Healthy Male Donors Research Articles

P‐glycoprotein activity in human Caucasian male lymphocytes does not follow its increased expression during aging

P-glycoprotein (P-gp) is a transmembrane protein that mediates the efflux of innumerous structurally unrelated compounds. It was initially found over-expressed in tumor cells, associated to a multidrug resistance phenotype (MDR). Then, P-gp was found constitutively expressed in excretory cells/tissues and in circulating cells, such as lymphocytes. Considering the importance of this transporter in the establishment of therapeutic protocols and the existence of contradictory results, this study aimed at evaluating the influence of aging in the expression and function of P-gp in human lymphocytes, comparing two different methodologies to assess both parameters. P-gp activity and expression were evaluated in lymphocytes isolated from whole blood samples of 65 healthy caucasian male donors, divided into two groups according to age (group 1: under 30-years old; group 2: above 60-years old). P-gp expression was assessed using the anti-P-gp monoclonal antibody, UIC2, in the presence and in absence of vinblastine (Vbl). P-gp activity was evaluated measuring the efflux rate of the fluorescent P-gp substrate rhodamine 123 (Rho 123) and also using UIC2 shift assay. Flow cytometric analysis was performed to assess all the proceedings. Furthermore, P-gp expression and each of the P-gp activity determination methods were compared, through correlation analysis and linear regression models. We observed a significant age-dependent increase in mean P-gp expression (p = 0.029), which was not reflected in the transporter's activity (p > 0.050). Statistical analysis allowed selection of UIC2 shift assay over Rho 123 efflux assay as a more selective method to assess P-gp activity. Despite the significant correlation between P-gp expression and P-gp activity found in lymphocytes (Gp1(group 1)-r = 0.609, p < 0.001; Gp2-r = 0.461, p = 0.012), using UIC2 shift assay, these data reinforce the need for P-gp activity assessment, rather than P-gp expression determination alone, when starting new therapeutic regimens with P-gp substrates, especially in men older than 60 years of age.

Isolation strategies of regulatory T cells for clinical trials: Phenotype, function, stability, and expansion capacity

Recent clinical results demonstrate the highly effective potency of regulatory T cells (Tregs) to control graft-versus-host disease (GvHD). In this presented study, we directly compared different Treg subpopulations in order to define the most promising Treg target cell population for cellular intervention studies with respect to their phenotype, functional properties, stability, and expansion capacity. Different Treg cell populations have been isolated from healthy donors and characterized by fluorescence activated cell sorting (FACS) analysis for their phenotypic marker and purity, functional properties by suppression assay, stability by Treg-specific demethylated region (TSDR) of the Foxp3 promoter and their invitro expansion capacity. The direct comparison of the respective Treg target cell populations identified CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs as the most promising Treg population for fresh cell infusions in clinical trials with respect to cell yield, phenotype, function, and stability. The CD4(+)CD25(hi) Tregs qualified as the best candidate for invitro expansion combining a highly stable phenotype with strong suppressive potential and attractive cell yield after repetitive stimulation. The suppressive capacity of freshly isolated CD4(+)CD25(hi)CD45RA(+) and CD49d(-)CD127(-) Tregs is comparable to freshly isolated CD4(+)CD25(hi), but inferior to CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs. Invitro expansion of CD4(+)CD25(hi)CD45RA(+) and CD49d(-)CD127(-) Tregs resulted in cell populations with less suppressive potency compared with CD4(+)CD25(hi) expanded Tregs correlating well with a higher TSDR demethylation level. In conclusion, future clinical trials should favor CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs for direct Treg cell transfer, whereas CD4(+)CD25(hi) Tregs qualify as best candidate for invitro expansion.

Comparison of unfractionated heparin, low-molecular-weight heparin, low-dose and high-dose rivaroxaban in preventing thrombus formation on mechanical heart valves: results of an in vitro study

