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Biomedical subjects

M Hirn

Publications and source records attributed to M Hirn.

At least 55 records · Page 3Linked to original sources

Management of perineal necrotizing fasciitis (Fournier's gangrene).

Eleven cases of perineal necrotizing fasciitis were treated in the Department of Surgery, University of Turku, Finland. In 10 cases the diagnosis was made on the basis of the fulminating progression of the infection to scrotal gangrene, identification of multiple underlying pathogenic organisms and toxaemia. In one female patient a corresponding infection developed in the labia majora. Each patient suffered nonspecific symptoms before the gangrene became evident. The management included surgical debridement of the necrotic tissue with incisions and drainage of the involved areas, antibiotic therapy and hyperbaric oxygen (HBO) treatment. The infection originated from the anorectal area in 5 patients, 1 patient had sustained scrotal trauma and in 5 cases the underlying condition was unknown. Colostomy was performed in 6 patients. One patient died 2 days after the admission. All the infections proved to be multimicrobial. Hyperbaric oxygenation was employed as a therapeutic adjunct in the present series, but it should neither replace nor delay surgical intervention. The key points in the management include early diagnosis with prompt surgical debridement and antibiotic therapy. After healthy granulation has appeared, the healing time can be shortened with reconstructive surgical procedures.

Adult↗

Anti LFA1 monoclonal antibody for the prevention of graft rejection after T cell-depleted HLA-matched bone marrow transplantation for leukemia in adults.

A mouse IgG monoclonal antibody (MoAb) directed against the human LFA1 molecule (25.3 MoAb) was used in nine adult leukemic patients to prevent graft rejection after T cell-depleted HLA matched bone marrow transplantation. Based on the results of a previous study in children 0.1 mg/kg of 25.3 was given on days -3, -1, +1, +3, +5 in addition to a standard conditioning regimen with cyclophosphamide (120 mg/kg) and fractionated total body irradiation. The marrow transplant was T cell-depleted using T101 Fab immunotoxin ricin A chain. Seven patients received post-graft immunosuppression with methotrexate and cyclosporine A; two patients received no immunosuppression post-graft. A mean T cell depletion of 98.3% (80-100%) was achieved. Tolerance to the infusions of 25.3 MoAb was excellent. No patient developed any form of graft-versus-host disease. However two patients failed to engraft and three patients had delayed graft failures. These results show that this regimen of anti LFA1 MoAb, which was extremely good at permitting engraftment of HLA mismatched T cell-depleted transplant in children with constitutional diseases, is not able to prevent graft failure and rejection of T cell-depleted HLA matched transplants in adults with leukemia. Further efforts are needed to overcome graft failures in this clinical situation.

Adult↗

Allogeneic manipulation of the GAT idiotypic cascade. Immunization of C57BL/6 mice by BALB/c anti-idiotypes stimulates similar strain-specific V genes as the original antigen.

Antibodies specific for the immunizing Ag (Ab1) (Id+ Ag+) and Ab3 (Id+ Ag+ or Id+ Ag-) of the (Glu60 Tyr10 Ala30) (GAT) idiotypic cascade express similar pGAT public determinants in BALB/c and C57BL/6 strains. These determinants have been shown to be dependent upon both VH and Vkappa encoded segments. The VH of the BALB/c Ab1 (germ-line gene H10) and that of the C57BL/6 Ab1 (germ-line gene V186-2) are only 75% homologous, whereas VK are much more conserved. C57BL/6 mice were immunized with BALB/c Ab2 (anti-idiotypic) antibodies and monoclonal Ab3 were derived after fusion of immunized spleen cells with the nonsecreting hybridoma cell line Sp/2.0-Ag. From 13 cell lines, five clones (four Id+ Ag- and one Id+ Ag+) were isolated and the mRNA V regions sequenced. Immunization with BALB/c anti-idiotypes elicits expression of the same or closely related C57BL/6 VH and Vkappa genes as when C57BL/6 mice were immunized with GAT, although functional VH BALB/c equivalents have been isolated in the B6 strain. Our results suggest that manipulation of the repertoire via antigenic or idiotypic stimulation both lead to the expression of different genes in different strains. They further confirm that the immune system is largely degenerate, for both idiotype expression and Ag recognition.

