Isotype determination of antibodies.

Frequently it is necessary to know the amount and serological class of antibodies made by an immunized animal, produced by hybridomas, or present in the serum of patients with inflammatory or neoplastic conditions. The immunologist's approach to such a problem is to consider the antibody or immunoglobulin molecules themselves as antigens and to use anti-immunoglobulin antibodies as the specific and sensitive agents of detection. This unit describes two methods for measurement and classification of supernatant or serum immunoglobulins: an ELISA and a method employing electrophoresis and immunofixation.

Isotype determination of antibodies.

Frequently it is necessary to know the amount and serological class of antibodies made by an immunized animal, produced by hybridomas, or present in the serum of patients with inflammatory or neoplastic conditions. The immunologist's approach to such a problem is to consider the antibody or immunoglobulin molecules themselves as antigens and to use anti-immunoglobulin antibodies as the specific and sensitive agents of detection. This unit describes two methods for measurement and classification of supernatant or serum immunoglobulins: an ELISA and a method employing electrophoresis and immunofixation.

Carbohydrate and lipid metabolism in endogenous hypercortisolism: shared features with metabolic syndrome X and NIDDM.

Carbohydrate and lipid metabolism was cross-sectionally assessed in 16 patients with endogenous hypercortisolism (endogenous Cushing syndrome). Five patients (31%) had fasting glucose levels over 6.6 mmol/l and a HbA1C over 7.5%. Six patients (38%) had diabetes mellitus based on an abnormal 75 g oral glucose tolerance test (OGTT) and two additional patients (13%) had impaired glucose tolerance based on an OGTT. Compared to obese individuals, patients with Cushing syndrome had an elevated glucose but no elevated insulin response to the OGTT. Regression analysis showed positive correlations between 24-h urinary free cortisol (UFC) and fasting blood glucose (P < 0.0005), UFC and OGTT glucose area under the curve (AUC) (P < 0.01), and UFC and HbA1C (P < 0.005). UFC levels were negatively correlated (P < 0.05) with OGTT insulin AUC and insulin/glucose ratios. Eleven (69%) patients required anti-hypertensive therapy for blood pressure control. Total cholesterol and triglycerides were elevated in patients with Cushing syndrome compared to obese controls, while LDL and HDL cholesterol, and Lp(a) were similar in the two groups. We conclude that impaired glucose tolerance and/or diabetes in patients with endogenous Cushing syndrome is due to the hyperglycemic effects of cortisol with relative insulinopenia. Thus, Cushing syndrome shares features with both the Metabolic Syndrome X and NIDDM, including impaired glucose uptake, hyperlipidemia and hypertension. However, in Cushing syndrome, a relative insulinopenia occurs, while in Metabolic Syndrome X and NIDDM, insulin excess is observed. In Cushing syndrome, as the hypercortisolemia exacerbates, insulinopenia becomes more paramount, suggesting that cortisol exerts a direct or indirect "toxic" effect on the beta-cell.

Blood-CSF barrier permeability and central nervous system immunoglobulin G in schizophrenia.

The ratio of albumin in cerebrospinal fluid (CSF) to serum may serve as an index of the integrity of the blood-CSF barrier, with increases in this ratio indicating increased permeability. The ratio of immunoglobulin G (IgG) in CSF to serum (divided by the albumin ratio to correct for variance in blood-CSF permeability) represents an index of the endogenous production of IgG in the central nervous system (CNS), with increases reflecting a possible infectious and/or autoimmune process stimulating central IgG synthesis. We analyzed simultaneously collected CSF and serum samples from 46 schizophrenic subjects, 8 of whom were studied both on and off neuroleptic treatment, and samples from 20 normal controls. The data indicated increases in CSF/serum albumin ratios or CSF/serum IgG indices in 22% and 20%, respectively, of the schizophrenic patients. Only 3 patients showed elevations in both indices. Comparison of values on and off neuroleptics indicated no significant effect of neuroleptics on these indices.

Hypoprebetalipoproteinemia, acanthocytosis, retinitis pigmentosa, and pallidal degeneration (HARP syndrome).

We describe the clinical and laboratory studies of an 11-year-old girl with prominent orofacial dyskinesia, dystonia, and progressive dementia. Investigations revealed hypoprebetalipoproteinemia, acanthocytosis, atypical retinitis pigmentosa, and evidence of iron deposition in the pallidal nuclei. Electroneuromyography and skin and sural nerve biopsies were normal. The "eye-of-the-tiger" sign, used to describe the pallidal nuclei in Hallervorden-Spatz syndrome, was present on T2-weighted MRIs (GE Signa, 1.5 T). Phase-contrast microscopy of whole blood showed 80 to 90% acanthocytes whose morphology was confirmed by electron microscopy. High-resolution lipoprotein electrophoresis demonstrated an absence of the pre-beta fraction. This case differs phenotypically from the previous reports of Hallervorden-Spatz disease with acanthocytosis by the presence of prominent orofacial dyskinesia and abnormal serum lipoproteins.

