Accumulation of PDGF+ cells and internalisation of the PDGF receptor at myotendinous junction following modified hindlimb muscle use in the rat

Morphological observations have shown previously that myotendinous junctions (MTJs) are sites where the associations between the cytoskeleton and the cell membrane are extensively remodelled during muscle growth and modified mechanical loading. The platelet derived growth factor (PDGF) molecule has been shown to induce cytoskeletal remodelling at focal contact sites of myoblasts in culture, the analogous structures of MTJs. The goals of the study were to determine whether PDGF is synthesised by mononuclear cells and whether PDGF receptors are internalised at the MTJs of the soleus muscle experiencing reloading. We also examined whether ED2⁺ macrophages that are nonphagocytic and activated inflammatory cells at MTJs during reloading secrete PDGF. Results obtained by immunohistochemistry showed that there was an increase in the number of cells expressing PDGF at remodelling MTJs and that the ED2⁺ macrophage population does not express PDGF at MTJs. According to morphological criteria, fibroblasts would be the logical candidates to secrete PDGF molecules near MTJs. Furthermore, the modification in muscle loading resulted in internalisation of PDGF receptors concentrated at the MTJ which accumulated predominantly around muscle nuclei. The enrichment of PDGF receptors and PDGF⁺ cells at MTJs and the internalisation of PDGF receptors during remodelling of MTJs suggest that PDGF may influence remodelling of MTJs following modified muscle use.     

The binding of human alloantibodies to recombinant glycophorin A

Chinese hamster ovary (CHO) cells were transfected with the wild-type, M allele of glycophorin A cDNA. The binding of human alloantibodies to recombinant glycophorin A was assessed with a modified hemagglutination-inhibition assay. Patient sera were incubated with acetone powders derived from CHO cells, and the adsorbed supernatants were tested in standard hemagglutination assays. Five M antibodies and one sample containing anti-En(a) bound to transfected CHO cells expressing glycophorin A but did not bind to untransfected CHO cells. Three N antibodies as well as 21 other alloantibodies (representing other major red cell blood group specificities) bound to neither CHO cell line. The M allele specificity of recombinant glycophorin A was further verified by the demonstration that a high-titer D alloantibody maintained the same titer of agglutination after incubation with recombinant glycophorin A. Transfected CHO cells thus express an M blood group antigen that appears to be serologically equivalent to that found on human red cells. A panel of cell lines expressing mutant glycophorin A molecules with defined variations in amino acid sequence and carbohydrate composition will be useful in studies of the fine specificity of human glycophorin alloantibodies. This approach may also provide an abundant source of artificial antigens for clinical use in blood group serology.     

COMMUNITY-ACQUIRED LOWER RESPIRATORY TRACT INFECTION: IMPLEMENTATION OF AN ANTIBIOTIC PROTOCOL

SUMMARY Enthusiastic formulation of clinical guidelines continues to increase but although theoretical difficulties in guideline implementation have been recognised, little attention has been paid to their effectiveness in everyday clinical practice. The introduction of a protocol for empirical treatment of lower respiratory tract infection (PETRI) to an acute medical take-in unit in Belfast is described. Early involvement of all relevant staff, preparation of user-friendly flow charts, and imaginative publicity, resulted in an initial implementation rate of 75%. The role of implementation as a significant rate-limiting step in the audit cycle is emphasised.     

Absence of strong linkage disequilibrium between odorant receptor alleles and the major histocompatibility complex

The odorant receptor (OR) genes constitute the largest gene family among vertebrates. While over 800 loci are present in the human genome, their allele diversity is still poorly characterized. It has been hypothesized that the products of OR genes can be relevant in the reproductive context, thereby interacting with products of genes of the major histocompatibility complex (MHC). Here we investigated the genetic diversity of the OR2W6P, OR2B8P, OR1F12 and OR12D2 genes, in order to define haplotypes and haplotype frequencies. We measured levels of linkage disequilibrium (LD) between these OR genes and the MHC genes HLA-A, HLA-B and HLA-DRB1. This was accomplished through the assessment of 30 single nucleotide polymorphisms (SNPs) in samples from 61 family trios. We characterized 26 alleles among the four OR genes and identified three SNPs that had not yet been reported. Based on our haplotype analysis, LD spanning the OR-HLA region is not very strong, and therefore not enough to enable selection regarding specific HLA-OR haplotypes.     

