Development of CD4+ T cell lines that suppress an antigen-specific immune response in vivo

It has been suggested for many years that the regulation of the immune system for the maintenance of peripheral tolerance may involve regulatory/suppressor T cells. In the past few years, several investigators have demonstrated that these cells can be generated in vitro. It has also been shown that they can inhibit the progression of various autoimmune disease models when infused into susceptible mice. We have generated two murine T cell lines in the presence of KLH-specific T cell clones from BALB/c or DBA2 mice. The lines are characterized by a low proliferative response to mitogens, the capacity to secrete high amounts of IL-10 and TGF-beta, and small amounts of IFN-gamma. Interestingly, these cells are unable to produce IL-2, IL-4 or IL-5. The study of the surface phenotype of both lines revealed CD4+, CD25high, CD44low and CTLA-4- cells. When injected intravenously in (CBy.D2) F1 mice, these cells were able to inhibit 50-100% of the TNP-specific antibody production, when the hapten was coupled to KLH. In the present study we offer another evidence for the existence of regulatory T cells in the T lymphocyte repertoire, suggesting that they can also regulate immune responses to foreign antigens. Furthermore, we demonstrate an alternative pathway to generate these cells different from approaches used thus far.     

The effects of distant and on-line visual information on the control of approach phase and step over an obstacle during locomotion

One of the goals of this study was to examine the nature and role of distant visual information sampled during locomotion in the feedforward control of leading and trailing limb while an individual is required to step over an obstacle in the travel path. In addition we were interested in whether or not on-line visual information available while the limb (lead or trail) is stepping over the obstacle influences limb trajectory control and whether the information provided during lead limb cross would be used to calibrate movement of the trail limb. Towards this end, we manipulated availability of vision following an initial dynamic sampling period during the approach phase in proximity to the obstacle and during the lead and trail limb stepping over the obstacle. Ten participants completed 40 trials of obstacle crossing in 8 testing conditions. Initial dynamic visual sampling was sufficient to ensure successful task performance in the absence of vision in the approach phase and during both lead and trail limb stepping over the obstacle. Despite successful task performance, foot placement of the lead and trail limb before obstacle crossing and limb elevation over the obstacle were increased after withdrawal of vision in the approach area. Furthermore, the correlation between toe clearance and foot placement was diminished. While both limbs require feedforward visual information to control the step over the obstacle, only lead limb elevation was influenced by availability of on-line visual information during obstacle crossing. Results were in agreement with the notion of primacy of information inherent in the optic array over those from static samples of the environment in guiding locomotion. It is suggested that the expected proprioceptive feedback information associated with the limb posture before the obstacle, reconstructed using visual memory from dynamic sampling of the environment, mismatched with those from the actual limb position. Accordingly, participants adopted a different strategy that enabled them to clear the obstacle with a higher safety margin.Financial assistance was provided by a grant from the Office of Naval Research, USA, NSERC/Canada, and CAPES/Brazil. We would like to thank Milad G. Ishac, Mike Greig, Zinat Shafaei-Shirazi, and Candida T. Goncalves for their assistance     

Functional characterization of human natural killer cells responding to Mycobacterium bovis bacille Calmette-Guérin

The kinetics of activation and induction of several effector functions of human natural killer (NK) cells in response to Mycobacterium bovis bacille Calmette-Guérin (BCG) were investigated. Owing to the central role of monocytes/macrophages (MM) in the initiation and maintenance of the immune response to pathogens, two different experimental culture conditions were analysed. In the first, monocyte-depleted nylon wool non-adherent (NW) cells from healthy donors were stimulated with autologous MM preinfected with BCG (intracellular BCG). In the second, the NW cells were directly incubated with BCG, which was therefore extracellular. In the presence of MM, CD4+ T lymphocytes were the cell subset mainly expressing the activation marker, CD25, and proliferating with a peak after 7 days of culture. In contrast, in response to extracellular BCG, the peak of the proliferative response was observed after 6 days of stimulation, and CD56+ CD3- cells (NK cells) were the cell subset preferentially involved. Such proliferation of NK cells did not require a prior sensitization to mycobacterial antigens, and appeared to be dependent upon contact between cell populations and bacteria. Following stimulation with extracellular BCG, the majority of interferon-gamma (IFN-gamma)-producing cells were NK cells, with a peak IFN-gamma production at 24-30 hr. Interleukin (IL)-2 and IL-4 were not detectable in NK cells or in CD3+ T lymphocytes at any time tested. IL-12 was not detectable in the culture supernatant of NW cells stimulated with extracellular BCG. Compared to the non-stimulated NW cells, the NW cells incubated for 16-20 hr with BCG induced the highest levels of expression of apoptotic/death marker on the NK-sensitive K562 cell line. BCG also induced expression of the activation marker, CD25, and proliferation, IFN-gamma production and cytotoxic activity, on negatively selected CD56+ CD3- cells. Altogether, the results of this study demonstrate that extracellular mycobacteria activate several NK-cell functions and suggest a possible alternative mechanism of NK-cell activation as the first line of defence against mycobacterial infections.     

