HPV11 mutant virus-like particles elicit immune responses that neutralize virus and delineate a novel neutralizing domain

Characterization of the regions of human papillomaviruses (HPVs) that elicit neutralizing immune responses supports studies on viral infectivity and provides insight for the development and evaluation of prophylactic vaccines. HPV11 is a major etiologic agent of genital warts and a likely vaccine candidate. A conformationally dependent epitope for the binding of three neutralizing monoclonal antibodies (mAbs) has been mapped to residues G(131)T(132) of the L1 major capsid protein. The mAbs bind L1 only when it is assembled into virions or into virus-like particles (VLPs) that mimic the capsid structure. We were interested in identifying other domains of L1 that elicit neutralizing responses. To this end, we have generated a panel of mAbs against VLPs derived from HPV11 L1 harboring a G131S substitution. The new mAbs are unlike the neutralizing mAbs previously mapped to residues G(131)T(132) in that they bind both prototype and HPV11:G131S mutant VLPs. Some of the new mAbs neutralized virus in vitro. We have mapped epitopes for three of these new mAbs, as well as a neutralizing mAb generated against HPV11 virions, by measuring binding to HPV6 VLPs substituted with HPV11-like amino acids. Two regions are critical: one defined by HPV11 L1 residues 263-290 and the other by residues 346-349. mAbs H11.H3 and H11.G131S.G3 bind HPV6 VLPs with substitutions derived from the 346-349 region; in addition, H11.G131S.G3 binds HPV6 VLPs with substitutions derived only from the 263-290 region. Although H11.H3 does not bind HPV6 VLPs with substitutions derived from the 263-290 region, binding to HPV6 VLPs is enhanced when both sets of substitutions are present. mAbs H11.G131S.I1 and H11.G131S.K5 bind HPV6 VLPs with the 263-290 substitutions, but show little binding to HPV6 VLPs with the 346-349 substitutions. However, binding to HPV6 VLPs is enhanced when substitutions at both regions are present. The 346-349 region has not previously been described as eliciting a neutralizing response for any HPV type. In addition, the work demonstrates a complex binding site contributed by two distinct regions of L1.     

A study on the establishment of Oesophagostomum dentatum in pigs following percutaneous exposure to third-stage larvae

We examined the possibility of establishment of patent infections after percutaneous exposure of parasite-naive pigs to 10,000 Oesophagostomum dentatum infective larvae (L₃). At 5 weeks after percutaneous exposure the pigs were slaughtered and low numbers of adult O. dentatum were recovered from the large intestine. In addition, exsheathed L₃ were intravenously injected into pigs, which resulted in the recovery of fourth-stage larvae (L₄) from nodules in the lungs as well as of L₄ and adult worms from the large intestine. This study demonstrates the ability of porcine nodular worms to establish in the large intestine when pigs have been exposed percutaneously to infective larvae. It is possible that the larvae reach the large intestine by invading blood veins or the lymphatic system at the site of penetration. Received: 24 February 1998 / Accepted: 13 May 1998     

Missense mutation in a von Willebrand factor type A domain of the alpha 3(VI) collagen gene (COL6A3) in a family with Bethlem myopathy

The Bethlem myopathy is a rare autosomal dominant proximal myopathy characterized by early childhood onset and joint contractures. Evidence for linkage and genetic heterogeneity has been established, with the majority of families linked to 21q22.3 and one large family linked to 2q37, implicating the three type VI collagen subunit genes, COL6A1 (chromosome 21), COL6A2 (chromosome 21) and COL6A3 (chromosome 2) as candidate genes. Mutations of the invariant glycine residues in the triple-helical domain-coding region of COL6A1 and COL6A2 have been reported previously in the chromosome 21-linked families. We report here the identification of a G-->A mutation in the N-terminal globular domain-coding region of COL6A3 in a large American pedigree (19 affected, 12 unaffected), leading to the substitution of glycine by glutamic acid in the N2 motif, which is homologous to the type A domains of the von Willebrand factor. This mutation segregated to all affected family members, to no unaffected family members, and was not identified in 338 unrelated Caucasian control chromosomes. Thus mutations in either the triple-helical domain or the globular domain of type VI collagen appear to cause Bethlem myopathy.      

doublecortin is the major gene causing X-linked subcortical laminar heterotopia (SCLH)

