Inverted terminal repeat sequences of adeno-associated virus enhance the antibody and CD8(+) responses to a HIV-1 p55Gag/LAMP DNA vaccine chimera

The immune responses to an HIV-1 p55Gag vaccine encoded as a DNA chimera with the lysosomal associated membrane protein-1 (LAMP) have been examined for the effect of the addition of the inverted terminal repeat (ITR) sequences of the adeno-associated virus (AAV) to the DNA plasmid construct, and of packaging the LAMP/gag gene as a recombinant AAV vector (rAAV). DNA plasmids encoding Gag and the LAMP/Gag protein chimera were constructed in two vectors, the pcDNA3.1 and a corresponding plasmid containing the ITR sequences (pITR) flanking the expression elements of the plasmid, and the pITR LAMP/gag DNA plasmid was encapsidated in the rAAV vector. Human 293 cells transfected in vitro with LAMP/gag plasmids either in pcDNA3.1 or pITR produced much Gag protein in cell extracts (1.6 and 2.2 ng of Gag/mg of protein, respectively). The immune responses of mice to immunization with these constructs were examined under three protocols: DNA prime/DNA boost, DNA prime/rAAV boost, and a single rAAV immunization. The results demonstrated that under DNA prime/DNA boost protocol, the "naked" DNA vaccines encoding the LAMP/gag chimera, either as pcDNA3.1 or pITR DNA plasmid constructs, elicited strong CD4(+) T cell responses. In contrast, significantly higher levels of CD8(+) and antibody responses were observed with the pITR-DNA constructs. Immunization with the rAAV vector under the DNA prime/rAAV boost protocol resulted in sustained T cell responses and a markedly increased antibody response, predominantly of the IgG(1) isotype resulting from the activation of the Th2 subset of CD4(+) T cells, that was sustained for at least 5 months after immunization.     

A survey of different high resolution visualization modes of a volumetric object with applications

Summary In view of the variety of 3D representation techniques, a clinical study was carried out in order to evaluate their respective usefulness. It appears that a single technique cannot be claimed to be valid for all clinical situations and that a combination of representations brings more relevant information. Among the different techniques a clear delineation must be established between those which allow the accurate definition of landmarks (multiplanar reformation, surface representation), and those which do not (integral shading, reconstructed radiology). The main point is the possibility to recognize anatomical landmarks on these latter modes and to choose oblique cut planes in relation to them. Visualization quality is strongly dependent upon the acquisition protocol which must provide a spatial resolution as isotropic as possible.

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Une revue de différents modes de visualisation en haute résolution d'un objet volumique avec des applications

Résumé Face à la variété des techniques de représentation 3D une étude clinique a été conduite pour évaluer leurs utilités respectives. Il apparait qu'une technique unique ne peut pas convenir à toutes les situations cliniques et qu'une combinaison de différents modes de présentation apporte une information plus pertinente. Parmi les différentes techniques une distinction claire doit être établie entre celles qui autorisent la prise de repères précis (reformatage multiplanaire, représentation de surface), et celles qui ne le permettent pas (ombrage intégral, radiologie reconstruite). Le point principal est la possibilité de reconnaître des repères anatomiques sur ces derniers modes et de choisir des plans de coupe en relation avec eux. La qualité de la visualisation dépend étroitement du protocole d'acquisition qui doit fournir une résolution aussi isotrope que possible.

     

Prevention of C3 deposition by capsular polysaccharide is a virulence mechanism of type III group B streptococci.

Strains of type III group B streptococci isolated from patients with neonatal sepsis are generally resistant to complement-mediated phagocytic killing in the absence of specific antibody. It has been suggested that the resistance of type III group B streptococci to phagocytosis results from inhibition of alternative-complement-pathway activation by sialic acid residues of the type III polysaccharide. To better define the relationship between structural features of the type III capsule and resistance of type III group B streptococci to complement-mediated phagocytic killing, we measured deposition of human C3 on group B streptococcal strains with altered capsule phenotypes. C3 binding was quantified by incubating bacteria with purified human 125I-C3 in 10% serum. Wild-type group B Streptococcus sp. strain COH1 bound eightfold fewer C3 molecules than did either of two isogenic mutant strains, one expressing a sialic acid-deficient capsule and the other lacking capsule completely. Similar results were obtained when the incubation with 125I-C3 was performed in serum chelated with Mg-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'- tetraacetic acid (MgEGTA), suggesting that the majority of C3 deposition occurred via the alternative pathway. In contrast to the wild-type strain, which was relatively resistant, both mutant strains were killed by human leukocytes in 10% serum with or without MgEGTA. We also measured C3 binding to 14 wild-type strains of type III group B streptococci expressing various amounts of capsule. Comparison of degree of encapsulation with C3 binding revealed a significant inverse correlation (r = -0.72; P less than 0.01). C3 fragments released by methylamine treatment of wild-type strain COH1 were predominantly in the form of C3bi, while those released from the acapsular mutant were predominantly C3b and those from the asialo mutant represented approximately equal amounts of C3b and C3bi. We conclude from these studies that the sialylated type III capsular polysaccharide inhibits alternative-pathway activation, prevents C3 deposition on group B streptococci, and protects the organisms from phagocytic killing.     

