Search for the antigen-specificity of homogeneous IgG components (H-IgG) after allogeneic bone marrow transplantation.

After allogeneic BMT, transient homogeneous Ig components (H-Ig) can be detected in the sera of most graft recipients. So far, data on the antigen-specificity and therefore the function of these H-Ig are not available. Such information may be important for our understanding of the underlying mechanisms that are responsible for these excessive clonal B cell expansions, and it may help to delineate the functional antibody repertoire after BMT. In the present study, sera of 98 paediatric BM graft recipients were investigated for the potential presence of H-Ig of IgG isotype (H-IgG) with specificity towards a panel of antigens, including vaccine and herpes virus antigens, auto-antigens and allo-antigens. The vast majority of H-IgG in sera of BM graft recipients were unreactive when tested for this panel of antigens. However, in four cases, antigen-specificity of H-IgG to tetanus toxoid could be demonstrated after vaccination with that antigen. An explanation for the negative findings may be either that a restricted antibody production had been elicited by other non-tested antigens, eg substances of colonizing and translocating bacteria or of food antigens, or that the H-IgG components may have anti-idiotype or anti-'self' specificity.

Effects of isoniazid (INH) on the BCG-induced local immune response after intravesical BCG therapy for superficial bladder cancer.

Because recent investigations showed that the use of isoniazid (INH) severely impaired the local immune reaction to intravesical bacillus Calmette-Guérin (BCG) in the bladder of guinea pigs, in this study the effect of INH in man has been investigated. Patients were treated with BCG with or without oral INH. The concentration of free INH in most urine samples of patients treated with BCG/INH was much higher (mean 38.0 +/- 60.9 micrograms INH/ml) than the minimal inhibitory concentration (MIC; 0.1 microgram INH/ml), suggesting at least a bacteriostatic potential of the INH present. However, in vitro studies showed that these urinary concentrations of INH did not kill BCG organisms effectively, even at a concentration of 150 micrograms/ml for 24 h. After the fifth and sixth BCG instillations a significant increase in the concentration of cytokines (IL2, IL6, IL8 and TNFa), IgG and IgA antibodies to BCG and the number of leukocytes in urine was observed. The leukocytes mainly consisted of granulocytes, besides monocytes/macrophages and, in lower amounts, T- and B-lymphocytes and natural killer (NK) cells. The absolute number of granulocytes and the concentration of IgG antibodies after BCG instillation were significantly suppressed by INH, whereas INH appeared to have no effect on the urinary cytokine and IgA antibody concentrations or the total number and phenotype of the leukocytes present. In conclusion, the results of this study indicate that INH does not impair the local immunological stimulation after BCG instillation in man as severely as was observed in the guinea pig and it may be expected that INH does not impair the antitumor efficacy of BCG.

Nephelometric measurements of human IgG subclasses and their reference ranges.

IgG subclass measurements are generally performed with the radial immunodiffusion (RID) technique. With this method, results are obtained after an incubation period of 48-72 hours. We developed nephelometric assays on the Behring Nephelometer Analyzer (BNA) that allow a quantification of IgG subclass concentrations in a large number of samples quickly (less than 15 minutes for a complete IgG subclass profile) and reproducibly (intra-assay variation 2.5-5.5%, interassay variation 3.4-6.0% and inter-lab variation 5.4-10.3%). The nephelometric method was compared with the RID technique by analyzing the IgG subclass levels in sixty selected samples. For all IgG subclasses identical results and high correlation coefficients (r > 0.93) were found. In addition, the detection limits of the nephelometric method for all four IgG subclasses were identical or lower than those of the RID technique. Furthermore, the interlab variations of the nephelometric IgG subclass assays are lower than those of the RID method. However, the major advantages of the nephelometric assay are the speed, the minimal workload (automated IgG subclass determinations) and the possibility for automated bidirectional data transmission. Recently we have established new reference ranges for the human IgG subclasses in sera of adults and children. In order to validate these reference values we have measured the IgG subclasses in sera from 112 healthy children with the nephelometric method. In 1992, more than 2000 patient sera were tested by the nephelometric assay. A predominance of IgG2 abnormality was observed. In 9.8% of these sera the IgG2 concentration was decreased. Elevated IgG2 concentrations were found in 1.9% of the sera. Furthermore, the sum of the quantitated four IgG subclasses was similar to that of total IgG (less than 20% difference).

Interleukin-8 during peritonitis in patients treated with CAPD; an in-vivo model of acute inflammation.

