Cloning and Mutagenesis of a Serotype-Specific DNA Region Involved in Encapsulation and Virulence of Actinobacillus pleuropneumoniae Serotype 5a: Concomitant Expression of Serotype 5a and 1 Capsular Polysaccharides in Recombinant A. pleuropneumoniae Serotype 1

A DNA region involved in Actinobacillus pleuropneumoniae serotype 5 capsular polysaccharide (CP) biosynthesis was identified and characterized by using a probe specific for the cpxD gene involved in CP export. The adjacent serotype 5-specific CP biosynthesis region was cloned from a 5.8-kb BamHI fragment and an 8.0-kb EcoRI fragment of strain J45 genomic DNA. DNA sequence analysis demonstrated that this region contained four complete open reading frames, cps5A, cps5B, cps5C, and cps5D. Cps5A, Cps5B, and Cps5C showed low homology with several bacterial glycosyltransferases involved in the biosynthesis of lipopolysaccharide or CP. However, Cps5D had high homology with KdsA proteins (3-deoxy-d-manno-2-octulosonic acid 8-phosphate synthetase) from other gram-negative bacteria. The G+C content of cps5ABC was substantially lower (28%) than that of cps5D and the rest of the A. pleuropneumoniae chromosome (42%). A 2.1-kb deletion spanning the cloned cps5ABC open reading frames was constructed and transferred into the J45 chromosome by homologous recombination with a kanamycin resistance cassette to produce mutant J45-100. Multiplex PCR confirmed the deletion in this region of J45-100 DNA. J45-100 did not produce intracellular or extracellular CP, indicating that cps5A, cps5B, and/or cps5C were involved in CP biosynthesis. However, biosynthesis of the Apx toxins, lipopolysaccharide, and membrane proteins was unaffected by the mutation. Besides lack of CP biosynthesis, and in contrast to J45, J45-100 grew faster, was sensitive to killing in precolostral calf serum, and was avirulent in pigs at an intratracheal challenge dose three times the 50% lethal dose (LD₅₀) of strain J45. At six times the J45 LD₅₀, J45-100 caused mild to moderate lung lesions but not death. Electroporation of cps5ABC into A. pleuropneumoniae serotype 1 strain 4074 generated strain 4074(pJMLCPS5), which expressed both serotype 1 and serotype 5 CP. However, serotype 1 capsule expression was diminished in 4074(pJMLCPS5) in comparison to 4074. The recombinant strain produced significantly less total CP (serotypes 1 and 5 CP combined) in log phase (P = 0.0012) but significantly more total CP in late stationary phase than 4074 (P < 0.0001). In addition, strain 4074(pJMLCPS5) caused less mortality and bacteremia in pigs and mice following respiratory challenge than strain 4074, indicating that virulence was affected by diminished capsule production. These results emphasize the importance of CP in the serum resistance and virulence of A. pleuropneumoniae.Actinobacillus pleuropneumoniae is an encapsulated, gram-negative bacterium that causes swine pleuropneumonia, a frequently fatal and highly contagious respiratory disease. There are 12 recognized serotypes of A. pleuropneumoniae that vary in virulence and geographic predominance (33). The capsular polysaccharide (CP) is responsible for serotype specificity and is required for virulence (19, 20). Nonencapsulated A. pleuropneumoniae mutants obtained by chemical mutagenesis have been shown to be effective vaccine candidates in that they are safe and highly protective (20). However, not all such mutants are stable, and the nature of the mutation(s) is unknown. Hence, characterization of the genes involved in CP export and biosynthesis would be desirable.We have previously reported cloning and sequencing an A. pleuropneumoniae DNA region involved in export of the CP of serotype 5a. This region consists of four genes, designated cpxABCD, which had a high degree of homology to the group II capsule export genes of Haemophilus influenzae type b (bexDCBA), Neisseria meningitidis group B (ctrABCD), and to a lesser extent, Escherichia coli K5 (kpsE and kpsMT) (54). This homology suggested that A. pleuropneumoniae also synthesized a group II capsule, whose organization predicted that the biosynthesis region would be upstream of cpxDCBA (12). We now report cloning and sequencing of four genes upstream of the cpx CP export region that correspond to capsular biosynthesis genes. A mutant with a deletion in three of these genes by allelic exchange was incapable of synthesizing CP and was avirulent in pigs. trans expression of these three genes in serotype 1 resulted in a chimeric strain producing both serotype 1 and 5 CP. However, expression of serotype 5 CP genes resulted in diminished production of serotype 1 CP, as well as diminished virulence in pigs and mice.     

