Endothelial cell adhesion to the fibronectin CS5 domain in artificial extracellular matrix proteins

This study examines the spreading and adhesion of human umbilical vein endothelial cells (HUVEC) on artificial extracellular matrix (aECM) proteins containing sequences derived from elastin and fibronectin. Three aECM variants were studied: aECM 1 contains lysine residues periodically spaced within the protein sequence and three repeats of the CS5 domain of fibronectin, aECM 2 contains periodically spaced lysines and three repeats of a scrambled CS5 sequence, and aECM 3 contains lysines at the protein termini and five CS5 repeats. Comparative cell binding and peptide inhibition assays confirm that the tetrapeptide sequence REDV is responsible for HUVEC adhesion to aECM proteins that contain the CS5 domain. Furthermore, more than 60% of adherent HUVEC were retained on aECM 1 after exposure to physiologically relevant shear stresses (相似文献   

The detection of heat-aggregated IgG (as a model for immune complexes) by reverse passive haemagglutination using a human monoclonal rheumatoid factor coupled to erythrocytes

A human monoclonal IgM rheumatoid factor (RF) produced in vitro by an Epstein-Barr virus (EBV)-immortalized cell line was purified by protein A-Sepharose adsorption and coupled by the chromic chloride method to human erythrocytes. The RF-coupled cells were incorporated in reverse passive haemagglutination (RPH) assays to detect immune complexes (IC) using heat-aggregated human IgG as a model system. The sensitivity of the RPH was comparable to an enzyme-linked immunosorbent assay (ELISA) using sheep C1q for the detection of ICs.     

Increased frequency of IgG heavy chain marker Glm(2) and of HLA-B8 in Lambert-Eaton myasthenic syndrome with and without associated lung carcinoma

In view of the evidence for an autoimmune pathogenesis of the Lambert-Eaton myasthenic syndrome, we have sought associations with IgG heavy chain allotypes (Gm) and HLA antigens in 30 patients, of whom 20 had evidence of lung carcinoma (histologically proven small ("oat") cell type in 17). A highly significant overall increase in frequency of Glm(2) (chi 2 = 10.95; p less than 0.001; n = 30) and of HLA-B8 (chi 2 = 19.07; p less than 0.001; n = 23) was observed. These two factors apparently occurred independently of each other. The Glm(2) frequency in 36 non-myasthenic small cell carcinoma cases was the same as in a control panel (n = 167). We conclude that Glm(2) and HLA-B8 both associate with increased susceptibility to the Lambert-Eaton myasthenic syndrome, and suggest that Glm(2) may be in linkage disequilibrium with a limited number of VH genes coding for antibodies to restricted antigenic determinants at the nerve terminals, which may be shared by the carcinoma cells.     

Pentoxifylline lessens the endotoxin-induced increase in albumin clearance across pulmonary artery endothelial monolayers with and without neutrophils.

Pentoxifylline, a methylxanthine with phosphodiesterase inhibitor activity, attenuates endotoxin-induced pulmonary vascular protein leak and decreases lung neutrophil accumulation in vivo. In vitro, pentoxifylline decreases neutrophil activation as measured by superoxide release and phagocytosis of latex beads. To test the hypothesis that the beneficial effect of pentoxifylline may be via a direct effect on the endothelial cells as well as via prevention of neutrophil activation, we incubated bovine pulmonary artery endothelial cell monolayers with endotoxin and pentoxifylline in the presence or absence of human neutrophils. Albumin clearance across the monolayers was used as an index of endothelial permeability. Endotoxin (1.0 micrograms/ml) increased albumin clearance in a dose- and time-dependent fashion (207.5 +/- 25%, P less than 0.05). Co-incubation with neutrophils enhanced this effect. Pentoxifylline significantly attenuated the endotoxin-induced increase in albumin clearance both with and without neutrophils, and lessened endotoxin-induced cell lysis (chromium release) and morphologic changes. Because increased endothelial cyclic adenosine monophosphate (cAMP) levels may decrease protein permeability and pentoxifylline increases cAMP in neutrophils, we measured cAMP levels in endothelial cells. Incubation with pentoxifylline failed to raise cAMP levels in endothelial cells, in contrast to incubation with aminophylline. In conclusion, pentoxifylline attenuates endotoxin-induced increase in albumin clearance across endothelial monolayers both in the presence and absence of neutrophils. These results suggest that part of the protective effect of pentoxifylline may be mediated via effects on endothelium. Furthermore, this pentoxifylline-mediated endothelial barrier effect appears to be independent of an effect on cAMP.     

