Diminished synthesis of immunoglobulin by peripheral lymphocytes of patients with idiopathic membranous glomerulonephropathy.

Some studies of animal models of serum-sickness nephritis have shown that the lesions of membranous nephropathy develop in animals exhibiting a poor antibody response to the administered antigen (if given in constant amounts). It is postulated that patients with idiopathic membranous nephropathy may share a similar characteristic, namely, a diminished capacity to produce sufficient amounts of antibody. To test this hypothesis, we examined the ability of lymphocytes isolated from 11 patients with this disorder to produce immunoglobulin (Ig)G and IgM on stimulation with a polyclonal B-cell activator, pokeweed mitogen. The peripheral blood lymphocytes (2 x 10(6) cells) from 24 normal individuals had geometric mean production rates of 1,779 ng for IgG, and 2,940 ng for IgM after 7 d of culture in the presence of pokeweed mitogen. By contrast, under identical conditions, lymphocytes from the 11 patients with membranous nephropathy produced significantly lower quantities of both immunoglobulins, with geometric mean concentrations of 511 ng for IgG and 439 ng for IgM. When lymphocytes from patients with membranous nephropathy were co-cultured with normal lymphocytes, the production of immunoglobulin by normal lymphocytes was depressed by 22-82%, suggesting that a population of suppressor cells was responsible for this disturbance in B-cell function. By co-culturing normal lymphocytes with patient lymphocytes depleted of either T cells or monocytes, the suppressor cell was identified as a monocyte.

Immune function of successfully treated lymphoma patients.

Immunologic function was evaluated in 12 patients with Hodgkin's disease and 5 patients with lymphocytic lymphoma who had been successfully treated with either chemotherapy, radiation therapy, or both of these modalities 3-42 mo previously. Only two of the patients were found to have total anergy to a battery of six recall skin test antigens and all were responsive to skin testing with phytohemagglutinin. However, 10 of 16 patients were unable to develop delayed cutaneous hypersensitivity to either of the neoantigens dinitrochlorobenzene or keyhole limpet hemocyanin. Four other patients developed reactivity to only one of these neoantigens for a total of 14 of 16 (88%) of the patients demonstrating some impairment in neoantigen response. Total lymphocyte, T-lymphocyte, B-lymphocyte, and null cell numbers, as well as serum immunoglobulins were quantitatively normal. Monocyte numbers, chemotaxis, and Fc receptor activity were normal. Monocyte staphylocidal activity at 60 min was modestly depressed and candidacidal activity was depressed in those receiving both chemotherapy and radiation therapy. Spontaneous (unstimulated) lymphocyte [3H]thymidine incorporation was low in the patients as a group and lymphoblastic transformation to specific antigens was impaired in 11 of 17 patients who had positive skin test reactions to the same antigen. Highly significant suppression of lymphoblastic transformation was noted after stimulation by the mitogens phytohemagglutinin, pokeweed, and concanavalin-A. The greatest impairment of mitogen response was seen in those patients receiving both chemotherapy and radiation therapy. These data demonstrate specific impairments of neoantigen processing, lymphocyte function, and to a lesser extent monocyte function in successfully treated patients with lymphoma. These impairments may contribute to the increased incidence of infections and second primary malignancies in these patients.

Uridine kinase activities in normal and neoplastic lymphoid cells.

Both adult (I) and embryonic (II) forms of uridine kinase have been identified in the transplantable EL-4 leukemia of C57BL/6 mice and in the P815Y mastocytoma of DBA/2 mice. Only Species I is found in primary tumor cells of lymphoid orgin (virus-induced feline lymphosarcoma, human acute and chronic lymphocytic leukemia) and in normal calf thymocytes and porcine peripheral blood lymphocytes; Species I was induced 4-fold upon stimulation of the normal blood lymphocytes with phytohemagglutinin. The level of uridine kinase activity in the feline lymphosarcoma of thymus-dependent lymphocyte orgin and childhood lymphocytic leukemia of possible thymus-dependent lymphocyte or null-cell origin was similar to the induced level in phytohemagglutinin-stimulated normal lymphocytes, i.e., thymus-dependent lymphocytes. In contrast lymphocytes of a patient with chronic lymphocytic leukemia of thymus-independent lymphocyte origin had a level of uridine kinase activity comparable to that of the unstimulated normal lymphocytes or thymocytes. The uridine kinase activity in the EL-4 tumor cells was repressed by acute treatment of the mice with 5-azacytidine.

