Identification of an idiotype on mouse monoclonal antibody directed to a 35 kDa glycoprotein from Entamoeba histolytica.
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Biomedical subjects
Publications and source records attributed to F Blanco.
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Our aim was to investigate links between systemic lupus erythematosus (SLE)-associated autoantibodies, idiotypes (Id) and genetic predisposition to their development. We studied four public Ids (16/6, WRI 176 beta, RT72 and RT84), identified the Km and Gm phenotypes and sought six selected autoantibodies in 32 SLE patients, 174 of their relatives and 15 spouses. Though anti-double-stranded DNA antibody was uncommon in the relatives (9%), the range of antinuclear reactivities was as broad in the relatives as in the probands. Antibodies to the synthetic peptide U1-RNP-A 35-38 were found in 56% of the patients, 28% of their relatives and 20% of the spouses, whereas antibodies to the Golgi apparatus was present in 7% of the patients, 26% of their relatives and 33% of the spouses. However, most of these family members were unaffected. RT84 Id was positively associated with antibodies to Sm-D peptide 1-20 and to Ro/SSA 60 kD peptide 304-324, but negatively associated with anti-dsDNA activity. The median of age was significantly lower in the RT84 Id-positive than in the RT84 Id-negative individuals. These data suggest that genetic as well as environmental factors are involved in the aetiology of SLE. In addition, RT84-carrying immunoglobulins (Ab2) might be directed to one of many cross-reactive Ids of dsDNA-binding antibodies (Ab1), perhaps down-regulating their production.
Articular cartilage chondrocytes have the unique ability to elaborate large amounts of extracellular pyrophosphate (PPi), and transforming growth factor beta (TGF beta) appears singular among cartilage regulatory factors in stimulating PPi production. TGF beta caused a time and dose-dependent increase in intracellular and extracellular PPi in human articular chondrocyte cultures. TGF beta and interleukin 1 beta (IL-1 beta) antagonistically regulate certain chondrocyte functions. IL-1 beta profoundly inhibited basal and TGF beta-induced PPi elaboration. To address mechanisms involved with the regulation of PPi synthesis by IL-1 beta and TGF beta, we analyzed the activity of the PPi-generating enzyme NTP pyrophosphohydrolase (NTPPPH) and the PPi-hydrolyzing enzyme alkaline phosphatase. Human chondrocyte NTPPPH activity was largely attributable to plasma cell membrane glycoprotein 1, PC-1. Furthermore, TGF beta induced comparable increases in the activity of extracellular PPi, intracellular PPi, and cellular NTPPPH and in the levels of PC-1 protein and mRNA in chondrocytes as well as a decrease in alkaline phosphatase. All of these TGF beta-induced responses were completely blocked by IL-1 beta. Thus, IL-1 beta may be an important regulator of mineralization in chondrocytes by inhibiting TGF beta-induced PPi production and PC-1 expression.
OBJECTIVE: To examine growth factor responses of human articular chondrocytes in aging and development. We have previously established a growth factor response profile for adult human articular chondrocytes and shown that transforming growth factor beta (TGF beta) is the most potent mitogen among a variety of factors tested. METHODS: Chondrocytes were isolated from human articular cartilage obtained from donors ages 11-83 years and tested in primary culture for proliferative responses to serum and recombinant preparations of the major chondrocyte growth factors. Proliferation was measured by 3H-thymidine incorporation and cell counting. Skeletal maturity of the young donors was determined by radiographic assessment of Risser's index. RESULTS: Chondrocytes showed a continuous age-related decline in the proliferative response to serum. Analysis of recombinant growth factors showed that with increasing donor age, there was a decrease in the levels of DNA synthesis in response to all factors tested. In chondrocytes from adult donors, there was no change in the relative potencies of the different growth factors. The decrease in the levels of DNA synthesis as measured by 3H-thymidine incorporation corresponded to a reduced rate of in vitro cell replication with increasing donor age. In addition to the quantitative changes in the proliferative responses of chondrocytes with increasing age, there was a qualitative change in the pattern of growth factor responses during development. Cells from young donors (ages 10-20) responded better to platelet-derived growth factor, AA chain homodimer (PDGF-AA) than to TGF beta 1, while the inverse pattern was seen in cells from adult donors. This decrease in the response to PDGF-AA was significantly correlated with increasing skeletal maturity. CONCLUSION: Chondrocyte growth factor responsiveness shows qualitative changes during development, and after skeletal maturity, there is a profound decline in the levels of DNA synthesis and cell replication in response to the known chondrocyte growth factors.