Subcortical laminar heterotopia (SCLH), or 'double cortex', is a cortical dysgenesis disorder associated with a defect in neuronal migration. Clinical manifestations are epilepsy and mental retardation. This disorder, which mainly affects females, can be inherited in a single pedigree with lissencephaly, a more severe disease which affects the male individuals. This clinical entity has been described as X- SCLH/LIS syndrome. Recently we have demonstrated that the doublecortin gene, which is localized on the X chromosome, is implicated in this disorder. We have now performed a systematic mutation analysis of doublecortin in 11 unrelated females with SCLH (one familial and 10 sporadic cases) and have identified mutations in 10/11 cases. The sequence differences include nonsense, splice site and missense mutations and these were found throughout the gene. These results provide strong evidence that loss of function of doublecortin is the major cause of SCLH. The absence of phenotype-genotype correlations suggests that X-inactivation patterns of neuronal precursor cells are likely to contribute to the variable clinical severity of this disorder in females.      

Effect of IL-2-Bax, a novel interleukin-2-receptor-targeted chimeric protein, on bleomycin lung injury

The role of lymphocytes in the pathogenesis of lung fibrosis is not clear, but the weight of the evidence supports a pro-fibrotic effect for lymphocytes. The high-affinity interleukin-2 receptor (haIL-2R) is expressed on activated, but not quiescent, T lymphocytes. This selective expression of haIL-2R provides the basis for therapeutic strategies that target IL-2R-expressing cells. We hypothesized that elimination of activated lymphocytes by IL-2R-targeted chimeric proteins might ameliorate lung fibrosis. We investigated the effects of IL-2-Bax, a novel apoptosis-inducing IL-2R-targeted chimeric protein, on bleomycin-induced lung injury in mice. Treatment groups included (i) a single intratracheal instillation of bleomycin and twice-daily intraperitoneal injections of IL-2-Bax; (ii) intratracheal bleomycin and intraperitoneal IL-2-PE66(4Glu), an older-generation chimeric protein; (iii) intratracheal bleomycin/intraperitoneal PBS; (iv) intratracheal saline/intraperitoneal PBS. Lung injury was evaluated 14 days after intratracheal instillation by cell count in bronchoalveolar lavage (BAL) fluid, semi-quantitative and quantitative histomorphological measurements and by biochemical analysis of lung hydroxyproline. Bleomycin induced a BAL lymphocytosis that was significantly attenuated by IL-2-Bax and IL-2-PE66(4Glu). However, morphometric parameters and lung hydroxyproline were unaffected by the chimeric proteins. These results show that IL-2-Bax reduces the lymphocytic infiltration of the lungs in response to bleomycin, but this effect is not accompanied by a decrease in lung fibrosis.     

3-Methylglutaconic aciduria type III in a non-Iraqi-Jewish kindred: clinical and molecular findings

Type III 3-methylglutaconic aciduria (MGA) (MIM 258501) consists of early bilateral optic atrophy, later development of spasticity, extrapyramidal dysfunction and occasionally cognitive deficits, and urinary excretion of 3-methylglutaconic acid and 3-methylglutaric acid. The presence of the disorder in an Iraqi-Jewish genetic isolate led to mapping of the OPA3 gene to chromosome 19q13.2-q13.3, followed by isolation of the gene itself. OPA3 consists of two exons and codes for a peptide of 179 amino acids. Iraqi-Jewish patients with type III MGA are homozygous for a splice site founder mutation in OPA3 (IVS1-1G>C) which abolishes mRNA expression in fibroblasts. Here we report a novel mutation in OPA3 (320-337del) in a Kurdish-Turkish patient with optic atrophy and 3-methylglutaconic and 3-methylglutaric aciduria, previously carrying the diagnosis of type IV MGA. We conclude that type III MGA occurs in patients of non-Iraqi-Jewish ancestry, and should be considered in patients with type IV MGA that have optic atrophy and ataxia.     

Comparison of epidermal keratinocytes and dermal fibroblasts as potential target cells for somatic gene therapy of phenylketonuria

Phenylketonuria (PKU) is caused by deficiency of phenylalanine hydroxylase (PAH) and increased levels of phenylalanine. PAH requires the cofactor BH(4) to function and the rate-limiting step in the synthesis of BH(4) is GTP cyclohydrolase I (GTP-CH). The skin is a potential target tissue for PKU gene therapy. We have previously shown that overexpression of PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH(4) supplementation [Gene Ther. 7 (2000) 1971]. Here, we investigate the capacity of fibroblasts, another cell type from the skin, to metabolize phenylalanine. After retroviral gene transfer of PAH and GTP-CH both normal and PKU patient fibroblasts were able to metabolize phenylalanine, however, in lower amounts compared to genetically modified keratinocytes. Further comparative analyses between keratinocytes and fibroblasts revealed a higher copy number of transgenes in keratinocytes and also a higher metabolic capacity.     

