Control of interleukin 1 (IL-1) activity. I. Inhibition of IL-1 activity by soluble immune response suppressor (SIRS) in vitro.

Soluble immune response suppressor (SIRS), a nonspecific inhibitor of cellular and humoral immune responses and cellular proliferation, reversed IL-1-induced inhibition of autologous rosette formation by thymocytes. In addition, SIRS prevented the IL-1-induced increase in resistance of thymocytes to the lytic action of hydrocortisone. Kinetic experiments showed that the action of SIRS on thymocytes was rapid (less than 15 minutes), although a longer time was required to exert protective effects on thymocytes. SIRS also inhibited the stimulation of thymocyte proliferation induced by Con A and IL-1 a costimulatory assay of IL-1 activity. Moreover, SIRS inhibited the IL-1-stimulated expression of complement receptors on neonatal B cells. The inhibitory effects of SIRS were selectively directed towards IL-1, since SIRS did not interfere with induction of LAK cells by IL-2, and did not reverse inhibition of autologous rosette formation induced by factors other than IL-1, such as IL-4, a proline rich polypeptide and lactoferrin. The results presented in this report demonstrate that SIRS may be a selective inhibitor of IL-1 activity with respect to T and B cells, rendering them unresponsive to IL-1 activation and/or maturation signals.     

Novel HLA-B35 subtypes: Putative gene conversion events with donor sequences from alleles common in native americans (HLA-B*4002 or B*4801)

In a study of 523 normal subjects of differing ethnic groups, including 189 South American Indians, we have described novel hybridization patterns corresponding to 22 potentially new HLA-B locus alleles. Three of these alleles were subtypes of B35. The locally assigned alleles, B-3504v, B-3505v, and B-3508v have been sequenced and were officially designated as B*3512, B*3517, and B*3518, respectively. In addition, we determined the nucleotide sequence of another new variant, locally designated B-3509.2. B*3517, was found in 3 individuals (2 Hispanic, 1 Caucasian), it differs from B*3505 by 3 nucleotide substitutions that lead to changes in residues 94, 95, and 103. B*3517 differs from B*3501 in residues 97 and 103. B*3518 was found in 7 South American Indian individuals (6 of 124 Toba Indians, 1 of 18 Pilaga Indians). It differs from B*3509 by 2 silent nucleotide substitutions and by one nonsynonymous substitution in codon 156 (Arg → Leu). B*3512 differs from B*3504 by 3 nucleotides, one of them leading to a substitution in residue 103 (Val → Leu). B*3509 was observed in 3 individuals from the Wichi tribe. The nucleotide sequence of one of these was determined and was found to differ from B*35091 by two synonymous nucleotide substitutions.

The distinguishing amino acid substitutions in residues 95, 97, and 156 contribute to the structure of specificity pockets F, C, and E, and D and E respectively; therefore, it is possible that some of the new alleles may have different peptide binding profiles. It has been shown that differences at residue 156 may elicit different allorecognition and mediate graft-versus-host disease and rejection in bone marrow transplantation. The mechanisms for the generation of these novel alleles may involve gene conversion events in which short exon-3 segments from the common Native American alleles B*4002 or B*4801 were inserted in HLA-B35 backbone structures. The novel allele B*3518 is closely related to B*35092 and to B*3508. Two alternative hypotheses for its generation can be suggested, the most plausible one would involve B*35092, the putative progenitor of B*3518, since both alleles are prevalent in the same Indian tribes.     

Accuracy of quick Sequential Organ Failure Assessment (qSOFA) score and systemic inflammatory response syndrome (SIRS) criteria for predicting mortality in hospitalized patients with suspected infection: a meta-analysis of observational studies

ObjectiveTo identify sensitivity, specificity and predictive accuracy of quick sequential organ failure assessment (qSOFA) score and systemic inflammatory response syndrome (SIRS) criteria to predict in-hospital mortality in hospitalized patients with suspected infection.MethodsThis meta-analysis followed the Meta-analysis of Observational Studies in Epidemiology (MOOSE) group consensus statement for conducting and reporting the results of systematic review. PubMed and EMBASE were searched for the observational studies which reported predictive utility of qSOFA score for predicting mortality in patients with suspected or proven infection with the following search words: ‘qSOFA’, ‘q-SOFA’, ‘quick-SOFA’, ‘Quick Sequential Organ Failure Assessment’, ‘quick SOFA’. Sensitivity, specificity, area under receiver operating characteristic (ROC) curves with 95% confidence interval (CI) of qSOFA and SIRS criteria for predicting in-hospital mortality was collected for each study and a 2 × 2 table was created for each study.ResultsData of 406 802 patients from 45 observational studies were included in this meta-analysis. Pooled sensitivity (95% CI) and specificity (95% CI) of qSOFA ≥2 for predicting mortality in patients who were not in an intensive care unit (ICU) was 0.48 (0.41–0.55) and 0.83 (0.78–0.87), respectively. Pooled sensitivity (95% CI) of qSOFA ≥2 for predicting mortality in patients (both ICU and non-ICU settings) with suspected infection was 0.56 (0.47–0.65) and pooled specificity (95% CI) was 0.78 (0.71–0.83).ConclusionqSOFA has been found to be a poorly sensitive predictive marker for in-hospital mortality in hospitalized patients with suspected infection. It is reasonable to recommend developing another scoring system with higher sensitivity to identify high-risk patients with infection.     

