediated abnormalities, including chronic viral infections, tuberculosis, rheumatoid arthritis, allergies, cancer, and auto-immune diseases.

A randomised placebo-controlled trial of the efficacy of beta-sitosterol and its glucoside as adjuvants in the treatment of pulmonary tuberculosis.

OBJECTIVE: To evaluate the adjuvant effect of beta-sitosterol and its glucoside in the treatment of culture proven pulmonary tuberculosis (PTB). DESIGN: A blinded randomised placebo-controlled trial in culture proven drug sensitive PTB. Patients were hospitalised for the duration of treatment and evaluated at monthly intervals with regard to sputum culture positivity, chest radiography, weight gain, Mantoux test response, routine haematology and liver functions. STATISTICAL EVALUATION: General linear models for repeated measures (SAS GLM package) compared the interaction effects, group effects and time effects of findings in 19 patients receiving sitosterols with those in 18 patients receiving a placebo (talcum powder). Absolute values and change from baseline values were evaluated, although only the latter are reported. RESULTS: Weight gain was significantly greater in the sitosterol group (mean weight gain 8.9 kg) than the placebo group (mean gain 6.1 kg) (P = 0.0023 group effects; P = 0.0001 for time effects). Speed of achieving culture negativity, radiological improvement and induration on Mantoux testing was similar in the two groups. Change in lymphocyte counts from baseline was significantly higher in the sitosterol group (P = 0.0001 and P = 0.0001 for group and time effects) as was the increase in eosinophil counts (P = 0.0001 and P = 0.0137 for group and time effects). CONCLUSION: The study has shown significantly improved weight gain and higher lymphocyte and eosinophil counts in PTB patients receiving sitosterols in addition to an efficacious antituberculosis regimen. Sitosterols and their possible mode of action should now be evaluated in larger numbers of tuberculosis patients and in diseases with a similar immunopathogenesis.

beta-Sitosterol and beta-sitosterol glucoside stimulate human peripheral blood lymphocyte proliferation: implications for their use as an immunomodulatory vitamin combination.

The phytosterols, beta-sitosterol (BSS), and its glucoside (BSSG) enhance the in vitro proliferative response of T-cells stimulated by sub-optimal concentrations of phytohaemagglutinin (PHA) several fold at extremely low concentrations (femtogram level). A 100:1 (mass:mass) ratio of BSS:BSSG (termed essential sterolin formulation, ESF) showed higher stimulation than the individual sterols at the same concentration. In vivo activity of ESF was also demonstrated when volunteers ingested ESF for 4 weeks. Proliferation of their T-cells, stimulated maximally with PHA, was significantly enhanced (20-920%) when compared to baseline values. In vitro, ESF (1 microgram.ml) was able to significantly enhance the expression of CD25 and HLA-Dr activation antigens on T-cells and increased the secretion, into the medium, of IL-2 and gamma interferon. NK-cell activity was also increased by BSS and BSSG alone, but with EST a higher activity was always found at different effector:target ratios (100:1 12:1).

A phase I trial of hypoxoside as an oral prodrug for cancer therapy--absence of toxicity.

OBJECTIVE: To assess the toxicity of hypoxoside taken orally by 24 patients with lung cancer. DESIGN: Open study with patients taking 1,200-3,200 mg standardised Hypoxis plant extract (200 mg capsules) per day divided in 3 doses in order to maintain metabolite blood levels near 100 micrograms/ml. PARTICIPANTS AND SETTING: Patients with histologically proven squamous, large-cell or adenocarcinoma were hospitalised initially at the radiation oncology ward, Karl Bremer Hospital, Bellville, W. Cape. Thereafter they returned every 2 weeks for full clinical examinations. METHODS: Routine biochemical and haematological measurements were done. Patients underwent regular full clinical examinations including radiographs and computed tomography scanning according to the discretion of the principal investigator. RESULTS: Nineteen patients on hypoxoside therapy survived for an average of 4 months with progression of their primary tumours and metastases, while 5 survived for more than a year. One of them survived for 5 years and histological examination of the primary lesion showed absence of cancer. No toxic effects, in clinical examinations or biochemical or haematological measurements, were found that could be ascribed to the ingestion of hypoxoside. Only one occasion of possible drug intolerance, with anxiety, nausea, vomiting and diarrhoea, was noted. CONCLUSION: The absence of toxicity warrants further investigation of hypoxoside as an oral prodrug, especially in patients with slow-growing necrotising tumours that are inoperable and have high concentrations of beta-glucuronidase and sulphatase as well as a high sensitivity for rooperol.

Analysis of human sperm membrane antigens reacting with sera from antisperm antibody positive and negative patients by western blotting.

