Ezio Merler

Identification and characterization of subpopulations of lymphocytes in human peripheral blood after fractionation on discontinuous gradients of albumin. The cellular defect in X-linked agammaglobulinemia.

Normal human peripheral blood lymphocytes were separated on discontinuous gradients of 17-35% bovine serum albumin (BSA) into nine fractions. Three subpopulations of lymphocytes were obtained. One occupies the top third of the gradient (fractions 1-3, 17-23% BSA) and is rich in cells characterized by a high spontaneous rate of DNA synthesis and by the ability to give rise to colony-forming units. The middle portion of the gradient (fractions 4 and 5, 23-27% BSA) is rich in thymus-derived (T) lymphocytes identified by their vigorous response to mitogens and by their ability to form rosettes with sheep erythrocytes (E). The third subpopulation at the bottom of the gradient (fractions 6-9, 27-35% BSA) is rich in bone marrow-derived (B) lymphocytes capable of staining with fluorescent antiimmunoglobulin antisera and of forming rosettes with EAC1423. The peripheral blood lymphocytes of five boys with proved X-linked agammaglobulinemia and two with probable X-linked agammaglobulinemia were found to be totally deficient in B lymphocytes (fractions 6-9) and lacked the subpopulation identified by immunofluorescent staining or rosette formation with EAC1423. One boy with proved X-linked agammaglobulinemia and two with probable X-linked agammaglobulinemia possessed a normal amount of circulating B lymphocytes.

Metabolism of gamma globulin fragments in normal and agammaglobulinemic persons.

Abstract Two types of enzymatically digested human gamma-globulin preparations, a pepsin digest and a plasmin digest, were administered to six normal persons and to six patients with agammaglobulinemia. No untoward reactions were observed. The fate of the injected material was observed by serial measurement of gamma globulin and its fragments in plasma and urine. The pepsin digest, consisting principally of an F(ab′)2S 5 fragment, had a half-time of disappearance of 10 to 14 hours. The plasmin digest, consisting mainly of an S 6.5 fragment, had a much longer half-time of disappearance: 18 to 20 days. On theoretical grounds, therefore, plasmin-digested gamma globulin appears suitable for the establishment and maintenance of passive immunity to prevent infection in agammaglobulinemic patients. It deserves further study to assess its effectiveness and safety.

Allergy to Ragweed Antigen E: Effect of specific immunotherapy on the reactivity of human T lymphocytes in vitro☆

Abstract Purified populations of human T lymphocytes were used for in vitro studies of the immune response to Ragweed Antigen E (AgE). T lymphocyte proliferation to AgE, assessed by 3 H-thymidine incorporation, was detected in AgE-sensitive but not in AgE nonsensitive individuals. The T cell response to AgE was biphasic, with maxima at AgE concentrations of 20–50 μg/ml and 0.01–0.10 μg/ml. Specific immunotherapy markedly decreased the T cell response at high AgE concentrations and virtually abolished the response at low AgE concentrations.

Human lymphocyte mitogenic factor: Synthesis by sensitized thymus-derived lymphocytes, dependence of expression on the presence of antigen☆

Abstract Lymphocyte mitogenic factor (LMF) was obtained from human lymphocytes stimulated with tetanus toxoid. LMF proved to be a soluble lymphokine produced by a sensitized T lymphocyte in response to stimulation with antigen. LMF induces proliferation in cells which normally do not proliferate in response to antigen (thymocytes, B lymphocytes). This function is expressed only in the presence of antigen. LMF produced in response to stimulation with a specific antigen is able to cooperate with more than one antigen in recruiting cells into division. LMF is a nondialyzable protein different from other lymphokines as judged by the kinetics of its release. It elutes in the postalbumin fraction of Sephadex G200.

The response in vitro of human lymphocytes to phytohemagglutinin and to antigens after fractionation on discontinuous density gradients of albumin

Abstract Quantitative in vitro assays of antigen recognition, as measured by specific uptake of labeled antigen, and cellular proliferation, as measured by incorporation of 3 H-thymidine into a 10% TCA-precipitable fraction, taken to be protein and DNA, were used to study the immunologic function of lymphocytes derived from human tissues. In addition, some of these lymphoid cells were separated on discontinuous gradients of BSA and the morphology and functions of each fraction studied. The principal findings were as follows: 1. 1. Lymphocytes from human tonsils, thymus, spleen, and bone marrow, derived from children previously immunized with tetanus toxoid, took up 125 I-labeled tetanus toxoid. This specific uptake was quantitatively greater and qualitatively different from the uptake of control proteins, and could be inhibited by unlabeled antigen, specific antibody, and low temperature, but not by inhibitors of cellular metabolism. 2. 2. Lymphocytes from human tonsils and thymus proliferated in response to PHA, tetanus toxoid, and allogeneic cells. 3. 3. Suspensions of tonsillar and thymic lymphocytes centrifuged in discontinuous gradients of BSA separated into at least three populations. The first was enriched with large and medium-sized lymphoblasts and underwent spontaneous mitotic activity. The second contained more than 95% small lymphocytes and consistently proliferated in response to PHA, tetanus toxoid, and, when tested, allogeneic cells. The third contained somewhat more dense, small lymphocytes and responded poorly to mitogenic stimuli. In the case of tonsillar tissue, this third population contained cells that took up most antigens. In the case of thymic tissue, these cells constituted 50–75% of the total cell number and responded weakly or not at all in the tests employed. It is concluded that within human lymphoid organs there exist functionally heterogeneous populations of cells. Moreover, this phenomenon may be studied effectively by separating cells in discontinuous gradients of BSA.

