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PubMedDate not supplied

Electroblotting of proteins on agarose gel containing specific antibodies.

A method is described for electroblotting of proteins, separated by gradient polyacrylamide gel electrophoresis, onto an agarose gel matrix containing specific antibodies. Three proteins of different molecular weight, including human albumin, isolated and in plasma, human plasma transferrin and C3 complement were tested. Immunoblotting on agarose, compared with nitrocellulose, was quantitative and highly sensitive, with small amounts of protein (i.e., 100 pg) being detected. Moreover, albumin aggregates (i.e., dimer, trimer and tetramer) were blotted quantitatively in addition to the monomer, and their percentages were calculated. This method is sensitive, quantitative, reproducible, and includes fewer manipulations; furthermore, it is less expensive and does not require the use of toxic or carcinogenic agents.

Collodion↗

PubMed1986

Enzyme immunoassay of complement-binding rheumatoid factors.

We describe an enzyme immunoassay for the determination of complement-binding rheumatoid factors. Polystyrene tubes are coated with heat aggregated human IgG. The rheumatoid factors (RFs) of patients heat inactivated sera are allowed to bind to aggregated IgG and thereafter saturated with fresh human complement. The amount of C3 complement bound is measured by indirect enzyme immunoassay. The levels of complement binding RFs were measured in 30 patients with seropositive rheumatoid arthritis (RA), in 19 patients with systemic lupus erythematosus (SLE), and in 30 healthy control subjects. Compared to the controls high levels of complement-binding RFs were found both in RA and in SLE (P less than 0.0005). The mean level of the complement binding RFs was higher (P less than 0.05) in active than in inactive SLE. Even though the 19 S IgM RF bound complement, in RA no correlation was found between the level of complement binding RFs and Waaler-Rose titre, but the level of complement binding RF correlated with the levels of nonagglutinating IgM RF (r = 0.56, P less than 0.01) and IgG RF (r = 0.70, P less than 0.001) that were obtained by enzyme immunoassay.

Adult↗

PubMed1987

[Gamma 1 heavy chain disease with immune vasculitis and rheumatoid arthritis].

We are reporting about a case of gamma heavy-chain disease (Franklin's disease) with immunovasculitis and rheumatoid arthritis. The diagnosis was confirmed by the results of immunoelectrophoresis of the patient's serum and also by evidence of stimulated lymphocytes without light chain, but having gamma heavy-chain surface proteins. The immunofixation of the serum showed two protein bands of gamma heavy-chains with different loads. These results are confirmed by a two dimensional electrophoresis and isoelectric focussing of the serum proteins. The pathologic protein consists of at least two different heavy-chain proteins (mol wt 40,000 and 80,000) with isoelectric points between pH 5.5 and 7.3. In the urine of the patient pathological gamma heavy chain-protein was found only in a very low concentration. The predominant clinical symptom of the patient was a necrotizing vasculitis which became a therapeutical problem. In the immunofluorescence examination of the skin biopsy specimens, immunoglobulins and C3-complement could be detected in the stratum papillare. This fact would be compatible with the development of antibodies or immune complexes against deposited heavy-chain proteins. The arthropathy and the positive rheumatoid factor could similarly be explained by an immune complex mechanism.

Antigen-Antibody Complex↗

PubMed1978

Antibody formation and transient immune complex glomerulopathy in A-strain mice with C1300 neuroblastoma tumors.

One to three-month-old A-strain mice, inoculated subcutaneously with 2 x 10(6) viable syngeneic C1300 neuroblastoma cells (clone NB9R) developed a palpable tumor within 9-12 days and died within 28-30 days. A transient glomerulopathy developed after 16-24 days. Despite a normal histologic appearance, the nephropathy was clearly demonstrated by electron microscopy and was classified as a focal mesangiopathic glomerulonephritis. Deposits of host 7S-G immunoglobulins and C3 complement fragments were detected in these same kidneys by immunofluorescence. Radioimmunoprecipitin determinations on sera obtained from mice at different intervals from tumor cell inoculation, revealed that untreated mice contained circulating antibodies capable of reacting with 125I-labeled gp69-71 glycoprotein from Gross murine leukemia virus (MuLV). Antibodies to p30 MuLV antigen and to crude membrane antigen (s) (CMA) solubilized from NB9R cells were found in sera only after tumor cell inoculation. Circulating immune complexes formed by host 7S-G immunoglobulins were clearly detected from day 16 to 22. Antibodies eluted from kidneys with nephropathy were shown to react with NB9R cells in vitro and to react specifically with CMA and the p30 MuLV antigen.