Haplotype and interspersion analysis of the FMR1 CGG repeat identifies two different mutational pathways for the origin of the fragile X syndrome

To understand the origins of the fragile X syndrome and factors predisposing alleles to instability and hyperexpansion, we have compared the haplotype (using markers FRAXAC1, FRAXAC2, and DXS548) and AGG interspersion patterns of the FMR1 CGG repeat for 214 normal and 16 premutation chromosomes. Association testing between interspersion pattern and haplotype reveals a highly significant (P < 0.002) non- random distribution, indicating that all three markers are useful in phylogenetic reconstruction of mutational change. Parsimony analysis of the FMR1 CGG repeat substructure predicts that loss of AGG interruptions has occurred independently on many haplotypes associated with the fragile X syndrome, partially explaining the haplotype diversity of this disease. Among haplotypes found in linkage disequilibrium with the fragile X mutation, two different modes of mutation and predisposition to instability have been identified. One pathway has involved the frequent and recurrent loss of AGG interruptions from rare asymmetrical ancestral array structures. Intergenerational transmission studies suggest that these predisposed chromosomes progress relatively rapidly to the disease state. In contrast, the second mutational pathway involves a single haplotype which has maintained two AGG interruptions. Parsimony analysis of CGG repeat substructure within this haplotype suggests that larger alleles have been generated by gradual increments of CGG repeats distal to the most 3' interruption. Pedigree analysis of the intergenerational stability of alleles of this haplotype confirms a gradual progression toward instability thresholds. As a result, a large reservoir of chromosomes carrying large repeats on this haplotype exists. These chromosomes are predisposed to disease. The present data support a model in which there are at least two different mutational pathways predisposing alleles to instability and hyperexpansion associated with the fragile X syndrome.      

HLA-DQ beta 3.1 allele is a determinant of susceptibility to DR4-associated rheumatoid arthritis

Rheumatoid arthritis is associated with HLA-DR4 in several ethnic groups. Since DR4 haplotypes encode a diverse array of class II molecules, it is of interest to characterize the nature of the primary association. We have examined molecular polymorphisms of HLA class II gene products expressed by normals and rheumatoid arthritis patients using monoclonal antibodies and two-dimensional electrophoresis. Most homozygous DR4 rheumatoid arthritis patients express DR beta 1 molecules associated with Dw4 or Dw14 mixed lymphocyte culture determinants. In Caucasoids, two DR4-linked DQw3-associated beta-chain alleles are defined by two-dimensional electrophoresis. These variants, designated DQ beta 3.1 and 3.2, are associated with the serologic determinants DQw7 and DQw8, respectively. A panel of 40 DR4-positive normals was also examined for nucleotide sequence polymorphisms associated with DQB3.1 and 3.2 genes using the polymerase chain reaction and specific oligonucleotide probes. At the DQ beta level the rheumatoid arthritis panel was distinguished by enrichment for the DQ beta 3.1 allele with 100% of patients positive for DQw7. Results presented here suggest that specific DQ beta alleles may modify the effect of HLA-DR4 beta 1 alleles in conferring susceptibility to rheumatoid arthritis in a phenotype-specific fashion.     

Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP)

Abstract: We have developed a single DNA typing method which uses 144 sequence-specific primer (SSP) reactions to simultaneously detect all known HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 specificities in an allele specific or group specific manner using the same method, reagents, PCR parameters and protocols for all loci. The results from this integrated class I & II method can be visualized on a single photographic or electronic image and hence is described as "Phototyping". Phototyping has an overall resolution greater than or equivalent to good serology and results can be obtained in under 3 hours making the method suitable for genotyping potential cadaver donor peripheral blood without serological backup. This in turn produces the potential for reducing cold ischaemia times in renal transplantation as well as the application of prospective matching to cardiac and liver transplantation. The method has capacity to detect new alleles, for example, novel amplification patterns suggestive of 4 new HLA-B alleles have been detected. The Phototyping set has been used as the sole method of HLA typing for over 1010 individuals. Phototyping is not problem-free; deviations from the standard protocol, poor quality DNA and unsuitable PCR machines can result in individual PCR failures or in incorrect assignment of antigens. Approximately 5% of genotypes were repeated (either partially or fully) because of incomplete or equivocal results.     

