Architecture and regulation of a GDNF-GFRα1 synaptic adhesion assembly.

Glial-cell line derived neurotrophic factor (GDNF) bound to its co-receptor GFRα1 stimulates the RET receptor tyrosine kinase, promoting neuronal survival and neuroprotection. The GDNF-GFRα1 complex also supports synaptic cell adhesion independently of RET. Here, we describe the structure of a decameric GDNF-GFRα1 assembly determined by crystallography and electron microscopy, revealing two GFRα1 pentamers bridged by five GDNF dimers. We reconsitituted the assembly between adhering liposomes and used cryo-electron tomography to visualize how the complex fulfils its membrane adhesion function. The GFRα1:GFRα1 pentameric interface was further validated both in vitro by native PAGE and in cellulo by cell-clustering and dendritic spine assays. Finally, we provide biochemical and cell-based evidence that RET and heparan sulfate cooperate to prevent assembly of the adhesion complex by competing for the adhesion interface. Our results provide a mechanistic framework to understand GDNF-driven cell adhesion, its relationship to trophic signalling, and the central role played by GFRα1.

Antibiotic prescription patterns in paediatric wards of Rivers State university teaching hospital, southern Nigeria: a point prevalence survey

Antimicrobial resistance remains a threat to patient safety and healthcare outcomes and largely arises from inappropriate antimicrobial prescriptions. This study aimed to determine the pattern of antibiotic prescriptions in the Paediatrics department of Rivers State University Teaching Hospital, Port Harcourt.Method:A point prevalence survey was conducted in the Paediatric wards and Special Care Baby Unit (SCBU) on 13 November 2021. Records of all children admitted before or at 8:00a.m. on the day of the survey were descriptively analysed using the protocol and web-based management system of the Global Point Prevalence Survey of Antimicrobial Consumption and Resistance, University of Antwerp.Results: The antibiotic prevalence in this study was 77.4%. The most common indication(s) for antibiotic use in SCBU was infection prophylaxis (81.3%) and in paediatric wards: Pneumonia, Ear Nose Throat and Soft tissue infections accounted for (23.1%) each. Third-generation cephalosporins and aminoglycosides were predominantly used in all wards and were empirical-based prescriptions. Regarding antibiotic quality indicators of prescriptions: In SCBU: 19 (90.5%) had indication(s) for antibiotics documented, 10 (46.7%) were guideline compliant, and 1 (4.8%) had documented review/stop date. In the paediatric medical and surgical wards, 17(85.0%) vs. 4(100%) had indication(s) for antibiotics documented, 6(30.0%) vs. 0(0%) were guideline compliant, and 1(5.0%) vs. 4(100.0%) had a review/ stop date.Conclusion:High prevalence of antibiotic use, suboptimal antibiotic quality indicators and absence of laboratory evidence for antibiotic prescriptions were observed in the paediatric units. There is a need to reorientate prescribers and institute strategic measures to improve antimicrobial stewardship

C-Reactive Protein and Covid-19 Severity: A Systematic Review.

BACKGROUND: COVID-19 clinical course has been quite unpredictable and efforts have been made to identify reliable markers that will help in early disease progression, prognosis and severity detection. Objective: This study thus aimed to provide evidence that will guide clinical management by reviewing studies that assessed CRP concentration and COVID-19 severity/outcome. METHODS: Three electronic databases, PubMed/Medline, Google Scholar, and JSTOR were searched to identify studies available online as at 1st September 2020 which assessed COVID-19 clinical outcome and CRP concentration. The search strategy involved words combination like "C-reactive protein" OR "inflammatory markers" OR "acute phase reactants" and "coronavirus 2019" OR ''COVID-19" OR "2019-nCoV" OR "SARS-CoV-2". RESULTS: Sixty-one articles were systematically reviewed out of 812 studies identified after duplicates were removed. The 61 studies comprised 13,891 COVID-19 patients made of 7,840 (56.4%) males and 6,051 (43.6%) females. All the papers revised were observational studies except one case-control and they cut across fifteen countries. The result of the review demonstrated that the severe cases had higher levels of C - reactive protein when compared to the mild cases in all the studies (100%). The increase in C-reactive protein was statistically significant in 78.7% of the cases. CONCLUSION: High levels of CRP are associated with COVID-19 severity. Highlights: Severe cases of COVID-19 is characterized with higher CRP levels. COVID-19 cases should be screened regularly for CRP to monitor severity.