Animals↗

PubMed1977

Autoimmune phenomena in ocular cicatricial pemphigoid.

Twelve patients with various stages of ocular cicatricial pemphigoid were studied. Tear volumes measured by Schirmer tests and visual acuities were generally reduced in proportion to disease severity. Mannitol-positive staphylococcus was recovered from 67% (16/24) of the eyelids cultured. Immunoglobulins were bound to the basement membrane of clinically involved conjunctivae in 67% of the patients (8/12). Eighty percent (8/10) of the clinically normal conjunctivae had no immunoglobulins bound to the basement membrane or epithelium, further reinforcing the importance of tissue-fixed immunoglobulins in the pathogenesis of this condition. Four of eight patients with immunoglobulins bound to basement membrane also had C3 complement bound to basement membrane. Acute disease may be associated with the presence of complement in the conjunctiva. In addition, three of the eight patients with basement membrane staining, and three without, showed marked epithelial intercellular and intracellular staining for immunoglobulins. Indirect immunofluorescence of patients' sera showed no circulating antibodies to basement membrane, but three patients had circulating antibodies directed to the conjunctival epithelium in an intercellular or intracellular location. No significant association of ocular pemphigoid with other circulating autoantibodies. HLA aantigens, or abnormal serum immunoglobulin levels was realized.

Aged↗

PubMed1984

Detection of immune complexes in experimental allergic neuritis.

Circulating immune complexes were assayed in the sera of animals with experimental allergic neuritis (EAN), and immunofluorescent staining and immunohistological examination were performed to clarify the role of immune complexes in the pathogenesis of EAN. The level of immune complexes in the sera of animals with clinical signs of EAN was from 1:32 to 1:64. Control animals showed a titer of immune complexes from 1:2 to 1:4. Animals with EAN showed deposition of immune complexes, which were detected by FITC-conjugated anti-rat IgG or C3-complement, in the vessels of the peripheral nerve. These findings suggest that immune complexes may contribute to the immunopathogenesis of EAN.

Animals↗

PubMed1986

Disseminated vasculomyelinopathy in the peripheral nervous system mediated by immune complexes (ICs). Immunohistochemical studies of sciatic nerves in chronic serum sickness (CHSS) in rabbits.

Histological examination of 20 sciatic nerves from rabbits with experimental chronic serum sickness (CHSS) revealed patchy vasculitis of the vasa nervorum of various intensity. The vessel lesions ranged from endothelial proliferation to vessel wall necrosis with fibrinoid degeneration and infiltration by lymphocytes, plasma cells, macrophages and, sporadically, by neutrophils. Perivascularly, there were oedema, chronic infiltrates or small haemorrhages. The myelinated fibres in close relation to the vascular system were focally depleted and features of perivascular demyelination were found. Teased fibres showed paranodal and segmental demyelination, axonal degeneration and, sporadically, remyelination. In all cases, immunofluorescent deposits of bovine serum albumin (BSA), IgG and C3 complement were found in and around some vasa nervorum. Other indirect evidence for immune complex (IC) deposition was provided by ultrastructural examination where vascular and endoneurial osmophilic deposits were found; in 4 cases with paracrystalline organization resembling cryoglobulin component. IC-mediated vasculitis led to blood-nerve barrier impairment and leakage of serum proteins into the endoneurial space. The morphological and immunohistochemical changes in this model which develop after a latency period of 2 or more weeks, strongly resemble those observed in human acquired inflammatory demyelinating polyradiculoneuropathies or in connective tissue diseases.