Immunological infertility is thought to be caused by the binding of antibodies to 'fertility-related' antigen(s) on the sperm membrane. We compared antibody profiles in sera from 20 ASA(+) and ASA(-) men, using a sperm membrane extract as an antigen. Antigens were separated by SDS-PAGE under reducing conditions. The patients were classed as ASA(+) by the MAR (> 50%), d-IBT (> 20%) and TAT (> 1:64). The results showed that immunoreactive bands in both the ASA(+) and ASA(-) groups were heterogeneous and included bands covering the whole molecular weight range. Statistical analysis showed significantly more patients in the ASA(+) group having immunoreactive bands at molecular weights of 32 Kd (P = 0.006) and 79 Kd (P = 0.02) when compared to the ASA(-) group. In the ASA(-) group significantly more patients had reactive bands at 81 Kd (P = 0.01) when compared to the ASA(+) group. The 32 Kd antigen reacted only with sera from ASA(+) patients. We conclude that differences exist between the ASA(+) and ASA(-) groups when this extraction method is used and that the isolation and purification of the 32 Kd protein may justify further investigation.

Peripheral blood lymphocyte response in patients with superficial transitional cell carcinoma of the bladder treated with intravesical Bacillus Calmette-Guérin--a useful marker of response?

Intravesical instillation of Bacillus Calmette-Guérin (BCG) offers safe and effective short-term/long-term treatment for superficial transitional cell carcinoma (TCC) and TCC in situ of the bladder. However, 17 to 42% of patients may experience recurrence in spite of this therapy and a marker of effective treatment is of paramount importance. In this study the in vitro response of peripheral blood lymphocytes (PBL) to BCG was analysed in 10 patients with superficial TCC and TCC in situ before and during BCG instillations. The in vitro response of PBL to BCG, expressed as a stimulatory index (SI), revealed that 6 patients had a SI > 5 and 4 patients had a SI < 5. None of the former patients had recurrence of TCC during a mean follow-up of 17 months, while all of the latter patients experienced recurrence of TCC within 9 months. It was concluded that the in vitro response of PBL to BCG may be used as a marker of response to intravesical BCG treatment.

The influence of solubilized porcine zona pellucida protein on the binding capacity of human spermatozoa.

The acrosome reaction, sperm-zona pellucida binding, sperm-oolemma binding/fusion and subsequent fertilization are known to be influenced by homologous as well as heterologous follicular fluid and zona pellucida protein. In this study, the effect was investigated of different concentrations of solubilized porcine zona pellucida protein on the zona binding potential of human spermatozoa under hemizona assay conditions. Human spermatozoa incubated with 617 and 142 micrograms/ml porcine zona pellucida protein showed a statistically significant increase in zona binding when compared with control spermatozoa (106.5 +/- 18.0 versus 60.9 +/- 29.0, P less than 0.02 and 111.0 +/- 26.6 versus 63.0 +/- 25.5, P less than 0.0001, respectively). Concentrations of 67 micrograms/ml porcine zona pellucida protein did not show a significant increase in zona binding (78.7 +/- 21.7 versus 66.7 +/- 25.4, P greater than 0.05). Control zona binding values for different experiments did not differ significantly (60.9 +/- 29.0; 63.0 +/- 25.5; and 66.7 +/- 25.4, P greater than 0.6). In conclusion, it seems likely that a factor(s) present in the porcine zona pellucida might play a beneficial role during human sperm-oocyte binding. The results of the study might be used in future investigations to manipulate gamete interaction to such an extent that improved fertilization rates can be accomplished.

The hemizona assay (HZA) as an experimental model to evaluate the inhibition of sperm binding to the murine zona pellucida by isolated zona pellucida protein.

Compelling evidence has demonstrated that zona binding represents gamete recognition by sperm binding with high affinity and specificity to complex glycoproteins of the zona pellucida. In the present study we evaluated the hemizona assay (HZA) in the investigation of the interaction of mouse spermatozoa with unfertilized murine oocytes and hemizonae after exposure to solubilized murine zonae pellucidae proteins. The zonae pellucidae were isolated from ovarian tissue following described mincing techniques. The sperm binding characteristics of murine spermatozoa were studied by using SDS-PAGE isolated ZP2 (+/- 120 Kd) and ZP3 (+/- 83 Kd) components of the zona pellucida. Sperm receptor activity was examined in a competitive gamete binding fashion using the HZA as an indicator of sperm/zona interaction. The results illustrated that isolated, solubilized ZP2 and ZP3 glycoprotein moieties of the zona pellucida inhibited sperm binding to hemizonae and oocytes when compared to controls, and that the HZA can be utilized as an internally controlled homologous bioassay to evaluate the effects of zona pellucida proteins on tight binding of spermatozoa to the zona pellucida.

