The porcine Ig delta gene: unique chimeric splicing of the first constant region domain in its heavy chain transcripts.

The pig delta gene is located approximately 3.4 kb downstream of the second transmembrane exon of the micro gene and shows a similar genomic structure to its counterpart in cow with three exons encoding the CH1, CH2, and CH3 domains. The porcine genomic deltaCH1 exon has been replaced by a recent duplication of the micro CH1 and its flanking sequences, a genetic event that also led to the formation of a short switch delta region, immediately upstream of the delta gene. The deltaCH1 exhibits a 98.7% similarity (314 of 318 bp) to the micro CH1 at the DNA level, whereas the homologies between the deltaCH2 and micro CH3, and the deltaCH3 and micro CH4 are only 33.3 and 35.8%, respectively. Either of the two CH1 exons ( micro and delta) could be observed in the expressed porcine IgD H chain cDNA sequences VDJ- micro CH1-H-deltaCH2-deltaCH3 or VDJ-deltaCH1-H-deltaCH2-deltaCH3, showing a pattern that has not been observed previously in vertebrates. In addition, transfection of a human B cell line, using artificial constructs resembling the porcine C micro -Cdelta locus, also generated both VDJ- micro CH1-deltaCH1-H1-deltaCH2 and VDJ -deltaCH1-H1-deltaCH2 transcripts. An examination of the pig delta genomic sequence shows a putative, second hinge region-encoding exon. Due to the lack of a normal branchpoint sequence for RNA splicing, this exon is not present in the normal pig delta cDNA. However, the exon could be spliced into most of the expressed transcripts in vitro in cell transfection experiments after introduction of a single T nucleotide to restore the branchpoint sequence upstream of the putative H2 exon.

A novel L3-acute lymphoblastic leukemia cell line (BALM-27) carrying cytoplasmic immunoglobulin 8 chain but lacking expression of cell surface immunoglobulin chains.

A human acute lymphoblastic leukemia (ALL) cell line, BALM-27, was established from the peripheral blood specimen of a patient with B-cell ALL L3 type (ALL-L3) at diagnosis. As with the original leukemia cells, the established line was negative for all cell surface immunoglobulin (Ig) chains, but carrying only cytoplasmic Ig delta heavy chain. Southern blot analysis of the various Ig chain genes demonstrated homozygous deletion of the Jkappa gene, germ line configuration of the Jlambda and rearrangement of IgJH genes. Cytogenetic analysis of both primary leukemic bone marrow and BALM-27 cells showed the der(8;15)(q10;q10) chromosomal alteration, in addition to the t(8;22)(q24;q11) abnormality which is highly associated with ALL-L3 and Burkitt's lymphoma. The established cell line BALM-27 represents a rich resource of abundant, accessible, and manipulable cell material for analyzing the unique expression of Ig chain and for investigating the pathogenesis of B-cell malignancy. The scientific significance of BALM-27 lies in (1) the rarity of this type of leukemia cell lines, and (2) its unique chromosomal aberrations.

Germline transcription and recombination of a murine VDJmudeltagamma1 transgene.

To investigate the regulation of Ig switch recombination, we have constructed mice with a 56 kb VDJmudeltagamma1 transgene. This transgene included an anti-nitrophenyl VDJ segment, Smu, Cmu, Cdelta, Igamma1, Sgamma1, Cgamma1 and the Cgamma1 membrane exons from the murine Igh(a) haplotype. Two founder lines were produced, with very similar characteristics. Transgenic B cells expressed normal amounts of Cmu (which is >95% transgenic), Cdelta and other cell surface markers, and normal amounts of VDJ and Cmu RNA. Gamma1 germline transcription of the transgenes is properly regulated since stable transcripts were not expressed in B cells treated with lipopolysaccharide (LPS) alone, nor in thymus or non-lymphoid tissues, but were expressed after treatment of B cells with LPS + IL-4 or CD40L + IL-4. B cells from both lines of transgenic mice expressed transgenic gamma1a after in vitro culture with CD40L + IL-4, but not after culture with CD40L alone. However, the CD40L + IL-4 induced IgG1 precursor frequency is much lower for VDJmudeltagamma1 transgenic B cells (1:240-760) than for non-transgenic B cells (1:9). Analysis of DNA from transgenic hybridomas indicated that switch recombination can take place in switch (S) regions, but can also take place outside S regions. These results indicate that targeting of switch recombinase to S regions must include regulation beyond the S regions themselves and correct germline transcription. This additional regulation might include cis-acting elements or appropriate spacing or arrangement of the recombining elements.

Peripheral B cells with intracytoplasmic mu chains in HIV infection.

