Biomedical subjects
Publications and source records attributed to A Tarkowski.
Seventeen patients with moderately active SLE participated in a double-blind, crossover study on the effect of MaxEPA, using olive oil as the control substance. During the first 3 months, 8/17 on Max EPA but only 2/17 on the control substance clinically and serologically improved (p = 0.05), but at 6 months there was no difference. The beneficial effect (if any) of MaxEPA on the disease was short-lived.
The present study addresses the question of whether there is a difference in the frequencies of autoantibody-producing B-cell precursors in healthy compared with lupus-prone mouse strains. Spleen mononuclear cells (MNC) from 4-week-old (i.e. at the preclinical stage of lupus) mice were activated in vitro for 3 and 6 days with lipopolysaccharides (LPS) and the numbers of IgG, IgA and IgM autoantibody-producing cells were analysed by the ELISPOT assay. The results indicate a high frequency of IgM autoantibody-secreting cells after both 3 and 6 days in vitro stimulation. In spite of high frequencies of IgG-producing cells appearing late during the course of LPS stimulation, no IgG or IgA autoantibody producing cells were detected. No significant differences in the autoantibody repertoire were noted between healthy and lupus-prone mice, indicating that independent of the genetic background the immune system has the capacity to react with autoantibody production. Phenotypic analysis of LPS-induced, IgM-secreting B cells showed clearly that the majority of them were surface IgM+, CD5+ but Thy-1-.
The chronic inflammatory diseases in humans have been intensively investigated, however the immune mechanisms underlying diseases such as rheumatoid arthritis (RA), inflammatory bowel disease, and periodontal disease (PD) remain elusive. In this study, we have analyzed the distribution of IgM, IgG, and IgA secreting cells with emphasis on the IgG and IgA subclasses among mononuclear cell populations isolated from gingiva at different stages of PD. Surgically removed tissues were treated with Dispase to gently dissociate cells and the Ficoll-Hypaque gradient centrifugation was used to enrich for viable mononuclear cells rich in lymphocytes, macrophages, and plasma cells. The total numbers of plasma cells increased with the severity of disease. Immunofluorescence analysis showed that most Ig-containing cells were of the IgG isotype; however, significant numbers of IgA-positive cells but few IgM-positive cells were seen. This isolation procedure allowed analysis, at the single cell level, of the distribution of IgG and IgA subclasses of antibody-secreting cells with monoclonal antibodies to human IgG and IgA subclasses. For this, we selected four monoclonal anti-IgG subclass (anti-gamma 1, -gamma 2, -gamma 3, and -gamma 4) antibodies with no subclass cross reactivity for use in the enzyme-linked immunospot assay. Analysis of slight, moderate, and advanced stages of PD showed a progressive increase in spotforming cells (SFC) numbers, and the major isotype of SFC was IgG followed by IgA. The major IgG subclass SFC seen was IgG1 followed by IgG2 whereas similar numbers of IgG3 and IgG4 SFC were observed, a pattern also seen with cells from synovium of RA patients and in mitogen-triggered spleen and PBMC. In terms of the IgA subclass distribution, IgA1 predominated in moderate stages, whereas a selective increase in IgA2 SFC were seen in the more advanced stage of PD. These results show that significant numbers of viable plasma cells/Ig-secreting cells can be isolated from inflamed gingival tissues. Further, careful analysis has shown that IgG subclass responses in gingiva are similar to those found in synovia of RA subjects, and in stimulated PBMC and spleen. However, it should be noted that the number of IgG4- and IgA2-secreting cells increased in the advanced stage of PD.
Production of antibodies to IgG and to type I and type II collagen (CI and CII) was analyzed by enzyme-linked immunospot assay in patients with rheumatoid arthritis (RA) and patients with other inflammatory or degenerative joint diseases. Anti-CII-secreting cells, generally in high numbers, were found among mononuclear cells eluted from inflamed synovial tissue in 12 of 13 patients with seropositive RA and 9 of 14 patients with seronegative RA or with undetermined serum rheumatoid factor levels. In contrast, no anti-CII-producing B cells were present among synoviocytes from 4 patients with other joint diseases. In none of 7 RA sera did we find significant levels of anti-CII. Synovial B cells secreting antibodies specific for CI were observed less frequently in patients with RA. These results indicate that measurement of serum antibody levels is not adequate to assess actual autoantibody production in rheumatoid joints and that local autoimmune reactions to CII are common in RA, which implies that collagen-reactive T cells are present within the inflamed joints of RA patients. The possible role of a local collagen autoimmunity in RA is discussed, particularly in relation to its putative role in rheumatoid factor production.
