Association between active Wegener's granulomatosis and anticytoplasmic antibodies.

Autoantibodies reacting with the cytoplasm of granulocytes and monocytes (anticytoplasmic antibodies [ACPAs]) were found in 42 of 45 patients with active Wegener's granulomatosis (WG) (sensitivity, 93%). Specificity was tested in selected groups of patients with closely related diseases. Of 58 patients without a diagnosis of WG, 2 had ACPAs (specificity, 97%). The significance of ACPA titration for assessing or predicting disease activity was evaluated in a 16-month prospective study of 35 patients with WG. Seventeen relapses were observed and all were preceded by a significant rise of the ACPA titer. Anticytoplasmic antibodies are a specific and sensitive marker for active WG; a rising titer is a sensitive marker for the development of a relapse.

Cytomegalovirus antigenemia as a useful marker of symptomatic cytomegalovirus infection after renal transplantation--a report of 130 consecutive patients.

In earlier work we demonstrated that CMV immediate early antigens can be detected in peripheral blood leukocytes of patients with active CMV infection. We now report a comparison of the antigenemia assay and an anti-CMV ELISA in a prospective longitudinal study of 130 renal transplant recipients who were monitored for active CMV infection during the first 3 months after transplantation. Active CMV infection developed in 56 patients. The antigenemia assay had a sensitivity of 89% and a specificity of 93% in the diagnosis of active CMV infection; for the ELISA these figures were 95 and 100%, respectively. In 22 of the 56 patients a CMV syndrome occurred. Antigenemia was demonstrated in all 22 patients while an antibody response occurred in 21 of them. The antigenemia assay became positive 8 +/- 7 days before the onset of symptoms while the antibody response was observed 4 +/- 9 days after the onset of symptoms. The pattern of antigenemia was helpful for monitoring the course of the infection. The maximum level of antigenemia was significantly higher and its duration significantly longer in symptomatic than asymptomatic infection. We conclude that CMV antigenemia is a sensitive, specific, and early marker of CMV infection. The antigenemia assay is of great value in monitoring patients with a high risk of CMV infection.

Prediction of recurrent cytomegalovirus disease after treatment with ganciclovir in solid-organ transplant recipients.

CMV disease often recurs after initially successful antiviral therapy. We retrospectively determined in a group of 36 organ transplant patients whether clinical, virological, or immunological parameters during or shortly after cessation of antiviral therapy can identify those at high risk of relapse. Eleven of 36 patients had recurrent CMV disease after ganciclovir therapy. Neither donor or recipient CMV serostatus, type of baseline immunosuppression, antirejection treatment, indication for antiviral treatment, nor presence of CMV in the blood during or after therapy (as detected by antigenemia, viremia, or a positive polymerase-chain-reaction signal) were helpful in identification of patients with subsequent relapse. However, quantitative monitoring of antigenemia fascilitated early diagnosis of relapse since 10 of 11 patients with > or = 10 antigen-positive cells per 50,000 PMNs relapsed (99.1%, 95% CI 58.7-99.8). IgM and IgG responses against CMV during primary infection were comparable in relapsing and nonrelapsing patients. During secondary infection relapse occurred only in the 4 patients with the lowest IgG responses. The number of activated CD8bright lymphocytes in the peripheral blood as determined by flow cytometry at the end of antiviral therapy was a strong risk factor for the subsequent clinical course: 6 of 7 patients (85.7%, 95% CI 42.1-99.6%) with < 100 x 10(3) HLADR+CD8bright cells/ml blood relapsed, while 8 of 8 (100%, 95% CI 63-100) with activated CD8bright cells above that level remained asymptomatic (P < .025). These data show that patients with a high risk of relapse of CMV disease can be identified at the end of antiviral therapy.

The serum IgG subclass levels in healthy infants of 13--62 weeks of age.