HCV quasispecies evolution during treatment with interferon alfa-2b and ribavirin in two children coinfected with HCV and HIV-1

Two children who acquired hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) infection by mother-to-child transmission were monitored during interferon alfa-2b and ribavirin treatment. In Patient C1, CD4(+) T cell counts were within normal range and HIV-1 viral load was undetectable. HCV viral load declined slightly following treatment initiation while novel variants rapidly emerged, indicative of quasispecies diversification. In Patient C2, CD4(+) T cell counts were low and HIV-1 replication was not fully controlled by antiretroviral therapy. HCV viral load rose during treatment and a striking conservation of the variant spectrum was observed. In both cases, there was no decline in quasispecies complexity following treatment initiation and sustained virological response was not achieved. These results suggest that reduction in quasispecies complexity, which is observed in adult responders following interferon treatment, may be mechanistically unrelated with evolution of the variant profile and/or selective pressure exerted on HCV.     

Clonal chromosome abnormalities in human breast carcinomas I. Twenty-eight cases with primary disease

Cytogenetic analysis was performed on a selected series of short-term cultures of primary breast carcinomas from 28 patients. All patients had histopathologically confirmed malignancies, with the majority (25/28 cases) demonstrating infiltrating ductal carcinoma. All 28 cases evidenced clonal chromosome abnormalities, with 10/28 displaying only numeric aberrations, whereas 18/28 displayed clonal structural alterations. In near-diploid tumors the most common numeric changes were — 17 and — 19. However, trisomy 7 was the only numeric change in two near-diploid tumors. Structural chromosome alterations were primarily isochromosomes, apparent terminal deletions, and unbalanced non-reciprocal translocations. Chromosomes 1 (10/18–56%) and 6 (8/18–44%) were most frequently altered in this series. Breakpoints of clonal structural abnormalities were shown to cluster to several chromosome segments, including 1p22-q11, 3p11, 6p11–13, 7p11-q11, 8p11-q11, and 19q13. Analysis of the gain or loss of specific chromosome segments revealed that the most consistent tendency was over-representation of 1q, 3q, and 6p. © 1993 Wiley-Liss, Inc.     

Phage-displayed mimotopes recognizing a biologically active anti-HIV-1 gp120 murine monoclonal antibody

Antibody-dependent cellular cytotoxicity (ADCC) is a host defense mechanism in which Fc receptor-bearing effector cells in combination with antigen-specific antibodies recognize and kill antigen-expressing target cells. The authors previously described a murine monoclonal antibody (MAb-ID6) that mediated ADCC activity against HIV-infected cells. It was demonstrated that the specificity of MAb-ID6 maps to the first 204 amino acids of gp120; however, the exact epitope was not identified. In the present work, by screening phage display libraries with MAb-ID6, the authors have mapped the corresponding epitope to amino acids 86-100 (HIV-1 gp120 sequence). This epitope lies within the C1 region of gp120 and is highly conserved among all subtypes and circulating recombinant forms of HIV-1. Thus, these phage mimotopes of C1 may serve as components of a vaccine for the induction of gp120-specific antibodies mimicking MAb-ID6.     

