Complete subtyping of the HLA-A locus by sequence-specific amplification followed by direct sequencing or single-strand conformation polymorphism analysis

Abstract: A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA-A locus. The method is based on a sequence-specific amplification identifying the serologically defined HLA-A specificities. The PCR products generated by these group-specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single-strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1-A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1-A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid-phase sequencing or SSCP analysis of the group-specific PCR fragments allowed complete subtyping of the HLA-A locus. This strategy can identify all 48 HLA-A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA-A locus. Only 4 out of these 1128 allele combinations remained unresolved.     

The amyloid proteins of Alzheimer's disease as potential targets for drug therapy

Two amyloid proteins accumulate in Alzheimer's disease. These proteins, beta amyloid protein and paired helical filament protein, are present in the hallmark lesions of Alzheimer's disease, neuritic plaques and neurofibrillary tangles. Although the amino acid sequences of these two proteins are likely to be different, they nevertheless share certain physical characteristics which define each as belonging to a common class of proteins, amyloid proteins. Since these proteins are probably important in the pathology of Alzheimer's disease, drugs that prevent their accumulation should have therapeutic utility. Based on the amyloidoses associated with other diseases, three mechanisms for amyloid formation have emerged. These mechanisms form a framework for studying Alzheimer amyloids and designing interventions. One mechanism involves posttranslational events which render a normal protein amyloidogenic. Proteolysis, phosphorylation, glycosylation, and transglutamination may be relevant posttranslational events in Alzheimer's disease. If more conclusive evidence can be generated suggesting that these events are involved in the abnormal formation of amyloid in Alzheimer's disease, then these events will become viable targets for drug therapy. Another mechanism for amyloid formation results from expression of an abnormal gene which, in the case of familial Alzheimer's disease, may be an important etiological component. A third mechanism involves the accumulation of a normal protein to a threshold concentration that spontaneously forms amyloid. An effective therapeutic approach for these last two mechanisms could likely include pharmacological manipulation of gene expression.     

Chromosomal aberrations and micronucleus frequency in nurses occupationally exposed to cytotoxic drugs

In this study, we evaluated the effect of low level occupationalexposure of nurses in a medical oncology unit in Cairo, Egypt,to anticancer drugs. Twenty nurses who constantly handled thesedrugs and 20 controls, matched according to age and sex, wereexamined. Metaphase chromosomes were studied. Percentages ofmetaphases with chromosomal aberrations were significantly higher(P < 0.001) in the exposed group (6.1 ± 2.7) versusthe controls (2.6 ± 1.6). The detected chromosomal aberrationswere in the form of chromatid gaps, chromatid breaks and acentricfragments. Micronucleated peripheral blood lymphocytes werealso analyzed in cytochalasin B treated binucleated lymphocytes.There was significant increase in cells with micronuclei (P< 0.001) in nurses (10.05 ± 4.71) in comparison tothe matched control (5.42 ± 2.22) (P < 0.001). Nursesexposed to the cytotoxic drugs for     

Tumor necrosis factor alpha enhances the adenoviral transduction of CD34+ hematopoietic progenitor cells

The purpose of this study was to improve the transduction efficiency of adenoviral vectors (Ad) in human CD34+ hematopoietic progenitor cells. CD34+ cells from cord blood or mobilized peripheral blood were incubated with tumor necrosis factor-alpha (TNF-alpha). After removal of free TNF-alpha, the cells were infected with an Ad encoding green fluorescent protein (GFP). One day later, viable cells were counted and analyzed for GFP and CD34 by flow cytometry. To visualize vectoral trafficking, CD34+ cells were incubated with fluorophore-conjugated Ad. Plating efficiencies of hematopoietic progenitors before and after transduction were evaluated by methylcellulose assays. Pretreatment with TNF-alpha increased the transduction efficiency more than twofold (39.2% versus 15.5%) in a dose-dependent manner and strongly improved the survival of GFP-positive CD34+ cells. Time course experiments showed that TNF-alpha incubation times as short as 10 minutes were still effective. Neutralizing antibodies to TNF receptor II and RGD peptides diminished the TNF-alpha-dependent increase in transduction efficiency. No TNF-alpha-dependent increase in adenoviral receptors (coxsackie-adenovirus receptor, alphavbeta3-integrin) occurred. Analysis of viral binding demonstrated a significantly higher incidence of local concentrations of Ad along the cell surface (caps) in virus-positive cells of the TNF-alpha-treated group. Plating efficiency, especially the formation of granulocyte-macrophage colony forming units, was enhanced by TNF-alpha pretreatment. We conclude that brief incubation with TNF-alpha before addition of the Ad significantly increased the Ad transduction efficiency in CD34+ cells, and improved post-transduction survival of progenitors of the granulocyte-macrophage lineage. This finding correlates with increased Ad capping at the cell surface and suggests an alteration of Ad trafficking.     

