Biomedical subjects
Publications and source records attributed to G Köhler.
A case of endometriosis of the large intestine is reported in a 41 years old woman. The endometrioma involved the Bauhin-valve, the distal ileum and the right adnexa. The symptoms and signs were thought of a caecal carcinoma causing a mechanical ileus. The morphologic aspects and the management of the diagnostic and therapeutic procedure are discussed with respect to the endometriosis of the intestine.
The rearranged mu and kappa genes from the 2,4,6-trinitrophenyl (TNP)-specific hybridoma Sp6 have been introduced into B cells from three different stages of differentiation as well as 5 non-B cell lines to determine the levels and modes of immunoglobulin (Ig) gene expression. In pre-B cells transformed with the mu and kappa genes, low levels of Sp6-specific mu RNA were produced and approximately 210-fold less mu and 800-fold less kappa proteins were produced than in the hybridoma Sp6. The Ig proteins were present intracellularly, but were not detected on the cell membrane. In mature surface sIg+ B cell transformants, higher levels of mu Sp6 and kappa Sp6 proteins and RNA were produced than in the pre-B cell transformants (12 X mu, 70 X kappa). These transformants displayed the mu Sp6 and kappa Sp6 proteins on the cell membrane and also secreted the transfected Ig product. Plasma cell transformants produced the highest amounts of mu Sp6 and kappa Sp6 proteins. These transformants secreted pentameric IgM but did not display detectable amounts of these proteins on the cell membrane. T cell and one fibroblast transformant produced Ig as normal sized mu Sp6 and kappa Sp6 proteins. All other mu Sp6 and kappa Sp6 non-B cell transformants (melanoma, teratoma and macrophage) failed to produce enough Ig to determine whether the Ig proteins were of the correct molecular weights. The T cell and fibroblast transformants that produced Ig proteins did not secrete or display detectable Ig on the cell membrane. The expression of Ig did not inhibit the expression of the T cell antigen Thy-1 in the T cell transformants.
From an IgM secreting hybridoma line we have isolated 16 spontaneous mutants that produce truncated IgM polypeptides. The size of the mu-mRNAs produced by these mutants is normal, but they express 3- to 100-fold less mu-mRNA and mutant mu-protein than the parental cell line. Nucleotide sequence analysis of cloned mu-genes and/or their mRNAs show frameshift mutations that generate in-phase chain termination codons. The extent of the reduction in mu-mRNA levels depends on the position of the nonsense codon within the gene.
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Fourteen patients with acute lumbar pain and cervico-brachial radicular pain were treated with a single oral dose of 100 mg ketoprofen. The analgesic effect of the substance was investigated, using an analog pain scale, in correlation with drug plasma levels. Blood samples were taken at zero, one-half, one, two, three, four, five, six, eight, and ten hours after oral administration of ketoprofen. On an average, pain was at its lowest two hours after the plasma level of ketoprofen was at its highest. Within the ten hour observation period, the maximum reduction in pain increased as the maximum plasma level rose. Five patients with a maximum plasma level greater than 9 microliter/ml experienced pain reduction of at least 50%.
Variants of four hybridoma lines secreting antibodies specific for either trinitrophenyl (TNP) or sheep erythrocytes (SRBC) were isolated which have lost either the specific heavy (H) chain or the specific light (L) chain. They were fused to lipopolysaccharide-stimulated mouse spleen cells and the resulting secondary hybridomas were screened for the restoration of the original antibody specificity. Antibody activity was 37 times more frequently restored with fusion lines donating H chain than with those donating L chain. We obtained a variety of different (spleen cell derived) L chains in association with one H chain and one specificity. We found that those L chains originally associated with a given H chain were rescued most often. Their frequencies were 1:74 and 1:490 for an anti-SRBC- and an anti-TNP-restoring kappa L chain, respectively. Two most commonly observed V-J kappa combinations in one anti-SRBC complementation group were detected with a 5- and 30-fold reduced frequency in two anti-TNP groups, indicating somatic diversification of kappa chains. It is shown that the Sp7/HK line resumes anti-TNP activity with a mutated, non-germline lambda 1 chain, which was found in 30% of lambda 1-expressing hybridomas.