Amino Acid Sequence↗

Quantitative analysis of rat brain neurons developing in primary cultures. II. Changes in the distribution of N-CAM associated to neuronal cell surfaces.

Cultured rat fetal brain cells underwent morphological differentiation, as quantitatively described in the companion paper. In the same system, biochemical and immunolabeling studies were performed to analyze the developmental changes in neural cell adhesion molecule (N-CAM) distribution and quantity at the cell surface of neurons. The cell surface-associated N-CAM, related to the culture protein content, remained stable during the two-week period under study, as demonstrated by 125I-protein A binding assays. Immunogold labeling experiments, both in transmission and scanning electron microscopy, indicated a dramatic decrease in N-CAM site density in each membrane compartment, perikarya and neurites. This temporal variation of N-CAM distribution was not accompanied by differences in N-CAM site density between these two membrane compartments. On the other hand, individual perikarya, observed in scanning electron microscopy, showed various levels of labeling. In addition, immunoblot experiments demonstrated the absence of chemical modulation of N-CAM during the period under study, since the high molecular weight (embryonic) form remained dominant. Moreover, an increase in the total N-CAM amount was detected, contrasting with the stable quantity of cell surface-associated N-CAM. This suggested the existence of an N-CAM intracellular pool in cultured neurons. Finally, since the neurite membrane surface area increased 9-fold (companion paper) and since only a 5-fold decrease in N-CAM site density was observed in this compartment, N-CAM supply to neurite membranes was postulated.

Animals↗

Prophylactic use of a monoclonal antibody (33B3.1) directed against interleukin 2 receptor following human renal transplantation.

33B3.1, a rat IgG2a monoclonal antibody (MAb) directed against interleukin 2 receptor, has been given in association with prednisone and azathioprine to prevent rejection in 30 recipients of a primary cadaveric kidney transplant. The MAb 33B3.1 had been administered intravenously (IV) at 10 mg per day for 2 weeks. Clinical and biological tolerance were excellent. Only one patient had a rejection episode (reversible) during the MAb treatment period. These results were significantly better than those recorded from a historical group of patients that received only prednisone and azathioprine, and were similar to those from patients who received antithymocyte globulin instead of 33B3.1. There was no life threatening infectious episode and all patients are alive. Ninety-seven percent of the patients have a functional graft (follow-up, 30 to 210 days). Enzyme-linked immunosorbent assay (ELISA) of 33B3.1 indicate that circulating trough blood level peaked at day 6 (30 nm). The majority of patients (85%) developed either IgG or IgM antibodies with a maximal response at day 24 for both isotypes. We conclude that this new treatment that targets a functional receptor present on activated lymphocytes only, is well tolerated and highly efficient in preventing early rejection after human renal transplantation.

Animals↗

HT-29 cells are an in vitro model for the generation of cell polarity in epithelia during embryonic differentiation.

A monoclonal antibody that recognizes a membrane glycoprotein specific for the apical membrane of human colonic epithelial cells has been used to follow the differentiation and polarization of a cell line, HT-29, derived from a human colon adenocarcinoma. When these cells formed a polarized epithelium, the antigen was concentrated at the apical plasma membrane. It was also found intracellularly in vesicles and vacuoles. When HT-29 cells were undifferentiated and unpolarized, the antigen was not expressed significantly at the plasma membrane but was found concentrated in the membranes of intracellular vacuoles. Cells not yet organized into an epithelium may thus synthesize a membrane protein specific for their future apical membranes and store it intracellularly until the polarization process takes place. Intermediary stages of differentiation were occasionally recognized. They are characterized by a small number of cells surrounding an intercellular lumen. These lumina displayed apical membrane features (the presence of the apical antigen, of some microvilli, and of junctional complexes), although the cells were not fully differentiated. The differentiation process in HT-29 cells is apparently similar to that observed during embryonic development of the intestine. Therefore, HT-29 cells represent a useful model system to study epithelial differentiation in vitro.