Polymorphic metabolizing genes and susceptibility to atherosclerosis among cigarette smokers

Atherosclerosis (AR) is the leading cause of morbidity and mortality in the US and cigarette smoking is a major contributing factor to the disease. Like cigarette smoking in lung cancer, genetic susceptibility may be an important factor in determining who is more likely to develop AR. However, the current emphasis has been on susceptibility based on altered cardiovascular homeostasis. In this investigation, we studied 120 AR patients and 90 matched controls to elucidate the association between polymorphisms in some metabolizing genes (GSTM1, GSTT1, CYP2E1, mEH, PON1, and MPO) and susceptibility to AR. We found that the GSTT1 null allele and the fast allele of mEH(*) (exon 4) are associated with risk for AR. Furthermore, the combined genotypes GSTM1 null/ CYP2E1(*)5B, GSTM1 null/mEH YY, and GSTT1 null/mEH YY are significantly associated with susceptibility to AR (OR = 15.42, 95% CI = 1.33-77.93, P = 0.021; OR = 3.48, 95% CI = 1.63-8.04, P = 0.0008; OR = 3.4; 95% CI = 0.99-17.38, P = 0.05; respectively). We have also conducted cytogenetic analysis to elucidate if induction of chromosome aberrations (CAs) is a biomarker of AR susceptibility. We found that among cigarette smokers (AR patients and smoker controls), individuals having the GSTM1 null allele had a significantly higher frequency of CAs compared to those with the normal allele (P < 0.05). This association was not found among nonsmokers. In addition, individuals who had inherited the CYP2E1(*)5B allele exhibited a significantly higher CA frequency (8.0 +/- 0.82) compared to those with the CYP2E1 wild-type genotype (4.31 +/- 0.35). Since the analysis of genetic susceptibility factors is still in its infancy, our study may stimulate additional investigations to understand the roles of genetic susceptibility and cigarette smoking in AR.     

Effects of tetracaine and procaine on skinned muscle fibres depend on free calcium

Summary The local anaesthetics, tetracaine and procaine have previously been found to block, induce or potentiate Ca²⁺ release from the sarcoplasmic reticulum (SR) of skeletal muscle depending on the preparation, experimental conditions and design. We now show that low concentrations of tetracaine and procaine block SR Ca²⁺ release whereas high concentrations induce release from the SR of amphibian and mammalian skinned fibres. Both actions depend on pCa, such that a shift in pCa can alter their effect from blocking to releasing Ca²⁺. In skinned fibres with Ca²⁺-loaded SR, tetracaine (1mm) produced a tonic contraction with a time to half-peak of 15–20 s and a magnitude reaching 80% of maximum force. Ca²⁺ release by tetracaine or procaine occured at pCa 6.5 and was not blocked by Ruthenium Red (RR) (25 m). This action of tetracaine was attributed to SR Ca²⁺ release rather than to a displacement of bound Ca²⁺ because fibres lacking a functional SR due to pre-treatment with quercetin (100 m), A 23187 (100 g ml^–1) or Triton X-100 (1%) did not contract after additions of tetracaine. Lower concentrations of tetracaine (0.5mm) and procaine (10mm) blocked contractions due to caffeine (at pCa 6.73), sulphydryl oxidizing agents, or Ca²⁺-induced Ca²⁺ release (CICR). The inhibition of CICR as a function of pCa was difficult to measure quantitatively since lowering pCa to elicit CICR twitches was sufficient to initiate tetracaine-induced tonic contractions.Experiments with isolated SR vesicles showed that 1mm tetracaine inhibited CICR, over a wide range of pCa but 3–5mm tetracaine induced rapid Ca²⁺ release. The opposite effects of tetracaine and procaine depend mostly on their concentration in SR vesicles and/or pCa in skinned fibres. Blockade of release seems to occur via the CICR pathway, and induction of release through an increase in SR membrane permeability.Abbreviations SR sarcoplasmic reticulum - HEPES N-2-hydroxy-ethylpiperazine-N¹-2-ethanesulphonic acid - EGTA ethylene glycol bis (-aminoethyl ether)-N,N,N¹,-N¹-tetraacetic acid - CICR Ca²⁺-induced Ca²⁺ release - MOPS morpholinopropane sulphonic acid - RR Ruthenium Red     

Toll- like receptor 2 polymorphism and IL-6 profile in relation to disease progression in chronic HBV infection: a case control study in Egyptian patients