The levels of IgG1, IgG2, IgG3, and IgG4 were determined in serum samples of 160 infants aged 13--62 weeks, and of their mothers. In addition the serum IgM, IgG, IgA, and IgD levels of the infants are presented. The results show that IgM, IgG1, and IgG3 slightly increase during the first year of life, whereas IgG2, IgG4, IgA, and IgD hardly do. This difference in the development of the various immunoglobulin isotypes reflects differences in the terminal maturation of subsets of B-lymphocytes into plasma cells. About 50% of the infants of this age had no detectable IgG4 and three children had no IgG2. These observations indicate that longitudinal investigations are needed in children suspected of a IgG2 or IgG4 subclass deficiency. No statistically significant influence of sex on the IgG subclasses could be demonstrated in these infants.

Isolation of highly purified lymphocyte subsets for functional studies by means of an indirect rosette technique.

An indirect rosette assay, utilizing ox erythrocytes (RBC) coupled with rabbit anti-mouse IgG and lymphocytes sensitized with monoclonal mouse antibodies against membrane markers, was used for purification of lymphocyte subsets that were functionally intact. Either peripheral blood mononuclear cells (PBMC) or T lymphocytes isolated by sheep RBC rosetting could be used as starting material for obtaining pure T-cell subsets (T4 or T8). The following steps of the method were evaluated: the procedure of coupling rabbit anti-mouse IgG to ox RBC via the CrCl3 method, the experimental conditions for specific rosetting, and the use of Percoll for the separation of rosettes from the non-rosetting cells. Under optimal experimental conditions the recovery of positively selected cells was 45-55% of the cells originally present in the PBMC. The purity of these cells reached a value of more than 95%, whereas the contamination of the depleted fraction was less than 3%. The functional integrity, manifesting itself as proliferation after mitogen stimulation and as regulatory influences on in vitro Ig synthesis, appeared to be unimpaired. The described technique may be applied to the purification of various cell subpopulations for functional studies, provided monoclonal antibodies against membrane antigens are available.

Characterization of the soluble immune complexes that are detected by three different techniques.

Three different tests which are based on different principles were used for the detection of soluble immune complexes (IC): (i) a PEG precipitation test, which is based on the solubility characteristics of IC; (ii) a solid-phase C1q binding assay, which is based on the complement binding property of IC, and uses peroxidase-linked anti-human IgG to detect the bound IC (C1q-ELISA); and (iii) the indirect granulocyte phagocytosis test (IGPT) which is based on the Fc R- and possibly the C3 R-binding of IC. Using heat-aggregated IgG (A-IgG) as a model for soluble IC all three tests showed a linear relation with the amount of A-IgG. The C1q-ELISA and the IGPT had a detection limit of less than 1 microgram/ml while the PEG test only detected quantities of more than 10 micrograms/ml. However, when using artificially produced soluble IC, which were prepared from human antibodies (ab) of different specificities and their respective antigens (ag) i.e., (i) tetanus toxoid, (ii) Helix pomatia hemocyanin (HPH), and (iii) dsDNA, and which consisted of the two components in a wide range of ag/ab ratios, distinct results were obtained with the three tests. Thus demonstrating that results obtained with A-IgG as a model for soluble IC can not simply be extrapolated to the behavior of real complexes in IC detection assays. No matter which ag was used, the composition of the IC, i.e., the ratio in which ag and ab were present, appeared to be the crucial factor for detectability in the different tests. The C1q-ELISA can detect IC over a wide range of ag/ab ratios, while it is particularly sensitive for IC formed in slight ag excess. The IGPT in contrast primarily detects, and is highly sensitive for, IC formed in ab excess. The PEG test appears to detect IC with freshly bound complement only. Another interesting finding has to be mentioned here: when increasing amounts of dsDNA were added to a SLE serum containing anti-DNA ab, the IC that had been detectable in the native serum with the IGPT completely disappeared, thus demonstrating that these complexes did consist of DNA and anti-DNA.

The subclasses of human IgG antibodies against tetanus toxoid.

