Establishment of fetomaternal tolerance through glycan-mediated B cell suppression.

Discrimination of self from non-self is fundamental to a wide range of immunological processes1. During pregnancy, the mother does not recognize the placenta as immunologically foreign because antigens expressed by trophoblasts, the placental cells that interface with the maternal immune system, do not activate maternal T cells2. Currently, these activation defects are thought to reflect suppression by regulatory T cells3. By contrast, mechanisms of B cell tolerance to trophoblast antigens have not been identified. Here we provide evidence that glycan-mediated B cell suppression has a key role in establishing fetomaternal tolerance in mice. B cells specific for a model trophoblast antigen are strongly suppressed through CD22-LYN inhibitory signalling, which in turn implicates the sialylated glycans of the antigen as key suppressive determinants. Moreover, B cells mediate the MHC-class-II-restricted presentation of antigens to CD4+ T cells, which leads to T cell suppression, and trophoblast-derived sialoglycoproteins are released into the maternal circulation during pregnancy in mice and humans. How protein glycosylation promotes non-immunogenic placental self-recognition may have relevance to immune-mediated pregnancy complications and to tumour immune evasion. We also anticipate that our findings will bolster efforts to harness glycan biology to control antigen-specific immune responses in autoimmune disease.

Are lateralized and bold fish optimistic or pessimistic?

Cognitive bias is defined as the influence of emotions on cognitive processes. The concept of the cognitive judgement bias has its origins in human psychology but has been applied to animals over the past 2 decades. In this study we were interested in determining if laterality and personality traits, which are known to influence learning style, might also be correlated with a cognitive bias in the three-spined sticklebacks (Gasterosteus aculeatus). We used the judgement bias test with the go/no-go procedure where fish were first trained to discriminate between a black and white card and, after reaching a minimum learning criterion, tested their response to an ambiguous card (grey). Optimistic subjects were expected to have a high expectation of reward associated with an ambiguous stimulus, whereas pessimistic subjects a high expectation of non-reward. We used an emergence and a mirror test to quantify boldness and laterality, respectively. We hypothesised that male, bolder and more strongly lateralized fish would be more optimistic than female, shy and less strongly lateralised fish. We found that males and more strongly lateralized fish were more optimistic than females and less strongly lateralized fish. In addition, bold males were more optimistic than shy males as we predicted, but females showed the opposite pattern. Finally, fish trained on the black colour card learned the training task faster than those trained on a white card. Our results indicate that both laterality and personality traits are linked to animals' internal states (pessimistic or optimistic outlooks) which likely has broad implications for understanding animal behaviour particularly in a welfare context.

Essential role of STAT3 in the control of the acute-phase response as revealed by inducible gene inactivation [correction of activation] in the liver.

We generated mice carrying a STAT3 allele amenable to Cre-mediated deletion and intercrossed them with Mx-Cre transgenic mice, in which the expression of Cre recombinase can be induced by type I interferon. Interferon-induced deletion of STAT3 occurred very efficiently (more than 90%) in the liver and slightly less efficiently (about 70%) in the bone marrow. Analysis of the induction of liver acute-phase genes in response to bacterial lipopolysaccharide unequivocally identifies STAT3 as a fundamental mediator of their induction. The different degrees of defectiveness displayed by the various genes allowed us to differentiate them into three separate groups according to their degree of dependence on STAT3. Induction was totally defective for group I genes, defective at 24 h but almost normal at earlier time points for group II genes, and only slightly defective for group III genes. This division was in good agreement with the known structures of the respective promoters. We also found that the overall induction of the transcription factors C/EBP beta and -delta was only minimally defective in the absence of STAT3. Finally, even though corticosterone levels and action were found to be normal in the conditional-mutant mice, production of both proinflammatory and antiinflammatory cytokines was increased and prolonged, probably as a result of STAT3 deletion in macrophages.

Gene electrotransfer results in a high-level transduction of rat skeletal muscle and corrects anemia of renal failure.

We have investigated the efficacy of a gene transfer strategy based on plasmid DNA electroinjection for the correction of anemia associated with renal failure. An expression plasmid encoding the rat erythropoietin (EPO) cDNA under the control of the CMV promoter as constructed and utilized for this work. Electroinjection of pCMV/rEPO in different rat muscles yielded sustained and long-term EPO production and secretion. The muscle-produced EPO corrected the anemia in five of six nephrectomized rats, used as a model of renal failure. The efficiency of muscle transduction was comparable in rats and mice injected with equivalent amounts of DNA per kilogram of body weight. These results demonstrate that gene electrotransfer can be applied to produce therapeutically significant levels of erythropoietin in chronic renal failure.

