Identification of two new HLA class II alleles: DRB1*01:50 and DQB1*05:18.

Two new human leukocyte antigen (HLA) class II alleles were identified during routine sequence-based typing.

Characterization of three new HLA-B alleles: B*35:05:03, B*52:29 and B*57:01:13.

Three new HLA-B alleles were identified during routine sequence-based typing.

Characterization of a new allele: HLA-A*03:134.

The new A*03:134 differs from A*03:01:01:01 by one amino acid change at codon 264.

Structure elucidation of sildenafil analogues in herbal products.

The structure of unknown compounds present in herbal products was elucidated using liquid chromatography-electrospray ionization-mass spectrometry, direct-infusion electrospray ionization-mass spectrometry, and nuclear magnetic resonance. Compounds 1-3 were identified as sildenafil analogues, 1 bearing an N-ethylpiperazine moiety instead of an N-methylpiperazine, and an acetyl group instead of the sulfonyl group, named acetildenafil, 2 bearing an N-ethylpiperazine moiety instead of an N-methylpiperazine (homosildenafil), and 3 bearing an N-hydroxylethylpiperazine moiety instead of an N-methylpiperazine, named hydroxyhomosildenafil. When analysing products marketed for penile erectile dysfunction or marketed as aphrodisiacs, attention should be given to the possible presence of these components.

Analysis of optically active compounds using conventional chromatography with a circular dichroism detector.

Analysis of optically active compounds in complex samples is often based on chiral chromatography or capillary electrophoresis in order to separate the enantiomers. This requires a chiral reagent, when using conventional chromatography, or an expensive chiral column, or a chiral selector, when using capillary electrophoresis. The type of column, reagent, or additive depends highly on the compound to be analysed. A simple and generally applicable method is using a conventional HPLC column coupled to a CD detector. Separation of enantiomers is not required, as they can be identified by a positive or negative peak. A racemate does not produce a peak; neither does an optically inactive compound. The application of HPLC-CD for the identification of pharmacologically active compounds, such as dexamphetamine, 5-hydroxytryptophan, (-)-huperzine A, and interferon, as standards, in registered drugs, in falsifications, and in food supplements is described.

A rapid solid-phase fluorimetric assay for measuring bacterial adherence, using DNA-binding stains.

In this report, we describe the validation of a rapid, single-step, microtiter plate method for quantifying bacterial adherence, based on fluorescent labeling of microorganisms with cell-permeable fluorescent DNA-binding probes. We have tested the binding to saliva-coated microtiter plates of bacteria, including Helicobacter pylori and viridans streptococci (S. mitis, S. gordonii, S. sanguis), known to interact with salivary components. Furthermore, we tested the short-term and longer-term temporal stability of a saliva-mediated adherence of these bacteria in a healthy population (N=30). The assay exhibited excellent reliability statistics, yielding within-assay variability coefficients ranging from 4.9% to 11%. A range of approximately 5 x 10(4)-1 x 10(7) cells could be detected. This method may be generally applicable to study surface binding of virtually any microbial species, while obviating the need of radioactive materials or specific antibodies for quantification, thus providing a procedure that is useful to both basic and clinical research.

Induction of herpes simplex virus gB-specific cytotoxic T lymphocytes in TAP1-deficient mice by genetic immunization but not HSV infection.

Loading of most endogenous peptides on major histocompatibility complex class I molecules is conditional on their transport into the endoplasmic reticulum (ER) by the peptide transporter TAP. We describe an HSV-2/1 cross-reactive cytotoxic T-cell (CTL) epitope that is processed in a TAP1-independent manner in vivo following immunization of TAP1-/- mice with naked DNA or a recombinant vaccinia virus. These data indicated that TAP1-independent processing of endogenous proteins is sufficient to prime CTLs in vivo. TAP1-independent processing of this epitope was not due to ER targeting by signal sequences and exogenous loading of MHC-I molecules and was not influenced by the amino acids flanking this epitope. In contrast, TAP1-/- mice infected with HSV-2 or HSV-2 mutants did not mount a CTL response against this epitope.

Limited antibody-dependent cellular cytotoxicity antibody response induced by a herpes simplex virus type 2 subunit vaccine.

The humoral response to a herpes simplex virus (HSV) type 2 subunit vaccine containing recombinant glycoproteins B (gB2) and D (gD2) was tested in 3 groups of patients. These included HSV-seronegative, HSV-1-seropositive, and HSV-2-seropositive individuals. There were excellent antibody responses, as measured by gB2- and gD2-specific ELISAs and HSV-2 neutralization assays. However, in 2 HSV-2 antibody-dependent cellular cytotoxicity (ADCC) assays, there were relatively low antibody responses, especially among HSV-seronegative individuals. The low ADCC responses may be associated with the poor efficacy of this vaccine observed in clinical trials.