The subclass of IgG antibodies against tetanus present in the serum of thirty-five human individuals, who received an injection with tetanus toxoid, was determined. Six successive serum samples were obtained from twenty-five normal individuals (laboratory personnel) 0, 3, 7, 14, 28 days and 2-3 months after the injection with tetanus toxoid had been given. Another ten serum samples were obtained from ten persons with a positive IgE-RAST, taken 2 weeks after the injection. Antibodies were determined with a quantitative immunofluorescence method known as the defined antigen substrate spheres (DASS) system. The normal individuals in whose serum a clearly positive IgG binding was found (nineteen) showed activity in all four subclasses. The binding activity in all individuals reached a maximum between 2 and 4 weeks after the injection. The antibody activity in the serum of four individuals whose serum gave weak IgG binding was confined to IgG1. Two individuals did not show any IgG binding activity at all. In the ten persons with a positive IgE-RAST and three of the normal individuals, who also had a positive IgE-RAST, the distribution of the antibodies over the subclasses was the same as in the others.

Fc gamma receptors on lymphocytes from normal donors and patients with lymphoproliferative diseases. Influence of incubation conditions.

The antibody-coated human erythrocyte and the antibody-coated ox erythrocyte rosette assays (EAHu and EAOx) were compared to detect Fc gamma receptors on human peripheral blood lymphocytes. Two incubation conditions were examined: 1 h at room temperature and overnight at 4 degrees C. In healthy persons, in patients with Hodgkin's disease and in patients with non-Hodgkin lymphoma (NHL) the mean percentage of EAHu-rosette-forming cells (EAHu-RFC) increased significantly when the incubation was carried out overnight at 4 degrees C instead of 1 h at room temperature. This increase was caused by Fc gamma receptor-bearing T cells. In the case of EAOx-RFC only a slight increase was found. The percentage of EAHu-RFC and EAOx-RFC differed significantly in the healthy group after the overnight incubation and in the NHL group after the 1-hour incubation. When comparing the mean percentage of EA-RFC in the patient groups with that of the healthy persons significant increases were observed: EAHu-RFC in patients with Hodgkin's disease in the overnight incubation and EAOx-RFC in patients with Hodgkin's disease and NHL in both incubation conditions. In patients with chronic lymphocytic leukemia (CLL, B-cell type) the mean percentage of EAHu-RFC was very low, however that of EAOx-RFC was moderate to high. It is concluded that in the two rosette assays the antigen-antibody complexes may have different avidities to different lymphocyte subpopulations, and that incubation conditions may have an influence on this avidity.

Circulating cytomegalovirus (CMV)-infected endothelial cells in patients with an active CMV infection.

In 10 of 14 patients with an active cytomegalovirus (CMV) infection, distinctive large cells (35-45 microns in diameter) were present in the peripheral blood. Morphologically these cells closely resembled the classic cytomegalic inclusion cells, generally regarded as a diagnostic hallmark of CMV infection. Moreover, these cells were shown to express CMV antigens belonging to all three stages of the viral replication cycle, indicating a productive CMV infection. In addition, immunologic staining with monoclonal antibodies directed against cell differentiation and marker proteins showed that these circulating cytomegalic cells were of endothelial origin. The presence of CMV-infected endothelial cells in the peripheral blood of patients with an active CMV infection indicates that such an infection might be accompanied by widespread occult vascular damage.

Ultrastructural analysis of circulating cytomegalic cells in patients with active cytomegalovirus infection: evidence for virus production and endothelial origin.

The presence of cytomegalic inclusion cells in the peripheral blood of patients with an active cytomegalovirus infection has recently been demonstrated. Immunologic staining showed that these cells were of endothelial origin. Study of circulating cytomegalic cells by transmission electron microscopy showed the cells to be productively infected with cytomegalovirus. Viral capsids were present in the nucleus and virus particles and dense bodies were found in the cytoplasm. The results indicate that these circulating cytomegalic cells could disseminate cytomegalovirus throughout the body. In addition, the finding of a cluster of cytomegalic cells in the peripheral blood linked together by zonula adherens type cell junctions is further evidence that these cells are of endothelial origin and suggests that the endothelial damage may be extensive.