Studies on the binding of C3b-coated microspheres to human neutrophils.

A method is described for the quantitation of C3b receptors on human neutrophils using a mixture of C3b-coated fluorescent and C3b-coated non-fluorescent microspheres. The method measures the "sterically available' C3b receptors on the cells, for example, the receptors available to opsonized bacteria. The use of mixtures of fluorescent and non-fluorescent microspheres resulted in lowered fluorescence intensities of the microsphere-coated neutrophils that were well within the fluorescence limitations of fluorescence activated cell analyzers or sorters used in the assay procedure. These mixtures also allowed the distribution of the C3b-coated microspheres around the neutrophils to be easily visualized in the fluorescence microscope. The binding of the C3b-coated microspheres to the neutrophils was shown to be receptor mediated by typical saturable binding kinetics, by complete inhibition by fluid phase C3b, but not by other proteins and by nearly complete inhibition by anti-C3b receptor antibody. Several parameters that could affect the binding of C3b-coated microspheres to neutrophils were studied; these included time and temperature of incubation of the microspheres with the cells, the diameter of the microspheres, the C3b content of the C3b-coated microspheres, the presence of metal ions, azide, EDTA, protein (BSA, IgG), soybean trypsin inhibitor in the buffers, and the method of isolation of the neutrophils. The C3b-coated microspheres were evenly distributed around the neutrophils in almost all of the cases; however, the neutrophils used in these studies were not activated and were not phagocytosing. The method is extremely reproducible and sensitive in detecting small changes in number of C3b receptors on cells.

Leukemic reticuloendotheliosis. A study of the origin of the malignant cell.

A highly pure preparation of neoplastic cells from the spleen of a patient with leukemic reticuloendotheliosis was studied for function, membrane characteristics and glucose metabolism. Glass adherence and phagocytosis of small particles (latex and carbon black) were demonstrated with phase contrast microscopy. Staphylocidal activity was similar to that of normal monocytes. Immunofluorescent assays revealed nonspecific uptake of antiserums to immunoglobulins G (IgG), M (IgM), A (IgA) and kappa and kappa and lambda light chains. Rosette assays indicated the presence of receptors for IgG on the surface of all cells but no receptors for complement (C3) or sheep red blood cells. Glucose metabolic studies revealed a pattern that differed from that of normal monocytes or lymphocytes with intermediate values for glycolysis, low hexose monophosphate shunt activity and high Krebs cycle activity. Increments in tritiated (3H)-thymidine uptake and glucose metabolism in response to phytohemagglutinin stimulation were minimal (5 per cent of normal lymphocyte values) and no response was noted with pokeweed mitogen stimulation. These findings suggest that the leukemic reticuloendotheliosis cell most closely resembles cells of the monocyte-histiocyte series.

Follicular center-cell lymphoma with plasmacytic differentiation, monoclonal paraprotein, and peripheral blood involvement. Recapitulation of normal B-cell development.

A patient with a nodular and diffuse small-cleaved follicular center-cell lymphoma that exhibited definite plasmacytic differentiation, a related monoclonal gammopathy, and circulating population of small lymphocytes is presented. Aside from showing that the presence of numerous plasma cells is not a reliable criterion for the diagnosis of a reactive follicular proliferation, the case is an example of a lymphoma without a block in maturation. The "neoplastic" B-cells show development to follicular center cells and beyond, to functioning plasma cells, and probably also to recirculating "memory" cells. It also suggests that plasmacytoid lymphocytic lymphomas (Lukes-Collins classification) might represent a heterogeneous group of lymphoid neoplasms with some closely related to follicular center-cell lymphomas and others more closely related to small lymphocytic lymphoma/B-cell chronic lymphocytic leukemia.