Thromboembolism and bleeding after mechanical heart valve replacement are still unsolved problems, particularly for patients requiring anticoagulative bridging therapy. The aim of this study was to investigate whether rivaroxaban, a new oral selective and direct coagulation factor Xa inhibitor, is as effective as enoxaparin and unfractionated heparin (UFH) in preventing thrombus formation on mechanical heart valves using an in vitro system. Blood from healthy male donors was anticoagulated with either UFH, enoxaparin, rivaroxaban at 300ng/ml, (n=10 each), or rivaroxaban at 30ng/ml (n=3). Mechanical aortic valve prostheses were placed into the in vitro testing system THIA II and exposed to the anticoagulant blood mixtures at a pulsatile flow for 60min. Overall thrombus weight, coagulation parameters, and electron microscopic features of thrombus formation on the valve surface were quantified as endpoints. The mean thrombus weights were 163±64mg for group 1 (UFH), 341±63mg for the group 2 (enoxaparin), 238±83mg for group 3 (rivaroxaban 300ng/ml) and 1.739±16mg for group 4 (rivaroxaban 30ng/ml). Whereas high-dosed rivaroxaban showed no significant differences compared to UFH or enoxaparin, low-dosed rivaroxaban generated a massive thrombus generation, thus differing significantly from all other treatment groups regarding the thrombus weight. We hypothesize that high-dose rivaroxaban is a competitive oral available alternative to UFH and LMWH's, that might be a worthwhile alternative for patients in need of anticoagulative bridging therapy. Prospective studies have to evaluate if rivaroxaban might even overcome the limitations of OAC in patients after implantation of artificial heart valves.

The effect of 17β-estradiol on cholesterol content in human macrophages is influenced by the lipoprotein milieu.

Estrogen and testosterone are thought to modulate coronary heart disease (CHD) risk. To examine how these hormones affect human macrophage cholesterol transport, a key factor in atherogenesis, we obtained monocytes from healthy male and postmenopausal female donors (age 50–70 years). Cells were allowed to differentiate in autologous serum. Human monocyte-derived macrophages (HMDMs) were exposed to estrogen, testosterone, or vehicle, during differentiation.Cells were cholesterol enriched with oxidized low-density lipoprotein (oxLDL) in the presence of treatment. Cell cholesterol mass, efflux, and the expression of proteins involved in HMDM cholesterol transport were examined.Estrogen significantly reduced cholesteryl ester (CE) content in both female and male HMDMs while having no measurable effect on cholesterol efflux. Testosterone did not affect cholesterol content or efflux. Both hormones significantly but modestly affected the gene expression of several proteins involved in HMDM transport, yet these effects did not translate into significant changes in protein expression. In THP-1 macrophages, the effect of estrogen on CE content was more potent in unloaded macrophages and was estrogen receptor dependent. A trend for a reduction in nonoxLDL uptake by estrogen was observed and was also found to be dependent upon estrogen receptor activation. Our data indicate that estrogen, but not testosterone, reduces CE accumulation in HMDMs obtained from a CHD age relevant population, independent of changes in the expression of proteins important to macrophage cholesterol transport. In THP-1 cells, this effect is reduced in the presence of oxLDL, indicating that a pro-atherogenic lipoprotein milieu is an important variable in sex hormone modulation of CHD.

An unusual case of a potentially clinically significant anti-M antibody in a healthy male blood donor without any history of blood transfusion.

Dear Sir, The prevalence of red blood cell (RBC) alloantibodies among general, hospital-based patients typically has averaged approximately 1% in various studies. The presence of irregular antibodies in healthy voluntary blood donors is uncommon. Anti-M is a fairly common, naturally occurring antibody. Most anti-M are not active at 37 oC and generally are ignored in transfusion practice1. Sometimes, however, they can be clinically significant and become a problem in the immunohaematology laboratory. A male volunteer donor age 32 years donated blood in August 2008. Blood cell grouping showed an “A” positive phenotype, but serum grouping showed a positive reaction (+3) with all the three reagent red cells: A cells, B cells and O cells. The tests were repeated on the ID-Card, DiaClon ABO/D + reverse grouping (gel card) and the same results were observed. When reverse grouping tubes were tested at three different temperatures, 4 oC, 22 oC and 37 oC, for 1 hour, the reaction remained the same at 22 oC and 37 oC but negative at 4 oC. Autocontrols and the direct antiglobuling test were negative. With an antibody screening cell panel, two out of three cells showed positive results and an antibody identification panel (ID-DiaPanel 1-11 by DiaMed, Gmbh 1785 cressier, FR, Switzerland) showed that the suspected antibody was anti-M. The most likely type of antibody was IgG as the plasma was reactive even after treatment with dithiothreitol, which destroys IgM antibodies2. The A1 and O cells used for reverse grouping were shown to be positive for the M antigen. The anti-M present in the sample was interfering in reverse grouping with A1 and O cells. The A1 and O cells treated with papain enzyme gave a negative reaction in reverse grouping, as papain destroys the M antigen. This further confirmed the presence of anti-M with a wide thermal range, reacting at room temperature as well as at 37 oC. The antibody should, therefore, be considered potentially clinically significant. In blood donors, the prevalence of naturally occurring anti-M detectable in microplates with saline suspended cells at room temperature is one in 2,500 with M+N– cells and one in 5,000 with M+N+ cells 3. Some anti-M antibodies are pH-dependent and react at the optimum pH of 6.5. Another feature of this antibody is its failure to react with ficin- or papain-premodified cells. Proteolytic enzymes, such as ficin and papain, cleave the red cell membrane at well-defined sites. Haemolytic disease of newborn, of varying degrees of severity, has been reported in association with anti-M4,5.