Monoclonal process in primary Sjögren's syndrome and cross-reactive idiotype associated with rheumatoid factor.

Monoclonal or oligoclonal B cell products have been described in the sera and urine of patients with primary Sjögren's syndrome (PSS). In addition, monoclonal expansion of plasma cells has been found in the exocrine glands of PSS patients with circulating monoclonal B cell products. The goal of this study was to raise an anti-idiotype to a cryoprecipitable monoclonal IgM kappa rheumatoid factor (RF) from a PSS patient. Using the F(ab')2 fragments of the rabbit IgG anti-idiotype, an idiotype-specific ELISA was developed and sera from 32 patients with PSS (13 with monoclonal IgM kappa), 33 with rheumatoid arthritis, three with rheumatoid arthritis + Sjögren's syndrome (SS), 30 with systemic lupus erythematosus, six with Waldenström's macroglobulinaemia, and 20 healthy controls were tested. The idiotype was primarily found in PSS patients with circulating monoclonal IgM kappa and more often in those who had a ratio of kappa: lambda intracytoplasmically positive plasma cells greater than 3:1 in the lymphocytic infiltrates of minor salivary glands, and systemic manifestations. The idiotype was also found in PSS and rheumatoid arthritis patients without circulating monoclonal cryoglobulins as well as in two of the six patients with Waldenstrom's macroglobulinaemia. Our results suggest that the monoclonal process observed in PSS could involve restricted idiotypic clones that are susceptible to malignant transformation.

Monoclonal immunoglobulins and autoantibodies in lymphoid malignancies.

The presence of serum monoclonal or oligoclonal immunoglobulins (paraproteins) was investigated in 38 non-Hodgkin's lymphoma and chronic lymphocytic leukemia patients, 33 patients with solid tumors and 33 healthy individuals. Seventy two percent of non-Hodgkin's lymphoma and 31% of chronic lymphocytic leukemia patients had serum paraproteins, in contrast to 21% and 15% of solid tumor patients and normal controls respectively. There was no significant prevalence of a certain isotype or light chain in the non-Hodgkin's lymphoma, chronic lymphocytic leukemia and solid tumor groups. In the healthy individuals all bands were monoclonal of the IgG isotype. No correlation was found between histologic grading of lymphoid malignancy or disease stage and serum monoclonality. No serologic or histologic autoimmune features were demonstrated in non-Hodgkin's lymphoma and chronic lymphocytic leukemia patients. In addition, no correlation was found between serum autoantibody activity and mono- or oligoclonal immunoglobulins.

Association of serum IgM kappa monoclonicity in patients with Sjögren's syndrome with an increased proportion of kappa positive plasma cells infiltrating the labial minor salivary glands.

Minor salivary gland biopsy specimens from 11 patients with primary Sjögren's syndrome with circulating monoclonal IgM kappa cryoglobulins, seven without cryoglobulins, and four patients with rheumatoid arthritis and Sjögren's syndrome (one with monoclonal and three with polyclonal cryoglobulins) were examined by the peroxidase antiperoxidase bridge technique, using antihuman kappa and lambda antibodies. In 6/11 patients with primary Sjögren's syndrome and in one patient with Sjögren's syndrome and rheumatoid arthritis with monoclonal cryoglobulins a predominance of plasma cells containing intracytoplasmic kappa light chains was found (kappa:lambda greater than 3:1). Two of those seven patients had immunohistological features of immunocytomas. In the other five patients with circulating monoclonal cryoglobulins the kappa:lambda ratio of positive cells did not exceed 3:1, while six out of seven patients without cryoglobulins and the patients with rheumatoid arthritis and Sjögren's syndrome with polyclonal cryoglobulins had almost equal numbers of kappa and lambda stained cells. One of seven patients with primary Sjögren's syndrome without cryoglobulins had an increased number of lambda light chain positive cells, indicating a non-secretory lambda monoclonal population. These findings suggest that the main area of B cell monoclonal expansion in primary Sjögren's syndrome may be the affected exocrine glands.

Oligoclonal immunoglobulin bands in serum in association with chronic viral hepatitis.