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The enzyme-linked immunospot assay was used to enumerate both the number and the frequency of spontaneous IgG, IgA, and IgM immunoglobulin-secreting cells and IgA- and IgM-rheumatoid factor (RF)-producing cells present in the gingivae and peripheral blood of adult periodontitis patients. Cells from 29 patients were incubated on plates coated with human IgG, Fc, or F(ab')2 fragments and on plates coated with class-specific anti-human antibodies and secreted antibodies were subsequently visualized by means of an immunoenzymatic procedure. The data indicate that (1) IgA-RF- and IgM-RF-secreting cells are frequently present in the gingiva of adult periodontitis patients; (2) production of RF in gingivae of adult periodontitis patients occurs in the absence of demonstrable RF production by simultaneously obtained peripheral blood mononuclear cells, suggesting that local autoimmune reactions may occur in this disease; and (3) lack of correlation between IgA-RF and IgM-RF production in diseased gingiva suggests that the two RF isotypes are regulated independently of each other.
The presence of the homozygous lpr gene (lpr/lpr) in MRL mice has been regarded as mandatory for the development of the early onset of lupus disease, T-cell dysfunction, and polyclonal B-cell activation. Congeneic MRL mice lacking the lpr gene (MRL +/+) display neither the lupus disease nor the immunological abnormalities within the first year of life. In this study we examined the cellular functions of MRL mice heterozygous at the lpr locus. The results indicate that MRL mice heterozygous at the lpr locus display intermediate delayed-type hypersensitivity compared with homozygous lpr/lpr mice on the one hand and congeneic +/+ mice on the other. Furthermore, proliferative responses to concanavalin A, measured by uptake of [3H]thymidine, were significantly lower in MRL mice heterozygous at the lpr locus than in +/+ mice, but significantly higher than in homozygous MRL lpr/lpr mice. Polyclonal B-cell activation, assessed by measurement of frequencies of IgG-secreting spleen cells, a prominent feature in MRL lpr/lpr mice, was significantly lower in lpr/+ mice and totally absent in +/+ mice. Furthermore, spleen cells spontaneously secreting auto-antibodies were found in large numbers in MRL lpr/lpr mice and in considerably lower but still significant numbers in heterozygous MRL +/lpr mice. In contrast, spleen cells from matched MRL +/+ mice did not display any spontaneous autoantibody production. Taken together, these data provide evidence for immunomodulatory properties of the heterozygous lpr gene.
An autoimmune disease in MRL lpr/lpr (MRL/l) mice is associated with a plethora of T-cell abnormalities, one of them being impaired delayed-type hypersensitivity (DTH). Three immunomodulating drugs, cyclophosphamide (Cy), cyclosporin A (CsA) and LS 2616, all with beneficial effects on autoimmune diseases, have been examined with regard to their potential effects on DTH in female MRL/l mice and in healthy control mice. All three drugs, given as a single dose at the time of sensitization, increased DTH reactivity of oxazolone in healthy control mice, while only two of them, Cy and LS 2616, significantly augmented the defective DTH response in MRL/l mice.
Lymphocytes obtained after enzymatic digestion of intestinal biopsies from patients with coeliac disease were examined for the presence of gliadin specific antibody secreting cells by means of the ELISPOT technique. This technique permits enumeration of gliadin antibody secreting immunocytes, differentiated with regard to immunoglobulin class. Patients with coeliac disease were found to have high (834/10(6) cells) numbers of antigliadin spot forming cells (SFC) in gut mucosa. IgG and IgM antigliadin antibody secreting cells were infrequently shown whereas IgA antigliadin SFC predominated in all patients tested (average 68% of total SFC). Ten control patients were investigated in parallel with the coeliac patients and showed only low numbers of gliadin antibody secreting cells in gut mucosa (49/10(6) isolated cells). Antigliadin antibody secretion by peripheral blood mononuclear cells was shown in only two of six coeliac patients tested and in none of the control patients. The findings suggest that the intestinal mucosa is a major site for antigliadin antibody production and that IgA is the dominating Ig-class of these antibodies. The high sensitivity and accuracy of the ELISPOT technique may provide a useful instrument for future studies of antibody production and regulation of the gut immune response to gluten and other alimentary antigens in coeliac and other intestinal diseases.
