Systematic design of mouse Vh gene family-specific oligonucleotides.

Kabat's database has often been used to design mouse Vh gene-specific 5' primers. The emphasis was mostly on constructing a universal (degenerate) 5' primer or 5' primer set, which would be able to match every mouse Vh gene. We were interested in finding oligonucleotides that could be used as primers or probes that can discriminate between the different Vh gene families. To this end, Kabat's database was reordered into Vh gene families based on more than 80% homology to prototype Vh gene family sequences. Rules were formulated for adequate annealing of putative primers to their respective genes. Putative primers were derived from the consensus sequences of the Vh gene families. A computer program was designed to systematically screen for the most optimal 5' and 3' Vh gene family-specific primers. This program enabled us to find a set of framework I-specific 5' primers, as well as a set of framework III-specific 3' primers. The found primers were also tested for their use as Vh gene family-specific probes.

RT7-defined alloantigens in rats are part of the leucocyte common antigen family.

Haemopoietic cells carry a variety of cell-surface molecules, some of which are known to have allotypic variation. In rats, the RT7 alloantigenic system has been well documented using alloantisera. We have produced the first mouse hybridoma cell line secreting an antibody, HIS41, which binds to leucocytes of rat strains carrying the RT7.2 but not the RT7.1 determinant. An IgG2b isotype switch variant (HIS41.2b) of the original HIS41 (IgG1 isotype) was also made. HIS41 showed a clear and discrete binding in immunofluorescent and histological experiments and has already been used in several studies on haemopoietic cell turnover and differentiation employing PVG rats congenic for RT7. The present study addresses the question of whether the RT7 gene products are members of the L-CA family, which has been a matter of controversy over the last decade. When using HIS41 for the analysis of tissue distribution and molecular weight of RT7 gene products, a strong similarity was evident with the data reported for the L-CA detected by MRC OX-1 and MRC OX-30. These two MoAb have been reported to bind to all members of the L-CA family. All haemopoietic cells, excluding erythrocytes and the more mature stages of erythropoiesis, stained with HIS41. The molecular weights of HIS41 binding molecules on thymocytes and peripheral T cells were comparable to the L-CA precipitated by MRC OX-1. Capping and sequential immunoprecipitation studies indicated that HIS41 and MRC OX-30-binding molecules were identical. MRC OX-1, however, appeared to bind only a subset of these molecules. Thus, our study confirms the identity of RT7.2 gene products and L-CA. It also revealed a difference between MRC OX-1 and MRC OX-30 not noticed previously.

[Immunomapping in localized bullous pemphigoid]. / Immunomapping in lokalisiertem bullösem Pemphigoid.

A 70-year-old Caucasian patient is described, who presented with a bullous dermatosis on an amputation stump. The bullous lesions had developed after practice with a Kondylen-Bettung-Münster (KBM) prosthesis. The lesions disappeared completely after prednisone therapy and replacement of the prosthesis with a Thomas splint. The patient has remained free of symptoms ever since, even though prednisone was completely withdrawn after 9 months. According to skin immunofluorescence criteria, the patient suffered from pemphigoid, rather than epidermolysis bullosa acquisita as strongly suggested by the anamnesis. In particular, the presence of linear IgG at the level of the lamina lucida of the epidermal basement membrane zone and the localization of laminin antigen at the blister floor are highly suggestive of pemphigoid.

Germinal centers develop oligoclonally.

As part of our studies into the role of germinal centers, we investigated whether each de novo generated germinal center (GC) develops from one single GC precursor cell (GCPC, monoclonal development), a few GCPC (oligoclonal development) or from many GCPC (polyclonal development). Thus, lethally (9 Gy) X-irradiated AO (RT1u) rats were reconstituted with 10(8) thoracic duct lymphocytes (TDL) containing mixtures of AO and AO X BN cells in various ratios. The AO TDL were tolerant for AO X BN cells by using TDL from AO----(AO X BN)F1 (RT1u/n) X-irradiation bone marrow chimeras. To induce GC formation in the spleen of TDL-reconstituted rats, animals were i.v. injected with 10(9) sheep red blood cells. Five days after reconstitution and antigenic challenge spleens were taken for analysis of cellular make up of de novo generated GC. Spleen sections were immunohistochemically stained with monoclonal antibody F17-23-2, recognizing major histocompatibility complex class II antigens of the RT1n haplotype but not the RT1u haplotype, to discriminate between B cells of AO and AO X BN origin. Analysis of the GC in spleens of rats reconstituted with a mixture of AO and AO X BN TDL revealed three types of GC: GC entirely composed of AO cells, GC entirely composed of AO X BN cells and GC containing a mixture of both. The relative frequencies of these three types of GC indicated that in our experimental system, de novo GC developed oligoclonally from one to three GCPC. These data strongly suggest that GC are sites of antigen-driven expansion of peripheral B cells to very large clones.

