The human fetal liver is an early site for B cell development. Pre B cells are first detectable in human fetal life at 8 weeks of gestation, when the rearrangement of the mu heavy chain genes starts. In this study we characterize the CDR3 region of rearranged alpha heavy chain transcripts from four human fetal livers ranging from 8 to 11 weeks of gestation. Each fetal liver showed a limited number of variations in CDR3 sequences compared with adult peripheral blood mononuclear cells (PBMC). Sequence analysis of 91 clones demonstrated that there was no preference for the usage of a certain J(H) gene segment, whereas a preference for usage of D-H family genes, DXP and DLR, was seen in most cases during early fetal life. This is the first study where rearranged alpha heavy chain genes in fetal liver have been characterized. Our data suggest that the usage of J(H) genes is random, while there is a preference for D-H family genes in human fetal liver.

Hepatocyte growth factor (HGF) and its receptor c-met are present in several human tissues but their expression in mesothelial cells has not been examined. In this study, we have investigated the expression of HGF and c-met in normal human mesothelial cells and 11 human malignant mesothelioma cell lines. Using RT-PCR and Western blotting we found that HGF is produced by 3/11 mesothelioma cell lines whereas c-met is expressed in 11/11 mesothelioma cell lines. In addition, c-met expression was also found in 6/6 cell samples obtained from pleural fluids of patients with mesothelioma. In contrast, neither normal cultured mesothelial cells nor mesothelial cells obtained directly from patients without mesothelioma expressed HGF nor c-met. We have also analysed the biological function of HGF and c-met in mesothelioma cell lines. Recombinant human (rh) HGF stimulated both directional (chemotactic) and random (chemokinetic) motility in all mesothelioma cell lines tested. Furthermore, mesothelioma serum free conditioned medium containing HGF stimulated mesothelioma cell migration. This effect could be blocked in the presence of neutralizing anti-HGF monoclonal antibodies (MAbs) in the assay. Addition of HGF to mesothelioma cells cultured on collagen type IV was associated with induction of bipolar shape and protrusion of prominent pseudopodia. We have also found that rhHGF was mitogenic for mesothelioma cells. Our findings suggest that expression of HGF/c-met is involved not only in mesothelioma progression but also in its growth and migration and that c-met expression found in mesothelioma cells taken directly from patients may be of diagnostic importance.

Immunoglobulin isotype switching to IgE in patients infected with Schistosoma mansoni and patients with atopic dermatitis was studied. Patients with parasitic infections or allergic diseases have a higher production of IgE. We found a four-fold increased production of I epsilon RNA in both patient groups as compared to control donors. The increased expression of germ-line transcripts correlates with higher serum IgE levels. Nested primer polymerase chain reaction was used to generate S mu/S epsilon fragments from DNA of patient peripheral blood mononuclear cells. Twenty-nine out of fourty sequenced switch fragments had undergone direct joining from S mu to S epsilon whereas seven fragments showed mono sequential switching from S mu via either S mu, S gamma2, S gamma4 or S epsilon to S epsilon and four fragments demonstrated double sequential switch: S mu/S mu/S gamma1/S epsilon, S mu/S gamma2/S epsilon/S epsilon or S mu/S gamma1/ S gamma2/S epsilon. The sequential switching had occurred either via deletions or inversions. Mapping of the breakpoints showed hot spots for recombination within S mu, S gamma1 and S epsilon. To our knowledge, this is the first in vivo study in humans demonstrating that switching to IgE can occur from sequential rearrangements via gamma1, gamma2 or gamma4.

IgA nephropathy (IgAN), the most common form of glomerulonephritis, is characterized by normal to elevated levels of serum IgA. In order to understand the molecular mechanism(s) involved in the production of IgA in IgAN, peripheral blood mononuclear cells (PBMC) from these patients were analysed in this study. IL-10, transforming growth factor-beta 1 (TGF-beta 1) and CD40 have previously been shown to be involved in IgA production. We show here that CD40L expression was increased three-fold in these patients. However, expression of TGF-beta 1 in serum levels was comparable to controls. In vitro stimulation of PBMC with a polyclonal activator resulted in a three-fold increase in synthesis of both IgA subclasses, with a preference for IgA1 RNA. In situ hybridization studies also showed a three-fold increase in the numbers of IgA1- and IgA2-producing cells, but the subclass distribution was similar to the controls. Furthermore, using the nested primer polymerase chain reaction (PCR) for amplifying switch (S mu/S alpha) breakpoints we could demonstrate that in unstimulated PBMC the switch frequency did not differ from that of control donors. Sequence analysis of the amplified switch breakpoints and the I alpha regulatory region from patients showed no structural abnormality. Although we have previously demonstrated a correlation to in vivo germ-line RNA expression and class switching, no I alpha transcripts were detected in unstimulated PBMC from these patients. However, stimulation of PBMC with TGF-beta 1 resulted in I alpha production. Taken together, results from in vivo and in vitro studies suggest that increased cytokine production and hyperresponsiveness to polyclonal stimulation may play an important role in the increased synthesis of IgA. The preference for IgA1 is due to increased production of IgA1 per cell, and the absence of I alpha RNA indicates that additional defect(s) in immune regulation may play an important role in the pathogenesis of IgAN.