Adenocarcinoma↗

Hyperbaric oxygen in the treatment of clostridial gas gangrene.

Thirty-two cases with clostridial gas gangrene were treated during the years 1971-87 in the Department of Surgery, Turku University Central Hospital. The presumptive diagnosis was made on the basis of the clinical appearance of the patient and presence of gram-positive bacilli on a smear. Each patient underwent surgical debridement, antibiotic therapy and hyperbaric oxygen treatment. Seventeen cases had diffuse spreading myonecrosis, 11 of whom survived. Fifteen patients developed clostridial cellulitis with toxicity, 12 survived. Thus the over-all mortality was 28.1%. All those patients who died had been transferred from other hospitals of the country and were already moribund on arrival. Twenty-two infections developed postoperatively, in 6 cases trauma was the antedecent cause and 4 were spontaneous infections. None of the patients with a posttraumatic infection died. The most common underlying disorders included arteriosclerosis, diabetes mellitus, malignancy and Buerger's disease. The addition of hyperbaric oxygenation to the treatment of gas gangrene--although strictly adjunctive to surgery, antibiotics and supportive therapy--has dramatically changed the surgical approach to treatment. Early diagnosis remains essential. Patient survival can be achieved if the disease is recognized early and appropriate therapy applied promptly.

Adolescent↗

Prevention of rejection of kidney transplants by monoclonal antibody directed against interleukin 2.

The effect of 33B3.1, a rat IgG2a monoclonal antibody (MAb) directed against interleukin 2 receptors on activated T lymphocytes, was studied during the first two weeks after transplantation in an attempt to prevent rejection in primary, cadaveric, kidney transplant recipients. 9 patients received 5 mg 33B3.1 daily by intravenous infusion for 14 days (group A) and 18 received 10 mg daily (group B). Both groups also received prednisone and azathioprine. In threatened rejection, rescue treatment consisted of anti-thymocyte globulin (ATG). 33B3.1 was well tolerated, with mild fever during the first 2 days being the most common side-effect. 3 patients in group A had a reversible acute rejection episode before day 14, whereas only 1 patient in group B had reversible rejection. 20 of 30 historical control patients (prednisone/azathioprine) and 2 of 55 patients previously treated with ATG had a rejection episode in a similar period after transplantation. Trough levels of 33B3.1 in the blood ranged from undetectable to 1.6 micrograms/ml in group A and from 0.3 to 9 micrograms/ml in group B. Most patients had antibodies against 33B3.1 by the end of treatment.

Acute Disease↗

Production and characterization of four monoclonal antibodies against porcine pancreatic colipase.

Four monoclonal antibodies directed against porcine colipase have been generated by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by binding to immobilized colipase in a solid-phase assay. Monoclonal antibodies were purified by affinity chromatography on colipase coupled to Sepharose. All monoclonal antibodies are of the IgG1 class with high affinity for the antigen. The dissociation constant of the complex formed in solution between porcine colipase and antibody varied from 1.1 X 10(-10) M to 1.8 X 10(-8) M. Epitope specificity was studied for each antibody and in pairs with an enzyme-linked immunosorbent assay (ELISA). Results indicate that the four monoclonal antibodies react with at least three different antigenic regions of colipase. Finally, three monoclonal antibodies were found to be potent inhibitors of colipase activity. Antiporcine monoclonal antibodies appear to be suitable probes for studying the lipid affinity site of the protein cofactor of pancreatic lipase.

Animals↗

A method for the production of highly specific polyclonal antibodies.