The Mrl lpr/lpr (Mrl/l) mouse is a model for systemic lupus erythematosus and rheumatoid arthritis in humans. The diet of Mrl/l mice was supplemented with EPA (see Introduction) in menhaden oil or in the commerical fish oil MaxEPA. The survival of mice was not affected by the dietary manipulations. The addition of menhaden oil decreased the severity of the renal pathology. However, MaxEPA containing the same amount of eicosapentaenoic acid was considerably less effective. A diet deficient in polyunsaturated fatty acids did not ameliorate any manifestations of the disease. Anti-DNA antibody levels in serum were not influenced by the therapy. Myocardial abscesses and/or ulcerating valvular lesions were observed in about one third of the mice, irrespective of the diet given.
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In this paper we report that treatment with 17 beta-oestradiol depresses cutaneous delayed-type hypersensitivity (DTH) to oxazolone (OXA) in female oophorectomized NZB/W mice. Analysis of the parental strains revealed that this oestrogen-induced inhibition of DTH mice is a trait inherited from the healthy NZW and not from the autoimmune NZB strain. The down-regulating effect of oestradiol on DTH in female NZB/W mice was found both in the sensitization and effector phase of the DTH response. Decreased DTH reactivity to OXA was demonstrated in oophorectomized NZB/W and NZW mice after administration of both low, physiological doses (s.c. injections) and high, pharmacological doses (siliac tube implantation) of oestradiol. In addition, s.c. implantation of testosterone containing siliac tubes suppressed DTH reactivity to OXA in castrated male NZW mice. This down-regulating effect was not found in male NZB or NZB/W mice similarly treated. Serological analysis revealed that high doses of oestradiol increased whereas testosterone depressed the IgG anti-OXA antibody responses in female NZB/W mice. Our results indicate that one possible contribution of the NZW parental strain to the lupus disease in NZB/W mice is a trait of oestrogen sensitivity leading to the acceleration of the autoimmune disease, the latter feature being inherited from NZB mice.
The influence of sex steroids on cutaneous delayed-type hypersensitivity (DTH) and antibody responses to oxazolone (OXA) in autoimmune and normal mouse strains has been investigated. The results show that: (i) treatment with 17 beta-oestradiol (E2) suppresses DTH responses and stimulates antibody responses in MRL, B6 and C3H mice, suppresses DTH in DBA/1 mice, while having no effects on DTH or antibody responses in BALB/c and NFR/N mice. (ii) Treatment with testosterone suppresses the antibody response in all studied strains (MRL, B6, BALB/c and DBA/1) while down-regulating the DTH response only in MRL and B6 but not in BALB/c or DBA/1. (iii) Neither the lympho-proliferative (lpr) gene, which accelerates autoimmune disease, nor the H-2 genes seem to be directly related to the effects of sex hormones on the immune system. (iv) Susceptibility to oestrogen- and testosterone-mediated suppression of DTH but not oestrogen-mediated enhancement of antibody response are inherited as dominant traits. The results are discussed in the context of certain autoimmune diseases known to be influenced by sex hormone manipulations.
Ig class-, and IgA and IgG subclass-specific immune responses to a 23 valent pneumococcal polysaccharide vaccine were studied at a single-cell level in the peripheral blood of systemically immunized adults. With a solid phase enzyme-linked immunospot (ELISPOT) assay, PBMC from immunized individuals were assayed for spontaneous Ag-specific antibody (Ab) production before, and on days 7, 14, and 28 after vaccination. On the day of immunization, no spontaneous Ag-specific Ab-secreting cells could be detected. On day 7 after vaccination, a high frequency of cells secreting Ab specific for pneumococcal polysaccharides (PPS) was observed. The IgA class comprised 79% (geometric mean) of the Ag-specific Ab-secreting cells, whereas IgG- and IgM-secreting cells accounted for 12% and 9%, respectively. The majority of Ag-specific IgA-secreting cells produced Ab of the IgA2 isotype. Serum, saliva, and tears collected before and on days 7, 14, and 28 after vaccination were assayed for specific Ab to the vaccine (anti-PPS Ab) by an ELISA. Serum IgA anti-PPS Ab showed the highest increase after vaccination with a 19-fold increase (geometric mean) which peaked on day 14. However, the ratio of Ag-specific polymeric vs monomeric IgA did not change after immunization. Serum IgG and IgM anti-PPS Ab displayed mean increases of 5.5-fold and 5.6-fold, respectively, on day 14. The most pronounced increase of salivary anti-PPS Ab was observed in the IgG class (4.5-fold on day 28) followed by IgM (4-fold on day 28), IgA (2.0-fold on day 14), IgA1 (2.4-fold on day 14) and IgA2 (2.0-fold on day 14). The levels of total IgA, IgG, and IgM in saliva did not change significantly throughout the course of immunization. IgG and IgM anti-PPS Ab levels in tears increased less than in saliva, whereas IgA behaved similarly as in saliva. There were no significant differences in the Ag-specific increase rates between the IgA, IgG, and IgM isotypes in tears.