Assessment of occupational exposures in a general population: comparison of different methods.

OBJECTIVES: To evaluate the relative merits of job specific questionnaires and various alternative assessment methods of occupational exposures often used in general population studies. METHODS: Subjects were participants in a hospital based case-control study of risk factors for male infertility. Estimates of exposure to organic solvents and chromium, based on job specific questionnaires, generic questionnaires, self reports of exposure, an external job exposure matrix (JEM), and a population specific JEM were compared with passive diffuse dosimeter results and measurements in urine. Urine samples from the end of the shift were analysed for metabolites of toluene, xylene, several glycol ethers, trichloroethylene, and chromium. Passive dosimeter date, metabolites of specific solvents, and urinary chromium concentrations were available for 89, 267, and 156 subjects, respectively. The alternative methods and measurements in urine were compared by means of the Cohen's kappa statistic and by computing the positive predictive value, sensitivity, and specificity of the alternative methods against measurements in urine. RESULTS: Passive dosimeter results indicated that exposure classifications with job specific questionnaire information could discriminate between high and low exposures. The kappa coefficients were < 0.4, so agreement between the various methods and measurements in urine was poor. Sensitivity of the methods ranged from 0.21 to 0.85, whereas specificity ranged from 0.34 to 0.94. Positive predictive values ranged from 0.19 to 0.58, with the highest values for job specific questionnaires. CONCLUSIONS: The results indicate that the implementation of job specific questionnaires in a general population study might be worth the extra expense it entails, bearing in mind the paramount importance of avoiding false positive exposure estimates when exposure prevalence is low.

Reduction of guinea pig pup isolation calls by anxiolytic and antidepressant drugs.

Guinea pigs possess central 5-HT1D receptors similar to humans but different from rats and mice. In order to study the role of this receptor on animal behaviour, it may be of interest to develop a paradigm measuring affective states in the guinea pig. Therefore we assessed the effects of a variety of psychotropic drugs on guinea pig pup isolation calls. Anxiolytic compounds such as the benzodiazepine receptor agonists diazepam and alprazolam, the full 5-HT1A receptor agonists 8-OH-DPAT and flesinoxan, and alcohol reduced isolation calling by the guinea pig pup. Moreover, mixed antidepressant/anxiolytic compounds like the 5-HT uptake inhibitors fluvoxamine and clomipramine or the MAO-inhibitor clorgyline as well as the antidepressant NA uptake inhibitors desipramine and maprotiline suppressed vocalizations. The 5-HT1D/1A receptor agonist 5-CT was also very effective in reducing separation calls. Remarkably, the partial 5-HT1A receptor agonists buspirone and BMY 7378 did not affect calling. The neuroleptic haloperidol, the psychostimulant d-amphetamine, the putative anxiogenics DMCM and m-CPP and the putative anxiolytics ondansetron and CI-988 had no effect on isolation calls of guinea pig pups. We propose this paradigm could be helpful to assess behavioural effects of anxiolytic and antidepressant drugs in a species different from rat or mouse, and in which the effects of 5-HT1D receptor ligands may possibly be established.

Human CD8+ herpes simplex virus-specific cytotoxic T-lymphocyte clones recognize diverse virion protein antigens.

The role of the HLA class I-restricted, CD8+, herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTL) in the control of human HSV infections is controversial because previous reports suggest that a substantial portion of the antigen-specific lytic response is mediated by CD4+ cells. To address this question directly, we isolated HSV-specific CD8+ CTL clones from a patient with recurrent genital herpes. These CTL were cloned by coculturing responder peripheral blood mononuclear cells (PBMC) with phytohemagglutinin-stimulated PBMC that had been infected with live HSV-2 and then irradiated prior to the addition of responder cells. After 1 week, CTL were cloned by limiting dilution using phytohemagglutinin stimulation and allogeneic feeder PBMC. Seven clones were isolated; all seven clones were CD8+ CD4- CD3+ DRbright, six lysed only HSV-2-infected targets, and one lysed both HSV-1- and HSV-2-infected targets. Antigen presentation was restricted by two to three different HLA class I loci. To determine the antigens recognized by these HSV-specific CTL, target cells were infected with HSV in the presence of acyclovir, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, or cycloheximide in a series of drug block/release protocols to limit the repertoire of viral gene expression to select transcriptional classes. Five of the clones exhibited a different pattern of cytotoxicity, suggesting that each recognized a distinct HSV antigen. One of the clones appears to be directed against an immediate-early antigen; six of the clones recognize virion proteins. Five of these clones recognized internal virion proteins that could be introduced into target cells by HSV infection in the absence of virus gene expression. Antigen specificity was further tested by using vaccinia virus vectors that express glycoproteins gD2 and gB2 or the tegument protein VP16. One clone lysed vaccinia virus/gD2-infected target cells; the remaining clones did not recognize any of these gene products. The diversity of the CD8+ response from a single individual indicated that several different antigens are recognized when presented in the context of a variety of class I HLA alleles, a pattern that markedly differs from that described for another human herpesvirus, cytomegalovirus.