We studied serum immunoglobulins in 54 patients with chronic viral hepatitis. The majority had hyperglobulinemia, with immunoglobulin G (IgG) being elevated the most. High resolution protein electrophoresis revealed the presence of oligoclonal bands within the gamma-globulin region of the electrophoretic pattern (oligoclonal immunoglobulin bands) in 32 (59%) patients. Between two and seven bands were noted in each sample. In order to analyze the significance of this observation, we performed stepwise discriminant analysis of clinical, demographic, serological, and serum biochemical features in patients with and without detectable oligoclonal immunoglobulin bands. The patient's ages and serum immunoglobulin G levels were found to be important predictors of the presence of oligoclonal immunoglobulin bands. No correlation was found with the type of hepatitis, disease severity, or duration of hepatitis. On follow-up, serum samples were retested in 20 patients, over 5 to 60 months. Significant change was noted in only one patient in whom oligoclonal immunoglobulin bands (initially very prominent) became progressively weaker and finally disappeared over a 5-yr period. This was associated with an improvement in his hepatitis after loss of hepatitis following loss of hepatitis B e antigen from serum. These observations suggest that the determination of oligoclonal immunoglobulin bands in serum may be a useful marker to study the course and activity of chronic viral hepatitis in selected patients.

Oligoclonal bands in acute schizophrenia: a negative search.

Numerous immunologic abnormalities have been reported in patients with schizophrenia, suggesting that this disorder may in some cases represent a viral or immunologic disorder of the brain. To further examine this question, evidence for intrathecal production of immunoglobulin (IgG) and immunoglobulin oligoclonal bands (OCB) was sought in patients with acute or early schizophrenia. Using high resolution zone electrophoresis and immunofixation, there was no evidence for intrathecal IgG generation in 5 of 5 patients tested. One of 9 patients had OCBs in CSF. This man, although he had a typical history of schizophrenia, tested positive for human immunodeficiency virus antibody in serum.

Prednisone effects on blood-brain barrier permeability and CNS IgG synthesis in healthy humans.

Corticosteroids reportedly decrease blood-brain barrier (BBB) permeability and/or IgG synthesis in patients with multiple sclerosis or brain tumors. However, these effects have not been studied in healthy humans. We investigated the effects of prednisone, 80 mg/day for five days, on the ratio of cerebrospinal fluid (CSF) albumin/serum albumin, a measure of blood-brain barrier (BBB) permeability, and on CSF and serum IgG levels in six healthy, normal volunteers. We found significant steroid-induced decreases in serum and CSF albumin levels and in serum IgG levels. However, we found only a nonsignificant decrease in BBB permeability and no significant change in CNS IgG synthesis. These findings, based on a small number of volunteers, suggest that it may be difficult to further decrease BBB permeability and CNS IgG synthesis in medically healthy subjects.

Oligoclonal immunoglobulins in serum of patients with chronic viral hepatitis.

Zone electrophoresis on agarose gel was performed in serum samples from patients with chronic viral hepatitis. On the basis of the clinical, serological, and biochemical evaluation, the patients were divided into three categories: chronic type B hepatitis; chronic type delta hepatitis; and chronic non-A-non-B (NANB) hepatitis. The results of the electrophoretic analysis showed a high incidence of oligoclonal immunoglobulin bands (OIBs) in patients with chronic B hepatitis and chronic delta hepatitis and a low incidence of OIBs in patients with NANB. Since other biochemical and serological characteristics are variable and do not correlate with clinical symptoms of disease, we suggest that oligoclonal immunoglobulins could be used as a marker to aid in studies of the natural history and pathologic course of the various types of chronic hepatitis.

Electrophoretic examination of proteinuria in Lowe's syndrome and other causes of renal tubular Fanconi syndrome.

Urine samples from 26 patients with five different causes of renal tubular Fanconi syndrome were examined by zone electrophoresis on agarose gel and immunofixation. The tubular disorders associated with Lowe's syndrome, cystinosis, and idiopathic Fanconi syndrome exhibited urine protein electrophoretic characteristics that differentiated them from normal and from each other. In particular, Lowe's syndrome urine exhibited four discrete bands in the gamma globulin zone. Electrophoresis of urinary proteins may be useful in distinguishing among the different metabolic disorders causing renal tubular Fanconi syndrome.

Polyclonal B-cell lymphocytosis and hypergammaglobulinemia in patients with Gaucher disease.