A patient was treated by tendon grafting following division of both flexor tendons of a digit. The graft was removed because of infection and a silicone rod was inserted in spite of the suppuration. After the replacement of the implant by a new tendon graft, the final result was satisfactory and the patient recovered good function.
We have identified and characterised three new idiotypes on human IgM McAbs generated from the splenocytes of a SLE patient with active disease. RT-6, which binds H1 and Sm/RNP, expresses essentially a private Id. Its expression is limited to a small number of human McAbs and the sera from patients with infectious diseases. In contrast RT-72Id and RT-84Id, expressed on McAbs which are polyreactive for two or more antigens, have a public distribution. RT-72Id and RT-84Id are found on McAbs from murine and human adult, and foetal tissues. In sera, significant numbers of SLE, RA and patients with other autoimmune diseases are positive for both Ids. RT-84Id is also elevated in SLE relatives and spouses, and in patients with Klebsiella infection. No correlation with disease activity, IgM or IgG levels was observed with either Id. However, RT-72Id was significantly associated with anti-ssDNA antibodies and RhF. RT-6Id and RT-72Id are located on the framework regions of the mu heavy chain, whereas RT-84Id is present on the kappa light chain, within the binding site. The McAbs are encoded by mainly germline genes: heavy chains of RT-6, RT-72 and RT-84 are encoded by the genes VH26, VH4.22 and VH4.21, respectively, and the light chain sequences of RT-6 and RT-72 are derived from DPL11 and HK102. Immunofluorescent staining revealed the presence of RT-72Id and RT-84Id positive immunoglobulin deposits in 18% and 45%, respectively, of the lupus renal sections compared with none in the disease control group, suggesting that these Ids may contribute to the pathology of the disease.
The use of a bone peg for fusion of the distal radio-ulnar joint as part of the Sauvé-Kapandji procedure is described. This has been used effectively in six patients, one of whom developed a painless pseudoarthrosis after a fall.
We describe a human monoclonal antibody designated WRI 176 beta and a common idiotype that it carries. This antibody was derived from the spleen of a patient with SLE. WRI 176 is an IgM kappa monoclonal reacting with ssDNA, dsDNA, poly(dT) and it is likely that mAb WRI 176 beta is a representative of the so-called natural autoantibodies. The common Id designated WRI 176 Id beta is located on the heavy chain of the mAb WRI 176 beta molecule and appears to be located outside the binding site. Sequence analysis of the WRI 176 beta heavy chain showed it to be highly homologous (97.3%) with a germline gene 56PI derived from a human fetus. In a retrospective analysis, although 44% of SLE patients had raised levels of the WRI 176 beta no correlation was found with the activity of the disease. The idiotype was also expressed frequently in a range of autoimmune rheumatic and infectious diseases and in some healthy first-degree relatives of SLE patients.
Porins isolated from Salmonella typhi have been demonstrated to protect against the challenge with this bacteria in mice. The mechanism has not been clarified, but could be associated with activation of both humoral and cellular immunity. In order to evaluate the induction of specific T cell responses, the lymphocytic proliferation to porins isolated from Salmonella typhimurium, Salmonella typhi and Escherichia coli was examined by 3H-thymidine incorporation assay in mice immunized with three different antigens: acetone-killed S. typhimurium, its porins, or outer-membrane proteins (OMPs) isolated from S. typhi. Higher proliferative responses were observed in mice immunized with porins and OMPs compared with those which received the acetone-killed bacteria. Although cross-reactivity was observed between porins, they were not mitogenic. Moreover, porins were able to activate T lymphocytes isolated from mice immunized with S. typhi OMPs. These results suggest that T cell activation, through the release of lymphokines, may play a role in the induction of protective immunity with porins.