Plasma cells and their precursors. IV. Cross-reacting and non-cross-reacting, sheep erythrocyte lysing IgG antibody clones induced in rabbits immunized with rat- and chicken-erythrocytes.

Rabbits were i.v. immunized with rat erythrocytes or chicken erythrocytes. The sera were investigated using the IEF technique. A number of the RRBC immunized animals and a high proportion of the CRBC immunized animals contained SRBC-lysing IgG antibody clones in addition to apparently normal anti-RRBC or anti-CRBC antibodies. Intravenous immunization with RRBC, followed by immunization with CRBC, yielded clones of SRBC-lysing IgG antibodies in almost every rabbit tested. By varying the interval between the RRBC- and CRBC injections, we established that the time needed for the RRBC-induced production of the responsible SRBC-specific IgG AFCP is approximately 5 days. These cells show an average functional half-life of 75 days. At least some of the RRBC- and CRBC-evoked SRBC-lysing IgG antibody clones are incapable of lysing either RRBC or CRBC. We suggest that the production of cross-reactive and non-cross-reactive, hetero-specific AFCP provides a natural explanation for the availability of preexistent AFCP responsible for the presence of the "early phase" of the IgG antibody response to several antigens.

Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis.

Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.

Plasma cells and their precursors. III. Late phases of anti-bovine serum albumin IgG antibody responses depend upon newly produced IgG B-memory cells.

The sequential appearance of the IgG antibody clones constituting the primary, secondary or tertiary response to BSA was studied in individual rabbits by relating antibody titers, antibody affinities, and clonal spectra obtained by IEF. The results showed that after a single i.v. injection of BSA, the primary response IgG antibodies peaked at day 11 and had a constant and low average affinity during the first 20 days. A slow rise of affinity was observed during the following 20 days. In this period, new IgG antibody clones appeared, though antibody titers decreased. A number of these newly appearing, so-called late-phase clones were isolated by preparative IEF. Their affinities to BSA were high. Secondary responses showed the rapid rise of both titers and antibody affinities typical for the activity of B-memory AFC. One animal immunized for a tertiary response showed a still further increase of antibody affinity in its late phase. These results, together with those described in two earlier papers (1, 2), demonstrate that primary immunization, apart from triggering preexisting IgG AFCP into production of antibodies having low affinity, elicits the origination of IgG (B-memory)-AFCP capable of producing high affinity IgG upon antigenic triggering, and show that some of these may already be triggered during the ongoing response, thereby giving rise to the so-called late phase in the primary IgG antibody response.

Plasma cells and their precursors. II. Kinetics of B-memory cell production in rabbits.

Rabbits were irradiated with 4.5 Gy in order to eliminate completely preexisting antibody-forming cell precursors. Sheep red blood cells were administered 24 h or 8 days after irradiation in order to induce the production of IgG B-memory AFCP. Resulting B-memory cells were triggered into antibody synthesis by a second dose of SRBC given 8 days after the challenge; the resulting IgG antibody clones were analyzed by isoelectric focusing. Memory IgG antibody clones were detectable from the third day after secondary immunization onward. It is concluded that antigen administered as early as 24 h after the irradiation induces B-memory cell production equally well as primary immunization 8 days after the irradiation. This B-memory cell production proceeds in the absence of detectable primary IgG antibody formation. Irradiated non-immunized rabbits showed spontaneous reappearance of IgG-AFCP with specificities to SRBC. In sharp contrast to the specifically induced production of B-memory IgG-AFCP mentioned above, this process took more than two months to reach potentialities comparable to those of "preexistent" AFCP present in normal, control rabbits.

Plasma cells and their precursors. I. Preexistent antibody-forming cell precursors dominate the anti-sheep red blood cell and anti-human gamma globulin IgG antibody response.

Rabbits were immunized by a single i.v. injection of SRBC or HGG and were bled serially. Sera were analyzed by IEF using the gel overlay technique to investigate clonal patterns of circulating IgG antibodies. Clonal patterns of primary-response IgG antibodies were quite stable over a period of months, showing that this response is dominated by plasma-cell precursors already present at the time of antigenic stimulation. In the late phase of the primary IgG response, new bands were occasionally observed in the antibody spectra, suggesting the presence and triggering of newly formed plasma-cell precursors. Primary-response antisera had individually unique clonal patterns. In secondary-response antisera elicited by a second single i.v. injection, the IEF spectra were found to be more complex and could be individually distinguished only with difficulty.

Rabbit IgM: its isolation in high yield by a convenient procedure using serum from trypanosome-infected animals.

Rabbit sera contain little IgM. This is a major obstacle for its isolation. However, levels of IgM up to 10 mg/ml are found in rabbits infected with living trypanosomes. A simple procedure, involving gel filtration, standing in the cold, and agarose block electrophoresis is described which allows the isolation of IgM from these sera in 60% yield, with a purity of better than 90%.