IgG subclasses and antibody response to pneumococcal capsular polysaccharides in children with severe sinopulmonary infections and asthma.

A prospective open study was carried out on 30 pediatric patients with most severe chest disease whose serum immunoglobulin levels were normal. The patients entered into the study had had two or more documented episodes of pneumonia, and/or six episodes of bronchitis with fever within a year, and/or severe asthma (steroid-dependent), and/or hospitalization for chest disease for more than 30 days within the year preceding the study. Eleven patients had sinopulmonary infections, 19 had asthma. Twenty patients had low levels of one or two IgG subclasses: 11 were deficient in IgG3, three in IgG4, three in IgG3 + IgG4, and three in IgG2 + IgG4. Patients with low IgG subclass levels were distributed throughout the different clinical entities. These children had significantly longer periods of hospitalization than the patients in whom all IgG subclasses were within the normal range. They suffered more often from sinopulmonary infections. Asthmatic children with low levels of an IgG subclass reported more days with wheezing and needed more steroids than the children without subclass deficiencies.

Analysis of CD3-antibody-mediated inhibition of T-cell activation.

In this study the influence of a non-mitogenic anti-CD3 antibody on accessory cell-dependent antigen and mitogen-induced T-lymphocyte proliferation has been investigated. The antibody was found to completely inhibit PHA, Con A, PWM, and tetanus toxoid stimulation, with no effect on the proliferation induced by the calcium ionophore A23187. VIT3 completely abrogated the production of IL-2 by lectin-stimulated T cells. It had no effect, however, on the IL-2-dependent proliferation of preactivated T-cell blasts. In addition, the antibody was able to elevate free cytoplasmic Ca2+ levels within minutes after the addition to T cells. Detailed time kinetic analyses revealed that the time interval critical for inhibition was significantly dependent on the interaction between T cells and accessory cells. Under standard conditions, in the presence of 10% non-T cells as accessory cells 50% inhibition was still achieved when VIT3 was added to PHA-stimulated T cells as late as 8 hr after the onset of culture. Delayed addition or a decrease in the number of added accessory cells significantly prolonged this time period. Lectin-stimulated T cells can thus obviously be inhibited via CD3 as long as they have not received all signals including those delivered by accessory cells. Although the underlying mechanisms are not clear so far, the observation that VIT3 at the same time triggers an early cytoplasmic Ca2+ response might indicate that it thereby actively interferes with antigen and lectin-initiated activation processes.

Cells reacting with the monoclonal anti-IL-2 receptor antibody AMT-13 in the regenerating thymus of irradiated mice.

The proportion and anatomical localization in murine thymus of T cell subpopulations, including those which are defined by the monoclonal anti-interleukin 2 (anti-IL-2) receptor antibody AMT-13, were studied by flow cytofluorometry and by immunohistochemical methods, both in irradiated and in normal mice. As a consequence of irradiation the proportion of AMT-13 positive cells and that of Lyt-1 positive cells were markedly enhanced, while the proportion of Lyt-2 positive cells was reduced. The vast majority of the AMT-13 positive cells both in normal and in irradiated thymi were located in the subcapsular area of the thymic cortex, whereas the irradiation resistant Lyt-1 positive cells were located in the medulla. These findings are compatible with the view that, similar to the developing thymus in the mouse embryo, in the regenerating adult thymus, AMT-13+ cells include the activated pro-thymocytes that repopulate the irradiated thymus.

Lymphocytes of haemophilia patients treated with clotting factor concentrates display activation-linked cell-surface antigens.

Peripheral blood lymphocytes from 30 patients with haemophilia A were investigated for the expression of six activation-linked cell surface antigens as well as with regard to the relative proportions and total numbers of Leu-3a and Leu-2a positive cells. Twenty-nine of the haemophilia patients showed no clinical symptoms of immunodeficiency or infection whereas one patient presented the typical symptomatology of the acquired immunodeficiency syndrome (AIDS). The proportions and total numbers of circulating lymphocytes displaying Ia antigens, the p45 protein and/or the two recently defined surface antigens VIP-4 and VIP-5 were significantly increased in haemophilia patients when compared to healthy individuals of the same age group. No such increases could be observed for transferrin receptor and IL-2 receptor expression. After the observation of depressed helper/suppressor T-cell ratios in many haemophiliacs, the expression of activation linked surface antigens represents a further lymphocyte abnormality which resembles the findings in AIDS and its prodromal stages and can also be found in certain viral and parasitic diseases.

Kinetics of activation antigen expression by in vitro-stimulated human T lymphocytes.