The sera of 34 acquired immune deficiency syndrome (AIDS) patients and 20 healthy male homosexuals were examined for the presence of elevated levels of IgA and IgM rheumatoid factor (RF) and compared with results obtained with sera from 23 healthy laboratory volunteers. IgA RF levels were elevated (greater than 3 standard deviation units) in 9 of 34 (26%) patients with AIDS as compared to the panel of laboratory controls. Levels of IgM RF did not differ significantly in the AIDS patients and in the controls. There were no differences in levels of either IgA RF or IgM RF when the homosexual controls were compared with the laboratory volunteers. Sucrose-gradient ultracentrifugation experiments and assays using monoclonal reagents specific for IgA subclasses indicated that the IgA RF was predominantly of the polymeric configuration and restricted to the IgA1 subclass, respectively. Polyethylene glycol (PEG) precipitates of serum enriched for circulating immune complexes (CIC) were also assayed for the presence of IgA RF and IgM RF. Although levels of IgA RF in serum and in PEG precipitates did not correlate with levels of IgA- or IgA/IgG-containing CIC in AIDS patients, levels of IgA RF in both serum and CIC-enriched material were significantly elevated in the AIDS population when compared with the control panel. In contrast, levels of IgM RF in both serum and CIC-enriched material were low and not significantly different from those in healthy controls. These results indicate that both IgA-containing CIC and IgA RF occur in many AIDS patients and raise the possibility that IgA RF may contribute significantly to the formation of immune complexes in this disease.
The autoimmune MRL/Mp-lpr/lpr (MRL/l) mouse spontaneously develops sialadenitis with a morphological and phenotypical pattern similar to that seen in human Sjögren's syndrome (SS). This makes the MRL/l mouse a suitable model for therapeutical studies of autoimmune sialadenitis. We have, by histological and immunohistochemical techniques, analyzed the therapeutical effect of treatment with LS2616, a recently synthesized oxokinolinamide derivative, on sialadenitis in submandibular glands of MRL/l mice. The results were compared with effects obtained after treatment with cyclophosphamide (CY) and physiologic saline. Administration of both LS2616 and CY to MRL/l mice has previously been found to result in prolongation of survival and amelioration of organ pathology. However, only CY treatment reduced sialadenitis, while LS2616 increased the semiquantitatively assessed focal inflammation of salivary glands in 6 months old mice. No differences in T-cell phenotypes of infiltrating lymphoid cells in salivary glands between different treatment regims could be noted. However, the frequency of B-cells in the sialadenitis was decreased in the CY treated group. In contrast, CY but not LS2616 treatment normalized expression of T-helper and cytotoxic T-cell phenotypes as well as reduced the B-cell portion in lymph nodes. It is concluded that CY treatment can suppress sialadenitis although both LS2616 and CY are effective in prolongation lifespan of MRL/l mice. This may implicate different immunopathogenic mechanisms for development of sialadenitis versus other organ lesions in the autoimmune disease of MRL/l mice.
The MRL/1 and New Zealand black-New Zealand white cross (NZB x W) mice spontaneously develop a disease similar to systemic lupus erythematosus in man. The effect of antihypertensive treatment on MRL/1 and NZB x W mice was studied with respect to survival, blood pressure, proteinuria, haematuria and renal histopathology. The treatment consisted of angiotensin converting enzyme (ACE) inhibitors (captopril and enalapril) and bretylium, a sympathetic blocker. Tail systolic blood pressure was measured with a strain gauge technique. All antihypertensive drugs caused a reduction in blood pressure in both strains. In MRL/1 bretylium and both ACE inhibitors improved renal histopathology, but only captopril prolonged survival, and it decreased proteinuria and haematuria. In NZB x W captopril decreased proteinuria but did not influence survival or renal histopathology. Bretylium was without any effect on these parameters. At the doses used captopril improves survival in MRL/1 mice and decreases proteinuria and haematuria in both MRL/1 and NZB x W mice. Since bretylium and enalapril lack this property despite a similar blood pressure reduction, it seems to be a drug-specific action.