IgA deficiency is the most common humoral defect in man and results in an increased susceptibility to respiratory tract and gastrointestinal infections. Both clinical and genetic data support a close relationship with common variable immunodeficiency, a disease which involves not only IgA and IgG production, but also, in half of the patients, IgM. It is likely that the two disorders represent an allelic condition with a variable expression of a common gene defect which is thought to be involved in the regulation of immunoglobulin class switching. It is possible that a single, autosomally inherited gene with a limited penetrance is responsible for the development of both these defects.

Immunological ReviewsVolume 138, Issue 1 p. 159-183 X-Linked Agammaglobulinemia and Other Immunoglobulin Deficiencies C. I. Edvard Smith, Corresponding Author C. I. Edvard Smith Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden Division of Clinical Immunology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden.C.I.E. Smith, Center for Bio Technology, Karolinska Insitute, NOVUM, S-141 57 Huddinge, Sweden.Search for more papers by this authorKhalid B. Islam, Khalid B. Islam Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden Division of Clinical Immunology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden.Search for more papers by this authorIgor Vořechovský, Igor Vořechovský Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, SwedenSearch for more papers by this authorOlle Olerup, Olle Olerup Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden Division of Clinical Immunology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden.Search for more papers by this authorErik Wallin, Erik Wallin Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, SwedenSearch for more papers by this authorHodjattallah Rabbani, Hodjattallah Rabbani Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden Division of Clinical Immunology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden.Search for more papers by this authorBerivan Baskin, Berivan Baskin Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden Division of Clinical Immunology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden.Search for more papers by this authorLennart Hammarström, Lennart Hammarström Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden Division of Clinical Immunology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden.Search for more papers by this author C. I. Edvard Smith, Corresponding Author C. I. Edvard Smith Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden Division of Clinical Immunology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden.C.I.E. Smith, Center for Bio Technology, Karolinska Insitute, NOVUM, S-141 57 Huddinge, Sweden.Search for more papers by this authorKhalid B. Islam, Khalid B. Islam Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden Division of Clinical Immunology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden.Search for more papers by this authorIgor Vořechovský, Igor Vořechovský Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, SwedenSearch for more papers by this authorOlle Olerup, Olle Olerup Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden Division of Clinical Immunology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden.Search for more papers by this authorErik Wallin, Erik Wallin Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, SwedenSearch for more papers by this authorHodjattallah Rabbani, Hodjattallah Rabbani Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden Division of Clinical Immunology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden.Search for more papers by this authorBerivan Baskin, Berivan Baskin Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden Division of Clinical Immunology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden.Search for more papers by this authorLennart Hammarström, Lennart Hammarström Center for Bio Technology, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden Division of Clinical Immunology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden.Search for more papers by this author First published: April 1994 https://doi.org/10.1111/j.1600-065X.1994.tb00851.xCitations: 94Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume138, Issue1April 1994Pages 159-183 RelatedInformation

The most common form of primary immunodeficiency is IgA deficiency (IgAD). However, the molecular basis of this disease remains elusive. Therefore, to address this issue we made a systematic analysis of the molecular events leading to IgA production. B lymphocytes that produce IgA have undergone somatic rearrangement that joins the switch (S) mu to S alpha region with deletion of the intervening sequences. Examination of the resulting S mu/S alpha junctions in unstimulated PBMC from IgAD patients by nested primer PCR revealed a significant decrease in the number of the S mu/S alpha fragments. To obtain the antisense primers to generate the S mu/S alpha fragments, we sequenced the human S alpha 1 and the downstream region extending to the C alpha 1 locus. Similar to previously reported switch sequences, we also found the S alpha 1 to be predominantly composed of pentameric repeats GAGCT and GGGCT. The decrease in the number of S mu/S alpha fragments is consistent with a profound decrease in the C alpha membrane mRNA expression in unstimulated PBMC, as well as in the C alpha mRNA levels and IgA production in PWM-stimulated PBMC. Sequence analysis of the switch junctions from IgA-producing cell lines, control donors, and an IgAD patient showed direct joining in 8 of 9 cases examined. TGF-beta 1, previously shown to be the switch factor for human and mouse IgA, was also examined. No difference in the TGF-beta 1 mRNA levels in unstimulated PBMC between the control subjects and the IgAD patients were detected. Our findings indicate that the failure to switch to IgA-producing B lymphocytes, or an impaired survival of such cells, may be an important molecular mechanism in IgAD.

