Charles F. Lange

Experimental autoimmune pinealitis in the rat : ultrastructure and quantitative immunocytochemical characterization of mononuclear infiltrate and MHC class II expression

Lewis rats immunized with Peptide M (an oligopeptide epitope of the S-antigen protein) developed experimental autoimmune uveoretinitis (EAU) and experimental autoimmune pinealitis (EAP). Temporal changes in mononuclear infiltrate to the pineal gland were quantitated by computer image analysis of sections immunostained with monoclonal antibodies to specific mononuclear populations. T helper/inducer cells (W3/25+) and monocyte/macrophages (OX-42+) were elevated during the early phases of inflammation (day 15) while cytotoxic/suppressor T cells (OX-8+) were elevated at days 15 and 21. Expression of MHC class II (OX-6) was markedly enhanced on pineal glia, but was not present on vascular endothelia during EAP. Ultrastructurally, many capillaries exhibited thickenings of the endothelia and basal lamina. EAP had little effect on the fine structure of pinealocytes and glia and there was little evidence of cellular destruction by day 21, in contrast to the extensive retinal destruction resulting from EAU. These findings suggest fundamental differences between EAU and EAP related to mechanisms of antigen processing/recognition in autoimmune diseases. Our study further indicates the importance of EAP as a model to investigate neuroendocrine-immune interactions.

Acetylcholinesterase in cultured human leukemia/lymphoma cell lines.

Fifty-two cultured leukemia/lymphoma cell lines were studied for their acetylcholinesterase activity. There was a striking effect of maturity on enzyme activity, only the most mature cells showing significant activity. Mature T cells exhibited far more enzyme activity than mature B cells, paralleling results on normal T and B cells.

Age related carbohydrate content of mouse kidney glomerular basement membrane and its reactivity to antistreptococcal membrane antisera

Abstract Rabbit antisera to group A, type 12 streptococcal cell membrane (SCM) and human glomerular or glomerular basement membrane (GBM) preparations were found to immunologically react with adult mouse GBM by an indirect fluorescent antibody test. Such cross-reactivity was enhanced by enzymatic cleavage of GBM carbohydrate. In comparative tests with neonatal and adult kidneys, the antisera were more reactive with the former than the latter. GBM carbohydrate cleavage had little or no effect on the reactivity of neonatal GBM with the antisera. Quantitative carbohydrate analyses revealed that adult mouse GBM contained a greater content of carbohydrate than did neonatal GBM. Thus the differential reactivities of adult and neonatal mouse GBM with anti-SCM antisera are directly related to their respective carbohydrate contents.

Prospective management of a child with neonatal citrullinemia

A patient with neonatal citrullinemia caused by severe deficiency of argininosuccinate synthetase was treated prospectively according to the currently accepted protocol. We gradually reduced the doses and then discontinued treatment with sodium benzoate and phenylacetate; blood glutamine levels were maintained in the normal range, but ammonia was mildly elevated. Growth and development progressed normally through 31 months of age. Some patients with citrullinemia can be successfully managed without daily sodium benzoate and phenylacetate therapy.

BEHCET SYNDROME: With Immunologic Evaluation

A case of Behcet syndrome with immunologic evaluation, including screening of a vulvar ulcer for IgG, IgM, IgA, and fibrinogen by direct fluorescent microscopy is presented. Attempts were made to demonstrate cellular and humoral immune responses to mucosal antigens by lymphoblast transformation in the presence of cadaver esophageal mucosal extracts and indirect immunofluorescence using autologous serum and mucosal tissue. Serial measurements of percentages of total T, active T, and B lymphocyte populations, and lymphocyte response to phytohemagglutinin (PHA) stimulation during the course of Behcet syndrome are also presented. Clinical evaluation, histology of a Behcet vulvar ulcer, and a 2-year followup with good response to chlorambucil are reviewed.

Isolation and partial characterization of antigens from basement membranes and streptococcal cell membrane (SCM) employing anti-SCM monoclonal antibody

Monoclonal antibodies (mAb) against streptococcal cell membrane (SCM) antigen were used to identify specific cross-reactive peptides prepared by trypsin digestion of purified glomerular basement membrane (GBM) and lung basement membrane (LBM). Anti-SCM mAb-coupled HPLC columns were used to affinity isolate soluble LBM, GBM, and SCM antigens which then were sized by HPLC. Alternatively, SCM, GBM, and LBM digests were subjected to an initial separation by HPLC into component polypeptides, followed by affinity purification and ELISA of these fractions using anti-SCM mAb. Comparison of the antigenic reactivities by ELISA of the sized polypeptides on a nanomolar basis permitted the estimation of their individual relative epitope densities. The results for SCM antigens showed increasing epitope density with increasing molecular size, which suggests that intact SCM consists of repeating epitopes. Low mol. wt GBM polypeptides in nanogram amounts inhibited mAb binding to SCM, indicating that these small GBM polypeptides may similarly contain more than a single cross-reactive epitope. The identification of these cross-reactive epitopes in LBM and GBM has important implications for the etiology of post-streptococcal sequelae.