Besides the major alteration of T lymphocytes, B-cell anomalies have been reported in HIV infection, related to late stages of B-cell maturation, and considered to result from the dysregulation of T/B interactions. Because T cells are also involved in the control of lymphopoiesis and/or because of specific alterations of the B lineage, anomalies of B-cell maturation could occur in HIV-infected patients. We investigated the presence of immature pre-B lymphocytes, characterized by cytoplasmic mu chains, in 35 peripheral blood samples from healthy controls, 82 from HIV-positive/non-AIDS patients, and 45 from AIDS patients. Significant numbers of such cells were observed in 48% of HIV-seropositive patients and in 40% of the patients with AIDS disease. The presence of pre-B cells correlated with higher numbers of CD8+ and/or CD57+ cells and of peripheral lymphocytes. These data suggest that B-cell dysregulation in HIV infection may lead to the abnormal release of immature B cells in the peripheral blood. This observation may be interpreted as a sign of bone marrow activity.

Premature termination of variable gene rearrangement in B lymphocytes from X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) results from failure of B lymphocyte development. Immature B cells from a patient with XLA were found to produce truncated mu and delta immunoglobulin H chains encoded by D-JH-C (mu delta). The 5' terminal sequence of cDNA encoding the H chains is composed of D-JH with the characteristic GGTTTGAAG/CACTGTG consensus sequence utilized for VH gene rearrangement upstream, and a leader sequence that serves for translation of this intermediate stage of rearrangement. Failure of variable region gene rearrangement may underlie the failure of B lymphoid development in XLA.

Nonsecretory IgD (kappa) multiple myeloma. Report of a case and review of the literature.

The authors have reviewed five cases of the nonsecretory IgD multiple myeloma (MM), including a newly observed case investigated in the authors' laboratory. The salient features of these five cases were summarized to allow a better understanding of this entity. The following clinical points should be noted: (1) All nonsecretory IgD MMs reported had kappa light chains, which are rarely seen in usual IgD MM. (2) All the cases were found in women. (3) The presence of monoclonal B-lymphocytes bearing surface IgD (kappa) in the peripheral blood was demonstrated in three of four patients whose B-cell subpopulation was mentioned. (4) Generally speaking, nonsecretory IgD (kappa) MM showed the combined features of usual IgD MM and nonsecretory MM. It seems likely that IgD (kappa), nonsecretory MM is more than might be expected to occur by chance. The pathogenesis of this entity is discussed in this report.

S-phase lymphocytes in chronic lymphocytic leukemia (CLL) in relation to immunoglobulin isotypes on the leukemic clone and to disease activity.

The fraction of blood S-phase (S+) lymphocytes from 41 patients with chronic lymphocytic leukemia of B cell type was determined by flow cytometry. The patients were grouped according to the smig isotype pattern of the leukemic cells. Patients with IgM as the predominant smig had higher numbers of S+ lymphocytes than patients with a leukemic clone co-expressing IgM and IgD (p less than 0.001). High relative as well as total numbers of S+ lymphocytes were associated with short therapy-free and overall survival. T cell proliferation was low although significantly higher in active than in indolent disease.

Expression of IgD on B cell malignancy. An immunopathological study of 50 cases.

Expression of IgD was studied immunopathologically on 50 cases of B cell lymphomas (B MLs), together with various B cell markers including IgM, kappa and lambda chains, B1, B2, (OK)B2, (OB)B7, (OK)T10, NUB1, Leu 1, Leu 14, and PCA. IgD was demonstrated on 19 cases heavily and on 8 weakly. It associated well with expression of two antigens, B2 (C3d receptor molecule) and Leu1 (pan-T antigen), besides IgM, while, B2 was closely related in expression on B MLs to (OK)B2, (OK)B7, and NUB1. lambda chain was dominant on IgD heavily-stained cases. Histopathologically, IgD positive MLs were distributed in various types. All of diffuse intermediate type were shown to be IgD positive (4/4, 3 heavily). As cases of this type were shown to express most of other B cell antigens present on non-tumorous primary follicle B cells or mantle zone B cells, this type of MLs is speculated to be a neoplastic counterpart of such non-tumorous B cells. Eight out of 18 cases of diffuse large cell type were IgD heavily positive, suggesting some unusual mechanisms in IgD expression on these neoplastic large cells, as non-tumorous B cells lose most of their surface IgD soon after blastoid transformation. Other types of MLs were discussed with special emphasis on expression of IgD.

Surface immunoglobulin restriction in B-non Hodgkin's lymphomas in cell suspension and on frozen tissue sections.

The diagnostic relevance of different tests for detection of surface immunoglobulin on tumour cells of B-type non-Hodgkin's lymphomas (B-NHL) was investigated by comparison of the direct antiglobulin rosetting reaction (DARR) in suspension with two-colour direct immunofluorescence (DIF) on frozen tissue sections. In benign lymph nodes (n = 27) the kappa/lambda ratio by DARR test ranged from 0.9 to 2.8. Tested by suspension and frozen tissue analysis, light chain restriction was found in 24 and 27 of 31 cases of B-NHL, respectively. Heavy chain restriction was found in half of the cases (14 of 26) studied in suspension and in almost all (28 of 31) tested on sections. In 9 cases DARR tests showed restriction of more than one Ig class on tumour cells, which was infrequent (2 of 28) in frozen section analysis. Although both tests appeared valuable for routine diagnostic purposes, we found the DIF analysis on tissue sections somewhat more discriminative, especially in detection of heavy chain restriction in B-NHL.

mIgM:mIgD ratios on B cells: mean mIgD expression exceeds mIgM by 10-fold on most splenic B cells.

