Genetic variations in androgen metabolism genes and associations with prostate cancer in South African men.

Background. In South Africa white men have the highest incidence of prostate cancer (PCa), coloured (mixed ancestry) men have an intermediate incidence, and low incidences are reported for black and Asian men. It has been suggested that ethnic differences in incidence and mortality of PCa are related to genetic variations in genes that regulate androgen metabolism. We investigated the role of genetic variants in the androgen metabolism genes and the probability of developing PCa in South African coloured and white men. Methods. Genotype and allele counts and frequencies of single nucleotide polymorphisms (SNPs) in CYP3A5, CYP3A4 and CYP3A43 were assessed in coloured men (160 case individuals, 146 control individuals) and white men (121 case individuals, 141 control individuals). Results. A genetic association indicating an increased probability of developing PCa was observed with the G allele of the SNP rs2740574 in CYP3A4 in coloured men, the A allele of rs776746 (CYP3A5) and the G allele of rs2740574 (CYP3A4) in white men, and the G allele of rs2740574 and the C allele of rs501275 (CYP3A43) in the combined ethnic groups analysis. In addition, we identified allele combinations (termed haplotypes) with significantly higher frequencies in the PCa case individuals than in the control individuals. Conclusions. The findings support the role of variants in genes that regulate androgen metabolism and the probability of developing PCa. The study paves the way to identify other genetic associations in South African men, and to establish genetic profiles that could be used to determine disease progression and prognosis.

Problems with prostate specific antigen screening for prostate cancer in the primary healthcare setting in South Africa.

OBJECTIVES: To assess the feasibility of detecting early-stage prostate cancer in the primary healthcare setting, and to investigate whether there is a higher incidence of prostate cancer in Black African men. PATIENTS AND METHODS: The study was a collaboration with registrars in the authors' institutions and primary healthcare centres serving mainly a Black African or mixed ancestry (Coloured) population in the semi-urban Cape Town metropolitan area of South Africa. Men aged 50-70 years attending the clinics were counselled about prostate cancer and invited to have a digital rectal examination (DRE), serum prostate-specific antigen (PSA) assay and transrectal ultrasonography-guided sextant prostate biopsy if the DRE was clinically suspicious of malignancy or the serum PSA was > or = 4.0 ng/mL. An American Urological Association Symptom Index (AUA-SI) was obtained, and urine analysed using dipsticks. RESULTS: From May 2000 to November 2001, 660 men were assessed (mean age 59.4 years, range 30-82); 60.6% were Black African, 37.3% mixed (Coloured), 1.8% White (Caucasian) and 0.2% Asian (Indian). The mean (range) AUA-SI was 5.98 (0-35) in the whole group; the DRE was recorded as clinically suspicious of malignancy in 3.2%. The mean PSA was 20.39 (0.04-10 000) ng/mL in the whole group, but when two outliers (1865 and 10 000 ng/mL) were disregarded, it was 2.4 ng/mL. In Black patients the mean PSA was 31.8 (0.04-10 000) ng/mL, and without the outliers, 2.1 ng/mL; in Coloured patients it was 2.94 (0.05-50) ng/mL. The PSA was > or = 4.0 ng/mL in 9.6% of the whole group, in 7.8% of Black and in 13% of Coloured patients. Prostate biopsies were taken in 21 patients (3.2% of the whole group and a third of those with a PSA of > or = 4.0 ng/mL); in Black patients, biopsies were taken in 1.5% and 19.4%, respectively, and in Coloured patients in 6.1% and 46.9%, respectively. The prostate biopsy showed cancer in 43% of the whole group, in a third of Black and in 47% of Coloured patients; prostate cancer was detected in 1.4%, 0.5% and 2.8%, respectively. CONCLUSIONS: That prostate biopsies were obtained in only 19% of Black and in only 47% of Coloured men with a serum PSA of > or = 4.0 ng/mL is of concern. This indicates that there is a significant problem in getting men with an elevated serum PSA level to undergo a prostate biopsy in the primary healthcare setting in South Africa.