Helicobacter hepaticus hydrogenase mutants are deficient in hydrogen-supported amino acid uptake and in causing liver lesions in A/J mice

Helicobacter hepaticus, a causative agent of chronic hepatitis and hepatocellular carcinoma in mice, expresses a nickel-containing hydrogen-oxidizing hydrogenase enzyme. Growth of a hyaB gene-targeted mutant was unaffected by the presence of hydrogen, unlike the wild-type strain, which showed an enhanced growth rate when supplied with H(2). Hydrogenase activities in H. hepaticus were constitutive and not dependent on the inclusion of H(2) during growth. Addition of nickel during growth significantly stimulated both urease (for wild-type and hyaB) and hydrogenase (for wild-type) activities. In a 5-h period, the extent of (14)C-labeled amino acid uptake by the wild type was markedly enhanced in the presence of hydrogen and was >5-fold greater than that of the hyaB mutant strain. In the presence of H(2), the short-term whole-cell amino acid uptake V(max) of the parent strain was about 2.2-fold greater than for the mutant, but the half-saturation affinity for amino acid transport was the same for the parent and mutant strain. The liver- and cecum-colonizing abilities of the strains was estimated by real-time PCR quantitation of the H. hepaticus-specific cytolethal distending toxin gene and showed similar animal colonization for the hyaB mutant and the wild type. However, at 21 weeks postinoculation, the livers from mice inoculated with wild type exhibited moderate lobular lymphoplasmacytic hepatitis with hepatocytic coagulative necrosis, but the hydrogenase mutants exhibited no histological evidence of lobular inflammation or necrosis.     

Growth of the human metatarsal bones in the fetal period (13–24 weeks postconception): a quantitative study

Summary We studied metatarsal growth in 600 metatarsal bones (60 pairs of feet) taken from 60 human fetuses (35 males and 25 females) ranging in age from 13 to 24 weeks postconception. The data obtained for the total length (TL) and for the ossified metatarsal length (OML) were correlated to fetal crown-rump length (C-R). The ossified metatarsal length presented a growth rate greater than the total length growth rate. There was no statistical difference between either the right and left metatarsals or males and females in total length and ossified metatarsal length growth during the period studied. We believe that metatarsal growth curves could be used to monitor fetal foot growth.

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Résumé Nous avons étudié la croissance métatarsienne sur 600 métatarsiens (60 paires de pieds) provenant de 60 foetus humains (35 masculins et 25 féminins) dont l'âge varie de 13 à 24 semaines après la conception. Les données concernant la Longueur Totale et la Longueur Osseuse ont été corrélées à la Longueur Vertex-Coccyx. La Longueur Osseuse a présenté un rythme de croissance plus rapide que la Longueur Totale. Il n'y avait pas de différences significatives pour la croissance de la Longueur Osseuse ou de la Longueur Totale ni entre les métatarsiens droits et gauches, ni entre les foetus masculins et féminins durant la période étudiée. Nous pensons que les courbes de croissance métarsienne pourraient être utilisées pour suivre la croissance du pied fetal.