Significance of detecting anti-HBc among Egyptian male blood donors negative for HBsAg*

Screening for hepatitis B virus surface antigen (HBsAg) reduces the risk of transfusion-transmitted hepatitis B viral (HBV) infection. However, the absence of HBsAg in the blood of apparently healthy individuals may not be sufficient to ensure the lack of circulating HBV. Blood containing anti-hepatitis B core antibody (anti-HBc) without detectable presence of HBsAg might be infectious; therefore, screening for anti-HBc has been implemented in some countries resulting in a decrease in the risk of post-transfusion HBV infection. To study the seroprevalence of anti-HBc. The relationship between anti-HBc positivity and the presence of circulating HBV among healthy blood donors negative for HBsAg will be helpful to decide whether supplemental testing may bring additional safety to blood products. A total of 1026 serum samples collected from HBsAg-negative Egyptian healthy male donors were tested for the presence of anti-HBc (both IgM and IgG types) using the competitive enzyme-linked immunosorbent assay technique. Anti-HBc-positive samples were subjected to real-time polymerase chain reaction to confirm the presence of HBV DNA. Of the 1026 samples tested, 80 (7·8%) blood samples were found to be reactive to anti-HBc. Of those, HBV DNA was detected in five of the samples (6·25%). The levels of detected viraemia were variable among the five donors. This study shows the insufficient effectiveness of HBsAg screening in protecting blood recipients from HBV infection. Inclusion of anti-HBc testing should be considered in the screening of blood donors.

Normal and Cut-Off Values of the Cytokinesis-Block Micronucleus Assay on Peripheral Blood Lymphocytes in the Croatian General Population

The cytokinesis-block micronucleus (CBMN) assay on peripheral blood lymphocytes is one of the most important methods employed in cytogenetic biomonitoring. For the purposes of biological dosimetry, it is important to know the spontaneous frequency of a biomarker and its normal values in general population. These values are used for population databases, which should be updated regularly. In this study, MN levels were investigated in cytokinesis-blocked lymphocytes of 200 healthy male and female blood donors selected at random from the general population of Croatia. The aim was to assess the variability and determine possible influences of external and/or internal factors on the background levels of MN and to establish the cut-off value for the CBMN assay. The background frequency of MN was (6.90+/-3.32) MN (median 7 MN) and the range was 0 to 18 MN per 1000 binuclear lymphocytes. The cut-off value, which corresponds to 95th percentile of the distribution of 200 individual values, was 12.5 MN. Spontaneous formation of MN was influenced by sex, age, and smoking. Women had higher MN levels than men. However, only age and smoking significantly increased the values of all parameters evaluated by the CBMN assay. Since the existing literature data on smoking-related formation of MN are contradictory, we will continue these investigations to resolve how the number of cigarettes smoked per day and the duration of smoking in years influence the results of the CBMN assay. Our results are consistent with the background MN frequencies reported by other cytogenetic laboratories worldwide. Normal and cut-off values estimated in this study will be used to update the current general population data and as reference for occupationally or accidental exposure.