血管畸形与血管瘤：神经束的存在是诊断血管畸形的一个线索

动静脉血管畸形和血管瘤都是良性的血管性疾病，这两种疾病在临床上不仅难以区分，而且在组织病理学上也容易混淆，但这两种疾病的治疗是不同的。早期主要是通过组织化学染色动脉的存在来区分二者。为了寻找另外有助于诊断和区分这些病变的方法，作者分析了167例颅内良性血管性病变，其中66例为动静脉血管畸形，101例为以前诊断的血管瘤。     

The PCR-SSP Manager computer program: a tool for maintaining sequence alignments and automatically updating the specificities of PCR-SSP primers and primer mixes

Abstract: An emerging problem of molecular typing methods such as PCR amplification using sequence-specific primers (PCR-SSP) is that they frequently require updating as new alleles are constantly being described which potentially affect the specificity of every PCR-SSP reaction. PCR-SSP uses pairs of primers to detect cis -linked polymorphisms and thus each new allele described must be compared to each individual primer pair. Furthermore, sequence homology between the various loci for class I and class Il means that, for example, new HLA-A sequences have to be compared with HLA-B and HLA-C primer mixes to rule out cross-locus amplification. We have developed a computer program known as SSP Manager which is capable of aligning HLA class I and class TI sequences obtained from Internet-accessible databases such as GenBank. The program then updates all individual primer specificities held in its database before updating the specificities of all primer mixes. Sets of primer mixes can then be combined from the primer mix directory to create PCR-SSP typing trays which are subsequently analysed by the program. A report is generated which stipulates whether all known sequences are amplified and the reason for apparent failure to test for individual alleles, e.g. a lack of relevant sequence information. SSP Manager has the flexibility to cope with unusual sequences (deletions and insertions), primers with internal mismatches and primers with a deliberate mismatch. The program also has many tools for developing new primer mixes, such as the facility to search for novel reactions using Boolean operators. The organisation and operational use of the SSP Manager program is described and its uses are illustrated with an updated allele list for our previously described Phototyping PCR-SSP class I and class TI typing set. The SSP Manager is available on request from the authors.     

Class II HLA associations with autoantibodies in scleroderma: a highly significant role for HLA-DP

Scleroderma is a condition of variable phenotype characterised by fibrosis of the skin and internal organs. There is a range of disease-specific autoantibodies found in the sera of patients. The aims of this study were to: (1) investigate the role of the MHC and particularly HLA-DP in the production of autoantibodies; (2) investigate clinical associations with autoantibodies. We have performed HLA class II typing using PCR with sequence-specific primers on DNA samples from 202 scleroderma patients and 307 UK control subjects. All patients had well defined clinical phenotypes. Sera from patients were examined for the presence of disease specific autoantibodies in particular the anti-topoisomerase autoantibody (ATA), the anti-centromere autoantibody (ACA) and the anti-RNA polymerase autoantibody (ARA). There was a striking association between HLA-DPB1*1301 and ATA (Pcorr = 0.0001). In addition, ATA was associated with HLA-DRB1*11 and the anticentromere autoantibody (ACA) with HLA-DRB1*04, HLA-DRB1*08 (P = 0.001) and HLA-DQB1 alleles with a glycine residue at position 26. Very strong associations were detected between clinical phenotypes and autoantibodies. ATA was associated with pulmonary fibrosis (P = 0.00002), anti-RNA polymerase autoantibody (ARA) with renal involvement (P = 0.0000006) and diffuse skin disease (P = 0.00001), and ACA with limited skin involvement (P = 0.00002) and protection against pulmonary fibrosis (P = 0.0000003). We have identified a significant association between the ATA and HLA-DPB1*1301 which may provide an insight into how this autoantibody is formed. Patient clinical characteristics depend on the autoantibodies they carry.