Antimurine antibody formation following OKT3 therapy.

OKT3 is an IgG2a murine monoclonal antibody directed against the CD3 antigen receptor of human T lymphocytes. A major concern with OKT3 treatment in solid organ transplant recipients is the development of antimouse antibody, which may preclude retreatment with this agent. We have administered OKT3 on 215 occasions (150 renal, 34 hepatic, 26 cardiac, 5 pancreatic) in 179 patients between April 1982 and December 1988. The mean duration of treatment was 10.5 days (range, 2-22 days). Antimouse antibody data were analyzed on the most recent 133 treatment courses where the antibody status was available pretreatment. Determination of antimouse antibody production was elicited by ELISA technology at days 0, 7, 14, and 28 of OKT3 treatment. Patients were categorized according to the antibody response as follows: (a) absence of antibody; (b) low titer (1:100); or (c) high titer (greater than or equal to 1:1000). Our earlier experience has demonstrated that retreatment with OKT3 is successful in groups a and b. The development of antimurine antibodies was analyzed with regard to the following parameters: (1) The duration of OKT3 treatment; (2) treatment type (prophylactic, primary, or secondary); (3) primary treatment or retreatment; (4) concomitant immunosuppressive regimen (double or triple therapy); (5) dosage of concomitant immunosuppressive drugs; and (6) transplant organ type. The following results were obtained. (1) Duration of treatment had no effect on antibody production (11.0 days in antibody negative and 10.0 days in antibody positive). (2) There was no difference in antibody formation rates for the first treatment of OKT3 when it was used as prophylaxis (26%), primary (19%), or secondary (27%) therapy. (3) Antibody formation rate with first treatment was 29%; with retreatment, patients who were antibody negative following first treatment became positive in 28% of cases, and retreated patients who were low titer positive following first treatment converted to high titer in 57% of cases. (4) Antibody formation was higher in patients receiving double immunosuppressive therapy (36%) than in those receiving triple immunosuppressive therapy (21%) during OKT3 treatment. (5) Concomitant immunosuppression was lower in the antibody-positive group during OKT3 therapy: steroids, 61 mg/day vs. 52 mg/day; azathioprine, 89 mg/day vs. 66 mg/day; CsA, 317 mg/day vs. 186 mg/day. (6) Antibody formation rates were lower in non-renal transplants following first treatment with OKT3 (liver 17%, heart 17%, kidney 28%); this reflects the higher doses of concomitant immunosuppressive therapy used in nonrenal transplants.(ABSTRACT TRUNCATED AT 400 WORDS)

Successful retreatment of allograft rejection with OKT3.

OKT3 is a murine monoclonal antibody to the CD3 antigen of human T lymphocytes. The production of human antimurine antibodies after treatment with OKT3 has been perceived as a major limitation to its extended use and reuse. Treatment of 142 patients with 168 courses of OKT3 resulted in the development of antimouse antibody in 28% of the patients. Twenty-six patients (16 kidney, 6 liver, 3 heart, 1 pancreas) have been retreated with 27 courses of OKT3. Eighteen patients had no antimurine antibodies present, and the rejection reversal rate was 83% (15/18). Six patients had a low-titer antimurine antibody present, and rejection reversal occurred in 5 (83%). Rejection was not reversed in 2 patients with a high-titer antibody. Development of antimurine antibody was more frequent in renal transplant recipients (33%) than in hepatic (12%) or cardiac transplant recipients (18%). We believe that this reflects the fact that concomitant immunosuppressive therapy is more likely to be reduced during OKT3 therapy in renal transplant recipients than in hepatic or cardiac transplant recipients. Retreatment of patients with no anti-OKT3 antibody resulted in depletion of CD3+ cells from the peripheral blood, but it took longer than in patients being treated with OKT3 for the first time. Similarly, serum OKT3 levels rose more slowly in retreated patients compared to first treatment. In retreating patients with a low-titer antimurine antibody, it often was necessary to increase the dose of OKT3 in order to achieve adequate serum OKT3 levels and to deplete CD3+ cells. De novo antimurine antibody developed in 4 of the 18 (22%) antibody-negative patients who were retreated. In conclusion, retreatment with OKT3 should not be considered unless the antibody status of the patient is known. Development of low-titer antibodies does not preclude successful retreatment with OKT3; however, alternate antirejection therapy should be used in patients with high-titer antimurine responses.