Contexte: L'évolution clinique du COVID-19 a été assez imprévisible et des efforts ont été faits pour identifier des marqueurs fiables qui aideront à la progression précoce de la maladie, au pronostic et à la détection de la gravité. Objectif : Cette étude visait donc à fournir des preuves qui guideront la gestion clinique en passant en revue les études qui ont évalué la concentration de CRP et la gravité/l'issue du COVID-19. Méthodes: Trois bases de données électroniques, PubMed/Medline, Google Scholar et JSTOR, ont été consultées pour identifier les études disponibles en ligne au 1er septembre 2020 qui évaluaient le résultat clinique du COVID-19 et la concentration de CRP. La stratégie de recherche comportait des combinaisons de mots comme "protéine Créactive" OU "marqueurs inflammatoires" OU "réactifs de phase aiguë" et "coronavirus 2019" OU "COVID-19" OU "2019-nCoV" OU "SARS-CoV-2". Résultats: Soixante et un articles ont été systématiquement examinés sur les 812 études identifiées après suppression des doublons. Les 61 études comprenaient 13 891 patients atteints de COVID-19, dont 7 840 (56,4 %) hommes et 6 051 (43,6 %) femmes. Tous les articles examinés étaient des études d'observation, à l'exception d'un cas-témoin, et ils couvraient quinze pays. Le résultat de l'examen a démontré que les cas graves avaient des niveaux plus élevés de protéine C-réactive par rapport aux cas légers dans toutes les études (100%). L'augmentation de la protéine C-réactive était statistiquement significative dans 78,7% des cas. Conclusion: Des niveaux élevés de CRP sont associés à la sévérité du COVID-19. Mots clés: Protéine C-réactive, COVID-19, SRAS-COV-2 et Coronavirus. Points forts: Les cas graves de COVID-19 sont caractérisés par des niveaux de CRP plus élevés. · Les cas de COVID-19 doivent faire l'objet d'un dépistage régulier de la CRP afin de surveiller la gravité de la maladie.

Significant IgG subclass heterogeneity in HLA-specific antibodies: Implications for pathogenicity, prognosis, and the rejection response.

IgG subclasses differ in their ability to fix complement and bind Fc receptors. This study describes a detailed analysis of the distribution of HLA-specific IgG subclasses in order to define how this varies in sensitised waiting-list patients. We found significant variation in the level, presence and combinations of each HLA-specific IgG subclass between and within individuals and this is influenced by the type of sensitising event. Graft failure in particular provokes higher levels of IgG1 (vs transfusion, p=0.071 and pregnancy, p=0.042), IgG2 (vs transfusion, p=0.001 and pregnancy, p=0.016), and IgG4 (vs transfusion, p=0.052). Both graft failure and pregnancy tend to stimulate multiple IgG subclass responses against HLA, whereas transfusion stimulated antibodies are dominated by responses limited to IgG1 (p=0.033) and have a low incidence of IgG4 (p=0.046). In marked contrast, IgG4 characterised nearly all HLA DQ-specific antibodies stimulated by graft rejection (p=0.006). Such widely varying IgG subclass heterogeneity is likely to be due to underlying immunological processes dependent on the route of sensitisation. This diversity, which implies functional variation, may help explain why HLA-specific antibodies are an obstacle to transplantation in some circumstances but not others. The subclass association with rejection has potential as a biomarker for chronic rejection.

Redox-mediated substrate recognition by Sdp1 defines a new group of tyrosine phosphatases.

Reactive oxygen species trigger cellular responses by activation of stress-responsive mitogen-activated protein kinase (MAPK) signalling pathways. Reversal of MAPK activation requires the transcriptional induction of specialized cysteine-based phosphatases that mediate MAPK dephosphorylation. Paradoxically, oxidative stresses generally inactivate cysteine-based phosphatases by thiol modification and thus could lead to sustained or uncontrolled MAPK activation. Here we describe how the stress-inducible MAPK phosphatase, Sdp1, presents an unusual solution to this apparent paradox by acquiring enhanced catalytic activity under oxidative conditions. Structural and biochemical evidence reveals that Sdp1 employs an intramolecular disulphide bridge and an invariant histidine side chain to selectively recognize a tyrosine-phosphorylated MAPK substrate. Optimal activity critically requires the disulphide bridge, and thus, to the best of our knowledge, Sdp1 is the first example of a cysteine-dependent phosphatase that couples oxidative stress with substrate recognition. We show that Sdp1, and its paralogue Msg5, have similar properties and belong to a new group of phosphatases unique to yeast and fungal taxa.