Intergenerational instability of the CAG repeat of the gene for Machado- Joseph disease (MJD1) is affected by the genotype of the normal chromosome: implications for the molecular mechanisms of the instability of the CAG repeat

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by unstable expansion of a CAG repeat in the MJD1 gene at 14q32.1. To identify elements affecting the intergenerational instability of the CAG repeat, we investigated whether the CGG/GGG polymorphism at the 3' end of the CAG repeat affects intergenerational instability of the CAG repeat. The [expanded (CAG)n-CGG]/[normal (CAG)n- GGG] haplotypes were found to result in significantly greater instability of the CAG repeat compared to the [expanded (CAG)n- CGG]/[normal (CAG)n-CGG] or [expanded (CAG)nGGG]/[normal (CAG)n-GGG] haplotypes. Multiple stepwise logistic regression analysis revealed that the relative risk for a large intergenerational change in the number of CAG repeat units (< -2 or > 2) is 7.7-fold (95% CI: 2.5-23.9) higher in the case of paternal transmission than in that of maternal transmission and 7.4-fold (95% CI: 2.4-23.3) higher in the case of transmission from a parent with the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes than in that of transmission from a parent with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)n- GGG]/[normal (CAG)n-GGG] haplotypes. The combination of paternal transmission and the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes resulted in a 75.2-fold (95% CI: 9.0-625.0) increase in the relative risk compared with that of maternal transmission and the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)n- GGG]/[normal (CAG)n-GGG] haplotypes. The results suggest that an inter- allelic interaction is involved in the intergenerational instability of the expanded CAG repeat.      

Functional consequences of ROMK mutants linked to antenatal Bartter's syndrome and implications for treatment

The antenatal variant of Bartter's syndrome is an autosomal recessive kidney disease characterized by polyhydramnios, premature delivery, hypokalemic alkalosis and hypercalciuria. It is genetically heterogeneous, having been linked recently to mutations in an ATP- sensitive, renal outer medullary K+channel, ROMK, and earlier to mutations in the Na-K-2Cl co-transporter, NKCC2. We characterized four of the mutations reported in three heterozygous ROMK variants of antenatal Bartter's and found that each expressed a distinct phenotype in Sf9 cells. One mutation expressed normal function and appears to be an allelic polymorphism. The other three mutations produced channels with significantly reduced K+fluxes. However, the mechanisms in each case were different and reflected abnormalities in phosphorylation, proteolytic processing or protein trafficking. The different mechanisms may be important in the design of appropriate therapy for patients with this disease.      

Detecting pre-ovulatory luteinizing hormone surges in urine

The study objectives were to determine (i) if pre-ovulatory luteinizing hormone (LH) surges, undetected in urine by two immunoradiometric assays (IRMA), were detectable by an ultrasensitive immunofluorometric assay (IFMA) and (ii) the influence of creatinine adjustment on the detection and timing of the urinary LH surges. Daily urine specimens were contributed by healthy 25-36 year old volunteers during 14 ovulatory menstrual cycles for an epidemiological study conducted in 1983-1985. Specimens were selected as having been previously assayed by two IRMA without consistently detecting LH surges. These urine specimens were remeasured using an IFMA and adjusted for creatinine concentration. IFMA measurements revealed unambiguous LH surges in all cycles. Adjusting IRMA urinary LH values for creatinine concentrations revealed previously undetected LH surges in four of eight cycles. Creatinine adjustment also altered the timing of IRMA and IFMA LH surges by 1-5 days. These results demonstrate an IFMA that detects pre- ovulatory LH surges in unpreserved, frozen urine from cycles where such surges were previously undetectable. Further, creatinine adjustment can markedly affect detection and timing of the onset and peak of the urinary LH surge. While our analysis suggests that this adjustment improves the validity of the LH measure, this requires further investigation.      

<Emphasis Type="Italic">Trypanosoma evansi</Emphasis> in inbred and Swiss-Webster mice: distinct aspects of pathogenesis

Trypanosoma evansi (Trypanosomatidae, Kinetoplastida) is a salivarian trypanosomatid that infects eight mammal orders spread over America, Europe and Asia. In Brazil, T. evansi is the etiological agent of Mal de Cadeiras, a horse disease very often described in the region known as Pantanal do Mato Grosso. Few data concerning the genetic diversity and biology of subpopulations of T. evansi that circulate in Brazil are available. The factors that modulate the interaction of this parasite with its hosts also remain to be elucidated. Here we evaluated the course of experimental infection of six T. evansi isolates derived from domestic and wild animals in Swiss-Webster mice and three Mus musculus lineages. The follow-up included biological, immunological as well as biochemical and hematological parameters. The same isolates as well as three others were characterized by pulsed-field electrophoresis. Our results showed that T. evansi isolates displayed significant differences regarding behavior and morbidity patterns in the distinct mouse lineages. Nevertheless, these differences could not be correlated with pulsed-field electrophoresis profiles. Indeed, concerning this molecular marker, only microheterogeneity was observed. Moreover, we observed that the outcome of the infection is defined by both host genetic background and peculiarities (virulence factors) of the distinct T. evansi isolates. Anemia and hypoglycemia were the only features that could be observed in all mouse lineages, independently of the inoculated T. evansi subpopulation. In addition, our data also show that Mus musculus is a suitable model host for the study of the different pathogenetic features of T. evansi infection.     

Characterization and partial purification of a lectin from the hemolymph of the white shrimp Litopenaeus schmitti

The agglutinating activity of the hemolymph of Litopenaeus schmitti is insensitive to calcium and specific for acetylated sugars, particularly sialic acid (Neu5Ac) and O-sialoglycoconjugates (bovine submaxillary mucin) and has varying specificity for different LPS, which may suggest a putative role in microorganism recognition. Affinity chromatography on fetuin-agarose of the agglutinin resulted in a 220 kDa band (lectin), and a 82.5 kDa band, which probably is hemocyanin. The 220 kDa protein consists of 31 and 34 kDa subunits, suggesting that this lectin is multimeric. The lectin molecular mass was estimated by gel filtration to be 153+/-10 kDa. The hemolymph of L. schmitti comprises at least another soluble lectin, with distinct chemical and carbohydrate specificity than the 220 kDa lectin.