Importance of human leukocyte antigen (HLA) class I and II alleles on the risk of multiple sclerosis

Multiple sclerosis (MS) is a complex disease of the central nervous system of unknown etiology. The human leukocyte antigen (HLA) locus on chromosome 6 confers a considerable part of the susceptibility to MS, and the most important factor is the class II allele HLA-DRB1*15:01. In addition, we and others have previously established a protective effect of HLA-A*02. Here, we genotyped 1,784 patients and 1,660 healthy controls from Scandinavia for the HLA-A, HLA-B, HLA-C and HLA-DRB1 genes and investigated their effects on MS risk by logistic regression. Several allele groups were found to exert effects independently of DRB1*15 and A*02, in particular DRB1*01 (OR = 0.82, p = 0.034) and B*12 (including B*44/45, OR = 0.76, p = 0.0028), confirming previous reports. Furthermore, we observed interaction between allele groups: DRB1*15 and DRB1*01 (multiplicative: OR = 0.54, p = 0.0041; additive: AP = 0.47, p = 4 × 10(-06)), DRB1*15 and C*12 (multiplicative: OR = 0.37, p = 0.00035; additive: AP = 0.58, p = 2.6 × 10(-05)), indicating that the effect size of these allele groups varies when taking DRB1*15 into account. Analysis of inferred haplotypes showed that almost all DRB1*15 bearing haplotypes were risk haplotypes, and that all A*02 bearing haplotypes were protective as long as they did not carry DRB1*15. In contrast, we found one class I haplotype, carrying A*02-C*05-B*12, which abolished the risk of DRB1*15. In conclusion, these results confirms a complex role of HLA class I and II genes that goes beyond DRB1*15 and A*02, in particular by including all three classical HLA class I genes as well as functional interactions between DRB1*15 and several alleles of DRB1 and class I genes.     

Induction of the 72-kilodalton heat shock protein and protection from ultraviolet B-induced cell death in human keratinocytes by repetitive exposure to heat shock or 15-deoxy-delta(12,14)-prostaglandin J2

It has been demonstrated that hyperthermia protects keratinocytes from ultraviolet B (UVB)-induced cell death in culture and in vivo. This effect is mediated by the antiapoptotic effect of heat shock proteins that are transiently induced after exposure to heat at sublethal temperatures. Consequently, induction of Hsp has been proposed as a novel means of photoprotection. However, in the face of daily UVB exposure of human skin in vivo, this approach would not be useful if keratinocytes become less sensitive to Hsp induction with repeated exposure to the inducing agent. The aim of this study was to investigate whether repeated exposure to hyperthermia or to the stress protein activating cyclopentenone prostaglandin 15-deoxy-delta(12,14)-prostaglandin J2 (15dPGJ2) leads to adaptation of the cells, attenuation of the heat shock response, and abrogation of the protective effect. Normal human epidermal keratinocytes (NHEK) and the carcinoma-derived cell line A431 were exposed to either 42 degrees C or to 15dPGJ2 for 4 hours at 24-hour intervals for 4 consecutive days. The intracellular level of the 72-kDa heat shock protein (Hsp72) was determined by enzyme-linked immunosorbent assay (ELISA). Cells were exposed to UVB from a metal halide source after the last heat or 15dPGJ2 treatment, and survival was determined 24 hours after exposure by a MTT assay. Our results demonstrate that (1) heat shock and 15dPGJ2 are potent inducers of Hsp72 expression and lead to increased resistance to UVB-induced cell death in human keratinocytes; (2) re-exposure to heat shock leads to a superinduction without attenuation of the absolute increase in Hsp72 and of its UVB-protective effect; (3) the UVB tolerance induced by 15dPGJ2 is enhanced by repeated exposure without a further increase of Hsp72; (4) repeated heat shock and 15dPGJ2 up to a concentration of 1 microg/mL have no influence on cell growth over a period of 4 days. We conclude that through repeated exposure to Hsp-inducing factors, stress tolerance can be maintained without additional toxicity in human keratinocytes. These results provide a basis for the development of nontoxic Hsp inducers that can be repeatedly applied without loss of effect.     