Identification of a new HLA-DRB5 allele, DRB5*0112, by routine PCR-SSP.

We describe the identification of a new HLA-DRB5 allele found in two members of a British Caucasoid family. Genomic analysis of exon 2 revealed a novel sequence. The name DRB5*0112 has been assigned to this allele by the WHO Nomenclature Committee, and the EMBL sequence database accession number for this is AJ427352. DRB5*0112 has several unique sequence features when compared to other DRB5 alleles, and comparison with all known DRB1/3/4/5 alleles indicates this to have arisen as a result of intraexonic recombination between a DRB5-containing haplotype and one carrying DRB1*09.

The distribution of 13 killer-cell immunoglobulin-like receptor loci in UK blood donors from three ethnic groups.

Killer-cell immunoglobulin-like receptors (KIRs) can inhibit the killing activity of natural killer (NK) cells if they interact with their ligand, class I HLA. Using a modified polymerase chain reaction-sequence-specific primers (PCR-SSP) method for typing KIRs using genomic DNA, we compared KIR frequencies in three ethnic populations drawn from UK blood donors. We found a significantly lower frequency of the inhibitory KIRs KIR2DS1 and 3DS1 in Afro-Caribbean blood donors. Despite this, there was a (non-significant) increase in the B haplotype in Afro-Caribbean and Indian Asian donors. Some donors from each group tested negative for all non-inhibitory KIRs. In addition, we describe several new KIR profiles. Analysis of linkage disequilibrium was consistent with previously published findings.

Used leucodepletion filters as a source of large quantities of DNA suitable for the study of genetic variations in human populations.

UNLABELLED: A simple technique for developing large control panels with large quantities of DNA suitable for studies in population genetics was established. BACKGROUND AND OBJECTIVES: Both a lack of suitable controls and insufficient quantities of DNA for repeated analysis of the same control group often hamper the investigation of genetic markers for disease. MATERIALS AND METHODS: Using a waste product from routine blood donation, we describe a simple method that allows the investigator to extract large amounts of DNA. RESULTS: A mean of 1520 microg of DNA per sample was obtained. The DNA obtained remains suitable for polymerase chain reaction and sequencing techniques after 2 years of storage at both 4 degrees C and -40 degrees C. CONCLUSION: This technique allows the development of a large panel of controls with sufficient quantities of genomic DNA for thousands of tests.

Rapid haplotyping of mutations in the Duffy gene using the polymerase chain reaction and sequence-specific primers.

The molecular basis for the antigenic variation and red cell expression of the Duffy antigen system has recently been elucidated. We have developed a simple one-step method for genotyping the single nucleotide polymorphisms in the promoter and exon of the Duffy gene using the polymerase chain reaction and sequence-specific primers (PCR-SSP). This method is also capable of haplotyping alleles at the two polymorphisms as being in cis or trans orientation. Twenty-four serologically typed Caucasoid and Afro-Caribbean samples were examined to validate the method, with absolute correlation between phenotype and genotype. A further 30 Gambian samples were genotyped, confirming homozygosity for the FY*null-FY*B haplotype. Allele, gene and haplotype frequencies were examined in 100 Caucasoid controls. This method permits the rapid genotyping of large numbers of samples and will prove useful as a clinical and research tool.

DNA allelic alterations within VNTR loci of scleroderma families.