Animals↗

PubMed2005

Comparative immune responses in Japanese flounder, Paralichthys olivaceus after vaccination with viral hemorrhagic septicemia virus (VHSV) recombinant glycoprotein and DNA vaccine using a microarray analysis.

A viral hemorrhagic septicemia virus (VHSV), recombinant glycoprotein vaccine (VHSVg) and a DNA vaccine (pCMV-VHSg) were injected in Japanese flounder (Paralichthys olivaceus). Each fish was injected with 10 microg vaccine dissolved in 50 microl phosphate buffer saline (PBS). One month after the vaccination, the fish were challenged intraperitoneally with either 1 x 10(2) or 1 x 10(3) TCID(50) of virus. Fish that received the VHSg DNA vaccine were highly protected against virus infection over a 21-day observation period, with cumulative mortalities ranging from 4 to 10%. However, the recombinant protein vaccine group appeared to have low survival rates. Using microarray analysis, humoral defense-related genes such as complement component C3, complement regulatory plasma proteins, IgM, IgD, MHC class II-associated invariant chain and CD20 receptor were observed to be up-regulated by the VHSg recombinant protein vaccine at 1 or 21 days post vaccination. On the other hand, cellular defense-related genes such as CD8 alpha chain, T-cell immune regulator, MIP1-alpha and apoptosis-associated protein were not detected. Specific antibodies against VHSVg protein were detected in both vaccinated groups at a titer of 1:40 at 28 days post vaccination.

Animals↗

PubMed1980

[Asymptomatic haematuria (author's transl)].

Mesangioproliferative glomerulonephritis of varying severity was found by renal biopsy in 86.2% among 130 patients with asymptomatic haematuria. 3.2% had benign nephrosclerosis, 3.2% had benign nephrosclerosis, 1.6% had interstitial nephritis and one patient showed a previously undiagnosed perimembranous glomerulonephritis. Normal renal parenchyma was observed in only 6.9% of cases. Iummunohistological findings were positive in 25.7% of investigated cases. The majority were IgA deposits combined with IgG and C3 complement. The intensity of haematuria is not correlated with the type and severity of histological changes. "Physiological" haematuria (erythrocytes less than 7/ml) which is usually considered normal must be re-evaluated as a consequence of these histological findings. After exclusion of urological or extrarenal disease only histological investigation of the kidneys will bring the final diagnosis. Repeated radiographical or urological investigations can thus be avoided. The risks of percutaneous renal biopsy under fluoroscopic or sonographic control are decidedly less than the information gained from histological evaluation. The different histological findings indicate that isolated haematuria should be considered only as a symptom and not as a uniform disease indicating focal nephritis.

Adolescent↗

PubMed2005

Protective antibody responses elicited by a meningococcal outer membrane vesicle vaccine with overexpressed genome-derived neisserial antigen 1870.

Background. Meningococcal outer membrane vesicle (OMV) vaccines are efficacious in humans but have serosubtype-specific serum bactericidal antibody responses directed at the porin protein PorA and the potential for immune selection of PorA-escape mutants.Methods. We prepared an OMV vaccine from a Neisseria meningitidis strain engineered to overexpress genome-derived neisserial antigen (GNA) 1870, a lipoprotein discovered by genome mining that is being investigated for use in a vaccine.Results. Mice immunized with the modified GNA1870-OMV vaccine developed broader serum bactericidal antibody responses than control mice immunized with a recombinant GNA1870 protein vaccine or an OMV vaccine prepared from wild-type N. meningitidis or a combination of vaccines prepared from wild-type N. meningitidis and recombinant protein. Antiserum from mice immunized with the modified GNA1870-OMV vaccine also elicited greater deposition of human C3 complement on the surface of live N. meningitidis bacteria and greater passive protective activity against meningococcal bacteremia in infant rats. A N. meningitidis mutant with decreased expression of PorA was more susceptible to bactericidal activity of anti-GNA1870 antibodies.Conclusions. The modified GNA1870-OMV vaccine elicits broader protection against meningococcal disease than recombinant GNA1870 protein or conventional OMV vaccines and also has less risk of selection of PorA-escape mutants than a conventional OMV vaccine.

