Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells

We aimed to determine whether three-dimensional (3D) cartilage could be engineered from umbilical cord blood (CB) cells and compare it with both engineered fetal cartilage and native tissue. Ovine mesenchymal progenitor cells were isolated from CB samples (n=4) harvested at 80-120 days of gestation by low-density fractionation, expanded, and seeded onto polyglycolic acid scaffolds. Constructs (n=28) were maintained in a rotating bioreactor with serum-free medium supplemented with transforming growth factor-beta1 for 4-12 weeks. Similar constructs seeded with fetal chondrocytes (n=13) were cultured in parallel for 8 weeks. All specimens were analyzed and compared with native fetal cartilage samples (n=10). Statistical analysis was by analysis of variance and Student's t-test (p<.01). At 12 weeks, CB constructs exhibited chondrogenic differentiation by both standard and matrix-specific staining. In the CB constructs, there was a significant time-dependent increase in extracellular matrix levels of glycosaminoglycans (GAGs) and type-II collagen (C-II) but not of elastin (EL). Fetal chondrocyte and CB constructs had similar GAG and C-II contents, but CB constructs had less EL. Compared with both hyaline and elastic native fetal cartilage, C-II and EL levels were, respectively, similar and lower in the CB constructs, which had correspondingly lower and similar GAG levels than native hyaline and elastic fetal cartilage. We conclude that CB mesenchymal progenitor cells can be successfully used for the engineering of 3D cartilaginous tissue in vitro, displaying select histological and functional properties of both native and engineered fetal cartilage. Cartilage engineered from CB may prove useful for the treatment of select congenital anomalies.     

Interaction of adenosine and its phosphorylated derivatives with putative purinergic receptors in the gill vascular bed of rainbow trout

1. The haemodynamic responses of trout gill to pulses of adenosine and related nucleotides were recorded in isolated trout head preparations. 2. Pulses of adenosine and related nucleotides induced a vasoconstriction of arterial gill vessels. Theophylline antagonized the resonse to adenosine but had not influence on its metabolism. 3. Dipyridamole and two adenosine deaminase inhibitors [deoxycoformycin and erythro-9(2-hydroxy-3-nonyl) adenine] had no effect on either the haemodynamic response of adenosine or its deamination and its uptake by gill tissues. 4. The adenosine response was neither mediated by cholinergic nor adrenergic receptors. 5. These results suggest the existence of extracellular "purinergic receptors" in the gills of trout.     

Genotypic characteristics of two serotypes of Bartonella henselae

The study of 16S rRNA gene sequences of all isolates of Bartonella henselae obtained in our laboratory and others from human patients or cats has revealed two genotypes according to the sequence of the 16S rRNA gene. Two isolates of these genotypes have previously been related to two different serotypes, and lack of cross-protection of the two serotypes has been demonstrated in cats. We investigated the grouping of eight strains of B. henselae on the basis of 16S ribosomal DNA, 35-kDa protein, Pap 31 protein, and internal transcribed spacer (ITS) gene sequencing; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles; and monoclonal antibody reactivity studies. Houston-1, 90-615, and SA2 strains showed the same patterns in SDS-PAGE, but they differed from the patterns of B. henselae isolates URBHLLY8, URBHLIE9, Cat6, Fizz, and CAL-1. Nine monoclonal antibodies derived from BALB/c mice immunized with B. henselae Houston-1 strain reacted only with strains Houston-1, 90-615, and SA2, and not with any other Bartonella strains. The two serogroups corresponded with two genotypes based on differences in the sequences of the genes encoding 16S rRNA, 35-kDa protein, and Pap 31 protein. Sequences of ITS genes were highly divergent among strains, as each had a unique sequence and the subdivision was not supported by DNA-DNA relatedness study. Study of 22 additional strains of B. henselae isolated from French bacteremic cats demonstrated that they all belong to one or the other of the proposed serotype or genotype.     

Two large French pedigrees with non syndromic sensorineural deafness and the mitochondrial DNA T7511C mutation: evidence for a modulatory factor

Hearing impairment is the most frequent sensory defect in children, with a genetic basis in about 50% of cases. Several point mutations and deletions in mitochondrial DNA (mtDNA) have been identified in non-syndromic sensorineural hearing loss (NSSNHL). Beside the frequent A1555G mutation, a number of mutations in tRNAs have been reported recently, but their incidence remains unknown. We identified the T7511C mutation in the tRNASer(UCN) gene in two French families with isolated deafness. Maternal transmission was obvious in both. The 15 patients with hearing impairment exhibited a variable disease phenotype in terms of onset, severity, and progression. T7511C was present in all the patients screened. Homoplasmic and heteroplasmic levels were observed and did not correlate with the severity of the disease. T7511C was also present in 12 hearing offspring of the oldest deaf mothers, confirming the existence of modulatory factors. Our data suggest that this mtDNA mutation should be screened for in all cases of familial NSSNHL compatible with maternal transmission.     