We have characterized genetic alterations at the molecular level in 49 scleroderma and 45 control families using variable number tandem repeats (VNTRs). Additionally, paired fibroblast cell lines from the 'affected' and 'unaffected' skin and peripheral blood leucocytes of 30 patients were also examined. All families in this study were typed for Class I Cw alleles and Class II-DRB, -DQA and -DQB to confirm family membership. There were significant rises in the level of VNTR mutations in scleroderma patients (36.7%, n = 18), their siblings (16.3%, n = 13) and offspring (21.7%, n = 15). The level of VNTR mutations in the control group was 0.6% (n = 5). These mutations did not correlate with the presence of autoantibodies and no patient was taking a known clastogenic drug. The most common VNTR site for mutation was pYNZ22 (17p13.4). Differences were also seen in the VNTR alleles between fibroblast and lymphocyte DNA from the same patient, as measured by size alteration of one of the alleles. We have found that VNTRs are unstable in scleroderma patients, relatives and offspring. The reason for the genomic changes remains unknown, but previous studies have implicated the presence of a clastogen.

Telomere reduction in scleroderma patients: a possible cause for chromosomal instability.

We have hypothesized that the chromosomal instability observed in scleroderma patients and their family members may result from the loss of long stretches of the telomeric repeat which is found at the ends of all linear chromosomes. We examined the telomere lengths in scleroderma (SSc) patients (n = 43), their family members (n = 182) and in age-matched controls (n = 96) using restriction fragment length polymorphism (RFLP) and chemiluminescent labelled probes. The average loss of telomeric DNA in SSc patients and family members was found to be 3 kb when compared to the controls. This loss was not related to age or the duration of the disease. These results may reflect a genetic predisposition for chromosomal instability in these families, or exposure to a common environmental agent. A wide variety of common environmental agents are known to produce chromosomal aberrations: these include fungicides, pesticides, air pollutants and drugs. Scleroderma-like syndromes may be induced by some of these agents.

Class II MHC typing in pemphigoid gestationis.

Pemphigoid gestationis (PG) is a rare, autoimmune skin disease associated with pregnancy or the immediate post-partum period, previously shown to be associated with the HLA class II antigens DR3 and DR4. Advances in molecular analytical techniques now allow the identification of HLA alleles previously difficult to define by serological assays. Unsuspected polymorphism within the HLA-DR3 and DR4 classes can, therefore, be identified. The aim of our study was to apply these newer techniques to the question of genetic predisposition in PG by re-evaluating the association with DR3 and by studying a possible link with DQ. We have investigated by restriction fragment length polymorphism, the DQA, and by sequence specified oligonucleotide probing the DQB and DRB1 (HLA DR) specificities of 41 women with immunofluorescence-confirmed PG. The principal finding of this study is that there is an association between PG and DRB1*0301 (DR3) and DRB1*0401/040X (DR4). Although there is also an increase (P = 0.06) in the concurrent presence of both antigens, this appears to be due to the association with either antigen alone. We also found an increase in the frequency of DQA1*2 (P = 0.016 vs. control) and a decrease in frequency of DQB1*0201 (P = 0.022 vs. controls) and DQB1*0602 (P = 0.026 vs. controls).

Familial scleroderma--evidence for environmental versus genetic trigger.

Families with more than one case of scleroderma are unusual. Four families each with two members (in one case monozygotic twins) with scleroderma (systemic sclerosis, SSc) were identified. Clinical, immunogenetic and autoantibody studies were carried out. Multicase SSc families cited in the literature were reviewed. Each family pair shared cutaneous subset of disease severity, and SSc-associated autoantibody. HLA typing showed two pairs shared an HLA-DR allele associated with scleroderma (DR3 or DR5), while one also had alleles reported in association with their SSc-specific autoantibody. Review of dates and ages of onset suggested that the timing of onset of scleroderma is more likely to have an environmental trigger than to be encoded genetically.

Anti-centromere antibodies (ACA) in systemic sclerosis patients and their relatives: a serological and HLA study.

Autoantibody reactivity to centromere proteins CENP-A, CENP-B and CENP-C was examined in 58 patients with systemic sclerosis (SSc), 218 first degree relatives and 22 spouses. HLA class II typing for HLA-DRB1 and HLA-DQA1 was performed by restriction fragment length polymorphism (RFLP) analysis in 50 families, and HLA-DRB1, HLA-DQA1 and HLA-DQB1 typing was performed by olignucleotide typing in 44 families. Eleven probands and two relatives had ACA. The two relatives with ACA also had SSc. One relative was an identical twin sister of a proband with ACA and the other relative was a sister of a proband with ACA. All ACA-positive probands and relatives were female, and all recognized CENP-A, CENP-B and CENP-C. The presence of at least one HLA-DQB1 allele not coding for leucine at position 26 of the first domain appeared necessary, although not sufficient for the generation of ACA. Therefore within SSc families ACA is strongly associated with female gender and disease phenotype, and is at least in part genetically determined.