Genomic instability after targeted irradiation of human lymphocytes: Evidence for inter-individual differences under bystander conditions

Environmental 222radon exposure is a human health concern, and many studies demonstrate that very low doses of high LET α-particle irradiation initiate deleterious genetic consequences in both irradiated and non-irradiated bystander cells. One consequence, radiation-induced genomic instability (RIGI), is a hallmark of tumorigenesis and is often assessed by measuring delayed chromosomal aberrations. We utilised a technique that facilitates transient immobilization of primary lymphocytes for targeted microbeam irradiation and have reported that environmentally relevant doses, e.g. a single 3He 2+ particle traversal to a single cell, are sufficient to induce RIGI. Herein we sought to determine differences in radiation response in lymphocytes isolated from five healthy male donors. Primary lymphocytes were irradiated with a single particle per cell nucleus. We found evidence for inter-individual variation in radiation response (RIGI, measured as delayed chromosome aberrations). Although this was not highly significant, it was possibly masked by high levels of intra-individual variation. While there are many studies showing a link between genetic predisposition and RIGI, there are few studies linking genetic background with bystander effects in normal human lymphocytes. In an attempt to investigate inter-individual variation in the induction of bystander effects, primary lymphocytes were irradiated with a single particle under conditions where fractions of the population were traversed. We showed a marked genotype-dependent bystander response in one donor after exposure to 15% of the population. The findings may also be regarded as a radiation-induced genotype-dependent bystander effect triggering an instability phenotype.

Evaluation of 14 Y-chromosomal Short Tandem Repeat Haplotype with Focus on DYS449, DYS456, and DYS458: Czech Population Sample

To evaluate the novel triplex polymerase chain reaction (PCR) assay for the analysis of polymorphic Y-chromosomal short tandem repeat loci (Y-STR). A total of 14 Y-STR loci was analyzed. Allele frequencies for 3 tetrameric Y-STR loci (DYS449, DYS456, and DYS458) and extended haplotype loci typed by Y-PLEXTM 12 system were investigated in a sample of 50 unrelated healthy Czech male donors. We computed the relevant intra-population statistic parameters for our data (gene diversity, average gene diversity over loci, and mean number of pairwise differences) and compared our sample set with other Central European populations using RST pairwise genetic distance. We focused on the comparison of genetic diversity between the Y-STR extended haplotype loci and that of the 3 additional loci, and on the benefit of using DYS449, DYS456, and DYS458 in forensic and population genetics applications. Total gene diversity in our sample set was 0.998367 when using all 14 loci. Our data analysis revealed very high genetic diversity at DYS449 locus (0.876735), which surpasses even the diversity at DYS385a/b (0.819592). Population comparison showed no difference between Czech, Bavarian, Austrian, and Saxon sample set. A minor difference was found between Czech and Polish sample set. Typing of 3 Y-chromosomal microsatellite polymorphisms may provide a useful complement to already established sets of Y-STRs.

Application of Similarity Coefficients to Predict Disease Using Volatile Organic Compounds

Measures of similarity are used to provide evidence in support of the conjecture that the presence and absence profile of a subset of volatile organic compounds found in urine differ between healthy (non-prostate cancer) male donors and prostate cancer donors.

The first successful adult right-lobe living donor liver transplantation in Hungary

The authors report on their experiences related to the first adult live donor liver transplantation performed in Hungary. The transplantation was done between brother and sister on 19th of November, 2007. The right lobe of the 33-year-old healthy male's donor liver (segments 5-8) was removed and implanted into the 23-year-old female suffering from cirrhosis on the ground of autoimmune hepatitis. The implantation of the right liver lobe was done after own hepatectomy in orthotopic position. Liver function has improved rapidly following the transplantation. The donor was discharged on the 10th post-operative day with stable liver function. He had full rehabilitation, got back to work, and control check-ups showed a significant liver regeneration. Two years after transplantation, the recipient also lives an active life with compensated liver function and she is under regular medical check-up. With the case report, authors overview the indications and techniques of living donor right-lobe liver transplantation.

A functional nonsynonymous toll-like receptor 4 gene polymorphism is associated with metabolic syndrome, surrogates of insulin resistance, and syndromes of lipid accumulation