Immunofluorescence and immunoperoxidase observations of anti-lactic dehydrogenase-1 antibody.

For immunoelectron-microscopic demonstration of lactic dehydrogenase (LDH), anti-subunit-B antibody was produced in rabbits by immunization with purified pig LDH-1 (B4) isoenzyme. Immunoglobin fragment Fab' was obtained from anti-LDH serum immunoglobulin G and conjugated with horseradish peroxidase. Subunit-B-containing LDH was present diffusely in the sarcoplasm of the pig heart muscle. There was concentration of LDH in the 1-band. LDH reaction was also present in the mitochondrial intermembranous space. The present immunoelectron-microscopic examination is in general agreement with the previous cytochemical demonstration of LDH using thiocarbamylnitro blue tetrazolium.

Expression of hematopoietic progenitor cell associated antigen CD34 in chronic myeloid leukemia.

The expression of progenitor cell associated antigen CD34 was investigated in cells from 28 patients with chronic myeloid leukemia (CML). The CD34 positivity varied from 0-26% in patients with chronic phases CML (n = 17); from 6-64% in patients with accelerated phase CML (n = 4); and from 27-97% in the patients with blastic crisis of CML (n = 8). The difference in CD34 positivity between chronic (mean 10.1 +/- 2.3%), accelerated (37.7 +/- 13.3%) and blastic (58.0 +/- 7.3%) phases of CML is statistically significant (p less than 0.05), however, the number of patients studied, especially in accelerated and blastic phases is very small. There was no difference in the CD34 positivity of the cells in the peripheral blood and in the bone marrow. CD34 positivity was higher in patients with chronic phase CML at diagnosis (untreated patients) than in those who were studied during treatment. The possible importance of serially studying CD34 positivity in patients with CML is discussed in the paper.

Immunohistochemical classification of acute leukemias using peripheral blood smears.

Immunophenotypic classification of the acute leukemias (AcL) is of well documented value in those of lymphoid or uncertain origin and of increasing importance in those of nonlymphoid origin. Most of these studies have been performed on viable cell suspensions. To study the efficacy of a simpler immunohistochemical approach to the classification of the acute leukemias requiring only peripheral blood smears, 15 AcL (including three CGL-BC) were studied using an immunoalkaline phosphatase method and a panel of anti-lymphoid and anti-myeloid monoclonal antibodies. Routine cytochemistries were also performed (Sudan black, PAS). Using immunohistochemistry, five cases marked as common ALL (four were undifferentiated by cytochemistry, one ALL), eight cases as ANLL (all ANLL by cytochemistry) and two cases marked only with anti-HLA-DR (AUL by cytochemistry). These results show that immunophenotypic analysis of AUL, ALL and ANLL can be successfully performed even when only air dried peripheral blood smears are available.

Flow-cytometric DNA analysis of hematopoietic and lymphoid proliferations: a comparison of fresh, formalin-fixed and B5-fixed tissues.

Flow-cytometric DNA analysis of human tumors using paraffin-embedded tissue samples is becoming an increasingly popular method of determining ploidy and proliferative rate. Particularly with hematopoietic/lymphoid proliferations, little is known about how these data compare with data from fresh suspension studies, or how B5-fixed tissues compare with those fixed in formalin. For these reasons, flow-cytometric DNA ploidy and cell cycle analysis was performed on 16 hyperplastic tonsils and 28 lymphoid/hematopoietic neoplasms using fresh (or fresh-frozen) cell suspensions (FR), B5-fixed paraffin-embedded (B5), and formalin-fixed paraffin-embedded (FO) tissue. Ploidy analysis showed all tonsil specimens to be diploid regardless of preparative method; however, for the neoplastic cases the FO and B5 preparations agreed with the FR preparations in 50% and 61% of the cases, respectively. Complete agreement between all three tissue preparation methods was present in only 36% of the neoplastic cases. Percent S or S + G2M phase fractions showed relatively poor correlation between the three preparative methods, with the best correlations found between the FR and B5 samples (S phase, r = 0.44; S + G2M, r = 0.43). In conclusion, while potentially useful data can be obtained from flow-cytometric DNA analysis of fixed, paraffin-embedded lymphoid/hematopoietic tissues, the specific limitations of such analyses must be recognized. Ploidy and proliferative phase data from fixed tissue preparations are not equivalent to information obtained from fresh suspension studies. B5-fixed tissue provides an acceptable alternative to formalin-fixed tissue for such analyses.