Functional interactions between the VRP1–LAS17 and RHO3–RHO4 genes involved in actin cytoskeleton organization in Saccharomyces cerevisiae

The RGD1 gene from Saccharomyces cerevisiae, which encodes a GTPase-activating protein for the Rho3 and Rho4 small G proteins, exhibits synthetic lethality with the VRP1 and LAS17 genes. Their products are proline-rich proteins that interact with both actin and myosins to ensure polarized growth. By testing functional links, we found that the VRP1 and LAS17 genes are potent suppressors of the rho3Delta mutation. In particular, they restore the polarization of actin patches in rho3Delta cells. Moreover, the vrp1Delta and las17Delta mutations were found to display a similar pattern of genetic interactions with specific actin-linked genes. These mutations also increase the sensitivity to activated forms of both Rho3p and Rho4p. These data support our working model, in which the VRP1 and LAS17 genes define a cellular complex that works in concert with the RHO3-RHO4 signaling pathway in yeast polarized growth. In addition, other observations lead us to propose that Rvs167p may act as a linking protein between the two cellular elements.     

Immune status and uptake of antiretroviral interventions to prevent mother-to-child transmission of HIV-1 in Africa

The aim of this study performed in Abidjan, C?te d'Ivoire, was to describe the distribution of CD4+ T-cell lymphocytes (CD4) in HIV-1-infected (HIV+) pregnant women diagnosed during prenatal voluntary counseling and testing and to assess whether HIV-related immunodeficiency influenced the acceptance of an antiretroviral (ARV) package (zidovudine beginning at 36 weeks of amenorrhea plus intrapartum nevirapine) to prevent mother-to-child transmission. Between April and June 2002, a CD4 count was systematically performed in all HIV+ women (n=221) in 5 antenatal clinics carrying out voluntary counseling and testing. No difference in CD4 count was found in HIV+ women who did not return for their test result (n=50) and those who were informed of their positive serostatus (n=171) (median CD4 count: 389/mm3 vs. 420/mm3; P=0.19). We also found a lack of difference in CD4 count in those who accepted ARV (n=72) and those who did not but knew their HIV status (n=99) (median CD4 count: 405/mm3 vs. 425/mm3; P=0.47). The overall uptake of the intervention (31.9%) appeared to be independent of the maternal immune status.     

Ovarian senescence in the rhesus monkey (Macaca mulatta)

BACKGROUND: A decline in fertility is evident in human females past their middle thirties. This 'reproductive senescence', marked by a sharp decline in pregnancy rates, may be attributed to reductions in numbers of available oocytes and their quality. Because Old World primates exhibit ovarian morphology and physiological control and timing of menstrual cycles closely resembling those of humans, the current study investigated the rhesus macaque as a potential model for human reproductive senescence. METHODS: Ovaries collected from females aged 1-25 years and divided into five age groups were analysed histologically. RESULTS: General ovarian morphology demonstrated significant changes as the females approached menopause. The proportions of primordial and primary follicles all demonstrated significant differences across age groups (primordial: 77.1, 79.9, 69.7, 62.9, 55.1%; primary: 21.5, 18.8, 28.5, 35.2, 43.1% for age groups 1 to 5 respectively; P<0.0001 for both). Samples from females approaching or undergoing the menopausal transition (aged 20-25 years) demonstrated evidence of ovarian senescence, having scattered and atretic follicles, low numbers of primordial follicles and reduced stromal tissue. CONCLUSION: This study supports the value of the rhesus monkey as a model for reproductive ageing because its ovary undergoes follicular reservoir depletion similar to that seen in humans.     

Isodicentric/pseudoisodicentric chromosome 21 amplification in four cases of acute myelocytic leukemia or myelodysplasia

The bone marrow karyotypes of three patients with acute myelocytic leukemia (AML) or myelodysplastic syndrome (MDS) were studied at diagnosis and revealed, multiple copies of the same chromosomal anomaly, considered as psu idic(21)(q22) associated with other rearrangement(s). The karyotype of a fourth patient with MDS in transformation showed one copy of a dicentric marker presumably derived from a similar psu idic(21) by (tandem?) interstitial amplification of part of its structure, resembling a "homogeneous staining region", and described as der(21)psu idic(21)(q22)hsr(21)(q22). This rearrangement, previously described in isolated cases only, might be considered as recurrent in AML/MDS and associated with an unfavorable prognosis. It is most probably a secondary change, because it was never observed as sole abnormality and the main association, as for trisomy 21, was with del(5q). In the four cases, the number of partial supernumerary segmental 21pter-->21q22 copies, ranged from 2 to 10. The AML1 gene did not appear to be the common target of this amplification because this locus had been lost by the psu idic(21) in one patient     

Multispacer typing of Rickettsia prowazekii enabling epidemiological studies of epidemic typhus

Currently, there is no tool for typing Rickettsia prowazekii, the causative agent of epidemic typhus, currently considered a potential bioterrorism agent, at the strain level. To test if the multispacer typing (MST) method could differentiate strains of R. prowazekii, we amplified and sequenced the 25 most variable intergenic spacers between the R. prowazekii and R. conorii genomes in five strains and 10 body louse amplicons of R. prowazekii from various geographic origins. Two intergenic spacers, i.e., rpmE/tRNA(fMet) and serS/virB4, were variable among tested R. prowazekii isolates and allowed identification of three and two genotypes, respectively. When the genotypes obtained from the two spacers were combined, we identified four different genotypes. MST demonstrated that several R. prowazekii strains circulated in human body lice during an outbreak of epidemic typhus in Burundi. This may help to discriminate between natural and intentional outbreaks. Our study supports the usefulness of MST as a versatile method for rickettsial strain genotyping.