Toll-like receptor 4 (TLR4) plays a key role in the activation of innate immune responses. Loss-of-function mutations in TLR4 prevent diet-induced obesity and insulin resistance (IR). We conducted a population cross-sectional study to evaluate whether Asp299Gly (rs4986790) TLR4 gene polymorphism is associated with metabolic syndrome (MS), surrogates of IR, and syndromes of lipid accumulation (SLAs) in Argentinean healthy male subjects. rs4986790 was genotyped in 621 healthy unrelated male blood donors. National Cholesterol Education Program/Adult Treatment Panel III–MS (NCEP/ATP III-MS); SLAs such as enlarged waist elevated triglyceride syndrome (EWET), hypertriglyceridemic waist (HW), and overweight-lipid syndrome (OLS); and surrogates of IR were assessed. The prevalence of MS, OLS, and EWET was significantly higher among Asp299Asp carriers ( P < .05). These findings were confirmed using 32 000 bootstrap samples. Surrogate markers of IR were also significantly higher in Asp299Asp carriers ( P < .05). Most findings were especially strengthened among individuals with C-reactive protein below the 95th percentile and/or total cholesterol to high-density lipoprotein cholesterol ratio ≥5. This is the first report to find, in Argentinean healthy male blood donors, associations between the Asp299Asp genotype of rs4986790 TLR4 gene polymorphism and high risk for NCEP/ATP III-MS, SLAs, and surrogates of IR. These findings are consistent with previous functional and observational studies showing that Asp299 allele, in comparison with Gly299, is associated with increased TLR4 activation, higher levels of inflammatory cytokines, acute-phase reactants and soluble adhesion molecules, and higher risk of atherosclerosis.

Naturally acquired tolerance and sensitization to minor histocompatibility antigens in healthy family members

AbstractBidirectional cell transfer during pregnancy frequently leads to postpartum persistence of allogeneic cells and alloimmune responses in both the mother and in her offspring. The life-long consequences of naturally acquired alloimmune reactivity are probably of importance for the outcome of allogeneic stem cell transplantation. We investigated the presence of CD8pos minor histocompatibility (H) antigen–specific cytotoxic T lymphocytes (TCTL) and CD8pos minor H antigen–specific T regulator cells (TREG) in peripheral blood cells obtained from 17 minor H antigen–disparate mother-offspring pairs. Absence of minor H antigen–specific TREG, as marked by the feasibility to expand TCTL from isolated tetramerpos populations, was observed in 6 mothers and 1 son. The presence of minor H alloantigen–specific TREG was observed in 4 mothers and 5 sons. These TREG were detected within isolated tetramerdim staining fractions and functioned in a CTLA-4–dependent fashion. Our study indicates that both TCTL and TREG mediated alloimmunity against minor H antigens may be present in healthy female and male hematopoietic stem cell donors, potentially influencing graft-versus-host reactivity in different ways.

Human papillomavirus DNA detected in peripheral blood samples from healthy Australian male blood donors

Recent studies have shown that human papillomavirus (HPV) DNA can be found in circulating blood, including peripheral blood mononuclear cells (PBMCs), sera, plasma, and arterial cord blood. In light of these findings, DNA extracted from PBMCs from healthy blood donors were examined in order to determine how common HPV DNA is in blood of healthy individuals. Blood samples were collected from 180 healthy male blood donors (18-76 years old) through the Australian Red Cross Blood Services. Genomic DNA was extracted and specimens were tested for HPV DNA by PCR using a broad range primer pair. Positive samples were HPV-type determined by cloning and sequencing. HPV DNA was found in 8.3% (15/180) of the blood donors. A wide variety of different HPV types were isolated from the PBMCs; belonging to the cutaneous beta and gamma papillomavirus genera and mucosal alpha papillomaviruses. High-risk HPV types that are linked to cancer development were detected in 1.7% (3/180) of the PBMCs. Blood was also collected from a healthy HPV-positive 44-year-old male on four different occasions in order to determine which blood cell fractions harbor HPV. PBMCs treated with trypsin were negative for HPV, while non-trypsinized PBMCs were HPV-positive. This suggests that the HPV in blood is attached to the outside of blood cells via a protein-containing moiety. HPV was also isolated in the B cells, dendritic cells, NK cells, and neutrophils. To conclude, HPV present in PBMCs could represent a reservoir of virus and a potential new route of transmission.

Changes in the Plasma Sex Hormone Profile in Males with Severe Concomitant Injury