Peroxisomal Disorders: Genotype, Phenotype, Major Neuropathologic Lesions, and Pathogenesis

Neurological dysfunction is a prominent feature of most peroxisomal disorders. Enormous progress in defining their gene defects has been achieved. The genes and gene products, peroxins (PEX), in five of the complementation groups have been defined. These studies confirm that Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD) are a disease continuum. The gene defect in adreno-leukodystrophy (ALD) / adrenomyeloneuropathy (AMN) involves an integral peroxisomal membrane protein. Neuropathologic lesions are of three major classes: (i) abnormalities in neuronal migration or differentiation, (ii) defects in the formation or maintenance of central white matter, and (iii) postdevelopmental neuronal degenerations. The central white matter lesions are those of: (i) inflammatory demyelination, (ii) non-inflammatory dysmyelination, and (iii) nonspecific reductions in myelin volume or staining with or without reactive astrocytosis. The neuronal degenerations are of two major types: (i) the axonopathy of AMN involving ascending and descending tracts of the spinal cord, and (ii) cerebellar atrophy in rhizomelic chondrodysplasia punctata and probably IRD. We postulate that the abnormal fatty acids in peroxisomal disorders, particularly very long chain fatty acids and phytanic acid, are incorporated into cell membranes and perturb their microenvironments resulting in dysfunction, atrophy and death of vulnerable cells. The advent of mouse models for ZS and ALD is anticipated to provide even greater pathogenetic insights into the peroxisomal disorders.     

Fludarabine-based conditioning secures engraftment of second hematopoietic stem cell allografts (HSCT) in the treatment of initial graft failure.

Graft failure is associated with a high mortality rate. To date, regimens invoked for second transplants have resulted in inconsistent engraftment with high transplant-related mortality (TRM). We here report 16 consecutive patients, aged 4-59 years, who received second HSCT (HSCT-2) at a median of 45 days following primary or secondary failure of an initial unmodified (N = 3) or T cell-depleted (TCD) (N = 13) HSCT (HSCT-1). HSCT-1 was administered after myeloablative total body irradiation (TBI)- or alkylator-based conditioning for acute leukemias (N = 7), MDS (N = 6), CML (N = 2), and Fanconi anemia (N = 1). All patients experienced 1 or more infectious complications between HSCT-1 and HSCT-2, and 10 patients had active infections at the time of HSCT-2. Cytoreduction regimens used for HSCT-2 included fludarabine (Flu) in combination with cyclophosphamide (CTX) (N = 9), or thiotepa (Thio) (N = 5). In addition, 1 patient received Flu alone and 1 patient Thio combined with CTX. Antithymocyte globulin (ATG) (N = 11) or Alemtuzumab (N = 3) was added pretransplant to prevent rejection. For HSCT-2, donors included HLA-matched (N = 3) or mismatched (N = 8) related, or matched (N = 2) or mismatched (N = 3) unrelated donors. The primary graft donor was used in 6 of 16 cases. The grafts administered were unmodified peripheral blood stem cell transplantation (PBSCT) (N = 5) or bone marrow transplantation (BMT) (N = 3), TCD PBSCT (N = 8). All patients achieved engraftment at a median of 12 days and evaluable patients achieved complete donor chimerism. Six patients are alive with a median follow-up of 49 months, including 4/9 conditioned with Flu/CTX. In this series, outcome was statistically superior for younger patients (相似文献   

Timing of deletion of autoreactive V{beta}6+ cells and down-modulation of either CD4 or CD8 on phenotypically distinct CD4+8+ subsets of thymocytes expressing intermediate or high levels of T cell receptor

In this paper we describe a differentiation sequence amongst adult murine thymocytes which goes from CD4+8+3lo(low) to CD4+8+3int(intermediate) to CD4+8+3hi(high) and then to mature single positive CD3hi thymocytes. Phenotypic characterization of CD4+8+3int/hi cells for a number of other surface markers is consistent with them being in transition from CD4+8+3lo phenotype to mature phenotype. The same observation was made for sensitivity towards ionomycin-mediated apoptosis. In the thymus of Mls-1a mice, where autoreactive TCR-V beta 6+ cells are negatively selected, deletion of TCR-V beta 6+ cells was first detected in the CD4+8+3int subset, and was complete by the CD4+8+3hi stage, suggesting that up-regulation of the TCR/CD3 complex is required for deletion of Mls-1a autoreactive thymocytes. No sign of apoptosis was detected among any fresh thymocyte subsets suggesting that apoptotic cells are rapidly cleared from the thymus. The CD4+8+3int/CD4+8+3hi cells are therefore populations in transit from the typical cortical thymocytes to the mature T-cells.     