Role of nuclear proteins on [H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Secondary metabolites in transgenic tobacco and potato: high accumulation of 4-hydroxybenzoic acid glucosides results from high expression of the bacterial gene ubiC

The bacterial gene ubiC encodes chorismate pyruvate-lyase (CPL), which converts chorismate to 4-hydroxybenzoate (4HB). The ubiC gene was expressed in tobacco (Nicotiana tabacum L., Solanaceae) and potato (Solanum tuberosum L., Solanaceae) under the control of the very strong constitutive plant promotor (ocs₃) mas. High accumulation of 4HB glucosides as new, artificial secondary metabolites was observed in the transgenic plants. 4HB glucoside content reached 5.1% of dry weight in tobacco cell cultures and 4.0% of dry weight in the leaves of potato shoots. This is the highest content of an artificial secondary metabolite produced by genetic engineering of plants reported so far. Surprisingly, no growth retardation and no phenotypical changes were observed in the transgenic cell cultures and plants. Glucosylation of 4HB was achieved by endogeneous, constitutively expressed glucosyltransferases. The total amount of 4HB glucoside acccumulated showed a strict linear dependence on the expression level of ubiC.     

A Whole Virus Pandemic Influenza H1N1 Vaccine Is Highly Immunogenic and Protective in Active Immunization and Passive Protection Mouse Models

The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID) mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine.     

Design and characterization of a hybrid miniprotein that specifically inhibits porcine pancreatic elastase

Studying protease/peptide inhibitor interactions is a useful tool for understanding molecular recognition in general and is particularly relevant for the rational design of inhibitors with therapeutic potential. An inhibitory peptide (PMTLEYR) derived from the third domain of turkey ovomucoid inhibitor and optimized for specific porcine pancreatic elastase inhibition was introduced into an inhibitor scaffold to increase the proteolytic stability of the peptide. The trypsin-specific squash inhibitor EETI II from Ecballium elaterium was chosen as the scaffold. The resulting hybrid inhibitor HEI-TOE I (hybrid inhibitor from E. elaterium and the optimized binding loop of the third domain of turkey ovomucoid inhibitor) shows a specificity and affinity to porcine pancreatic elastase similar to the free inhibitory peptide but with significantly higher proteolytic stability. Isothermal titration calorimetry revealed that elastase binding of HEI-TOE I occurs with a small unfavorable positive enthalpy contribution, a large favorable positive entropy change, and a large negative heat capacity change. In addition, the inhibitory peptide and the hybrid inhibitor HEI-TOE I protected endothelial cells against degradation following treatment with porcine pancreatic elastase.     

Interaction of the disintegrin and cysteine-rich domains of ADAM12 with integrin alpha7beta1

We describe a novel interaction between the disintegrin and cysteine-rich (DC) domains of ADAM12 and the integrin alpha7beta1. Integrin alpha7beta1 extracted from human embryonic kidney 293 cells transfected with alpha7 cDNA was retained on an affinity column containing immobilized DC domain of ADAM12. 293 cells stably transfected with alpha7 cDNA adhered to DC-coated wells, and this adhesion was partially inhibited by 6A11 integrin alpha7 function-blocking antibody. The X1 and the X2 extracellular splice variants of integrin alpha7 supported equally well adhesion to the DC protein. Integrin alpha7beta1-mediated cell adhesion to DC had different requirements for Mn2+ than adhesion to laminin. Furthermore, integrin alpha7beta1-mediated cell adhesion to laminin, but not to DC, resulted in efficient cell spreading and phosphorylation of focal adhesion kinase (FAK) at Tyr397. We also show that adhesion of L6 myoblasts to DC is mediated in part by the endogenous integrin alpha7beta1 expressed in these cells. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulate that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle development, function, and disease. We also conclude that laminin and the DC domain of ADAM12 represent two functional ligands for integrin alpha7beta1, and adhesion to each of these two ligands via integrin alpha7beta1 triggers different cellular responses.     