Two polyclonal antibodies directed against paramyosin and tropomyosin from Owenia fusiformis (a marine polychete annelid) were obtained using a new method of immunization. After purification by two-dimensional gel electrophoresis, proteins were transferred onto a nitrocellulose sheet using the Western blot technique. The proteins bound to their cellulose support were injected into rabbits without Freund's adjuvant and without solubilization of nitrocellulose with dimethyl sulfoxide. Highly specific polyclonal antibodies were generated.

Animals↗

Apical membrane marker is expressed early in colonic epithelial cells.

We have identified and characterized a membrane glycoprotein located at the apical plasma membrane of adult human colon epithelial cells, by the use of the monoclonal antibody technique in combination with immunocytochemical and biochemical methods. Analysis of membranes extracted with Triton X-114 and treated with specific hydrolases indicated that the antigen was an integral membrane glycoprotein. In the colon, the antigen was expressed in differentiated cells and along the entire crypt. It was also expressed at the apical membrane of the crypt cells of the distal ileum. It was not found in the proximal ileum, jejunum, or duodenum. In contrast, the antigen was found in all segments of the intestine of a 24-week-old embryo. Furthermore, the antigen had different apparent molecular weights in the adult ileum (200 kDa), adult colon (200 kDa and 301 kDa), and embryo (170 kDa). Therefore, this antigen should prove to be a useful marker to study the appearance of epithelial cell polarity during embryogenesis.

Antibodies, Monoclonal↗

Immunological evidence for the involvement of cell wall proteins in phosphate uptake in the yeast Saccharomyces cerevisiae.

Immunological cross-reactivity between cell wall proteins obtained from two yeast genera (Candida tropicalis and Saccharomyces cerevisiae) is reported. Specific retention of two cell wall proteins from Saccharomyces cerevisiae by an immunoabsorbent column coupled with antibodies against phosphate binding protein 2(PiBP2) from Candida tropicalis allowed to generate antibodies against the proteins from S. cerevisiae. These antibodies were effective in inhibiting phosphate uptake by S. cerevisiae cells. The proteins from S. cerevisiae displayed a phosphate binding activity which was inhibited in the presence of the forementioned antibodies. These results and the observation that the amount of these proteins in the shock fluid was dependent of the growth conditions (i.e., in the presence or in the absence of phosphate) support the idea that these proteins are involved in the high affinity phosphate transport system.

Antibodies, Fungal↗

Differential expression of mouse neural cell-adhesion molecule (N-CAM) mRNA species during brain development and in neural cell lines.

The cell-adhesion molecules N-CAM (neural cell-adhesion molecule) are ligands in the formation of cell-cell bonds and have been shown to play important roles during neuro-ontogenesis. They exist in several molecular forms which differ at the protein and carbohydrate levels. The regulation of the expression of these different forms is an important issue that bears on such questions as to how adhesive interactions between cells are modulated during morphogenesis. In the present study we have used N-CAM cDNA clones to investigate the expression of the cognate mRNAs in the mouse and rat brain and in 2 neural cell lines. The results were compared with the levels of the different N-CAM proteins. We made the following observations. A complex set of 5 size classes of mRNAs--which show developmental, regional, and cell-type-dependent variations in their expression--hybridize to 1 of our cDNA probes. While embryonic brain contains N-CAM gene transcripts 7.4, 6.7, and 4.3 kilobases (kb) in length, 2 additional mRNAs of 5.2 and 2.9 kb appear postnatally. Transformed brain cells of an astrocytic character express predominantly mRNAs of 6.7, 4.3, and 2.9 kb and a neuroblastoma line those of 7.4, 6.7, 4.3, and 2.9 kb. There are important quantitative changes in the amount of N-CAM message expressed during brain development, with a peak around birth, suggesting that N-CAM synthesis is controlled at the transcriptional level. A comparison of N-CAM protein and mRNA levels reveals a striking correlation between the relative concentrations of the Mr 120,000 N-CAM protein (N-CAM120) and the 5.2 kb transcript.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Isolation of mouse N-CAM-related cDNA: detection and cloning using monoclonal antibodies.