Increased distribution and expression of CD64 on blood polymorphonuclear cells from patients with the systemic inflammatory response syndrome (SIRS)

Evidence is growing to suggest that the multiple organ damage of the systemic inflammatory response syndrome (SIRS) arises from the untoward activity of blood polymorphonuclear cells (PMNs), which upon activation acquire the IgG high affinity receptor, CD64. In the current study, flow cytometry was used to assess the prevalence of CD64-bearing PMNs and the intensity of expression of CD64 in whole blood samples from 32 SIRS patients, 11 healthy normal subjects and from eight non-SIRS patients in the intensive care unit (ICU). The percentage of PMNs expressing CD64 was higher in SIRS patients (mean 65%) than in non-SIRS patients (mean 42%; P < 0.02) and in healthy controls (mean 19%; P < 0.001) and was particularly evident in patients with SIRS and sepsis (mean 71%; P < 0.02) as opposed to SIRS alone (mean 55%). There were more CD64 molecules expressed on PMNs from patients with SIRS (median 1331 molecules/cell) in comparison with PMNs from healthy subjects (median 678 molecules/cell; P < 0.01). The highest intensity of CD64 expression was associated with PMNs from patients with both SIRS and sepsis. Functional studies revealed that the supranormal binding of PMNs from patients with SIRS to endothelial monolayers treated with TNFalpha was impeded by anti-CD64 antibodies (mean 24% inhibition; P < 0.01). Monitoring the distribution of CD64+ PMNs and their level of CD64 expression could be of assistance in the rapid discrimination of patients with SIRS from other ICU patients and in the identification of PMNs which are likely to participate in the pathological manifestations of the disease.     

Structural implication of the induced immune response by Bacillus thuringiensis Cry proteins: role of the N-terminal region

The potential role of the regions (carboxil and amino) of the Cry proteins in the ability of these proteins to elicit strong immune responses was investigated. Intraperitoneal immunization of mice with the homologous Cry1A protoxins (130–133 kDa), with the long C-terminal half gave rise mostly to similar, strong serum and mucosal IgG and IgM antibody response but a lower induction of these Ab by intranasal route. Remarkably, Cry3A protoxin, devoid of C-terminal half was able to induce a significant mucosal IgG, and IgM Ab as well as Cry1A protoxins, suggesting us that immunogenic abilities are not restricted to C-terminal half but N-terminal half itself could be involved. In fact, this assumption was strengthen by the strong immunogenic abilities of the Cry1A toxins, specially IgG and IgA Ab induced by both routes in different mucosal sites. These data indicate that immunogenic abilities of the Bt Cry proteins reside and depends of the N-terminal half.     

Generation of a soluble immune response suppressor factor (IRSF) by unstimulated leukocytes of healthy subjects.

Supernatants derived from unstimulated cultures of mononuclear leucocytes obtained from healthy subjects contained a factor(s) which consistently suppressed lymphocyte proliferative responses to antigens and mitogens. This factor(s) is produced by a non-adherent cell and was generated in vitro after 4 hr of culture. Cells from almost all healthy subjects produced this substance(s). Cell number and viability were not affected by it. Kinetic studies suggested that interference with antigen presentation was not the mechanism of its action. The release of this immune response suppressor factor(s) (IRSF) was not blocked by indomethacin and its biological activity was unrelated to levels of prostaglandin E2. Experiments with low-specific-activity thymidine showed that suppression was not due to release of unlabelled nucleotide. Preliminary characterization of IRSF revealed that it is heat-stable and partially dialysable through membranes with an exclusion size of 12,000 daltons. IRSF differs from previously reported soluble suppressor substances and may play a role in immunoregulation in health.     