Yolk sac tumors with pure and mixed polyvesicular vitelline patterns

Four cases of polyvesicular vitelline tumor are presented; two were of a previously unreported pure type, and the other two were mixed with endodermal sinus tumor.The morphologic features of the vesicles favor an endodermal origin, as originally proposed by Teilum. Marked specialization of the vesicular lining cells, seen ultrastructurally, suggests a differentiation toward gut structures and mature yolk sac. One case of pure polyvesicular vitelline tumor showed massive erythropoiesis. We propose that the pure tumor reflects an intermediate degree of differentiation within the selectively endodermal yolk sac tumor group, that is, a further stage of organization than the endodermal sinus tumor.In our cases of pure polyvesicular vitelline tumor, the marked degree of differentiation was correlated with an improved prognosis, as in the case of the possible homologue of this tumor, the yolk sac tumor of the infant testis. In contrast, the two cases of the tumor admixed with endodermal sinus tumor illustrated the low survival rate expected in the pure endodermal sinus tumor; in these cases the metastases had no polyvesicular component. Because of the significance of such a difference in prognosis we emphasize the importance of an accurate diagnosis, suggesting that a large number of sections be taken in order to demonstrate any endodermal sinus tumor component that may be present, and that the possibility of pure polyvesicular vitelline tumor always be considered in the differential diagnosis of multicystic ovarian tumors.     

Human papillomavirus type 18 variant lineages in United States populations characterized by sequence analysis of LCR-E6, E2, and L1 regions

While HPV 16 variant lineages have been well characterized, the knowledge about HPV 18 variants is limited. In this study, HPV 18 nucleotide variations in the E2 hinge region were characterized by sequence analysis in 47 control and 51 tumor specimens. Fifty of these specimens were randomly selected for sequencing of an LCR-E6 segment and 20 samples representative of LCR-E6 and E2 sequence variants were examined across the L1 region. A total of 2770 nucleotides per HPV 18 variant genome were considered in this study. HPV 18 variant nucleotides were linked among all gene segments analyzed and grouped into three main branches: Asian-American (AA), European (E), and African (Af). These three branches were equally distributed among controls and cases and when stratified by Hispanic and non-Hispanic ethnicities. Among invasive cervical cancer cases, no significant differences in the three HPV variant branches were observed among ethnic groups or when stratified by histopathology (squamous vs. adenocarcinoma). The Af branch showed the greatest nucleotide variability when compared to the HPV 18 reference sequence and was more closely related to HPV 45 than either AA or E branches. Our data also characterize nucleotide and amino acid variations in the L1 capsid gene among HPV 18 variants, which may be relevant to vaccine strategies and subsequent studies of naturally occurring HPV 18 variants. Several novel HPV 18 nucleotide variations were identified in this study.     

Risk factors for Epstein–Barr virus reactivation after renal transplantation: Results of a large,multi-centre study

Epstein–Barr virus (EBV) reactivation is a very common and potentially lethal complication of renal transplantation. However, its risk factors and effects on transplant outcome are not well known. Here, we have analysed a large, multi-centre cohort (N = 512) in which 18.4% of the patients experienced EBV reactivation during the first post-transplant year. The patients were characterized pre-transplant and two weeks post-transplant by a multi-level biomarker panel. EBV reactivation was episodic for most patients, only 12 patients showed prolonged viraemia for over four months. Pre-transplant EBV shedding and male sex were associated with significantly increased incidence of post-transplant EBV reactivation. Importantly, we also identified a significant association of post-transplant EBV with acute rejection and with decreased haemoglobin levels. No further severe complications associated with EBV, either episodic or chronic, could be detected. Our data suggest that despite relatively frequent EBV reactivation, it had no association with serious complications during the first post-transplantation year. EBV shedding prior to transplantation could be employed as biomarkers for personalized immunosuppressive therapy. In summary, our results support the employed immunosuppressive regimes as relatively safe with regard to EBV. However, long-term studies are paramount to support these conclusions.