Seventeen monoclonal rat antibodies with specificities for mouse mu heavy chain recognize seven distinguishable determinants that are located on the four constant region domains. All determinants are present on secreted, intracellular and membrane bound IgM, and all but two are expressed on isolated mu heavy chain. One antibody, specific for a site in the first constant region domain, recognizes a determinant that is present on IgM of AKR, C3H/HeJ, C57BL/6J, SJL, DBA/2, BALB/c, NZB and CBA/J mouse strains, but not on IgM of A.TH, A/J and A.CA strains of mice.
In this paper we summarize experiments which were undertaken to create altered antibody molecules. Three different approaches were used. Established hybridoma lines were re-hybridized to mouse spleen cells to generate arrays of secondary hybridomas which express one particular heavy chain and one specificity together with a multitude of different light chains. In such hybrids the influence of light chains to the antibody combining site and the influence of affinity to antibody effector functions can be studied. Another way to obtain altered antibodies was the selection of cells producing less lytic IgM. With this technique we obtained (among many other variants) a series of mu-deletion products which were used to map the fine specificity of rat anti-mouse mu monoclonal antibodies. Both the anti-mu antibodies and the deletion variants were used to assign the Clq binding to the fourth C mu-domain demonstrating the power of mutant IgM in the structure-function analysis. In a third series of experiments we show the feasibility of generating new antibody combining sites by the methods of molecular genetics. The variable region gene of a heavy chain was placed in front of a kappa-constant region gene. The plasmid construct was transferred into mouse myeloma lines which stably express a variable heavy-constant light chain protein. Upon fusion with a light chain producing line, chimaeric light chain dimers with a functional antibody combining site were secreted. These experiments demonstrate that new series of man-made antibody molecules can be made in the future.
Estradiol-17 beta, progesterone, LH and FSH levels in plasma were measured simultaneously by radioimmunoassay and BBT was recorded in order to investigate the effect of a single midcycle oral dose of 0.5 or 2.0 mg dienogest (17 alpha-cyanomethyl-17 beta-hydroxy-estra-4,9-dien-3-one, VEB Jenapharm, Jena/GDR) on pituitary and ovarian function in 18 healthy fertile females. After application of 0.5 mg dienogest in the follicular phase the cycles appeared to be anovulatory. Administration of 2.0 mg two days prior to the expected LH-surge produced a delay of the LH-peak combined with an absence of ovulation as well as the absence of the normal subsequent increase of progesterone. With 0.5 mg the delay of the LH-surge was followed by an ovulation with normal corpus luteum function. The application of 0.5 or 2.0 mg one day before or during the rising LH-peak lowered the LH-surge but ovulation and luteal phase were not altered. Administration of both dosages during the LH-peak could neither prevent ovulation nor disturb corpus luteum function. Postovulatory ingestion of 0.5 or 2.0 mg dienogest during the BBT-rise produced no alteration of the further cycle. These results demonstrate that dienogest in a single-dose-administration in midcycle can alter pituitary and ovarian function depending on the time interval between application and the day of LH-surge.
Using a mouse hybridoma system, we have developed methods of isolating a variety of mutant cell lines in which immunoglobulin function or synthesis is defective. The analysis of mutants defective in kappa chain synthesis has defined a class of murine transposons. The deletion mutants produce immunoglobulin M (IgM) bearing mu heavy chain fragments and provide information on the requirements of IgM assembly and mu gene expression. We also describe a transfer system for the mu and kappa genes which will be useful in analyzing the structural basis of IgM function.
The proportion of T-lymphocytes was estimated by the alpha-naphthylacetate-esterase-reaction as a T-cell marker in blood smears of 40 patients with gynecologic malignancies and 30 healthy controls. In addition the red and white blood cell counts were determined. A lymphopenia and a reduction of T-lymphocytes were found in the peripheral blood of the tumorgroup, on the other hand the number of B-lymphocytes was elevated in this patients. This method of lymphocyte differentiation may be of value for controls in patients suffering from malignant tumors.