In this study a panel of monoclonal antibodies was used to investigate the kinetics of the appearance of activation-linked surface determinants as well as cytoplasmic and nuclear determinants in human T cells following lectin stimulation. Well known activation markers, such as Ia/DR, transferrin receptor, IL-2 receptor, T10, and gp24, were compared and investigated together with the T13 structure, recently found in this laboratory. T13, not demonstrable on resting T cells, could be seen within 24 hr after lectin stimulation. Kinetics of the appearance were similar to IL-2 receptor and transferrin receptor expression. Ia/DR synthesis was investigated separately for each polypeptide and the cytoplasmic invariant gamma-chain expression could be demonstrated for the first time with a gamma-chain-specific monoclonal antibody VIC-Y1. Moreover, gamma-chain synthesis seems to precede alpha- and beta-chain occurrence in human T cells. In addition, data from quantitative studies on antigenic densities are presented.

Immunological features of nonimmunogenic hyperthyroidism.

Blood lymphocyte subpopulations (Leu 4+ cells = pan-T cells, Leu 3a+ cells = helper/inducer cells, and Leu 2a+ cells = suppressor/cytotoxic cells), thyroid-stimulating immunoglobulins, microsomal antibodies and antibodies against thyroglobulin were determined in 10 patients with hyperthyroidism due to single autonomously functioning thyroid nodules (ATN), 11 patients with hyperthyroidism due to Graves' disease (GD) and in 20 normal subjects. Thyroidectomy was performed in 8 of the patients with ATN and in 6 of those with GD after 3 weeks of antithyroid drug treatment with methimazole. Lymphocytic infiltration of thyroid tissue, the amount of the various lymphocyte subsets (Leu 4+, Leu 3a+, and Leu 2a+ T cells as well as B+ B cells) in the thyroid gland, as well as the expression of the histocompatibility antigen HLA-DR on thyrocytes and intrathyroidal lymphocytes were examined. Blood Leu 4+ cells were reduced due to a lack of Leu 2a+ cells in patients with ATN and GD when compared to normal subjects. Thyroid-stimulating immunoglobulins were detected in all patients with ATN and GD, but in none of the normal subjects. Lymphocytic infiltration of thyroid tissue was present in patients with ATN and GD. The various lymphocyte subsets in the thyroid gland did not differ between the two patient groups. DR expression on thyrocytes was seen in 6 of the patients operated for ATN and in 5 of those who underwent surgery for GD. Infiltration with DR+-T lymphocytes was found in all thyroid glands investigated. Thus immunological findings usually classified as proof for the autoimmune origin of GD exist also in patients with ATN. An overlap in the pathogenetic background of both diseases seems possible.

Alterations of immunologic parameters in pregnancies complicated by EPH-gestosis.

To characterize the cellular and humoral immune situation in pregnancies with EPH-gestosis (n = 44), percentages of peripheral T-lymphocytes, ratio of helper and suppressor T-lymphocytes further circulating immuncomplexes (CIC), beta 2-Microglobulin (beta 2-MG) and lysozyme (Lz) were determined. 50 pregnant women without EPH-gestosis were examined as controls. The mean counts of total lymphocytes decreased with increasing gestosis index in contrast to T-lymphocyte percentages. The mean T-helper/T-suppressorcell ratio indicated a severe imbalance in a few cases with severe gestosis. CIC were present in all serum samples of patients with moderate and severe gestosis. Lz was found to be elevated in EPH-gestosis in correlation to severity of disease. beta 2-MG levels in serum were only increased in women with reduced renal function. This parameter was not strictly correlated to the severity of EPH-gestosis but only to the degree of kidney dysfunctioning. EPH-gestosis represents a heterogeneous group with uniform symptomatology. Only in severe gestosis an imbalance of T-lymphocyte subpopulations as well as CIC and elevated Lz levels were detected. In mild gestosis no alterations of the used parameters were found.

Exposure by desialylation of myeloid antigens on acute lymphoblastic leukemia cells.

The 3-fucosyl-N-acetyllactosamine structure, a sugar sequence contained in the human milk oligosaccharide lacto-N-fucopentaose III, is recognized by most of the granulocyte-specific monoclonal antibodies (MoAb) reported in the literature, including the six MoAb from our laboratory. Blast cells from patients with acute myeloblastic leukemia (AML) displayed a heterogeneous reaction pattern when they were exposed to MoAb against this moiety, and the proportion of reactive cells in individual cell samples was highly variable. The intensity of the reaction was strongly enhanced by neuraminidase treatment of AML blasts, and reactive structures were exposed on previously negative AML blast cells. Surprisingly, this granulocyte-associated antigen was exposed by desialylation not only on malignant myeloid precursor cells but also on common acute lymphoblastic leukemia cells. No such effect was seen when normal peripheral blood lymphocytes, lymphocytes from patients with chronic lymphatic leukemia, or blast cells from patients with B-cell acute lymphoblastic leukemia, acute erythroid leukemia, and acute megakaryoblastic leukemia were treated with neuraminidase.