The current studies were undertaken to assess the role of the porins and outer membrane proteins (OMP) in the human immune response to Salmonella typhi 9, 12 Vi:d. Experiments were performed to determinate the lymphocyte activation response to porins in individuals who had been vaccinated against typhoid fever. 10 healthy volunteers were studied before and 10 days after oral or subcutaneous immunisation. Five patients with typhoid fever were also studied. Lymphocyte activation was measured by the 3H thymidine incorporation assay. Individuals with typhoid fever as well as those immunised with oral vaccine responded well to porins and outer membrane proteins, as opposed to those immunised with the subcutaneous vaccine. These results suggest that the porins and OMP play a role in the cellular immune response against Salmonella typhi.
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In this study the concentration of the different subclasses of IgG in sera from patients with a range of autoimmune rheumatic diseases (ARD) was detected by radial immunodiffusion. In the second part the IgG subclasses of autoantibodies that recognize single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), Ro, La, Sm and RNP in patients with ARD were measured by enzyme-linked immunosorbent assay. We studied 15 patients with lupus, 20 patients each with primary and secondary Sjögren's syndrome (SS) and 10 each with rheumatoid arthritis (RA), scleroderma and myositis. Twenty healthy controls were also measured. The serum concentration of IgG2 in ARD patients was generally reduced. In contrast, the concentrations of IgG1, IgG3 and IgG4 subclasses were normal or raised. A high degree of correspondence in the IgG1, IgG2 and IgG3 responses to dsDNA and ssDNA in SLE was found. Notable differences in the IgG1 anti-Ro and ssDNA responses compared to the other subclasses were seen in 1 degree and 2 degrees SS. In addition, an unexpected high level of IgG4 antibodies to ssDNA in 1 degree SS (65%) and IgG4 antibodies to Sm/RNP in RA was observed.
In 71 patients with fever and bacteremia without complications, a prospective study of acute-phase reactants is done. Raises in haptoglobin, ceruloplasmin, alpha-1-antitrypsin, protein C, beta-2-microglobulin, IgA and ferritin serum levels, together with leucocytosis and GSR, were very significant when diagnosis was done. Fibronectin, sideremia and transferrin were lowered. After 3 and 6 days of treatment haptoglobins, alpha-1-antitrypsin, protein C, ferritin, leucocytosis and GSR are lowered, while immunoglobulins, sideremia, transferrin and fibronectin raised, the latter until normalization. Fibronectin as well as changes in iron metabolism were very reliable parameters of inflammation and favorable evolution.
The frequency and clinical associations of anti-RNA antibodies measured by ELISA were assessed in 138 patients with systemic lupus erythematosus (SLE). Of the sera from these patients 9.4% had anti-RNA antibodies but no distinguishing features, clinical, serological or immunogenetic, between those with or without these antibodies could be identified. However, investigations of patients with other autoimmune rheumatic diseases did not reveal any anti-RNA positivity, which indicates a marked disease specificity for anti-RNA antibodies in SLE. The initial anti-RNA antibody screen used a soluble yeast extract as test antigen. The positive sera were further tested against a range of RNAs from 10 different types of rat tissue. In essence few differences were observed, suggesting that the anti-RNA response is directed against common, highly conserved epitopes.