Sera from 23 individuals with Gaucher disease (GD) were analyzed for hypergammaglobulinemia and oligoclonal and monoclonal gammopathies. Serum IgG level was elevated in 15/23 (65%) patients, and a diffuse hypergammaglobulinemia was present in 10/23 (43%) patients. An oligoclonal gammopathy was noted in six patients, and a monoclonal gammopathy in two. Lymphocyte subset analysis was also carried out in eight individuals with GD. Four of five individuals showed increased surface Ig-positive lymphocytes, while 7/7 were positive for either increased CD19- and/or CD20-positive lymphocytes. An eighth patient was found to have a B-cell leukemia. Statistical analysis of kappa and lambda histograms were suggestive of a monoclonal excess. However, restriction enzyme analysis of four individuals with GD and increased B cells failed to show any evidence of Ig gene rearrangements. Serum Ig abnormalities and perhaps B-cell lymphocytosis appear to be common in the GD patient population and are not associated with circulating monoclonal lymphocytes.

Monoclonal immunoglobulins in the serum of patients with primary Sjögren's syndrome.

Monoclonal immunoglobulins (M Igl) were detected in the serum of 10 of 20 patients with primary Sjögren's syndrome (prim. SS), by using high resolution electrophoretic technique combined with immunofixation. The finding of three free lambda chains and two IgG lambda suggests that the lambda-expressing B-clones are involved in this process, but the class distribution is different from one series to another, and from one country to another. The prim. SS patients with multiogran disease have the higher incidence of M Igl.

Identification of HIV-specific oligoclonal immunoglobulins in serum of carriers of HIV antibody.

Zone electrophoresis on agarose gel was performed on serum samples from HIV-antibody carriers and negative controls. Nitrocellulose strips precoated with an HIV preparation were then placed on top of the gels and developed by an immunoblotting procedure. A positive reaction was demonstrated between the HIV antigens and the HIV-antibody-positive serum samples with hypergammaglobulinemia and oligoclonal IgG bands. A negative reaction was found between the HIV antigens and HIV-antibody-negative serum samples from a normal person and a patient with monoclonal gammopathy. The presence of oligoclonal IgG bands in the serum of HIV-antibody carriers, and their positive identification with HIV antigens, indicates a specific immune response of the host to the HIV infection and supports the use of oligoclonal IgG bands as markers to follow the course of HIV infection.

CD5-expressing B lymphocytes in the blood and salivary glands of patients with primary Sjögren's syndrome.

CD5, the human counterpart of Ly-1 molecules in the mouse, are detectable but weakly expressed on a minute fraction of circulating B cells. The number of CD5 + B cells in the blood of patients with Sjögren's syndrome was slightly higher than in control blood, but it became statistically significant after treatment of the cells with phorbol myristic acetate. These numbers were even higher in patients with homogeneous serum bands than in the others. A few scattered cells were stained with anti-human IgM antibody on salivary gland sections, and among them 5-10% were found to be positive for anti-CD5.

[Monoclonal cryoglobulins in primary Sjögren's syndrome]. / Monoklonal'nye krioglobuliny pri pervichnom sindrome Shegrena.

Cryoglobulins were studied in the sera of 40 unselected pSS patients. The nature of the cryoglobulins was defined using a very fast and sensitive resolution electrophoresis technique combined with immunofixation. Thirty-five per cent of SS patients were shown to have circulating IgMk mixed monoclonal cryoglobulins. On the contrary, all the other tested cryoprecipitates were found to be mixed polyclonal, with the exception of one patient with SLE. The presence of cryoglobulins in the sera of SS patients correlated with extraglandular disease and with autoantibodies to Ro (SSA) cellular antigen and rheumatoid factor. Patients with cryoglobulins had lower serum C4 levels compared to patients without cryoglobulins. These findings suggest that SS in addition to polyclonal B-cell hyperreactivity expresses a monoclonal process preceding the development of lymphoid neoplasia. Extraglandular manifestations of the syndrome may be associated with an immune complex mediated pathology.

Monoclonal and oligoclonal immunoglobulins in the serum of patients with B-chronic lymphocytic leukemia.

The incidence of monoclonal and oligoclonal immunoglobulins (paraproteins) was determined in serum samples of 45 chronic lymphocytic leukemia (B-CLL) patients using the high-resolution agarose gel electrophoresis technique combined with immunofixation. Paraproteins were identified in 25 of the 45 patients tested. Twelve paraproteins were monoclonal and 13 oligoclonal. IgG kappa and/or lambda immunoglobulin isotypes were found in 17/25 of these patients. No correlation of the lymphocyte cell morphology and the presence of paraproteins was demonstrated. The high frequency of serum oligoclonal immunoglobulins in B-CLL indicates that more than one lymphocyte clone may be present in this disease.