M2, a novel myelomonocytic cell surface antigen and its distribution on leukemic cells.

The selectivity of a novel myelomonocytic cell surface antigen, designated M2, has been assessed in a series of 208 leukemias. The M2 antigen is defined by a monoclonal antibody (VIM-2) of the IgM class. Its expression within the normal hemopoietic system is restricted to myelomonocytic cells. Lymphocytes, erythrocytes, thrombocytes and their morphologically recognizable precursors are negative. Sixty of the 66 acute myeloblastic leukemias (= 91%) and 28 of the 30 myeloid blast crises of CML patients (= 93%) were M2-positive. As expected from our findings with normal myeloid cells, the myeloid cells found in stable phase of CML were also in all instances, M2-positive. Quite in contrast, lymphoid cells from patients with B-CLL, T-CLL, prolymphocytic leukemia, hairy-cell leukemia, lymphoblastic lymphoma, Sézary syndrome, from CML patients in lymphoid blast crisis and from the majority of patients with ALL, were completely M2-negative. Also negative were the blast cells of patients with acute megakaryoblastic leukemia and acute erythroleukemia. A direct comparison of M2 expression with the display of the 3-fucosyl-N-acetyllactosamine determinant, the structure recognized by most of the anti-myeloid monoclonal antibodies reported so far, shows that more AMLs are M2-positive and the proportion of M2-positive blast cells in individual AML samples is higher.

A human Ia cytoplasmic determinant located on multiple forms of invariant chain (gamma, gamma 2, gamma 3).

An antigenic determinant present in the cytoplasm, but not on the surface membrane of human Ia+ cells, is defined by a monoclonal antibody (VIC-Y1) and is shown by immunoprecipitation and by NEPHGE to be expressed by Ia oligomers. Immunoprecipitations of cellfree translates and of purified Ia subunits indicate that the VIC-Y1 determinant is located on the Ia gamma-(invariant) chain, as well as on two other related molecules, provisionally termed gamma 2 and gamma 3. Within our experimental conditions, the three forms of gamma-chains co-precipitate exclusively with Ia oligomers. As detected by VIC-Y1 and in the limits of our assays, gamma-chains could not be found at the cell surface; their tissue distribution, determined by cytoplasmic indirect immunofluorescence with VIC-Y1, closely resembles that of Ia antigens, with the possible exception of acute lymphatic leukemia cells (Ia+, gamma-chain-).

Monoclonal antibodies to human myelomonocyte differentiation antigens in the diagnosis of acute myeloid leukemia.

The immunological definition of malignant lymphatic cells has already been routinely applied by many laboratories over a number of years. Today it is clear and undisputed that the phenotypic data obtained in that way are often very useful and supporting. The immunological definition of non-lymphoid leukemias is not yet as far advanced. Additional surface markers for the recognition of poorly differentiated non-lymphoid cells are clearly needed. In this paper ten monoclonal antibodies to myeloid surface antigens are described. It is hoped that they are a useful addition to the presently available relatively small panel of well studied myeloid surface markers which are displayed by immature malignant blast cells.

Glycophorin A expression in malignant hematopoiesis.

Two hundred twenty-nine patients with hematopoietic malignancies were tested for reactivity with a monoclonal anti-human glycophorin A antibody. One hundred twenty-three of these cases were classified as acute leukemias of either the myeloid, lymphoid, erythroid, or undifferentiated type. The monoclonal antibody we used (VIE-G4) was obtained after immunization with a human thymocyte suspension. It selectively reacts with glycophorin A (GpA) and strongly binds to 40% of K-562 cells and all morphologically recognizable erythroid precursor cells. Apart from two cases with acute erythroid leukemia, this antibody reacted with none of the malignant cells in the 229 tested hematopoietic malignancies, including the 121 nonerythroid acute leukemias. This finding seems to contradict the earlier observations by L. Andersson and colleagues that a considerable proportion of acute leukemias express GpA on their surface. One reason for this discrepancy might be the fact that VIE-G4 detects only complete glycosylated GpA. If this is the sole explanation, this would mean that the poorly differentiated cells in these cases express incompletely glycosylated GpA.

T-cell alterations in hemophiliacs treated with commercial clotting factor concentrates.