Objective: to perform a complex study of typical plasma sex hormone changes and their functional significance in males with severe concomitant injury (SCI). Subjects and methods. Fifty-nine males aged 18—49 years who had SCI were enrolled in the study. The admission severity was an APACHE II score of 18.6±2.4. According to the outcome of the disease, all the patients were divided into 2 groups: A) survivors; B) deceased persons. A control comprised 12 healthy male donors aged 19-36 years, in whom the levels of 8 sex steroids were measured. The standard procedures were used to comparatively analyze the concentrations of pituitary reproductive hormones and aldosterone. Hormonal concentrations were studied over time on posttraumatic days 1, 3, 5, 7, 10, and 15. The plasma hormone profile was examined by test kits (BSL, USA) on a Stat Fax 2100 device (Awareness Technology Inc., USA) for enzyme immunoassay. Prolactin, luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P), 17-hydroxyprogesterone (17-OH-P), dehydroepiandrosterone sulfate (DHEA-S), androstendione (A), testosterone (T), dihydrotestosterone (DHT), estrone (E1), estradiol (E2), and aldosterone were determined. Results. The complex study of phasic changes in the profile of 11 plasma sex hormones was first conducted in males in the posttraumatic period. Moreover, the typical plasma hormonal changes were elevated prolactin levels and their phasic variations, normal LH and FSH levels with a tendency for further phasic LH changes and FSH reduction. After the injury, the plasma concentration of P was increased and that of 17-OH-P was decreased. The levels of A and DHEA-S varied in the normal range with a tendency for DHEA-S to be lower during the process. In the posttraumatic period, the plasma content of T and DHT was substantially reduced and that of E1 and E2 was increased. The deceased patients generally showed higher levels of A, DHEA-S, and estrogens as a reflection of dysregulatory pathology and complications. The changes revealed in hormonal levels are of significance in understanding the pathogenesis of SCT and its sequels. This may serve as a basis for the development of new therapy methods using sex hormones as adaptogens in the postresuscitative period. Key words: severe concomitant injury, sex hormones, prolactin, luteinizing hormone, follicle-stimulating hormone, progesterone, 17-hydroxyprogesterone, androgens, estrogens.

Multimarker Circulating DNA Assay for Assessing Blood of Prostate Cancer Patients

Prostate cancer (PCa) detection using serum-based prostate specific antigen (PSA) is limited by frequent false-positive and -negative results. Genetic aberrations such as allelic imbalance (AI) and epigenetic changes such as promoter hypermethylation have been detected in circulating DNA of cancer patients. We hypothesized that circulating multimarker DNA assays detecting both genetic and epigenetic markers in serum would be useful in assessing PCa patients. We assayed blood from healthy male donors (n = 40) and 83 patients with American Joint Cancer Committee (AJCC) stage I-IV PCa. DNA was assayed for AI of 6 genome microsatellites. We assessed methylation of RASSF1, RARB2, and GSTP1 using a methylation-specific PCR assay and analyzed the sensitivity of each assay for the detection of genetic or epigenetic changes in circulating DNA. The relation between circulating tumor-related DNA detection and prognostic factors was investigated. The proportion of patients demonstrating AI for > or =1 marker was 47% (38 of 81 patients). Methylation biomarkers were detected in 24 of 83 patients (28%). By combining 2 DNA assays, the number of PCa patients positive for > or =1 methylated or LOH marker increased (52 of 83; 63%). The combined assays detected PCa in 15 of 24 patients (63%) with normal PSA concentrations. The combination of the DNA assays detected the presence of PCa regardless of AJCC stage or PSA concentration. Combination of the DNA and PSA assays gave 89% sensitivity. This pilot study demonstrates that the combined circulating DNA multimarker assay identifies patients with PCa and may yield information independent of AJCC stage or PSA concentration.

Polymorphisms of mtDNA control region in Tunisian and Moroccan populations: An enrichment of forensic mtDNA databases with Northern Africa data

Current forensic mitochondrial (mt)DNA databases are limited in representative population data of African origin. We investigated HVS-I/HVS-II sequences of 120 Tunisian and Moroccan healthy male donors applying stringent quality criteria to assure high quality of the data and phylogenetic alignment and notation of the sequences. Among 64 Tunisians, 56 different haplotypes were observed and the most common haplotype (16187T 16189C 16223T 16264T 16270T 16278T 16293G 16311C 73G 152C 182T 185T 195C 247A 263G 309.1C 315.1C; haplogroup (hg) L1b) was shared by four individuals. 56 Moroccans could be assigned to 52 different haplotypes where the most common haplotype was of West Eurasian origin with the hg H sequence motif 263G 315.1C and variations in the HVS-II polyC-stretch (309.1C 309.2C) shared by six samples. The majority of the observed haplotypes belong to the west Eurasian phylogeny (50% in Tunisians and 62.5% in Moroccans). Our data are consistent with the current phylogeographic knowledge displaying the occurrence of sub-Saharan haplogroup L sequences, found in 48.4% of Tunisians and 25% of Moroccans as well as the presence of the two re-migrated haplogroups U6 (7.8% and 1.8% in Tunisians and Moroccans, respectively) and M1 (1.6% in Tunisians and 8.9% in Moroccans).