Multilobated lymphoma of B cell type: a multiparameter investigation.

Multilobated lymphomas were originally described as T-cell neoplasms, but many of B-cell type have subsequently been reported. A case of B-cell origin is reported in which both immunophenotypic and genotypic studies performed on a cell suspension of the lymphoma gave inconclusive and potentially misleading information, while paraffin and frozen section immunohistologic studies, as well as genotypic studies performed on DNA obtained from snap-frozen tissue, were definitive. Thus, this case illustrates some of the problems that may be encountered using cell suspensions as a source for immunophenotypic, and even the much more sensitive genotypic, studies.

CD8-positive B-cell chronic lymphocytic leukemia. A report of two cases.

Most B-cell chronic lymphocytic leukemias and a small normal subset of B lymphocytes express the T-cell-associated CD5 antigen; expression of other T-cell antigens has been reported only rarely. The authors report two cases of typical B-cell chronic lymphocytic leukemia, seen during 1 year, in which two-color flow cytometric analysis documented expression of the T-cell-associated CD8 antigen by the monoclonal B cells. Genotypic studies showing immunoglobulin but not T-cell-receptor gene rearrangements confirmed the B-cell origin of the neoplastic cells. The true frequency of the CD8-positive B-cell chronic lymphocytic leukemia, any clinical implications, and the possibility of a normal subset of CD5-positive CD8-positive B cells remain to be determined.

Immunoglobulin gene rearrangement in abnormal lymph node hyperplasia.

The histologic designation "abnormal lymphoid hyperplasia" is applied to lymph nodes demonstrating varying degrees of architectural effacement and/or cytologic atypia. Although some of these cases may be suggestive of non-Hodgkin's lymphoma, a definitive diagnosis is not possible despite careful morphologic and immunophenotypic studies. Because the demonstration of immunoglobulin and T-cell receptor gene rearrangements by Southern blot analysis provides a sensitive marker of lineage and clonality in lymphoid malignant conditions, the frequency with which such gene rearrangements could be identified in abnormal hyperplasia and their significance were studied. DNA samples from lymph node biopsy samples of 11 patients with abnormal lymphoid hyperplasia were analyzed for rearrangements of immunoglobulin and T-cell receptor genes by Southern blot hybridization. Six of these patients had monoclonal B-cell populations identified by immunoglobulin gene rearrangements; all were found subsequently to have non-Hodgkin's lymphoma by repeated biopsy from 8 days to 46 months later. Two patients with negative Southern blot studies also developed lymphoma, one a T-cell non-Hodgkin's lymphoma and one a cutaneous B-cell non-Hodgkin's lymphoma. Three patients without detectable gene rearrangements showed no evidence of malignant lymphoma at 36-, 45-, and 60-month follow-up evaluations. Southern blot analysis thus identified monoclonal B-cell lymphoid populations in a subset of patients with abnormal lymphoid hyperplasia; the presence of clonal immunoglobulin gene rearrangement predicted progression to overt non-Hodgkin's lymphoma.

Sarcoid lymphocytes: B- and T-cell quantitation.