CAPD-related peritonitis was used as an in-vivo model to study Il-8 during peritoneal inflammation. Eleven episodes were studied in nine patients, who were followed on 8 consecutive days from the start of peritonitis and once after recovery (control). Il-8 was measured in dialysate (night dwells) and serum. The Il-8 time course was compared to Il-6 and TNF alpha. In addition, an in-vivo relationship between dialysate Il-8 and intraperitoneal accumulation of neutrophils was studied. A highly increased peritoneal appearance rate of Il-8 was found in the acute phase that decreased to control values during recovery. A remarkable parallelism was observed for dialysate Il-8 and Il-6 with respect to the time course and the peritoneal appearance rate. In contrast, the appearance rate of TNF alpha was much less and had a different time course. In three of four cases where the dialysate Il-8 peak occurred on day 2, the dialysate Il-6 peak still coincided with Il-8, in contrast to TNF alpha (always day 1). Dialysate Il-8 generally exceeded serum concentrations during the entire follow-up, indicating intraperitoneal Il-8 synthesis. A positive correlation was present between the dialysate Il-8 peak and the maximal number of neutrophils in dialysate. This relationship was absent for Il-6 and TNF alpha. In five of six episodes where neutrophils were quantified on both day 1 and 2, the Il-8 peak occurred simultaneously with the neutrophil peak. These findings suggest that Il-8 is involved in the recruitment of neutrophils towards the dialysate during peritonitis.

Interlaboratory variability of the antiperinuclear factor (APF) test for rheumatoid arthritis.

The antiperinuclear factor (APF) test is as sensitive and specific for rheumatoid arthritis as the classical rheumatoid factor tests. Five European laboratories decided to perform a consensus study on the interlaboratory variability of the APF test. They used different donor cells, different conjugates and different criteria for positivity. However, the results differed by not more than one two-fold dilution step, when expressed in international units using WHO reference rheumatoid arthritis serum as the standard.

Long-term effect of human growth hormone therapy on the prevalence of autoantibodies in Turner syndrome. The Dutch Growth Hormone Working Group.

Abnormalities of immune status, particularly a high prevalence (about 50%) of thyroid autoantibodies, have been reported before in Turner syndrome. Results are conflicting as regards other abnormalities of immune function. Growth hormone (GH) has immunomodulatory effects, but results of its effects on GH-deficient children are inconsistent. In this study, 42 girls with Turner syndrome, aged 7.3-19 years, are investigated before, during and after 4 years of human GH therapy. Girls over 12 years old also received ethinyl oestradiol. The prevalence of antithyroid antibodies was 16.7% initially, 35.3% after 24-45 months and 48% after 4 years of therapy though, as there was no control group, it was difficult to conclude that GH was enhancing their appearance. Hypothyroidism was extremely uncommon, and the growth response was no different in those who had the antibodies from those who had not. There were no dramatic increases in prevalence of any of the other antibodies investigated, though the prevalence of parietal cell antibodies was higher than expected.

Necessity of the assessment of health status in human immunogerontological studies: evaluation of the SENIEUR protocol.

Disease is frequent in ageing, and the many conflicting results in studies of the ageing process can be due to the presence of factors such as underlying disease or the use of medication. For immunogerontology, a solution to this problem was initiated in 1984 by a working party of EURAGE, the European Community's Concerted Action Programme on Ageing and Diseases. A protocol defining strict admission criteria to studies of ageing, the SENIEUR protocol, was elaborated. This protocol intends to limit the influence of disease and/or medication and to standardize admission criteria to immunogerontological studies. In subjects fulfilling the SENIEUR criteria, we found less immunological defects with ageing than generally stated. This could mean that many studies performed in not-optimally healthy subjects describe defects that are not a consequence of the ageing process, but could be a result of underlying disease or of the influence of medication. For lymphocyte subsets, certain changes are only found in the comparison of SENIEUR groups of young and aged, while other changes are only found when non-healthy groups are compared. The occurrence of monoclonal gammopathies and autoantibodies was increased in ageing, but was also influenced by health status. Experience of other groups, and the objections against the protocol are discussed.

Serum immunoglobulins, including IgG subclasses, in Vietnamese leprosy patients.

Levels of serum immunoglobulins were measured in healthy Vietnamese and in leprosy patients. Healthy Vietnamese had higher levels of IgG, IgA, IgM, and IgE than did healthy Dutch controls, as well as higher levels of three of the four subclasses of IgG (IgG1, IgG2, IgG3). Lepromatous leprosy patients had significant increases in all classes and subclasses of immunoglobulins, except for IgG2, in comparison with local controls. Tuberculoid leprosy patients had more IgG1, IgG3, and IgG4 than did local controls and had higher total IgG levels. The patients had no increase in autoantibodies against tissue antigens compared to local or Dutch controls.

Immunofluorescence detection of liver cell membrane autoantibodies using PLC/PRF/5 cells.

Detection of liver cell membrane autoantibodies is routinely performed by immunofluorescence testing of patient sera on rabbit hepatocyte suspensions. We have investigated the possible use of cells from the PLC/PRF/5 human hepatoma cell-line. These cells were employed as substrate in an immunofluorescence test which was compared with the conventional rabbit hepatocyte assay. We found a close correlation between the results obtained with these different substrates on visual reading. We furthermore compared visual reading of immunofluorescence preparations with flow-cytometrical analysis of immunostained cell suspensions. The results with these different methods were largely confirmatory. The PLC/PRF/5 cells are easily available and should therefore be regarded as a highly valuable new substrate for detection of liver cell membrane autoantibodies. Flow cytometry appears to be a technically simple and reliable method for quantitative analysis.