Investigating the Importance of Haemoglobin Measurement for Selection of Blood Donors in Libya

In Libya, haemoglobin estimation is not used routinely to assess the fitness of blood donors. We examined the importance of including this parameter in donor selection. Venous blood samples were collected from the bleed line at the end of the donation process from 1339 healthy adult male blood donors, aged 18-60 years, in Al-Jala Women Hospital in Tripoli, Libya. Haemoglobin concentration was measured using an automated haematology analyzer. The main donor age categories were 21-30 and 31-40 years (49.5% and 37%, respectively). Blood donation was mainly a replacement donation. The mean ±2SD range for haemoglobin was 10.8-17.0 g/dl. 27.2% of the donors had haemoglobin concentration of less than 13 g/dl, 16.9% of them had less than 12.5 g/dl and 1.9% had less than 10.8.5 g/dl. Our results suggest that it is important to include haemoglobin estimation as a routine procedure in selection of blood donors.

Pro12Ala Polymorphism of the Peroxisome ProliferatorActivated Receptor-.GAMMA. Gene is Associated With Metabolic Syndrome and Surrogate Measures of Insulin Resistance in Healthy Men Interaction With Smoking Status

The Pro12Ala polymorphism (rs1801282), a nonsynonymous substitution of peroxisome proliferator-activated receptor-gamma (PPARG), has been robustly associated with type 2 diabetes. However, its role in metabolic syndrome (MetS) remains poorly understood. The associations among rs1801282, MetS and surrogate measures of insulin resistance (IR) were investigated in the present study. A cross-sectional population-based survey of 572 unrelated healthy male Argentinian blood donors with normal findings on medical examination and not taking any medication was conducted. MetS was assessed using the National Cholesterol Education Program/Adult Treatment Panel III (NCEP/ATP III) criteria, and the HOMA-IR, and QUICKI were calculated. Genotyping of rs1801282 was performed using RFLP-PCR. The prevalence of MetS was 26.2%. The Pro/Ala genotype (and the Ala12 allele) was associated with a high risk for MetS (odds ratio (OR) 1.67 [95% confidence interval (CI) 1.03-2.72], P=0.0394). This was highlighted among nonsmokers (OR 2.20 [95%CI 1.25-3.88], P=0.0059). ANCOVA confirmed an interaction between smoking status and this association (P=0.031). Ala12 carriers had a higher waist circumference than noncarriers (P=0.0065). Among nonsmokers, surrogates of IR, such as HOMA-IR, were significantly higher in Ala12 carriers than in noncarriers (P<0.05). Healthy men, in particular nonsmokers, carrying the Ala12 allele of PPARG rs1801282 polymorphism, have a high risk for MetS and IR.

“Natural” Human Leukocyte Antigen Antibodies Found in Nonalloimmunized Healthy Males

Human leukocyte antigen (HLA) antibodies were normally not found in subjects who have not been immunized by pregnancies, transfusions, or transplants. But with new methodology, we now see that HLA antibodies are often found in nonalloimmunized males. The sera of 424 healthy male donors were tested with single antigen Luminex beads. Human leukocyte antigen antibodies were detected in 63% of 424 male blood donors when a fluorescent value of more than 1000 was used as the cutoff. Antibodies to class I was found in 42%, class II in 11% and both in 12%. Five males who were tested eight times over a 6-month period consistently had the same specificity at similar strength levels at each testing. The antibodies reacted with specificities that are rare in the general population: 18.9% had antibodies to A*3002; more than 10% had antibodies to A*3101, B76, B*8201, and Cw*1701. About half of the donors with antibodies had one or two specificities; the other half had three or more specificities. Among those with class II specificities, 20.5% had antibodies to DPA1*0201/DPB1*0101, and 10.8% to DQA1*0503/DQB1*0301. Because the above data were obtained by testing sera of 424 Mexican donors, as a check, 29 males in Los Angeles were tested and shown to have similar specificities at roughly similar frequencies. Normal males were found to have HLA antibodies to infrequent HLA specificities. It is likely that these HLA antibodies are produced to cross-reactive epitopes found in microorganisms, ingested proteins and allergens-making them natural antibodies.