Large doses of estrogens employed for postcoital contraception have definitely been shown to be effective, but estrogens used in such high doses would result in a high incidence of side-effects. Therefore this method should be reserved for the occasional emergency. Postcoital estrogen-progestogen-combination is suitable for women exposed to an isolated act of intercourse in midcycle. The advantage of this method is the possibility of a delayed administration after intercourse. Progestogens alone can be used after each coital exposure. One of the most frequent problems is the alteration of the menstrual pattern. Continued use can be recommended for women with occasional intercourse in cycle. Possible mechanisms of action of steroidal postcoital contraceptives are discussed involving problems of this contraceptive method.
A case of bilateral gonadoblastomas in a 45,XO patient with features of Turner's syndrome is presented. The case stresses the importance of a careful laparoscopic evaluation of the pelvic cavity and a biopsy of both streak gonades is strongly recommended also in patients with 45,XO karyotype.
Nonsteroidal anti-inflammatory drugs are an important part of the therapy of rheumatoid arthritis. For optimal dosage it would be necessary to know the levels of such compounds in inflamed tissue and not only in blood because the drugs inhibit prostaglandin synthesis in all cells of the body. In the present study, the levels of indomethacin in blood, synovial fluid, synovial membrane, muscle, fat, bone and spinal fluid of patients with rheumatoid arthritis have been measured at 1, 3, 6, and 12 hours after administration of 50 mg indomethacin (Amuno, Merck Sharp & Dohme). At 3, 6, 9 and 12 hours after administration levels in synovial fluid and tissue were higher than in blood but the differences were not significant. Levels in the other tissues investigated did not differ much from blood. In experiments with carrageenan-induced inflammation in rabbits, levels of indomethacin in blood and exudate have been measured after administration of 7.5 mg/kg i.v. Between 3 and 8 hours after administration levels in exudate were significantly higher than in blood. From the time course of indomethacin in blood compared to synovial fluid or exudate and from the comparison of the elimination half lives it can be concluded, that the transport of indomethacin into the inflamed area is mainly a process of diffusion.
The therapeutic activity of antiinflammatory agents in rheumatic joint disease is related to their presence at the target site of action, i.e. the joints. Tissue concentrations of such agents have been previously determined in patients with rheumatic disorders under long-term treatment with acemetacin and indomethacin (Köhler et al., 1981). The purpose of the present study was to determine concentrations of ketoprofen and acetylsalicylic acid in blood as well as synovial fluid, synovial membrane and periarticular bone and adipose tissue, three hours after administration of a single dose. Drug concentrations found in each of these tissues following a single intramuscular injection were sufficient to ensure therapeutic efficiency.
B14-2-14 is a monoclonal cytotoxic IgM antibody which reacts with thymocytes of all mouse strains tested. The fraction of positive cells (by visual immunofluorescence) varies between strains from about 25-45% in A.CA to 65-85% in C57BL/6, and high levels are dominant in F1 hybrids. In the periphery, the antigen is found on a few percent of lymph node and not on splenic T cells, and it is absent in nude mice. Among thymocytes, the distribution of the B14 determinant largely overlaps with that of the TL antigen and of molecules binding peanut agglutinin. The B14 antibody reacts only minimally with hydrocortisone-resistant thymus cells. Biochemical analysis shows that B14 antibody, anti-TL antibody and peanut agglutinin bind to separate molecules. The target of the B14 antibody may be either an immature, thymic form of Thy-1, or another molecule associated with it. Two polypeptides, of 40 and 35 kDa are precipitated by both B14 and anti-Thy-1 antibodies from biosynthetically labeled thymus cell lysates, and two others, of 27 and 17 kDa, from surface-iodinated thymus cell preparations. B14-2-14 offers an additional method for identification and selection of thymocytes at different stages of differentiation, and should also be useful for studies of the Thy-1 antigen.
The rearranged immunoglobulin heavy (mu) and light (kappa) chain genes cloned from the Sp6 hybridoma cell line producing immunoglobulin M specific for the hapten 2,4, 6-trinitrophenyl were inserted into the transfer vector pSV2-neo and introduced into various plasmacytoma and hybridoma cell lines. The transfer of the mu and kappa genes resulted in the production of pentameric, hapten-specific, functional IgM.