Animals↗

PubMed1978

Therapy with essential amino acids and their nitrogen-free analogues in severe renal failure.

The effects of therapy with essential amino acids and their nitrogen-free hydroxy and keto precursers on nitrogen and amino acid metabolism of patients with chronic renal failure were examined. Data obtained under treatment with essential amino acids and alpha-keto acid analogues showed higher levels of plasma amino acids, C3-complement, and transferrin than with a low-protein diet alone. Nitrogen balance became positive and plasma urea levels fell. With keto acid analogue therapy, these effects could be obtained with a reduced nitrogen intake. Supplementation of a low protein diet with alpha-hydroxy acid analogues was associated with more positive nitrogen balance and a rise in plasma histidine and methionine. However, plasma levels of three other essential amino acids fell.

Amino Acids↗

PubMed1981

Effect of renutrition on humoral and cell-mediated immunity in severely malnourished children.

Forty-three Colombian children suffering from either kwashiorkor (21), combined protein-calorie malnutrition (11), or maramus (11) were hospitalized and provided a high protein, high calorie diet for 4 to 5 wk. Improvement in clinical and nutritional status was accompanied by significant increases in levels of serum immunoglobulins G and M and C3 complement and by significant decreases in serum immunoglobulin A concentrations, especially in infants with kwashiorkor. Skin test reactions to purified protein derivative and candidin improved during renutrition. Lymphocyte blastogenesis after stimulation in vitro with phytohemagglutinin and pokeweed mitogen increased rapidly during hospitalization. After 1 yr posttreatment, cell-mediated immune responses, both in vivo and in vitro, had diminished. These results indicate that some aspects of the immune response are affected to a different degree in kwashiorkor, maramus, and combined malnutrition. Short-term nutritional rehabilitation has a differential effect on the long-term restoration of various aspects of immunity.

Antibody Formation↗

PubMed1993

Arthritis in Mediterranean spotted fever. An immune complex mediated synovitis.

Arthromyalgia are frequent in Mediterranean spotted fever (MSF) (16-76%) but arthritis is rare. We report on a 54-year-old woman who, 1 day after suffering from fever, headache and malaise, developed a painful and swollen left knee. A maculopapular rash and the characteristic 'tache noire' skin lesion appeared 5 days later. Immune complexes were detected in serum and in the SF and normalized following improvement of clinic manifestations. These findings along with a low C3 complement level in the SF suggest that arthritis was mediated by immune complex deposits.

Antigen-Antibody Complex↗

PubMed1975

Thymectomy as immunosuppression in uremic patients.

Five uremic patients awaiting renal transplantation underwent transcervical thymectomy in an evaluation of the immunosuppressive effect of removal of the thymus in such transplantations. A number of immunological parameters, including lymphocyte transformation tests, were followed in these patients for up to 30 weeks after thymectomy. The number of B lymphocytes in the blood, stem cells in bone marrow, and T lymphocytes in lymph nodes decreased, whereas IgG, IgA, and IgM in four cases and C3 complement in all five cases increased. Blood leucocyte and lymphocyte counts did not show any characteristic changes. The T cell response of circulating lymphocytes was determined after stimulation with mitogens, specific antigens, and allogeneic cells in mixed lymphocyte culture, and showed a large increase. Thymectomy of uremic patients results in a considerable increase in immunocompetence in the first 30 weeks, indicating that it is not suitable as an immunosuppressive treatment.

Adult↗

PubMed2000

Insight into the physiological function(s) of uteroglobin by gene-knockout and antisense-transgenic approaches.