Chronic hepatitis B (CHB) has a wide range of outcomes depending on host immune responses mainly Toll-like receptors (TLRs) signaling and released cytokines. Toll-like receptor 2 (TLR2) single nucleotide polymorphisms (SNPs) and interleukin 6 (IL-6) may influence the course of CHB. We aimed to elucidate the relation between TLR-2 polymorphism, IL-6 profile, and CHB progression. We analyzed TLR-2 polymorphism (SNP; rs3804099) in 185 CHB patients and 60 controls using TaqMan allelic discrimination assay. Serum IL-6 levels were assessed by ELISA. IL-6 levels were considerably higher in active CHB and cirrhotic patients compared with inactive carriers and controls (P < 0.001). IL-6 showed positive correlation with ALT and advanced fibrosis in active CHB patients (r = 0.31, P = 0.02). A significant positive correlation was noticed between IL-6 and HBV DNA PCR in all CHB groups. TT genotype of rs3804099/TLR-2 was significantly more prevalent in inactive carriers compared to active hepatitis patients (P = 0.04, OR = 0.39 and 95% CI: 0.16–0.95). Both heterozygous CT and mutant TT genotypes were significantly more frequent among inactive carriers compared to cirrhotic patients (P = 0.01, OR = 0.33, 95% CI: 0.13–0.81 and P = 0.009, OR = 0.32, 95% CI: 0.13–0.77). TT genotype was significantly related to lower IL-6 levels in active hepatitis and cirrhotic groups (P = 0.005 and P = 0.001, respectively) showing that TLR mutations would be associated with milder hepatitis activity and lower possibility for disease progression. There may be a positive association between TLR2 rs3804099 polymorphism and hepatitis B activity. IL-6 is a good indicator of CHB disease progression.

     

Mental health and nutritional status among the adult Serbian minority in Kosovo

CONTEXT: Since the beginning of the North Atlantic Treaty Organization intervention in Kosovo in June 1999, few objective data have been available on relevant health indicators for the Serbian ethnic minority in Kosovo. OBJECTIVE: To determine the prevalence of undernutrition among Serbian adults aged 60 years or older and psychiatric morbidity among the adult Serbian population in Kosovo. DESIGN, SETTING, AND PARTICIPANTS: A systematic random sample survey of 212 households was conducted between September 27 and October 2, 1999, in Pristina, the capital city, and in 10 towns in the rural municipality of Gnjilane in Kosovo. Of the 212 households surveyed, 204 adults aged 15 years or older completed the General Health Questionnaire-28 (GHQ-28) and anthropometric measurements were taken for 98 adults aged 60 years or older and for a comparison group of 51 adults aged 18 to 59 years. MAIN OUTCOME MEASURES: Body mass index of less than 18.5 kg/m(2) in older adults; nonspecific psychiatric morbidity among adults; and self-reported use of health care services, access to food rations, and primary sources of prewar and postwar income. RESULTS: Undernutrition was found in 11.2% (95% confidence interval [CI], 5.7%-19.2%) of Serbian adults aged 60 years or older compared with 2.0% (95% CI, 0.1%-11.8%) of Serbian adults aged 18 to 59 years. The mean (SE) total score for the GHQ-28 was 13.0 (0.52). In a comparison of the GHQ-28 scores of the Serbian adults with the Kosovar Albanian adults (data from a recent survey), the mean (SE) score adjusted for age and sex was 12.8 (0.52) vs 11.1 (0.58); P =.03, respectively. The GHQ-28 scores were also higher for the Serbians in the subcategories of social dysfunction (2.8 [0.17] vs 2.2 [0.13]; P =.008) and severe depression (1.9 [0.15] vs 0.9 [0. 09]; P<.001), respectively. Serbian women and persons living alone or in small family units were more prone to psychiatric morbidity. Of the 141 respondents reporting the need for health care services, 83 (57.6%) reported not obtaining such services; 204 of 212 (96.2%) households were on a food distribution list. The majority of prewar income came from government jobs compared with farming and humanitarian aid for postwar income. CONCLUSIONS: The undernutrition of older Serbian adults in Kosovo should be monitored. The high prevalence of symptoms of social dysfunction and severe depression suggest the need for implementation of mental health programs in the Serbian community. JAMA. 2000;284:578-584     

Surgical management of echinococcosis of the heart

A 23-year-old female with an echinococcal cyst in the atrioventricular groove of the heart is reported. The diagnosis and the location of the cyst were confirmed by echocardiography and cardiac catheter. Successful enucleating of the cyst with the aid of cardiopulmonary bypass and the length of follow-up is reported, along with a review of relevant literature.