Kurt Hirschhorn

LYMPHOCYTE INTERACTION: A POTENTIAL HISTOCOMPATIBILITY TEST IN VITRO.

Lymphocytes from two unrelated individuals, cultured together in the same tube, undergo morphological transformation to large cells and divide. Both of these parameters may be estimated quantitatively. There is a correlation between the degree of this response and the degree of cross-reactivity of grafts from the two individuals placed on a third unrelated recipient.

IMMUNE RESPONSE AND MITOSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES IN VITRO.

Phytohemagglutinin causes cultured lymphocytes to agglutinate, divide, and produce γ-globulin. Most cells are transformed into large lymphocytes, some resembling plasmocytes. Actinomycin D stops γ-globulin production after 2 hours. When specific antigens are added to lymphocytes from sensitized individuals, only some cells undergo morphological transformation, produce γ-globulin, and divide. When cells or cell extracts from an unrelated individual are added to a culture, a similar reaction occurs.

Deletion of short arms of chromosome 4–5 in a child with defects of midline fusion

We have described a case of a child with multiple congenital anomalies, including defects of midline fusion and abnormal dermatoglyphies. Chromosome analysis revealed deletion of part of the short arm of a chromosome in group 4--5. Although autoradiography studies were not done in this ease, it clinically resembles one in which the deleted chromosome belongs to the late replicating pair of group 4--5. This ease, therefore, not only differs from the cat cry syndrome in its clinical manifestations, but may be a result of a deletion of one of the other chromosome pair in the group.

ACID PHOSPHATASE-RICH GRANULES IN HUMAN LYMPHOCYTES INDUCED BY PHYTOHEMAGGLUTININ.

Human lymphocytes, cultured in the presence of phytohemagglutinin, undergo morphologic transformation and subsequent mitosis. Before mitosis (48 to 72 hours), a sharp increase in acid phosphatase activity occurs in cells stimulated with phytohemagglutinin. Histochemical examination of these cells demonstrates that innumerable granules containing acid phosphatase develop in the cytoplasm before mitosis. It is possible that enzymes present in granules which stain for acid phosphatase activity (lysosome-like) may play a role in phytohemagglutinin-stimulated cell division.

HISTOCOMPATIBILITY AND IMMUNOLOGIC COMPETENCE IN RENAL HOMOTRANSPLANTATION.

Cultures of peripheral lymphocytes may be used to assay histocompatibility. The lymphocytes from potential kidney recipients are cultured with those from potential donors and the percentage of large cells and mitoses stimulated is determined. The immunologic competence of cultured lymphocytes from recipients receiving immunosuppressive therapy is evaluated by determining their responsiveness to phytohemagglutinin. Suppression of response appears to be correlated with the absence of clinical signs of graft rejection.

Agammaglobulinemia: The Fundamental Defect

Addition of phytohemagglutinin and of streptolysin S to in vitro cultures of leukocytes of normal and agammaglobulinemic subjects resulted in mitosis of lymphocytes and their differentiation to plasma cells. In contrast, specific antigens induced mitosis and differentiation of lymphocytes of normal but not of agammaglobulinemic donors. The data suggest that the absence of plasma cells in agammaglobulinemia is not in itself responsible for failure of antibody production, but is rather the morphologic concomitant of the primary defect (failure of antibody production on exposure to antigenic stimulus).

Double Beta-Lipoprotein: A New Genetic Variant in Man

A β-lipoprotein variant, in which two bands appear after electrophoresis, has been found in three generations of a family. The variant, immunologically also a β-lipoprotein, differs in molecular size, density, and charge fromn normal β-lipoprotein. Individuals showing the variant appear to be heterozygotus for an ulncommon multant gene.

Incidence of Familial Hyperlipemia

Familial hyperlipemia is an inherited disease associated with early onset of coronary atherosclerosis. In a survey of a student population in Sweden, an estimated case incidence of 2 to 3 percent was discovered. This study also demonstrates that there is probably a heterogeneity of causes for the primary elevation of blood triglycerides in man.

THE RELATIONSHIP OF IN VITRO LYMPHOCYTE COMPATIBILITY TO HOMOGRAFT SENSITIVITY IN MAN

In another paper’ in this monograph, we have presented evidence that cultured human peripheral blood lymphocytes are immunologically capable and show immunological specificity. As outlined in that paper, their degree of response to specific and nonspecific stimuli can be quantitated by counting the number of cells which have enlarged in culture and those cells undergoing mitosis. Wilson2 has shown that if a n animal is immunized with a skin graft from an unrelated animal, thoracic duct lymphocytes from the recipient kill cultured renal cells derived from an animal of the donor’s strain. These lymphocytes will not attack renal cells from other strains known to have markedly different histocompatibility antigens, demonstrating the specificity of this cytotoxic effect. Many investigators have used lymphocytes to sensitize individuals, producing accelerated rejection of the skin graft from the donor of the lymphocytes to the immunized individual. This shows that the lymphocyte contains histocompatibility antigens and is capable of producing an immune reaction to these. Therefore, the lymphocyte appears to be representative of both the donor’s and the recipient’s contribution in transplantation. Brent and Medawar? have used lymphocytes injected subcutaneously to test for genetically determined histocompatibility differences between various animals. They have found no reaction to injected lymphocytes transferred within an inbred strain. This correlates with the finding by numerous investigators that transplants from one identical twin to the other are not rejected. These observations provide a basis for the assumption that differences in histocompatibility antigens are genetically determined. Bain et al.* have shown that if peripheral blood lymphocytes from two unrelated individuals are mixed in vitro, there is an increase in the incorporation of radioactive thymidine into DNA. Monozygous twins showed no such response, while among a series of dizygous twins, two pairs were found who also did not react with each other. We have done similar studies on lymphocyte mixtures. However, our method of quantitation depends on counting the percentage of cells which have become enlarged in culture, and adding to these the percentage of cells in mitosis, since these latter derive from enlarged cells. The details of the culture methods are described e l ~ e w h e r e . ~ A time curve of this response (FIGURE 1) demonstrates that the peak of observable reaction occurs between seven and eight days. The degree of this reaction varies from no increase over the spontaneous rate of enlargement and mitosis (approximately five per cent) in identical twins, to a response of 85 per cent observed in two individuals from widely divergent ethnic backgrounds. As a rule, blood relatives show less of a response. than unrelated

Lysosomenveränderungen bei kurzdauernder unspezifischer Stimulierung kleiner Blutlymphocyten in vitro

Kleine Blutlymphocyten werden in vitro durch Phytohamagglutinin (PHA) und andere „unspezifische“Wirkstoffe zu einer blastenartigen Transformation und erhohten Mitoseaktivitat angeregt [1]. Diese Transformation ist bei 50 bis 95% der Zellen zu beobachten, die hochste Mitoserate findet sich nach 2 bis 3 Tagen. Mit den morphologischen Veranderungen gehen erhebliche Stoffwechselalterationen einher, die z. T. bereits wenige Minuten nach Zusatz von PHA verifiziert werden konnen [2, 3, 4, 5]. Der Mechanismus der Lymphocytentransformation ist bisher unbekannt. Cyto- und biochemische Untersuchungen zeigten, das im Cytoplasma PHA-behandelter Lymphocyten wahrend der pramitotischen Phase vermehrt lysosomenartige Organellen und erhohte Aktivitaten lysosomaler Enzyme nachweisbar werden [6, 7, 8, 9]. Die vorliegenden Untersuchungen gingen von der Frage aus, ob auch wahrend der Fruhphase der Lymphocytenstimulierung Lysosomenveranderungen auftreten, die sich mit biochemischen Methoden erfassen lassen.