Clones coding for the mouse neural cell adhesion molecule (N-CAM) were isolated from a cDNA library prepared in the expression vector lambda gt 11 from mRNA extracted from a mouse neuroblastoma cell line. This library was screened with two anti-N-CAM monoclonal antibodies directed against different sites on the molecule and with rabbit anti-N-CAM serum. Two clones were identified with the first monoclonal antibody, three with the second one, none reacted with both. The relevance of these cDNA clones to N-CAM was confirmed by several observations. First, cDNA sequences detected with one monoclonal antibody cross-hybridized with those identified by the other antibody. Second, the different fusion proteins all bound the rabbit serum in addition to one monoclonal antibody. Finally, the probes hybridized to discrete mRNA species of sufficient lengths to code for the very large N-CAM polypeptides in RNA preparations from N-CAM-expressing, but not from N-CAM-negative cells. An additional mRNA species not seen in embryonic brain was expressed in adult mouse brain. Genomic blot experiments indicated that sequences corresponding to one of our probes are present only a few times in the mouse genome.

Animals↗

Structural and functional studies on N-CAM neural cell adhesion molecules.

The neural cell adhesion molecules N-CAM are to date the best characterized adhesion molecules of the nervous system. They have a high content of sialic acid residues which are present in the form of unusual sialic acid polymers. During development, a 3 fold decrease in the sialic acid content is observed. These changes in the degree of sialylation profoundly affect the binding properties of the molecules. A subpopulation of mouse brain N-CAM bears a carbohydrate determinant shared with other brain cell surface proteins and with the HNK-1 antigen of natural killer cells. Not only the carbohydrate side chains but also the protein moieties of the N-CAMs are heterogeneous. Three polypeptides of 180 K, 140 K and 120 K have been characterized in mouse brain. The 180 K and 140 K chains span the membrane. They differ mainly by the length of their cytoplasmic extensions. These intracellular domains are unusually long and contain phosphorylated serine residues. The 120 K chain exists in two forms, one membrane-bound and one soluble. Earlier studies had shown the presence of N-CAM on neurones and astrocytes of the mouse central nervous system, whereas cultured astrocytes had been reported to be N-CAM-negative. Recent results show that N-CAM is also expressed on astrocytes in culture. To study expression and heterogeneity of N-CAM polypeptides at the mRNA and gene level, cDNA clones for mouse N-CAM have been isolated. They reveal multiple mRNA species in mouse brain. By contrast, the corresponding sequences seem to be present only a few times, perhaps only once, in the mouse genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Studies on the transmembrane disposition of the neural cell adhesion molecule N-CAM. A monoclonal antibody recognizing a cytoplasmic domain and evidence for the presence of phosphoserine residues.

The N-CAMs are a group of surface glycoproteins involved in adhesive interactions of neurones. Related molecules of the mouse nervous system, identified in our laboratory, have been called BSP-2 and shown to act as ligands in adhesion of neuroblastoma cells. Results presented in this report show that they are immunochemically identical with N-CAM. A monoclonal anti-(N-CAM) antibody, that recognized a determinant accessible only after permeabilization of intact cells, was used to define the mode of association of the N-CAMs with the plasma membrane. This antibody bound a 35 000-Mr fragment in lysates of trypsin-treated neuroblastoma cells. It is concluded that the antibody reacts with a transmembrane or cytoplasmic domain of the molecules. The same antibody recognized the Mr-180 000 and Mr-140 000 proteins but not the Mr-120 000 chain, which co-purify from adult mouse brain. The latter polypeptide was detected in the cytosol and could be partially released from brain membranes by osmotic shock. Part or all of the Mr-120 000 protein may thus lack a transmembrane segment. Our conclusion that the N-CAM forms of higher Mr are transmembrane proteins was further corroborated by our finding that they contain phosphoserine residues, which can be labeled with (32P)phosphate in intact neuroblastoma cells.

Animals↗