The object of paper has been the study of amebic seroepidemiology in the northern region of Mexico, by means of an immunoenzymatic assay in solid phase (ELISA). The proteic antigen was obtained from axenic culture of E. histolytica's trophozoites of strain HM1:IMSS, by means of solubilization with phenoxy polyethoxyethanol (Triton X-100). Standardization of the method was obtained by analysis of 80 sera from patients with both a clinical and radiologic diagnosis of amebic hepatic abscess and 80 sera from healthy patients. The sensitivity reached 95% and the specificity was 90.7%. 15,457 sera from eight Mexican northern states were tested as part of the National Seroepidemiologic Survey. A semiautomatic BIOMEK 1,000 (Beckman Ins.) equipment was used. The positive ratio varied between 2.0% in Southern Baja California and 4.92% in Chihuahua, with an average ratio for the whole group of 3.34%. The first three places, in descending order, are Chihuahua (4.92%), Sinaloa (4.01%) and Northern Baja California (3.78%). The positive man: woman ratio was 0.87:1 and fluctuated between 0.38:1 in Coahuila and 1.76:1 in Nuevo Leon. In the whole sample the positive ratio of men was 3.00% and in women was 3.43%. The antibody profile related to age, is similar to diseases reported to be highly endemic; the percentage of positive sera rose progressively up to the age of 20 years (4.05%), at which a plateau occurred with a slight descending tendency up to 60 years of age (3.54%), after which a clear descent in the positive index is seen. No important variations were observed when comparing groups of ages and sexes. The antibody level seems to show an inverse relationship with the incidence rate of amebiasis.
There probably exist many different surface molecular polysaccharides with different compositions of their constituent sugars, in the different zymodemes. This fact might have a repercussion upon the resistance to the lysis of the complement. Seeking to prove this hypothesis, the ideal would be to obtain polysaccharide molecules of type LPFG of pathogenic and non-pathogenic zymodemes, demonstrating that there is a difference in the chemical composition of their polysaccharides. If this were true, a serologic classification, of the different zymodemes, would be possible in a fashion similar to gram-negative bacteria. Our results suggest the existence of structural differences between the polysaccharides of a virulent clone (C-A) and a non-virulent one (L-6).
Soluble glutamine synthetase activity (L-glutamate:ammonia ligase, ADP forming, EC 6.3.1.2) was purified to electrophoretic homogeneity from the filamentous non-N2-fixing cyanobacterium Phormidium laminosum (OH-1-p.Cl1) by using conventional purification procedures in the absence of stabilizing ligands. The pure enzyme showed a specific activity of 152 mumol of gamma-glutamylhydroxamate formed.min-1 (transferase activity), which corresponded to 4.4 mumol of Pi released.min-1 (biosynthetic activity). The relative molecular mass of the native enzyme was 602 kilodaltons and was composed of 12 identically sized subunits of 52 kilodaltons. Biosynthetic activity required the presence of Mg2+ as an essential activator, although Co2+ and Zn2+ were partially effective. The kinetics of activation by Mg2+, Co2+, and Zn2+ were sigmoidal, and concentrations required for half-maximal activity were 18 mM (h = 2.2), 6.3 mM (h = 5.6), and 6.3 mM (h = 2.45), respectively. However, transferase activity required Mn2+ (Ka = 3.5 microM), Cu2+, Co2+, or Mg2+ being less effective. The substrate affinities calculated for L-Glu, ammonium, ATP, L-Gln, and hydroxylamine were 15, 0.4, 1.9 (h = 0.75), 14, and 4.1 mM, respectively. Optimal pH and temperature were 7.2 and 55 degrees C for biosynthetic activity and 7.5 and 45 degrees C for transferase activity. The biosynthetic reaction mechanism proceeded according to an ordered three-reactant system, the binding order being ammonium, L-Glu, and ATP. The presence of Mn2+ or Mg2+ drastically affected the thermostability of transferase and biosynthetic activities. Heat inactivation of biosynthetic activity in the presence of Mn2+ obeyed first-order kinetics, with an Ea of 76.8 kcal (ca. 321 kJ) mol-1. Gly, L-Asp, L-Ala, L-Ser and, with lower efficiency, L-Lys and L-Met, L-Lys, and L-Glu inhibited only transferase activity. No cumulative inhibition was observed when mixtures of amino acids were used. Biosynthetic activity was inhibited by AMP (Ki= 7 mM), ADP (Ki= 2.3 mM), p-hydroxymercuribenzoate (Ki= 25 microM), and L-methionine-D, L-sulfoximine (Ki= 2 microM). The enzyme was not activated in vitro by chemically reduced Anabaena thioredoxin. This is the first report of glutamine synthetase activity purified from a filamentous non-N2-fixing cyanobacterium.