Identification of a 'disease-associated' antigen in pigeon breeder's disease by western blotting.

Sera from 9 symptomatic and 7 asymptomatic pigeon breeders were analyzed for their reactivity to pigeon serum by western blotting. All 9 symptomatic sera (9/9; 100%) and only 4 of 7 (57%) asymptomatic sera revealed specific antibodies. The immunoreactivity patterns of the sera varied: the majority of the sera reacted to antigens having molecular weights of 220 kD or more (9/16 sera), 98 kD (8/16 sera), and 86 kKD (8/16 sera). However, only sera from symptomatic breeders recognized an antigen of approximate molecular weight of 29-32 kD (9/9; 100% of symptomatics). We conclude that this antigen is 'disease associated' and may be useful in the diagnosis of pigeon breeder's disease.

Use of specific monoclonal antibodies to secretory IgA for the detection of spermatozoal antibodies in serum and seminal plasma by enzyme-linked immunosorbent assay.

The role of anti-sperm secretory IgA has recently received attention since some workers feel it plays an important role in the prognosis of the immunologically infertile couple. Current methods used in our laboratory cannot separately detect anti-sperm secretory IgA. An enzyme-linked immunosorbent assay (ELISA) utilizing specific monoclonal antibodies to secretory IgA was used to detect anti-sperm secretory IgA as well as anti-sperm monomeric IgA and IgG in serum and seminal plasma of a sperm-antibody-positive (ASA+) and sperm-antibody-negative (ASA-) group of men. Results showed significantly raised serum levels in the ASA+ group when compared to the ASA- group for anti-sperm secretory IgA (P less than .001), anti-sperm monomeric IgA (P less than .001), and anti-sperm IgG (P less than .01). Seminal plasma levels were also raised in the ASA+ group, but only significantly so for monomeric IgA (P less than .02). The performed ELISA has definite potential in research, especially with the use of monoclonal antibodies for the detection of anti-sperm secretory IgA, but cannot as yet be used as a prognostic predictor of fertility in the individual antibody-positive patient. Infertility specific antigens will have to be identified and isolated and subsequently used in the ELISA.

Autosperm antibodies in treated, pregnant and non-pregnant immunologically infertile patients.

Twenty-two immunologically infertile couples (male partners had proven autosperm antibodies-positive mixed antiglobulin reaction test [MAR] and direct immunobead test [d-IBT]) were treated with washed spermatozoa used either in the gamete intrafallopian tube transfer (GIFT) or artificial insemination (AIH) procedures. Sixteen of the 22 couples (72.2%) fell pregnant with an ongoing pregnancy rate of 54.5% (12/22). The pregnant and non-pregnant groups were compared with regards to the sperm antibodies detected on the spermatozoa (MAR, d-IBT and sperm cervical mucus contact [SCMC] test) and in the serum and seminal plasma of the male partners (tray agglutination test [TAT], indirect immunobead test [i-IBT], and enzyme-linked immunosorbent assay [ELISA]). The semen parameters, motility, forward progression, count/ml and normal morphological forms were also compared. Statistical analysis showed no difference between the two groups (pregnant and non-pregnant) with regards to the antibody tests performed on semen, serum and seminal plasma. No difference was also seen between the semen parameters of the two groups. The washing of spermatozoa for the GIFT or AIH procedures may therefore be a successful method of treatment for immunologically infertile couples. The results also indicate no difference in the fertility prognosis for the two groups since antibody levels and semen quality were not different between the pregnant and nonpregnant group.

Pigeon breeder's disease: the effect of antigen-specific antibodies on in vitro T cell responses of normal blood lymphocytes.

The effect(s) of affinity-purified antibodies to pigeon serum immunoglobulins on normal T cell responses in vitro was measured. Our results show that the antibodies partially inhibited the concanavalin-A-induced proliferation of normal lymphocytes (32% at 10 micrograms/ml concentration; p less than 0.001), whilst they had no significant effects at equivalent doses on the phytohemagglutinin-induced response. A study of the kinetics of this inhibition revealed that the antibodies exerted their effect(s) within the first 24 h of culture (p less than 0.001); this is probably due to their interference in early events intimately involved in the de novo synthesis and expression of activation antigens such as HLA-DR and Tac: antibody-treated cultures expressed 51.6 and 41.4% less HLA-DR and Tac, respectively. Two-colour immunofluorescence analysis showed that the specific antibodies bind to a subset of CD8+ cells only.