Establishment and characterization of a squamous cell carcinoma of the vulva in tissue culture and immunologic evaluation of the host

Abstract A continual epithelial cell culture has been established from a squamous cell carcinoma of the vulva metastatic to the right inguinal lymph node. Initial characterization of the cell line (designated LT2), including morphology, growth properties, karyotyping, and secretion of carcinoembryonic antigen (CEA) and the beta subunit of human chorionic gonadotropin (HCG-β) is reported. The LT2 cell line satisfies the following criteria of malignancy: (1) establishment in culture for more than one and one-half years exceeding more than 70 generations; (2) heteroploid karyotype; (3) abnormal cytology; (4) growth in soft agar suspension; and (5) tumorigenicity in nude mice. This cell line has also been found to be free from contamination by fibroblasts, mycoplasma, bacteria, and fungi. With the LT2 cell line, the specific tumor-host immunorelationship was assayed by autologous (host lymphocyte versus LT2 cells) cytotoxicity and lymphoblast transformation in the presence of tumor extracts or mitomycin C-treated LT2 cells. In vivo patient immunocompetence was evaluated specifically by skin testing with autologous tumor extracts and nonspecifically with bacterial recall skin test antigens and dinitrochlorobenzene. In vitro nonspecific immunocompetence was evaluated by total peripheral white blood cell and lymphocyte counts, T cell, active T cell and B cell determinations, lymphocyte response to the mitogen phytohemagglutinin, and spontaneous lymphocyte-mediated cytotoxicity versus cultured bronchogenic carcinoma cells. Correlation of immunocompetence and the status of the tumor-host relationship with the patients clinical course, and the potential significance of LT2 CEA and HCG-β production are discussed. The patients response to bacillus Calmette-Guerin immunotherapy is evaluated.

Epitope mapping of homologous and cross-reactive antigens by monoclonal antibodies to streptococcal cell membrane (mAb to SCM)

An approach to epitope mapping of a series of anti-streptococcal cell membrane (SCM) mAbs is described. Evaluations by enzyme-linked immunosorbent assay (ELISA) of one control mAb HB-35 and 13 different anti-SCM mAbs were made on homologous SCM antigen and human basement membrane antigens isolated from glomeruli (GBM) and lung (LBM). These anti-SCM mAbs were previously shown to be cross-reactive in a variety of systems with both GBM and LBM. The binding capacities were measured for all 14 mAbs on ELISA plates sensitized with SCM antigen or the cross-reactive GBM or LBM antigens, at 5 micrograms/ml or approximately 20 pM/well. From the 50% binding capacity dilution the pM of mAb bound/pM antigen-well was calculated which translated into an estimate of the ratio-number of epitopes bound. Observations with the homologous and cross-reactive antigens showed multiple reactive epitope ratios to eight mAbs whereas the other five yielded a ratio value of one or two on the tested antigens. Plates blocked with a specific dilution of one mAb evaluated the binding by a second mAb providing both binding and specificity data. One mAb (I-F-3) blocked all the other anti-SCM mAbs on all three antigen plates. An additive effect was noted by three mAbs, I-G-8, II-C-4 and II-D-8 with most of the other mAbs. The order of placement, however, made distinctive differences; II-F-4 showed an additive or enhancement effect on I-B-5 but no reciprocal effect was seen. A similar effect was made with I-G-8 and II-C-4 or I-F-7. Possible interpretations are that each mAb is binding different epitopes each fully exposed, and the order of placement of the mAbs makes no difference. Where an enhanced effect was observed it is suggested that the binding of the first mAb changed the conformation of the antigen, thereby opening and exposing additional epitope(s) to the second antibody. Or, in contrast, where the second mAb was blocked by the first, a fixing of the protein conformation is suggested thereby occluding the other epitope, as seen with I-F-3 and II-C-4. These epitope mapping procedures confirmed that all 13 anti-SCM mAbs were binding at different epitopes. The nature of basement membrane collagens and how this relates to post-streptococcal sequelae will be discussed.

A simplified batch method for the isolation of alpha1-glycoproteins from normal and pathological serums and urines

Abstract A method is presented for the rapid quantitative isolation of specific α1-glycoprotein from serum and urine. The immunological differences between the serum and urinary isolates are discussed.

Localization of [14C] Labeled Anti-Streptococcal Cell Membrane Monoclonal Antibodies (anti-SCM mAb) in Mice

Six different hybridomas secreting anti-SCM mAb and one control mAb were placed into adult mice along with [14C] amino acids for biosynthetically labeling. After sacrifice, the 14C mAb ascites along with serum, heart, kidney, lung and skeletal muscle were recovered. Tissue associated specific radio-activity (SpAc) and microscopic structural analyses were performed. Confirmation of mAb specificity was by both immunodot blots as well as Western blot analysis. Peritoneal injection of measured doses of anti-SCM mAb yielded tissue SpAc confirming their in vitro specificities. Two of the mAb showed strong reactivity to both renal (GBM) and lung basement membrane (LBM), two were mainly GBM reactive and two showed polyreactivity with a marked reactivity to a Z-line antigen. Autoradiographic light microscopy confirmed that the anti-SCM mAbs bound to both GBM and LBM and to Z-line antigen. Titrated doses of the mAb yielded autoradiographic confirmation in which the grain number on the GBM and LBM increased with increasing dose of mAb for all mAb except the control. This effect was not seen in the muscle tissues but anatomical localization at the Z-line was consistent. The major significance of these studies is the demonstration that circulating antibodies to SCM can react in vivo with normal mammalian antigens adding confirmation to the in vitro specificity of these cross-reactive anti-SCM mAbs.