Detection and characterization of latent HSV RNA by in situ and northern blot hybridization in guinea pigs.

Following intravaginal infection of guinea pigs, herpes simplex virus establishes a latent infection in the sensory lumbosacral ganglia. Using the techniques of in situ and Northern blot hybridization, we have characterized this latent HSV-2 virus and compared it to latent HSV-1 at the same anatomical site. For HSV-2, a single 1.8-kb latency-associated transcript (LAT) was detected. In contrast, as described for latent HSV-1 in the trigeminal ganglia of rabbits and mice, two HSV-1 LAT species were detected in the lumbosacral ganglia, an abundant transcript of 1.8 kb and a less abundant transcript of 1.55 kb. Despite these differences in LAT expression, the clinical course of the acute and recurrent genital disease was similar for both viruses. LAT was detected in 0.3-6.0% of the sensory neurons of sacral but not in lumbar ganglia. The abundance of LAT correlated with the severity of the initial infection, but not with the frequency of recurrent disease. Thus, vaccination strategies that substantially reduced or eliminated symptomatic disease following challenge infection appeared to block the establishment of a latent infection.

Evidence of gene amplification in tunicamycin-resistant Chinese hamster ovary cells.

Restriction digests of genomic DNA from tunicamycin-resistant Chinese hamster ovary cells, 3E11, were probed with the yeast transferase gene, ALG7. The data presented suggest moderate amplification of the N-acetylglucosaminyl-1-phosphate transferase gene occurred in these cells, consistent with the previously observed chromosomal translocations and increased enzymatic activity. This is the first example of gene amplification as a mechanism for aberrations in N-linked glycosylation.

Identification of a common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflammatory mediators.

Recently, cDNA sequences have been reported for both human and murine tumor necrosis factor (TNF; cachectin). The coding region of the TNF genes is highly conserved between man and mouse; 80% homology is apparent at the amino acid level. We now observe that a 33-nucleotide sequence, comprised entirely of A and T residues and located in the 3'-untranslated region, is conserved in toto in the murine and human TNF mRNAs. Since the 3'-untranslated region is normally not conserved, we reasoned that this sequence might play a regulatory role. We identified a consensus sequence (TTATTTAT) present in the 3'-untranslated region of both human and mouse TNF mRNAs, as well as the mRNAs encoding human lymphotoxin, human colony stimulating factor, human and mouse interleukin 1, human and rat fibronectin, and most of the sequenced human and mouse interferons. All of these mRNAs, except the lymphotoxin mRNA, lack homology to the TNF mRNAs in the coding region. The consensus sequence is uncommon among mammalian mRNAs in general, but it appears with a frequency greater than chance alone would dictate, suggesting that it may serve a specific regulatory function among the mRNAs in which it is found. It is particularly prevalent among mRNAs encoding proteins related to the inflammatory response.

Characterization of the polypeptide composition of human factor VIII:C and the nucleotide sequence and expression of the human kidney cDNA.

Human coagulation factor VIII:C has been purified approximately 5000-fold from commercial preparations with an average activity yield of 35%. Proteins of 92 kD and 77-80 kD enriched during purification are precipitated by a human serum polyclonal antibody which inhibits factor VIII:C activity. Evidence suggests that these polypeptides are linked by a calcium ion bridge. Partial amino acid sequence information from these proteins has been obtained from the intact polypeptides and from products of digestion with thrombin, endoproteinase lysC, or trypsin after citraconylation. An oligonucleotide probe designed from one of the amino acid sequences was used to isolate a partial genomic clone from a human 4X chromosome library in bacteriophage lambda. The genomic segment was used to isolate two cDNA molecules encompassing the entire human kidney factor VIII:C mRNA. Biologically active factor VIII:C has been produced in a mammalian cell line utilizing a complete cDNA construction.