     

DNA typing for class II HLA antigens with allele-specific or group-specific amplification. III. Typing for 24 alleles of HLA-DP

The second exon of HLA-DPB includes five polymorphic segments with extensive sharing of sequences between alleles. In order to facilitate assignment of specificities in heterozygous individuals, we have used group-specific amplification of two nonoverlapping sets of DPB alleles (here called group A and group B) with especially designed primers. Group A and group B polymerase chain reaction products were hybridized with sequence-specific oligonucleotide probes generating easily recognizable patterns which defined 24 distinct HLA-DPB alleles. We also established a routine procedure for distinguishing HLA-DP homozygosity from failed amplification in one of the alleles. Our results showed that when only one allele was detected, failure of amplification had occurred in less than 4% of the cases. DNA typing with this method correlated well with primed-lymphocyte typing for HLA-DP in the Tenth Workshop, as determined by us in assays performed on the workshop B-cell lines. Two normal panels of unrelated subjects were tested to obtain population frequencies. We conclude that this method is simple, relatively quick, and accurate. It is the method of choice for studies to determine the role of HLA-DP alleles in T cell reactions, in various diseases, and in transplantation.     

Escape efficiency of diploid and polyploid frogs: A comparison ofOdontophrynus cultripes (2n=22) andO. americanus (4n=44)

The escape efficiency of two closely related species of frogs,Odontophrynus cultripes(2n=22) and the tetraploidO. americanus (4n=44), were compared in a shuttle box and under simulated naturalistic conditions.O. americanus was generally superior toO. cultripes, and females tended to outperform males within both species. The relative inefficiency ofO. cultripes escape behavior was examined in light of the animals' having an elaborate, passive defense mechanism in the form of well-marked venom glands. Escape efficiency was highly variable in both species. Possessing twice the amount of DNA, the tetraploid behavioral variation was paradoxically less than that of the diploid, but compatible with what has been found for morphological characters in other organisms.This research was carried out at the Instituto Butantan with the support of ongoing grants from the Brazilian CNPq, FAPESP, FEDIB, and PNUD while the first author was a visiting professor in the Departamento de Genética Humana, Instituto de Biociências, Universidade de São Paulo, under the auspices of the Programa Multinacional de Genética, Organization of American States.     

Reproductive genetic counselling in non-mosaic 47,XXY patients: implications for preimplantation or prenatal diagnosis: Case report and review

With an incidence of approximately 1 in 500 male newborns, the 47,XXY genotype is one the most common sex chromosome anomalies. It is also the most frequent genetic cause of human infertility. Some non-mosaic 47,XXY patients have sperm production which allows infertility treatment to be offered by ICSI. Therefore, the risk of transmitting a chromosome anomaly to the next generation is an important problem in reproductive genetic counselling of these patients. Here, we report on a twin pregnancy where two karyotypically normal neonates 46,XX and 46,XY were born after the use of ICSI in assisted reproduction of a patient with a non-mosaic 47,XXY syndrome. To date, only 38 evolving pregnancies including the present cases, have been reported after ICSI using sperm from non-mosaic 47,XXY patients. Although these data are scarce, they suggest that the risk of chromosome anomaly in the offspring of these patients is low; hence, their reproductive genetic counselling can be reassuring, and management of the pregnancy can proceed with caution.     

Presence of the cfxA gene in Bacteroides distasonis

In this study we investigated the presence of the cfxA gene (encoding a class A beta-lactamase) in 73 strains of the Bacteroides fragilis group belonging to the species B. distasonis (34), B. vulgatus (14), B. thetaiotaomicron (8), B. merdae (6), B. caccae (9) and B. ovatus (2) isolated from human intestinal microflora of healthy children and adults. Employing specific primers to the cfxA gene, a 312-bp amplified fragment was obtained in 2 strains of B. vulgatus and 9 strains, the majority from children, of B. distasonis. The expression of this enzyme was analysed by determining the MICs to cefoxitin and cefotaxime and values varied from 2 to >256 microg/ml of both cefoxitin and cefotaxime. Sequence analysis of the amplicons corresponding to the cfxA gene from B. distasonis and B. vulgatus revealed identical sequences between these isolates and high similarity with other beta-lactamase genes of anaerobes such as cfxA of B. vulgatus (99%) and cfxA2 of Prevotella intermedia (99%), both sequences of which deposited in Genbank under accession numbers U38243 and AF118110, respectively. However, a fragment obtained from a B. distasonis strain (EC17-4) showed a unique RFLP profile and 87% nucleotide similarity with cfxA and cfxA2 genes. These results seem to suggest a dissemination of these resistance determinants among Bacteroides species.