The gene mutated in the human disease, X-linked agammaglobulinemia (XLA), is related to the Src gene family of cytoplasmic protein-tyrosine kinases and is designated Btk (Bruton's agammaglobulinemia tyrosine kinase; formerly Atk/Bpk; the human gene is denoted BTK, using capital letters according to the kinase nomenclature). We have recently reported that this gene is expressed in B lymphocytes and that the specific mRNA was undetectable in T cells using Northern blotting. Further analyses of different sources of B and T lymphocytes confirmed this pattern. However, BTK transcripts were undetectable in four plasmacytoma lines. Moreover, as virtually normal amounts of BTK transcripts were found in PBMC from two patients carrying a point mutation in BTK, despite low B cell numbers, we anticipated that the gene would also be expressed in cells of other lineages. The erythroleukemia cell line K-562, the promyelocytic line HL-60 and the histiocytic lymphoma line U-937 were found to have BTK mRNA levels comparable to B cells. BTK mRNA was also detected in monocytes from healthy donors as well as in the human immature basophilic cell line KU812, in the human mast cell leukemia cell line HMC-1 and in the CD34 expressing myeloblast KG-1. A similar expression pattern was obtained when BTK protein was analyzed by immunoprecipitation and Western blotting. Using a polymerase chain reaction-based analysis, a small amount (less than 1% of the level in B cells) of BTK mRNA was identified in T lymphocytes. Our findings are compatible with a general expression of the BTK gene in hematopoietic cells, except in T lymphocytes and plasma cells, in which the transcript level is selectively down-regulated.

SUMMARY Previous in vitro studies suggest that transcription of the unrearranged immunoglobulin switch region and its 5′ flanking region precedes isotype switching. These transcripts, which arc devoid of a variable region, contain unique exons and are called germ-line (GL) mRNA. A crucial point in this regard is whether such transcripts could be detected in vivo, and if their expression correlates with immunoglobulin class switching in health and disease. To understand the in vivo role of this transcriptional activity we have adapted the reverse transcription-polymerase chain reaction (PCR) to analyse the GL transcripts from unstimulated peripheral blood mononuclear cells (PBMC) in healthy individuals and in different immunological diseases. Furthermore, mononuclear cells from different human organs were also analysed. We report here that GL (Iα. Iγ and Iɛ used lo designate the GL mRNA for IgA. IgG and IgE, respectively) nRNA are expressed differentially during ontogeny of B cells. Unexpectedly, no difference of Iα mRNA expression between the PBMC and the secondary lymphoid organs was detected. Rather. the levels of GL transcripts were correlated to ihe number of sIgM+ cells. GL mKNA of all three isotypes could be detected in PBMC from healthy donors, whereas there was a decrease of specific GL transcript synthesis in individuals with Immunoglobulin deficiency. Furthermore, during the in vivo immune response in a parasitic infection, we could demonstrate an induction of GL k mRNA during in vivo immune response. Concomitantly. there was also increased synthesis of productive F. transcripts. These findings implicate a potential role of GL transcription during in vivo immunoglobulin class switching.

Immunoglobulin isotype switching is preceded by transcription of exons located 5' of the immunoglobulin switch regions. These exons are referred to as 'I-exons' and transcription of these regions is believed to be an essential step in switch recombination. Such mRNA species lack a variable portion and are denoted 'germline' transcripts. We have previously identified I-regions in man and we have now investigated the in vivo expression of human germline transcripts. Germline alpha transcripts (containing an I alpha exon) were expressed in various lymphoid organs, including peripheral blood mononuclear cells. Decreased levels of germline transcripts were found in immunoglobulin deficiency diseases. These findings are compatible with an in vivo role of germline transcription in isotype switching and may contribute to the understanding of mechanisms underlying deficiency diseases.