Human monoclonal islet specific autoantibodies share features of islet cell and 64 kDa antibodies.

The first human monoclonal islet cell antibodies of the IgG class (MICA 1-6) obtained from an individual with Type 1 (insulin-dependent) diabetes mellitus were cytoplasmic islet cell antibodies selected by the indirect immunofluorescence test on pancreas sections. Surprisingly, they all recognized the 64 kDa autoantigen glutamate decarboxylase. In this study we investigated which typical features of cytoplasmic islet cell antibodies are represented by these monoclonals. We show by double immunofluorescence testing that MICA 1-6 stain pancreatic beta cells which is in agreement with the beta-cell specific expression of glutamate decarboxylase. In contrast an islet-reactive IgM monoclonal antibody obtained from a pre-diabetic individual stained all islet cells but lacked the tissue specificity of MICA 1-6 and must therefore be considered as a polyreactive IgM-antibody. We further demonstrate that MICA 1-6 revealed typical features of epitope sensitivity to biochemical treatment of the target tissue which has been demonstrated for islet cell antibodies, and which has been used to argue for a lipid rather than a protein nature of target antigens. Our results provide direct evidence that the epitopes recognized by the MICA are destroyed by methanol/chloroform treatment but reveal a high stability to Pronase digestion compared to proinsulin epitopes. Conformational protein epitopes in glutamate decarboxylase therefore show a sensitivity to biochemical treatment of sections such as ganglioside epitopes. MICA 1-6 share typical features of islet cell and 64 kDa antibodies and reveal that glutamate decarboxylase-reactive islet cell antibodies represent a subgroup of islet cell antibodies present in islet cell antibody-positive sera.

Human monoclonal islet cell antibodies from a patient with insulin-dependent diabetes mellitus reveal glutamate decarboxylase as the target antigen.

The autoimmune phenomena associated with destruction of the beta cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

Immortalization of human lymphocytes by fusion with cytoplasts of transformed mouse L cells.

Fusion of mouse L929 cytoplasts with human peripheral blood lymphocytes induced lymphocyte proliferation that gave rise to lymphoid cell lines of B and T cell origin with unlimited growth potential. The immortalized cell lines were routinely grown in standard medium supplemented with fetal calf serum. Furthermore these cell lines could be propagated in chemically defined serum-free media. Each establishment of lymphoid cell lines was preceded by a proliferation phase 2 wk after cytoplast/cell fusion, which appears to be a necessary step in the immortalization process. The immortalized cells have a nearly normal human karyotype, do not form colonies in soft agar medium, and are not tumorigenic in nude mice. Cloned B cell lines produced human immunoglobulins of heavy and light chain types. No cross-reaction with DNA of herpes simplex virus, human cytomegalovirus, human T cell leukemia/lymphoma virus I and II, or polyoma virus was detected in the genome of immortalized cell lines by Southern blot hybridization. Furthermore B and T cell lines were established that appear to be free of Epstein-Barr virus genome.

Deviated lysis: lysis of unsensitized cells by complement. V. Generation of the activity of low pH or low ionic strength.

In serum exposed to acid pH (6.4), a serum activity was generated which lyzed unsensitized erythrocytes in the presence of EDTA. It was similar to the d.l. activity found following serum activation by inulin (2). In contrast to the d.l. generation by the classical or by the alternative pathway of C activation, the generation of d.l. by acid pH did not require C4 plus C2 or C3 plus factor B resp. It was, thus, not dependent on any hitherto known pathway of C activation. A similar activity appeared when NHS was centrifuged in a sucrose gradient at low ionic strength. Physicochemical alterations of the component proteins which influence their affinity for each other are seen as the basis for the activation of the attack phase of C.

Contribution of immunoglobulins M and G, complement, and properdin to the intracellular killing of Escherichia coli by polymorphonuclear leukocytes.

The effect of immunoglobulins and complement (C) on phagocytosis and intracellular killing of Escherichia coli was studied in vitro. The incubation system consisted of monolayers of human polymorphonuclear leukocytes and C-resistant, [3H]thymidine-labeled E. coli C source was human serum deprived of immunoglobulins and properdin by immunoabsorption. In the absence of C, only immunoglobulin G-coated bacteria were phagocytosed, whereas immunoglobulin M lacked opsonic activity. In the presence of C, phagocytosis was enhanced; however, immunoglobulin M was now more efficient than immunoglobulin G. Intracellular killing was notably augmented when C was activated by immunoglobulin G- or immunoglobulin M-coated bacteria; in contrast, the alternative activation of C by properdin had no effect on phagocytosis or intracellular killing. These results demonstrate the importance of immunoglobulins together with C not only for phagocytosis but also for efficient intracellular killing.