Diversity of alleles encoding HLA-B40: relative frequencies in united states populations and description of five novel alleles.

The frequency of each B*40 allele was determined by DNA sequencing in four major United States populations: Caucasians, African Americans, Asians/Pacific Islanders, and Hispanics. Thirty-two individuals from each ethnic group, who were previously described serologically as B40, B60, or B61, were randomly selected out of a pool of 82,979 unrelated individuals for allele characterization. Out of nine different B*40 alleles identified in this study, B*4001 and B*4002 were the two most frequent B*40 alleles in all the population groups. B*4001 was the primary B*40 allele seen in Caucasians (83%) and African Americans (76%), while B*4002 was found in the majority of Hispanics (62%). The distributions of both alleles were comparable in the Asian/Pacific Islander population. These two alleles were the only B*40 alleles detected in Caucasians while four to five additional B*40 alleles were seen in the other population groups. The other B*40 alleles detected in this study included: B*4003 and B*4010 in Asian/Pacific Islanders; B*4012 and B*4016 in African Americans; and B*4004, B*4006, and B*4027 in Hispanics. Analysis revealed significant differences between Hispanics and all other groups as well as between African Americans and Asian/Pacific Islanders. This report also describes five novel B*40 alleles: B*4019, B*4020, B*4024, B*4027, and B*4028.

Effect of naloxone on TRH-induced PRL and TSH response in normal man.

The effect of naloxone on PRL and TSH response to TRH was studied in 8 normal men. PRL response to TRH was unaffected by the administration of naloxone. TSH response to TRH was significantly decreased by naloxone, considering the maximum absolute increment (p less than 0.01) and the areas under curves (p less than 0.05). In conclusion, the PRL response to TRH may be unaffected by endogenous opioids or naloxone and we were unable to reveal a possible opioid effect in our experimental conditions. TSH response to TRH seems to be affected by endogenous opioids, possibly due to an indirect or direct action on TRH and/or on the pituitary.

[Determination of prolactin secretion in idiopathic galactorrhea]. / Valutazione della secrezione di prolattina nella galattorrea idiopatica.

We have studied 15 women with idiopathic galactorrhoea and normal serum PRL basal values, regularly menstruating, with normal x-ray of the sella turcica and mammography. The patients were not suffering from other endocrine diseases nor taking medications known to cause galactorrhoea. These subjects and 5 normal women were submitted to an evaluation of the serum PRL circadian rhythm, to TRH test for PRL and TSH, metoclopramide test for PRL, and basal determination of TT3 and TT4. The results did not show any significant difference between the two groups and did not reveal any abnormality of PRL secretion in the patients. These data, resulting from a homogeneous series of patients, support the hypothesis of an increased sensitivity of breast receptors to PRL, or of the secretion of heterogeneous forms of PRL, biologically active on mammary tissue, but not detected by current RIA methods for PRL.

Efficacy of part- and full-time early intervention.

The effectiveness of an early intervention program to remediate developmental delays in children age birth to 3 years was examined in part- and full-time groups (Study 1). Significant improvements on age-appropriate measures of developmental standing were observed for both groups, with the greatest gains observed for the full-time group. In Study 2, the stress of parents with developmentally delayed children was measured on the Parental Stress Inventory. Reductions in stress related to children's characteristics and dysfunctional parenting skills were observed on some subscales, supporting prior research which indicated extension of the outcomes of early intervention beyond the child was desirable.

[Isolated peripheral arterial ischaemia and medullary neurostimulation: case report]. / Ischemia arteriosa periferica isolata e neurostimolazione midollare: case report.

Isolated peripheral arterial ischaemia (IPAI) is an unusual pathology of dialysis and peritoneal patients which represents the first sign of a complication of uraemia known as calciphylaxis. Recent studies have revealed an increased incidence of this complication. Risk factors are known but there is no consensus on them: elevated CaxP product, female gender, elevated serum parathormone. We present here the case of a 65-year-old man with 21-year history of dialysis, distal isolated ulceration and without any signs of severe vasculopathy. Our clinical diagnosis was calciphylaxis. In this case, the role of early PTX is not clear and the use of steroids is recommended only in non-ulcerating cases. The therapy gives good results but not in all patients. Electrical stimulation of the posterior roots of the spinal cord is an alternative approach to this case. We hypothesised that the electrical action, through cutaneous vasodilatation of afferent dorsal fibres and release of calcitonin gene-releasing protein, determines the release of prostaglandin E sub 2 that may positively affect the proliferation and activity of epidermal fibroblasts.