Various immunological parameters were determined in 46 patients with severe hemophilia A and in 9 patients with severe hemophilia B. All patients were treated over many years with commercial factor VIII or IX concentrates. Patients with severe classic hemophilia had a significantly reduced relative and absolute number of T-helper cells and a significantly increased relative and absolute number of T-suppressor cells. About half of these patients had an inverse T-helper/suppressor cell ratio. Patients with moderate hemophilia A and severe hemophilia B did not show these abnormalities. Hemophiliacs with an inverse ratio had a significantly higher concentration of serum total protein, IgG and IgM. No relationship between the amount of factor VIII concentrate administered, the HLA-type of the patient, the presence or absence of CMV-antibodies, hepatitis markers, thrombocytopenia and abnormal liver function tests to the T-cell abnormalities could be established. Lymphadenopathy was frequently associated with an inverse ratio. Indirect evidence suggests that the alterations of the immune system began in 1979/80.

Surface antigens defined by monoclonal antibodies as tumor markers in human leukemia.

The detection of surface-linked antigenic determinants by heteroantisera has greatly contributed to a better understanding of the heterogeneity of benign and malignant hematopoietic cells. The difficulties encountered in rendering these heteroantisera specific for a unique cell surface component have been a major drawback to a more rapid development of immunologic cell typing. With the introduction of hybridoma technology, it became possible to obtain monoclonal antibodies and markedly improve immunologic cell typing. We have, therefore, used this new technology for the production of monoclonal antibodies against human leukocyte surface antigens. This paper describes four cell type-specific monoclonal antibodies, which turned out to be very useful reagents in leukemia diagnosis. One of these antibodies, VIM-D5, is directed against a myeloid cell surface antigen. VIL-A1 is specific for the common acute leukemia associated antigen. VIB-C5 recognizes B-cell differentiation antigen and VIE-G4 is specific for glycophorin A, and thus detects erythroid precursor cells.

[Diagnosis of leukemia with monoclonal antibodies]. / Leukämiediagnostik mit monoklonalen Antikörpern.

The diagnosis and classification of leukaemic diseases is still primarily based on morphological and cytochemical criteria. Interpretational difficulties occur quite frequently, especially in acute leukaemias. Subjective morphological cell-type characterization--which is acquired only after many years of experience--is rather unsatisfactory in the long run. Therefore in order to obtain uniform results in all haematological areas, new methods are needed. In principle, immunological cell-type characterization represents an alternative method. By applying requisite antibodies practically any cell component can be demonstrated and quantitated. Up to recently the major obstacle to a more rapid development of immunological cell typing was the lack of specific antisera, but with the introduction of hybridoma technology it became possible to obtain monoclonal antibodies and thus, markedly improve immunological cell typing virtually overnight. We have, therefore, used this new technology for the production of monoclonal antibodies to human leucocyte antigens. This paper describes six of the cell-type specific monoclonal antibodies obtained, their suitability for diagnostic purposes and the classification of human leukaemias. With the help of these antibodies the majority of human leukaemias can now be typed. Without claiming completeness and in full awareness of the fact that a number of problems remain to be solved, we nevertheless believe that the presented data point to the possibilities opened up by this technology for leukaemia diagnosis in the future.

VIL-A1, a monoclonal antibody reactive with common acute lymphatic leukemia cells.

The VIL-A1 monoclonal antibody raised against Reh cells reacts with common acute lymphatic leukemia (CALL) cells but not with normal or malignant B or T lymphocytes. It also shows no binding to normal or malignant myeloid, monocytic or erythroid cells, nor does it react with thrombocytes. The antibody is of IgM class and lyses CALL cells very efficiently in the presence of rabbit but not human complement. Immunoprecipitation experiments followed by SDS-polyacrylamide gel electrophoresis under reducing conditions revealed that VIL-A1 defines a 95,000 mol. wt membrane protein. Approximately 40% of it binds to lens culinaris lectin. Capping experiments showed that the membrane component defined by VIL-A1 co-caps with the one recognized by another recently described monoclonal antibody to CALL cells (J5).

Interaction of targeted liposomes with human peripheral mononuclear blood cells and lymphocytes of chronic lymphoid leukaemic patients.

Association of small unilamellar vesicles (SUV) can be effectively targeted to normal human peripheral mononuclear blood cells (PMBC). - Lymphocytes of chronic lymphoid leukaemic (CLL) patients show a low rate of liposome association, which cannot be improved by targeting with anti-lymphocyte globulin (ALG). Presence of serum evoked an enhanced association of liposomes to PMBC.