Linkage and physical mapping of X-linked lissencephaly/SBH (XLIS): a gene causing neuronal migration defects in human brain

While disorders of neuronal migration are associated with as much as 25% of recurrent childhood seizures, few of the genes required to establish neuronal position in cerebral cortex are known. Subcortical band heterotopia (SBH) and lissencephaly (LIS), two distinct neuronal migration disorders producing epilepsy and variable cognitive impairment, can be inherited alone or together in a single pedigree. Here we report a new genetic locus, XLIS, mapped by linkage analysis of five families and physical mapping of a balanced X;2 translocation in a girl with LIS. Linkage places the critical region in Xq21-q24, containing the breakpoint that maps to Xq22.3-q23 by high-resolution chromosome analysis. Markers used for somatic cell hybrid and fluorescence in situ hybridization analyses place the XLIS region within a 1 cM interval. These data suggest that SBH and X-linked lissencephaly are caused by mutation of a single gene, XLIS, that the milder SBH phenotype in females results from random X-inactivation (Lyonization), and that cloning of genes from the breakpoint region on X will yield XLIS.      

Effects of follicular fluid supplementation of in-vitro maturation medium on the fertilization and development of equine oocytes after in- vitro fertilization or intracytoplasmic sperm injection

The aim of this study was to compare the effect of the addition of follicular fluid (FF) collected from preovulatory follicles with that of oestrous mare serum (EMS) (acting as the control) to TCM-199 medium on the in-vitro maturation, fertilization and development of equine cumulus-enclosed oocytes. Oocytes (<30 mm in diameter) were obtained from the ovaries of slaughtered mares. After in-vitro maturation in the presence of the two supplements, their fertilization, cleavage and developmental potential were compared after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) using frozen-thawed spermatozoa. Follicular fluid did not increase the maturation of oocytes to metaphase II stage compared to control. After IVF, there was no difference in fertilization rates between FF- supplemented oocytes and controls (7/87, 8.4% of oocytes showing two pronuclei with FF versus 7/116, 6% with EMS; not significant). However, after ICSI, FF-supplemented oocytes showed significantly increased normal fertilization (32/85, 37.6% of two-pronuclear oocytes) and developmental potential (15/31, 48% cleavage) compared to the control oocytes (7/47, 14.9%, P < 0.01; and 2/48, 4%, P < 0.01, respectively). Overall, ICSI resulted in increased fertilization rates compared to IVF, regardless of the presence or absence of FF (39/132, 29.5% with ICSI versus 14/203, 6.9%). These results suggest that follicular fluid supplementation may improve the maturity of equine cumulus-enclosed oocytes sufficiently for the successful use of ICSI, but not sufficiently for normal sperm-egg interaction occurring during IVF.      

Length-dependent gametic CAG repeat instability in the Huntington's disease knock-in mouse

The CAG repeats in the human Huntington's disease (HD) gene exhibit striking length-dependent intergenerational instability, typically small size increases or decreases of one to a few CAGs, but little variation in somatic tissues. In a subset of male transmissions, larger size increases occur to produce extreme HD alleles that display somatic instability and cause juvenile onset of the disorder. Initial efforts to reproduce these features in a mouse model transgenic for HD exon 1 with 48 CAG repeats revealed only mild intergenerational instability ( approximately 2% of meioses). A similar pattern was obtained when this repeat was inserted into exon 1 of the mouse Hdh gene. However, lengthening the repeats in Hdh to 90 and 109 units produced a graded increase in the mutation frequency to >70%, with instability being more evident in female transmissions. No large jumps in CAG length were detected in either male or female transmissions. Instead, size changes were modest increases and decreases, with expansions typically emanating from males and contractions from females. Limited CAG variation in the somatic tissues gave way to marked mosaicism in liver and striatum for the longest repeats in older mice. These results indicate that gametogenesis is the primary source of inherited instability in the Hdh knock-in mouse, as it is in man, but that the underlying repeat length-dependent mechanism, which may or may not be related in the two species, operates at higher CAG numbers. Moreover, the large CAG repeat increases seen in a subset of male HD transmissions are not reproduced in the mouse, suggesting that these arise by a different fundamental mechanism than the small size fluctuations that are frequent during gametogenesis in both species.      