The relative frequency of mIgM and mIgD molecules on B cell surfaces is important in determining, in large part, the isotype involvement in antigen binding and signal transduction. Although it is generally assumed that on most mature B cells, mIgM and mIgD occur in roughly equal quantities, no formal analysis of this question has been reported. In this report, we describe such an analysis based on the quantitation of anti-Fab or anti-kappa specific immunofluorescence of splenic B cells before or after capping with rabbit anti-IgD or anti-IgM antibodies or both. The results indicate that, whereas mean expression of IgD exceeds IgM on splenocytes by threefold, members of the major B cell subpopulation (60 to 70% of cells) express 10-fold more IgD than IgM.

Structural heterogeneity of murine IgD during ontogeny.

We have studied the expression of the two membrane delta heavy chains (delta 1 and delta 2) and the two native IgD structures (IgDI and IgDII) in neonatal mice. Both delta-chains appear simultaneously during development and neonatal mice, like adults, express equal amounts of delta 1 and delta 2. IgDI and IgDII also appear simultaneously during ontogeny and in the same ratio as expressed by adult mice of the same strain. Thus, when IgD first appears during ontogeny it shows the same structural heterogeneity as observed in adult mice.

Migrant mu+ delta+ and static mu+ delta- B lymphocyte subsets.

Immunoglobulin isotype expression in isolated lymph node (LN), spleen and blood lymphocyte suspensions was assessed in rats. The proportion of mu+ delta- B cells in spleen (34%) was approximately twice that in blood and LN. Immunohistological examination of spleens showed the cells of the marginal zones to be predominantly mu+ delta-. On the other hand, mu+ delta+ B cells were mainly confined to the follicles in both spleen and LN. These follicles had a minor mu+ delta- component. The migratory properties of B cells with these two phenotypes were assessed by depleting lymphocytes migrating through the white pulp of rat spleen. This was achieved by placing a 32P-impregnated beta-emitting polythene strip over one half of the spleen. Examination of the nonirradiated half of the spleen, LN and peripheral blood after 12 days irradiation showed selective loss of delta + B cells. The mu + delta- cells of the splenic marginal zone were numerically unaltered. There was also a substantial residual mu + delta- B cell presence in the small lymphocyte compartment of follicles of LN and spleen in depleted animals. In addition, the blood selectively retained a mu + delta- B cell component. This was not derived from the spleen, as mu + delta- blood B cell numbers were sustained even where both halves of the spleen were irradiated. It is concluded that: (a) the major static B cell component of spleen and LN is mu + delta-, (b) that most if not all delta + B cells repeatedly migrate through the spleen and (c) there is a blood-born mu + delta- component which is resistant to depletion by splenic irradiation.,

[Delta-antigen in hepatitis B]. / Delta-Antigen bei Hepatitis B.

Among 571 liver biopsies delta-Ag was found in 10 of 365 HBsAg positive patients (9 with CAH, 1 with CPH). Delta-Ag was demonstrated in nuclei and cytoplasm of liver cells in both cryostat and paraffin sections. Follow-up studies revealed persistence of delta-Ag for as long as 6 years and temporary appearance or loss of Dane particle-associated parameters without a change in the concomitant inflammation. These findings are compatible with the hypothesis that delta-Ag represents a defective viral agent which modulates, by superinfection, the typical viral expression patterns of HBV infection.

IgG antibodies: in vitro blocking activity of IgE mediated reactions.

Sera from 177 atopic patients treated by immunotherapy were screened in order to detect IgD antibodies against a purified fraction of Lollium perenne (fractions C). IgD antibodies were found in 23% of the subjects. Eight of these sera were selected and their IgD isolated by means of immunoabsorption. The in vitro biological activity of the IgD antibodies against fraction C was tested by three methods: basophil degranulation, histamine release and RAST inhibition test. In all these assays, the data obtained indicated a possible blocking effect of the IgD antibodies upon the IgE in vitro mediated reactions.

Multiple heavy chain isotypes on the membrane of the small B lymphocytes in human blood.

Small lymphocytes from adult human blood were examined for the presence of membrane-associated alpha, gamma, delta and mu Ig isotypes by means of a direct immunofluorescence technique. Since less than 10% of the small lymphocytes in blood are B cells as defined by positive reactivity with an anti-Fab conjugate, our experiments were performed on a T cell-depleted fraction in which about 80% of the small lymphocytes were B cells. With the two-wavelength immunofluorescence method, all of the double-isotype combinations were found. The percentage of cells bearing more than two isotypes was deduced. The delta mu and the alpha delta mu combinations were the most common, as previously found on tonsillar lymphocytes. In contrast with the tonsils, no lymphocytes bearing only delta were observed and the proportions of alpha gamma- and alpha gamma mu-bearing lymphocytes were very small. The presence of lymphocytes bearing four isotypes could practically be excluded.