Genome Prediction of Putative Genome-Linked Viral Protein (VPg) of Astroviruses

Positive-sense single-stranded RNA (+ssRNA) viruses replicate by uncoating the RNA genome for translation to provide viral proteins essential for genome replication and the production of new viral particles. The viral proteins are synthesized from a polyprotein precursor, which is cleaved nascently. The synthesized proteins include viral RNA-dependent RNA polymerase (RdRP), viral genome-linked protein (VPg), and a helicase. VPg is covalently attached to the genomic form of +ssRNA viruses. Helicases and NTPase unwind the RNA before replication. VPg and helicases have been identified in +ssRNA families, however, the presence of VPg and helicase in the Astroviridae, another +ssRNA family, has not been fully elucidated. Computational tools were utilized to provide sequence analysis evidence for the presence and genomic location of astrovirus VPg and helicase. HMMER program v2.1.1 was used to build Hidden Markov Model (HMM) profile for calicivirus VPg to search for conserved motifs in the astrovirus genome. We performed phylogenetic analysis of two genomic regions of astroviruses and caliciviruses (encoding the RdRP and VPg). We identified a putative VPg coding region in astrovirus. This region was located in open reading frame 1a (ORF1 a) and included sites with high sequence similarity to the VPg coding regions of Caliciviridae, Piconaviridae, and Potyviridae. A region encoding a putative astrovirus helicase identified conserved motifs only with pestivirus helicase sequences. Sequence analysis and comparison to other +ssRNA viruses supports the presence of VPg in the Astroviridae. Further laboratory analysis will be necessary to confirm these findings.     

High-resolution sequence typing of HLA-DQA1 and -DQB1 exon 2 DNA with taxonomy-based sequence analysis (TBSA) allele assignment

High-resolution DNA sequencing of exon 2 of DQA1 and DQB1 genes that uses a taxonomy-based sequence analysis (TBSA) method to assign alleles was developed. The system uses fewer primers for polymerase chain reaction (PCR) amplification and sequencing than other methods and yields accurate DQA1 and DQB1 typing when either homozygous or heterozygous DNA samples are tested. The approach was initially corroborated by the correct typing of 10 blinded samples that had been previously typed by PCR using sequence-specific oligonucleotide probes (PCR-SSOP) or serology, and subsequently confirmed by sequencing of cloned PCR products. DNA from peripheral blood cell samples of 130 individuals enrolled in a case-control analysis of HLA determinants of abdominal aortic aneurysm were subsequently evaluated. Overall, 8 different DQA1 and 19 DQB1 alleles were identified. All 21 DQA1 heterozygous combinations and 45 of 49 DQB1 heterozygous combinations were successfully resolved with TBSA. The two pairs of heterozygous DQB1 combinations that were not unambiguously typed required sequence specific PCR amplification for correct allele identification. We conclude that the method provides precise analysis for HLA-DQ typing.     

Effects of K-76COOH (MX-1) on immune response: induction of suppressor T-cells by MX-1.

K-76COOH (MX-1), isolated from the cultured supernatant of a species of fungi imperfecti, Stachybotrys complement nov. sp. K-76, is an inhibitor of the complement component, C5. The effects of MX-1 on various immune responses were investigated. MX-1 enhanced the response of spleen cells to PHA and LPS: MX-1 at 0.01-250 micrograms/ml for PHA and at 10-250 micrograms/ml for LPS. In contrast, it inhibited the response to Con A: MX-1 at 0.01-500 micrograms/ml for spleen cells and at 100-500 micrograms/ml for thymocytes. MX-1 and IL-1 synergistically acted to enhance the Con A response of spleen and thymus cells from which accessory cells and Ia-positive cells had been removed by passing through Sephadex G-10 columns and treating with anti-Ia monoclonal antibody plus complement. T-cells pretreated with MX-1, IL-1 and Con A for 3 days suppressed not only the response of B-cells to LPS but also the production of anti-SRBC antibodies. In addition, MX-1 was found to increase CD8+ T-cells. These results suggest that MX-1 acts on T-cells to induce suppressor T-cells.     

Nucleotide sequence analysis of an HLA-B47 variant (HLA-B*4702)

The name B*4702 has been officially assigned by the WHO Nomenclature Committee in November 1996. This follows the agreed policy that, subject to the conditions staled in the most recent nomenclature report (1), names will be assigned to new sequences as they are identified. Lists of such names will be published in the following WHO nomenclature report. The nucleotide sequence data reported in this article will appear in E.M.B.L. under the accession number Y09118.     