Is the bioreactivity of vitamin-E-modified dialyzer an expression of increased plasmatic vitamin E concentration?

The present study was designed to test the biocompatibility of a new vitamin E-modified multilayer membrane compared with highly biocompatible polysulphone dialyzer and acrylonitrile dialyzer. Thirty patients (mean age 53.2 +/- 15.3 SD years; dialytic age 36 +/- 5.6 months) were selected for the study. The study was divided into three periods of 6 months (phases A, B and C). In the first phase (from Jan. 1999 to June 1999) patients undergoing maintenance bicarbonate dialysis were randomly divided into three filter groups composed, respectively, of 10 patients: acrylonitrile group, polysulphone group and vitamin E-coated dialyzer group. In the phase B (from July 1999 to Dec. 1999) and in the phase C (from Jan. 2000 to June 2000), all three groups changed their own dialysis membranes. Vitamin E-coated dialyzer causes significant decreases in beta(2)-microglobulin, ferritin and immunoglobulin G, a normalization of complement C3 and an increase of plasmatic vitamin E compared to other filters. In the VE group homocysteine decreases but not in a significant manner. In addition, this dialyzer seems not to influence lipid pattern and protein-energy malnutrition parameters. These results clearly show a positive effect of this new filter in influencing different biochemical parameters, perhaps saving vitamin E and reducing polymorphonuclear cell activation.

Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation.

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 microgram of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.

Novel HLA-A locus alleles including A*01012, A*0306, A*0308, A*2616, A*2617, A*3009, A*3206, A*3403, A*3602 and A*6604.

This paper describes 10 novel HLA-A alleles that have been characterized by DNA sequencing. Seven alleles, A*0308, A*2616, A*3009, A*3206, A*3403, A*3602 and A*6604 carry motifs observed in other HLA-A alleles, suggesting that gene conversion has created this diversity. The remaining three alleles, A*01012, A*0306 and A*2617, contain polymorphisms not previously found in any "classical" class I allele. All alleles were identified due to unexpected probe hybridization patterns during routine SSOP typing. Exons 2 and 3 of each allele were subsequently characterized by DNA sequencing.

Development of animal models for adeno-associated virus site-specific integration.

The adeno-associated virus (AAV) is unique in its ability to target viral DNA integration to a defined region of human chromosome 19 (AAVS1). Since AAVS1 sequences are not conserved in a rodent's genome, no animal model is currently available to study AAV-mediated site-specific integration. We describe here the generation of transgenic rats and mice that carry the AAVS1 3.5-kb DNA fragment. To test the response of the transgenic animals to Rep-mediated targeting, primary cultures of mouse fibroblasts, rat hepatocytes, and fibroblasts were infected with wild-type wt AAV. PCR amplification of the inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genome integrated into the AAVS1 site in fibroblasts and hepatocytes. Integration in rat fibroblasts was also observed upon transfection of a plasmid containing the rep gene under the control of the p5 and p19 promoters and a dicistronic cassette carrying the green fluorescent protein (GFP) and neomycin (neo) resistance gene between the ITRs of AAV. The localization of the GFP-Neo sequence in the AAVS1 region was determined by Southern blot and FISH analysis. Lastly, AAV genomic DNA integration into the AAVS1 site in vivo was assessed by virus injection into the quadriceps muscle of transgenic rats and mice. Rep-mediated targeting to the AAVS1 site was detected in several injected animals. These results indicate that the transgenic lines are proficient for Rep-mediated targeting. These animals should allow further characterization of the molecular aspects of site-specific integration and testing of the efficacy of targeted integration of AAV recombinant vectors designed for human gene therapy.

Novel HLA-B alleles associated with antigens in the 8C CREG.

This paper describes 13 novel HLA-B locus alleles, B*0809, B*0812, B*0813, B*0814, B*14062, B*3804, B*3806, B*3914, B*3915, B*3918, B*3919, B*3920, and B*3922 which represent new patterns of known polymorphic residues.

Twelve novel HLA-B*15 alleles carrying previously observed sequence motifs are placed into B*15 subgroups.

Twelve new B*15 alleles are described. All of the known B*15 alleles are divided into subgroups based on serologic assignments and/or nucleotide sequence polymorphisms. These groups might be used as a reference for DNA-based testing at an intermediate (i.e. "serologic") level of resolution.

Twenty-nine new HLA-B alleles associated with antigens in the 5C CREG.

This paper describes 29 novel HLA-B locus alleles identified during low-resolution typing. The majority of the novel alleles carry new patterns of previously known polymorphic motifs or codons. Three alleles carry alterations in the Bw4/Bw6 epitope. Five alleles carry novel substitutions.