Production of antibody by the central nervous system in subacute sclerosing panencephalitis

SUBACUTE Sclerosing Panencephalitis ( SSPE) is a relatively stereotyped disease of children and young adults. The incidence is low, only 12 cases having been seen at The Children’s Hospital Medical Center in the past decade. Antemortem diagnosis is facilitated by the finding of an elevated cerebrospinal fluid (CSF) gamma globulin concentration, with an associated abnormality in the colloidal gold reaction. The CSF globulin may, at times, exceed 50 per cent of the total protein concentration. A disproportionate elevation of CSF gamma globulin may result from a number of factors. First, there may be increased transfer of gamma globulin from serum to CSF, as a result of a selective increase in permeability, or specific transport across membranes separating blood and CSF. Secondly, fragmentation or enzymatic cleavage of gamma globulin may occur within the CSF compartment, leading to an apparent elevation of gamma globulin concentration when measured by conventional techniques. Thirdly, there may be local production of gamma globulin by cells within the central nervous system, meninges or cerebrospinal fluid. Such a mechanism has been postulated in patients with multiple sclerosis and neurosyphilis,’ and there is experimental evidence to support this po~ tu la t e .~ ,~ As a means of investigating these various possibilities, immunoglobulin-G ( IgG) exchange between serum and CSF was studied in two patients with SSPE with elevated CSF gamma globulin concentration, and in four control subjects with normal CSF gamma globulin concentration. These results have been presented in part elsewhere.4 11 Immunoglobulin-G, prepared from normal human serum, was iodinated with 1251 and injected intravenously. Serial samples of serum and lumbar fluid were withdrawn over the next 10 to 20 days and assayed for protein bound 1251 activity. IgG concentration was determined by an immunoprecipitin reaction employing rabbit anti-human IgG as the precipitating antiserum. Results were expressed as specific activity (cpm/mg IgG) relative to serum specific activity 15 minutes after injection. CSF IgG specific activity curves, plotted against time, were analyzed by the method of successive exponential subtraction. To measure the half-life of IgG, the daily excretion of radioactivity in the urine was measured and subtracted from total injected radioactivity to yield the total body retained radioactivity. In the control subjects, serum and CSF specific activity were approximately equal at a time when CSF specific activity was maximal (Fig. 1). This relationship indicated that in the controls, serum IgG was the precursor of CSF IgG.6 In the patients with SSPE, the maximum specific activity in CSF was only a fraction of that of serum. In the case illustrated (Fig.

The effect of Fc fragments of IgG on human mononuclear cell responses

Abstract Fc fragments of IgG increased the endogenous production of prostaglandin E by mononuclear cells in culture and monocyte monolayers. The proliferative response of lymphocytes to tetanus toxoid in the presence of Fc fragments or in supernatants derived from monocytes treated with Fc fragments was suppressed. Coculture with Fc fragments and indomethacin inhibited endogenous prostaglandin E production and increased the proliferative response to tetanus toxoid. Fc fragments also induced an increase in the production of IgM by peripheral blood cells and tonsil B cells. Antigen was not required for this increased immunologlobulin synthesis. Monomeric IgG, Fab fragments, and albumin had no effects on the immune response. Aggregated myeloma IgG, but not the monomeric IgG, produced effects similar to that of Fc fragments.

Failure of heavy chain glycosylation of IgG in some patients with common, variable agammaglobulinemia.

Four patients with common, variable agammaglobulinemia were preveiously reported to have normal numbers of circulating B lymphocytes which synthesized normal amounts of IgG in tissue culture but failed to secrete the newly synthesized IgG. The B lymphocytes of these patients fail to incorporate [3H]mannose and/or [3H]glucosamine into newly synthesized IgG, whereas such incorporation appears to occur just before IgG secretion in cultures of normal B lymphocytes.

Synthesis of an M Component by Circulating B Lymphocytes in Severe Combined Immunodeficiency

SEVERE combined immunodeficiency is a hereditary disease characterized by considerable impairment of both cellular and humoral immunity and is assumed to result from a defect in lymphoid stem-cell ...

Cooperation between human thymus-derived and bone marrow-derived lymphocytes in the antibody response to ragweed antigen E in vitro.

Human T lymphocytes from patients with ragweed hay fever, when exposed to ragweed antigen E (AgE) in vitro, produced an activity that, in the presence of antigen, induced B cells from AgE-sensitive donors to synthesize and secrete IgE and IgG antibodies to AgE. Anti-AgE specificity was assessed both in vitro and in vivo. B lymphocytes from ragweed-sensitive individuals exposed in vitro to AgE alone failed to transform or to secrete antibody to AgE. The T cells activity had no effect on B cells of individuals not sensitive to AgE. The results of this study suggest that the human reaginic antibody response requires T and B cell cooperation. The experimental approach used may be a useful model for the investigation of the antibody responses of allergic individuals.