Randomized, controlled human challenge study of the safety, immunogenicity, and protective efficacy of a single dose of Peru-15, a live attenuated oral cholera vaccine

Peru-15 is a live attenuated oral vaccine derived from a Vibrio cholerae O1 El Tor Inaba strain by a series of deletions and modifications, including deletion of the entire CT genetic element. Peru-15 is also a stable, motility-defective strain and is unable to recombine with homologous DNA. We wished to determine whether a single oral dose of Peru-15 was safe and immunogenic and whether it would provide significant protection against moderate and severe diarrhea in a randomized, double-blind, placebo-controlled human volunteer cholera challenge model. A total of 59 volunteers were randomly allocated to groups to receive either 2 x 10(8) CFU of reconstituted, lyophilized Peru-15 vaccine diluted in CeraVacx buffer or placebo (CeraVacx buffer alone). Approximately 3 months after vaccination, 36 of these volunteers were challenged with approximately 10(5) CFU of virulent V. cholerae O1 El Tor Inaba strain N16961, prepared from a standardized frozen inoculum. Among vaccinees, 98% showed at least a fourfold increase in vibriocidal antibody titers. After challenge, 5 (42%) of the 12 placebo recipients and none (0%) of the 24 vaccinees had moderate or severe diarrhea (> or = 3,000 g of diarrheal stool) (P = 0.002; protective efficacy, 100%; lower one-sided 95% confidence limit, 75%). A total of 7 (58%) of the 12 placebo recipients and 1 (4%) of the 24 vaccinees had any diarrhea (P < 0.001; protective efficacy, 93%; lower one-sided 95% confidence limit, 62%). The total number of diarrheal stools, weight of diarrheal stools, incidence of fever, and peak stool V. cholerae excretion among vaccinees were all significantly lower than in placebo recipients. Peru-15 is a well-tolerated and immunogenic oral cholera vaccine that affords protective efficacy against life-threatening cholera diarrhea in a human volunteer challenge model. This vaccine may therefore be a safe and effective tool to prevent cholera in travelers and is a strong candidate for further evaluation to prevent cholera in an area where cholera is endemic.     

Algorithms for sleep–wake identification using actigraphy: a comparative study and new results

The aim of this study was to investigate two new scoring algorithms employing artificial neural networks and decision trees for distinguishing sleep and wake states in infants using actigraphy and to validate and compare the performance of the proposed algorithms with known actigraphy scoring algorithms. The study employed previously recorded longitudinal physiological infant data set from the Collaborative Home Infant Monitoring Evaluation (CHIME) study conducted between 1994 and 1998 [ http://dccwww.bumc.bu.edu/ChimeNisp/Main_Chime.asp ; Sleep 26 (1997) 553 ] at five clinical sites around the USA. The original CHIME data set contains recordings of 1079 infants <1 year old. In our study, we used the overnight polysomnography scored data and ankle actimeter (Alice 3) raw data for 354 infants from this data set. The participants were heterogeneous and grouped into four categories: healthy term, preterm, siblings of SIDS and infants with apparent life‐threatening events (apnea of infancy). The selection of the most discriminant actigraphy features was carried out using Fisher’s discriminant analysis. Approximately 80% of all the epochs were used to train the artificial neural network and decision tree models. The models were then validated on the remaining 20% of the epochs. The use of artificial neural networks and decision trees was able to capture potentially nonlinear classification characteristics, when compared to the previously reported linear combination methods and hence showed improved performance. The quality of sleep–wake scoring was further improved by including more wake epochs in the training phase and by employing rescoring rules to remove artifacts. The large size of the database (approximately 337 000 epochs for 354 patients) provided a solid basis for determining the efficacy of actigraphy in sleep scoring. The study also suggested that artificial neural networks and decision trees could be much more routinely utilized in the context of clinical sleep search.