To determine the physiological function(s) of uteroglobin (UG), a steroid-inducible, homodimeric, secreted protein, we have generated transgenic mice that either are completely UG-deficient due to UG gene-knockout (UG-KO) or are partially UG-deficient due to the expression of UG antisense RNA (UG-AS). Both the UG-KO and UG-AS mice develop immunoglobulin A (IgA) nephropathy (IgAN), characterized by microhematuria, albuminuria, and renal glomerular deposition of IgA, fibronectin (Fn), collagen, and C3 complement. This phenotype of both UG-KO and UG-AS mice is virtually identical to that of human IgAN, the most common primary glomerulopathy worldwide. The molecular mechanism by which UG prevents this disease in mice appears to center around UG's interaction with Fn. Since Fn, IgA, and UG are present in circulation and high plasma levels of IgA-Fn complex have been reported in human IgAN, we sought to determine whether UG interacts with Fn and prevents Fn-Fn and/or IgA-Fn interactions, essential for abnormal tissue deposition of Fn and IgA. Our coimmunoprecipitation studies uncovered the formation of Fn-UG heteromers in vitro and these heteromers are detectable in the plasma of normal mice, but not UG-KO mice. Further, high plasma levels of IgA-Fn complex, a characteristic of human IgAN patients, were also found in UG-KO mice. Finally, coadministration of UG + Fn or UG + IgA to UG-KO mice prevented glomerular deposition of Fn and IgA, respectively. Our results define a possible molecular mechanism of IgAN and provide insight into at least one important physiological function of UG in maintaining normal renal function in mice.

Animals↗

PubMed1995

Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene.

In a previous study we cloned and determined the nucleotide sequence of the prtH gene from Porphyromonas gingivalis W83. This gene specifies a 97-kDa protease which is normally found in the membrane vesicles produced by P. gingivalis and which cleaves the C3 complement protein under defined conditions. We developed a novel ermF-ermAM antibiotic resistance gene cassette, which was used with the cloned prtH gene to prepare an insertionally inactivated allele of this gene. This genetic construct was introduced by electroporation into P. gingivalis W83 in order to create a protease-deficient mutant by recombinational allelic exchange. The mutant strain, designated V2296, was compared with the parent strain W83 for proteolytic activity and virulence. Extracellular protein preparations from V2296 showed decreased proteolytic activity compared with preparations from W83. Casein substrate zymography revealed that the 97-kDa proteolytic component as well as a 45-kDa protease was missing in the mutant. In in vivo experiments using a mouse model, V2296 was dramatically reduced in virulence compared with the wild-type W83 strain. A molecular survey of several clinical isolates of P. gingivalis using the prtH gene as a probe suggested that prtH gene sequences were conserved and that they may have been present in multiple copies. Two of 10 isolates did not hybridize with the prtH gene probe. These strains, like the V2296 mutant, also displayed decreased virulence in the mouse model. Taken together, these results suggest an important role for P. gingivalis proteases in soft tissue infections and specifically indicate that the prtH gene product is a virulence factor.

Alleles↗

PubMed1987

Gc and C3 polymorphisms in South Sardinia.

The distribution of phenotypes and gene frequencies of the group-specific component (Gc) and C3 complement were studied in South Sardinia. The gene frequencies were: Gc1 = 0.7346; C3F = 0.1963.

Complement C3↗

PubMed1988

C3 allotypes in pregnancy hypertension and eclampsia.

C3 allotyping has been performed on 424 Australian women, 203 with normotensive pregnancies, 161 with hypertensive noneclamptic pregnancies and 60 eclamptic women. The frequency of women heterozygous for 'rare' C3 alleles was 1% in the normotensive women and 3.7% in the hypertensive group. Three out of 25 (12%) of the women with proteinuric hypertension in pregnancy carried 'rare' C3 alleles. This suggested the hypothesis that pre-eclampsia/eclampsia is associated with a higher frequency of rare alleles. The sample of 60 eclamptic women collected to test the hypothesis had no rare alleles, refuting the hypothesis. The frequency of the common (C3F, C3S) alleles did not differ significantly between the three groups. We conclude that there is no evidence for any association between susceptibility to eclampsia and allotypes of the C3 complement component.

Complement C3↗