The “ART” of Linkage: Pre-Treatment Loss to Care after HIV Diagnosis at Two PEPFAR Sites in Durban,South Africa

Background

Although loss to follow-up after antiretroviral therapy (ART) initiation is increasingly recognized, little is known about pre-treatment losses to care (PTLC) after an initial positive HIV test. Our objective was to determine PTLC in newly identified HIV-infected individuals in South Africa.

Methodology/Principal Findings

We assembled the South African Test, Identify and Link (STIAL) Cohort of persons presenting for HIV testing at two sites offering HIV and CD4 count testing and HIV care in Durban, South Africa. We defined PTLC as failure to have a CD4 count within 8 weeks of HIV diagnosis. We performed multivariate analysis to identify factors associated with PTLC. From November 2006 to May 2007, of 712 persons who underwent HIV testing and received their test result, 454 (64%) were HIV-positive. Of those, 206 (45%) had PTLC. Infected patients were significantly more likely to have PTLC if they lived ≥10 kilometers from the testing center (RR = 1.37; 95% CI: 1.11–1.71), had a history of tuberculosis treatment (RR = 1.26; 95% CI: 1.00–1.58), or were referred for testing by a health care provider rather than self-referred (RR = 1.61; 95% CI: 1.22–2.13). Patients with one, two or three of these risks for PTLC were 1.88, 2.50 and 3.84 times more likely to have PTLC compared to those with no risk factors.

Conclusions/Significance

Nearly half of HIV-infected persons at two high prevalence sites in Durban, South Africa, failed to have CD4 counts following HIV diagnosis. These high rates of pre-treatment loss to care highlight the urgent need to improve rates of linkage to HIV care after an initial positive HIV test.     

Responses of the saucer scallop Amusium balloti to potential predators

A high natural mortality rate has been documented for the saucer scallop Amusium balloti, an Australian scallop found on sediment bottoms at 30–60 m deep, but little is known about the causes of mortality. We studied escape responses of A. balloti to five consumers common in the bycatch of the scallop fishery as a means of identifying potential predators. The scallop showed a negligible to weak response when touched with the sea star Pentaceraster regulus and the red portunid crab Portunus rubromarginatus. The response was similar to that observed when the scallop was touched with a plastic object. In contrast, A. balloti showed a consistent and vigorous swimming response to contact with the slipper lobster Thenus orientalis, the blue swimmer crab Portunus pelagicus and the coral crab Charybdis cruciata. This was not a generalized response to crustaceans, given the scallop's weak response to the red portunid crab. This is the first report of a scallop that has a strong swimming escape response to contact with decapod crustacean predators.     

Different propensity for spontaneous differentiation of cell clones isolated from the human ovarian surface epithelial cell line HOC-7

Abstract. Limiting dilution culture of cell fractions obtained by discontinuous density gradient centrifugation was used to establish six different cell clones from HOC-7 ovarian adenocarcinoma cells (D1-D3, N1-N3). Clones D1-D3 revealed a phenotype similar to that seen in parental cells exposed to differentiation inducers such as dimethyl sulfoxide (DMSO, 0.8% [v/v]). They were flattened, slowly growing cells (doubling times: 42–46 h). The cells developed long cytoplasmic extensions and adopted a complicated growth pattern. Fixed-cell enzyme-linked immunosorbent assay (ELISA) and Western blotting demonstrated that these cells contained high levels of epidermal growth factor-receptor (EGF-R), carbohydrate antigen 125 (CA 125), fibronectin and desmoplakin, but low levels of myc oncoproteins. However, untreated parental cells and clones N1–N3 were fastgrowing (doubling times: 23–28 h), regularly shaped, polygonal cells ("cobblestone'monolayer) with low levels of EGF-R, CA 125, fibronectin and desmoplakin, but relatively higher amounts of myc oncoproteins. The similarity of the sublines to either untreated or inducertreated parental cells indicated that clones D1–D3 represented spontaneously differentiated HOC-7 cells, whereas clones N1–N3 originated from less-differentiated cells. The features examined in this model cell system proved to be closely related to ovarian cancer cell proliferation and differentiation. The observation of a tumorinherent propensity for spontaneous differentiation suggests that exogenous stimulation of existing differentiation pathways may represent an alternative approach for tackling the problem of growth control and differentiation in malignant tissues.