Complement polymorphism in herpes gestationis: association with C4 null allele.

BACKGROUND: Herpes gestationis (HG) is a rare, pregnancy-related skin disease characterized by the production of an autoantibody to a component of the hemidesmosome. It is associated with the class II antigens HLA-DR3 and HLA-DR4, but its potential association with the "class III antigens" C2, C4, and factor B has not previously been studied. OBJECTIVE: Our purpose was to study complement polymorphism in HG. METHODS: Using electrophoresis and immunofixation techniques, we determined the allele frequencies of C4A, C4B, C3, and factor B in 42 patients with a history of HG. RESULTS: Ninety percent of patients carried a C4 null allele (C4*QO). No statistically significant association with C3 or factor B alleles was seen. CONCLUSION: HG is associated with the presence of a C4*QO. Whether the C4*QO is the primary genetic association, or whether the C4*QO is related to its linkage disequilibrium with DR3 and DR4 has yet to be determined.

Anti-HLA antibodies in pemphigoid gestationis (herpes gestationis).

Pemphigoid gestationis (PG; herpes gestationis) is a rare autoimmune disease associated with pregnancy, currently defined by the presence of complement deposition along the cutaneous basement membrane zone. It is known to be associated with HLA-DR3 and DR4, and an increase in anti-HLA antibodies in those with a history of PG has been reported. We have studied 39 patients with an immunofluorescence-confirmed diagnosis of PG for the presence and specificity of anti-HLA antibodies. Anti-HLA antibodies were found in all 39 patients. Specificity was against class I antigens in 98% (controls 10%; P < 0.001) and class II antigens in 25% (controls 8.5%; P < 0.001). Almost all anti-HLA antibodies were cytotoxic. The universal presence of anti-HLA antibodies in PG suggests that they may develop coincidently with antibasement membrane antibodies, and may reflect a common immunological event.

Genetic and environmental factors in scleroderma.

This review discusses possible markers of scleroderma, including fibronectin gene mutations, major histocompatibility complex class II antigens, anti-Scl-70, and the Fc receptor. The association between genetic and environmental factors is also reviewed, as is a mouse model of human disease.

Immunogenetic prediction of pulmonary fibrosis in systemic sclerosis.

75 systemic sclerosis patients were independently tested for pulmonary fibrosis, autoantibodies, and MHC class II genes. 24 of 42 (57%) patients with pulmonary fibrosis had either HLA DR3/DRw52a or anti-Scl-70 vs 2 of 33 (6%) patients without pulmonary fibrosis. The presence of DR3/DRw52a or anti-Scl-70 gives a relative risk of 16.7 for the development of pulmonary fibrosis in a patient with scleroderma--a risk substantial enough to require careful monitoring of these patients and treatment at an early stage of disease.

Influence of C4 null alleles on C4 activation in systemic lupus erythematosus.

Deficiencies of early components of the classical complement pathway are known to be associated with systemic lupus erythematosus (SLE). C4 null alleles, C4A Q0 and C4B Q0, are prime candidates for the major histocompatibility complex associated factor which determines susceptibility to SLE. There is poor correlation, however, between the presence of low concentrations of C4 and possession of C4 null alleles, and thus the basis of the association between C4A Q0, C4B Q0 and SLE remains obscure. The possibility that activation of C4 may be related to the possession of C4 null alleles was examined. C4 phenotypes were investigated, and C4 concentration and activation were estimated in patients with SLE. C4 activation was determined by measuring the concentration of C4d--a split product of C4. Twenty five of 35 patients had C4 phenotypes which include null alleles. No association between low C4 concentrations and C4 null alleles was found, but a significant association between low C4d concentrations and C4 phenotypes including null alleles, particularly those with C4A Q0, was noted. No correlation between concentrations of C4 and C4d was found. These results show an influence of C4 null alleles on the activation of the C4 molecule, which is independent of the concentration of C4. The possession of silent genes coding for C4 null alleles might predispose to SLE by conditioning poor C4 activation, a critical event for the clearance of immune complexes mediated by the classical complement pathway.