Antibody responses to human cytomegalovirus-specific polypeptides studied by immunoblotting in relation to viral load during cytomegalovirus infection.

The humoral immune response to individual proteins of human cytomegalovirus (CMV) was studied by immunoblotting. CMV polypeptides present in an extract of CMV-infected fibroblasts in a late stage of infection were recognized by sera of healthy seropositive individuals and transplant recipients who suffered from a primary or secondary CMV infection. The results showed that the sera reacted with a maximum number of 18 polypeptides ranging in molecular weight from 28-235 kDa. In 60% or more of the healthy seropositives, polypeptides were recognized with an apparent molecular weight of 150, 98, 94, 58, 50, 44, 38, and 32 kDa. In the patient group, the most immunogenic polypeptides were those with an apparent molecular weight of 150, 104, 94, 66, 50, 38, and 32 kDa. A correlation was found between the antibody levels in sera from the healthy seropositives and the number of recognized polypeptides but no such relationship was seen in the transplant recipients. However, sera of patients with a high virus load during their secondary CMV infection, as detected by the CMV-antigenemia test, appeared to react less frequently and less intensely with the polypeptides than those with low viremia. A high number of antigen-positive leukocytes in the CMV-antigenemia test was related to a low frequency of polypeptides with molecular weights of 85, 76, 66, 44, 38, and 32 kDa recognized before transplantation.

Class- and subclass-specific antibody response to hemocyanin in nonmalignant paraproteinemia.

IgM, IgG, and IgA class-specific, as well as IgG subclass-specific antibody titers against the primary immunogen HPH were measured with ELISA in 19 patients with nonmalignant paraproteinemia (eight with IgG1, two with IgG2, two with IgG4, four with IgM, and three with IgA) and in a simultaneously studied age- and sex-matched control group. After primary immunization only IgM and IgA anti-HPH titers were significantly lower in the patient group. Four patients with relatively high IgG or IgA serum paraprotein levels did not produce antibodies in some Ig classes or IgG subclasses, whereas all other patients and all controls developed antibody titers in all classes and IgG subclasses. Low or absent antibody titers did not occur preferentially in the Ig (sub)classes to which the paraproteins belonged. After secondary immunization the patients could not increase or maintain their antibody titers as well as the controls, and this was most clear in the IgM and IgA antibody class. A direct correlation between polyclonal serum IgM levels and IgM anti-HPH titers was present in the patients. Such a correlation was absent for IgA in the patients and for all classes in the controls. It is concluded that humoral immunosuppression as measured with a newly encountered antigen in patients with nonmalignant paraproteinemia is most clearly expressed in the IgM and IgA antibody class and that the paraprotein (sub)class is not preferentially involved.

Immune complexes in peripheral blood polymorphonuclear leucocytes of malignant melanoma patients.

Immune complexes in PMN cells of sixty-five patients with malignant melanoma were studied by scoring the cells for IgG and complement inclusions. Group (a), consisting of thirty-four patients free from tumour after surgical therapy, showed a median IgG score of 17, which was not statistically different from the control group (n = 11), with a median score of 10. However, the tumour-bearing group (b) (n = 31) showed a significantly higher median score of 22. When group (a) was subidivided, it was shown that the patients who had an invasive growing melanoma with a bad prognosis (n = 11) showed a significantly higher median score of 25, while the patients who had a superficial melanoma with good prognosis were not different from the controls. After subdividing group (b), it was shown that patients with localized disease (n = 13) had a median score of 12, which not different from that of the controls. However, the patients with regional lymphnode involvement (n = 9) or distant metastases (n = 9) showed significantly higher median scores of 69 and 24 respectively. Six out of sixteen patients showed a significant drop in PMN inclusions after surgery. Complement scores gave similar patterns.