Polymerase chain reaction in the diagnosis of urinary tract tuberculosis.

The polymerase chain reaction (PCR) is a technique that can be used to amplify a specific DNA genomic sequence, whereby the presence of an extremely small number of bacteria can be detected. The high sensitivity of PCR is particularly useful in paucibacillary situations such as non-pulmonary tuberculosis (TB). The aims of the present study were to establish a PCR assay for the rapid detection of Mycobacterium tuberculosis (MTb) in urine, to compare the sensitivity of PCR with routine culture technique (Bactec) and to determine the optimal type of urine specimen for PCR detection of MTb. In the first phase of the study, a total of 92 urine specimens were collected from 83 patients with suspected urinary tract TB. Two urine specimens in 2 patients were positive for TB by both PCR and Bactec, while 90 specimens from 81 patients were negative by both methods. Inhibition of PCR was present in nine urine specimens (10%). In the second phase of the study, a further seven patients were selected for intensive investigation to determine the optimal urine sampling for PCR detection of MTb. The conclusions of the study are that PCR can provide much faster confirmation of urinary TB (within 24-48 h) than Bactec urine culture (which may take several weeks). About 10% of urine specimens could not be evaluated by PCR due to the presence of inhibitory substances of unknown nature. MTb organisms were found to be excreted intermittently in the urine of infected patients, and single specimens were more likely to be false negative than a 24-h sample. The best method appeared to be the concentration of a large volume of urine, for instance 11 concentrated to 2 ml.

Sperm decondensation and semen parameters: utilization of a simple staining technique for the evaluation of human sperm decondensation.

The ability of spermatozoa to fertilize an oocyte depends on a sequence of events ending ultimately in the decondensation of the sperm chromatin on penetration of the oocyte. Knowledge of what percentage of sperm decondenses is useful, especially in patients where other functional tests and sperm quality fail to explain the reported poor in vitro fertilization (IVF) rates. The objective of this study was (1) to compare sperm decondensation induced by either SDS/EDTA or heparin with semen parameters (volume, concentration, motility and morphology), and (2) to evaluate the use of a simplified staining technique (Diff QuikR [DQ]) in comparison with the standard phase contrast method (Rose Bengal-[RB]). Randomly selected semen samples from 31 men attending an assisted reproductive programme were analysed for basic semen parameters and decondensation with SDS/EDTA and heparin. Two staining methods for the evaluation of decondensation were compared (phase contrast microscopy after Rose Bengal [RB] staining and light microscopy after Diff QuikR (DQ) staining). Moderate and grossly swollen sperm heads were recorded. Semen samples included both fertile and unfertile semen parameters. Sperm decondensation results showed poor to moderate correlations with semen parameters. The SDS/EDTA (DQ) (moderate forms) showed a significant negative correlation (r = -0.46) with seminal volume and a significant positive correlation (r = 0.41) with normal sperm morphology. The heparin (DQ) (moderate forms) decondensation showed a significant positive correlation with motility (r = 0.61) and sperm concentration (r = 0.43). The DQ method was preferred over the RB method due to its optical and storage advantage.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

In vitro reactivities of blood lymphocytes from symptomatic and asymptomatic pigeon breeders to antigen and mitogens.

The proliferation of blood lymphocytes in response to pigeon gammaglobulin in the presence or absence of IL2 was measured in 9 symptomatic and 12 asymptomatic pigeon breeders and 24 controls. The symptomatic breeders exhibited spontaneous lymphocyte proliferation (p less than 0.001) when compared with controls whilst lymphocytes from asymptomatic breeders required the simultaneous addition of IL2 in order to proliferate (p less than 0.001). IL2 alone did not induce cellular proliferation. Both patient groups had abnormal T-cell functions and response to lectins and, in the symptomatic group, there existed an inverse correlation (r = -0.70, p less than 0.001) between specific antibody titres to pigeon gammaglobulin and the Con A response. We conclude that both patient groups have circulating, presensitized antigen-specific lymphocytes but these require different stimulatory signals at the cellular level.