Mutations in the N-terminal globular domain of the type X collagen gene (COL10A1) in patients with Schmid metaphyseal chondrodysplasia

Schmid metaphyseal chondrodysplasia (SMCD) is a relatively common, heritable osteochondrodysplasia characterized by short-limbed short stature with normal facies, and generalized metaphyseal dysplasias of the long and short tubular bones. Several mutations of the type X collagen gene (COL10A1) have been reported in patients with SMCD, all in the C-terminal globular domain. To address whether mutations in other domains can cause SMCD, we examined the coding region of the COL10A1 gene in DNA samples from six Japanese families affected with SMCD, by direct sequencing. We detected novel mutations in three unrelated SMCD patients; one was a one-base deletion in the C-terminal globular domain and others were de novo missense mutations in the N-terminal globular domain. All three cases revealed a typical clinical phenotype for SMCD. Thus, we have demonstrated that mutations of COL10A1 in regions other than the C-terminal globular domain can cause SMCD, and the results suggest that the N-terminal globular domain also plays an important role in formation of type X collagen. Hum Mutat 9:131–135, 1997. © 1997 Wiley-Liss, Inc.     

Nucleotide sequence of the gene for perfringolysin O (theta-toxin) from Clostridium perfringens: significant homology with the genes for streptolysin O and pneumolysin.

The nucleotide sequence was determined for the gene encoding the thiol-activated cytolysin, perfringolysin O (theta-toxin), from Clostridium perfringens. The nucleotide-sequence-derived primary structure of perfringolysin O is 499 residues long and exhibits a 27-amino-acid signal peptide. The calculated molecular weight of the secreted (mature) form of perfringolysin O is 52,469. The deduced amino-terminal sequence of perfringolysin O is identical to that determined for purified perfringolysin O. Hydropathy analysis indicated that, except for the signal peptide, no major stretches of hydrophobic residues are present. Extensive amino acid sequence homology (65%) was detected with the low-molecular-weight form of streptolysin O, and a lesser amount (42%) was detected with pneumolysin. The nucleotide sequence of the perfringolysin O gene (pfo) exhibits approximately 60% homology with the streptolysin O gene (slo) and 48% homology with the pneumolysin gene (ply). All three toxins contain an identical region of 12 amino acids, which includes the essential cysteine of all three toxins. The location of these 12 residues was conserved in all three toxins when the primary sequences were aligned for maximum homology.     

Full-length cDNA nucleotide sequence of a serologically undetectable YLLA-DQA1 allele: HLA-DQA1*"LA"

This study describes the characterization of a serological HLA-DQ "blank" specificity that segregates with the HLA-A2, -B7, -DR14, -DR52 haplotype. Although conventional serological typing techniques could not detect an HLA-DQ product on the haplotype positive for the HLA-DQ "blank" specificity, sequence-specific oligonucleotide (SSO) dot-blot analysis demonstrated the presence of the HLA- DQA1*01 and HLA- DQB1*05 alleles. Full-length cDNA nucleotide sequence analysis revealed that the HLA- DQB1 allele that segregated with the HLA-DQ "blank" specificity was identical to HLA- DQB1*05031. As for the HLA- DQA1 allele, one nucleotide substitution distinguished the HLA- DQA1 "blank" allele from HLA- DQA1*0104. In exon 2 at nucleotide position 304 a C was substituted for a T (Arg→Cys). Pending official recognition by the WHO Nomenclature Committee, this HLA- DQA1 "blank" allele is termed HLA- DQA1*"LA". Furthermore, it is postulated that the introduction of cysteine at amino acid position 102 abrogates the classical HLA-DQ1 specificity.     

Complete genomic sequence and an infectious BAC clone of feline herpesvirus-1 (FHV-1)

Infection with feline herpesvirus-1 (FHV-1) is a major cause of upper respiratory and ocular diseases in Felidae. We report the first complete genomic sequence of FHV-1, as well as the construction and characterization of a bacterial artificial chromosome (BAC) clone of FHV-1, which contains the entire FHV-1 genome and has the BAC vector inserted at the left end of U_L. Complete genomic sequences were derived from both the FHV-1 BAC clone and purified virion DNA. The FHV-1 genome is 135,797 bp in size with an overall G + C content of 45%. A total of 78 open reading frames were predicted, encoding 74 distinct proteins. The gene arrangement is collinear with that of most sequenced varicelloviruses. The virus regenerated from the BAC was very similar to the parental C-27 strain in vitro in terms of plaque morphology and growth characteristics and highly virulent in cats in a preliminary in vivo study.     

A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

ANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and δ -rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, δ -rPR3-S176A does not. Culture supernatants of rPR3-S176A and δ -rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA⁺ sera were tested. With δ -rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.