Bone-marrow-derived B lymphocytes and thymus-dependent T lymphocytes were quantitated in a group of 38 patients with histologically confirmed sarcoidosis. B lymphocytes were identified by detecting surface immunoglobulins (Ig, IgG, IgM, and IgA) and complement receptors. T lymphocytes were identified by E-rosette assay. The untreated patients with both limited and disseminated disease had lymphopenia, reduced T-cell number, and low E/Ig cell ratios. Absolute numbers of circulating E-rosette lymphocytes did not show any correlation with cutaneous anergy. The numbers of Ig-bearing lymphocytes or the sum of the numbers of IgG, IgM, and IgA(GMA)-bearing lymphocytes were elevated in patients with disseminated disease, whereas the numbers of complement receptor lymphocytes were normal in all groups. It is proposed that this discrepancy of results on B-lymphocyte subpopulations might be explained by the presence of antibody or extrinsic antigen-antibody complexes bound to lymphocytes, as supported by elevated GMA/Ig ratios. The numbers of circulating B lymphocytes, as detected by any of three markers employed in this study, showed no correlation with the levels of serum immunoglobulins. The mechanisms of T-cell depletion and increase of immunoglobulin-bearing cells remain to be determined.

Anti-idiotypes and autoimmune disease.

Idiotypes represent structural sites located on the Fab fragment of antibodies. Even though these sites are combining sites for antigenic determinant, their uniqueness makes them antigenic, generating anti-Ids that have functionally performed as suppressive agents for the further production of the antibody. Jerne's network theory of idiotypes has provided a basis upon which investigators have searched for means to reduce, or explain the reduction of, Ab production. A principal area of this reduction of Ab expression concerns the autoAbs in autoimmune diseases. Experimental and clinical evidence suggests a role for anti-Ids in suppressing autoAb production and an inverse relationship between serum levels of anti-Ids and autoimmune disease activity.

Host response to mycobacterial infection in the alcoholic rat: male and female dimorphism.

Increased susceptibility to tuberculosis occurs in the alcoholic. One explanation for the altered susceptibility is a change in T-lymphocyte modulation. To evaluate this, 24 male and 24 female Sprague-Dawley rats were treated with either a Lieber-type liquid ethanol diet (LED) or an isocaloric control (LCD). After 2 weeks, half the subjects were infected with BCG (10(8) colony-forming units) and sacrificed after 42 days. Splenic helper (CD4) and suppressor/cytoxic (CD8) cells were quantitated by flow cytometry. By three-way analysis of variance, splenic cellularity was significantly increased by infection (p < 0.0001) but suppressed by LED (p = 0.0002). There was a marginal sexual difference (p = 0.065) with females exhibiting a 35% lower response while on alcohol. Examining lymphocyte subsets, the most significant changes were observed after infection (BCG) and alcohol treatment (LED). CD4 levels were diminished by LED (p = 0.0002) but markedly increased by infection (p < 0.0001), producing a highly significant interaction that affected both absolute number (p < 0.0001) and relative percent present (p = 0.0078). CD8 was influenced only by infection (p < 0.0001). This resulted in a infection-related increase in the CD4/CD8 ratio which was lower with LED (p = 0.0032). Splenic T-lymphocytes, predominately CD4, are involved in the host response to BCG hepatitis and are adversely influenced by LED, which may contribute to increased susceptibility.

Significance of DNA ploidy and cell proliferation in juvenile respiratory papillomatosis.

The clinical course of recurrent respiratory papillomatosis (RRP) in children is variable and unpredictable. At present there is no way to identify patients at risk for aggressive disease. The objective of this study was to evaluate whether DNA ploidy and cell proliferation analyses can predict the clinical course in children with RRP. Two different methods of estimating proliferation activity were compared. Nonembedded papilloma biopsy specimens from 18 pediatric patients were analyzed by flow cytometry providing DNA content with cell cycle analysis. The expression of the proliferative marker Ki-67 in papilloma tissue was quantified by immunohistochemistry. The patients were prospectively observed for 12 to 18 months. DNA content analysis and Ki-67 expression were compared to clinical information regarding number of disease sites, distal tracheobronchial spread, number of recurrences, need for tracheostomy, and disease remission. High S-phase fraction, proliferative index, and Ki-67 expression correlated with an aggressive clinical course. DNA ploidy analysis and immunodetection of proliferative markers may assist in predicting prognosis in children with RRP.