Quantitative comparison of intestinal invasion of zoonotic serotypes of Salmonella enterica in poultry

The aim of the present study was to compare the invasion of selected zoonotic Salmonella serotypes of poultry in an in vivo chicken intestinal loop model and also in vitro in epithelial cell cultures. Invasion was measured relative to a reference strain, Salmonella Typhimurium 4/74 inv H201::Tn phoA . Two serotypes demonstrated intracellular log 10 counts that differed significantly from all other serotypes tested: Salmonella Enteritidis PT4 being 1.5 log 10 colony forming units (CFU) (31-fold) higher, and Salmonella Tennessee being 0.7 log 10 CFU (fivefold) lower than the reference strain ( P h 0.0001). A group of serotypes, which can be vertically transmitted, showed significantly higher intracellular counts (fourfold to eightfold) than the reference strain. The group included S. Typhimurium 4/74, S. Typhimurium DT104 (poultry and porcine isolates), S. Enteritidis PT1, S. Enteritidis PT6, S. Enteritidis PT8, and Salmonella Berta. The serotypes Salmonella Hadar, Salmonella Virchow, S. 4,12:b:-, S. Typhimurium DT41, and Salmonella Infantis, most of which are considered horizontally transmitted, did not show significantly different intracellular counts from the reference strain. Results from the cell culture invasion studies agreed with the in vivo data, with the exception of S. Berta and the poultry isolate of S. Typhimurium DT104.     

Coagulation and complement activation

The purpose of this investigation was to assess the effect of heparin coating of a new stent construction (Stent Graft, Jomed Implantate GmbH, Germany) on platelet and coagulation activity. METHODS: Stent grafts with an ePTFE membrane interfoliated between two stents were deployed in tubings to form Chandler loops. Fresh human blood with a low concentration of heparin was rotated for 1 h, then collected and used for measurements of platelet number, thrombin-antithrombin complex (TAT), CD11b, C3a and C5b-9. There were five study groups: Group 1, conventional unmodified stents (n = 8); Group 2, untreated stent grafts (n = 8); Group 3, heparin-coated stents and untreated membrane (n = 7); Group 4, heparin-coated stents and membrane (n = 8); Group 5, heparin-coated PVC tubings with no stents (n = 8). RESULTS: There was a significant drop in platelet count, increase in TAT-values and CD11b expression in Groups 1-3 but not in Group 4 compared to Group 5. Examination by scanning electron microscopy revealed extensive activation on non-modified stents but almost no deposition of thrombotic material on heparin-modified stent grafts. CONCLUSIONS: With unmodified stents and membrane there were signs of significant activation of platelets and coagulation. In contrast, the heparin-coated stent graft induced much less alterations, indicating improved blood compatibility.     

Cloning and characterization of a protective outer membrane lipoprotein of Actinobacillus pleuropneumoniae serotype 5.

The gene encoding an outer membrane lipoprotein (omlA) of Actinobacillus pleuropneumoniae serotype 5 was cloned, and the protein was expressed in Escherichia coli. One open reading frame of 1,104 bp was detected that encoded a protein (OmlA) with a predicted molecular mass of 40 kDa. A comparison with the omlA gene and the corresponding protein of A. pleuropneumoniae serotype 1 (G.-F. Gerlach, C. Anderson, S. Klashinsky, A. Rossi-Kampos, A.A. Potter, and P.J. Wilson, Infect. Immun. 61:565-572, 1993) revealed that the nucleic acid sequences had an overall sequence identity of 62.9% and the deduced amino acid sequences showed a sequence agreement of 57.3%. Both proteins were antigenically distinct. In a Western blot (immunoblot) analysis using a specific antiserum against A. pleuropneumoniae serotype 5 OmlA, a homologous protein was detected in the reference strains of A. pleuropneumoniae serotypes 5A, 5B, and 10. Pigs immunized with this recombinant protein were protected from death in an aerosol challenge experiment with an A. pleuropneumoniae serotype 5 isolate.