Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains

Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow cytometry and were clonal by Southern blotting. Surprisingly, two of the mu/lambda-expressing cell lines contained both kappa alleles in germline configuration, and synthesis/expression of conventional lambda L chains was directly proven by immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in one of them. Thus, human fetal bone marrow B lineage cells harbor the capacity to make functional lambda L chain gene rearrangements without rearranging or deleting either kappa allele. A third unusual cell line, designated 30.30, was observed to coexpress cell surface kappa and lambda L chains associated with mu H chains. The 30.30 cell line had a diploid karyotype, a single H chain rearrangement, both kappa alleles rearranged, and a single lambda rearrangement. Immunoprecipitation/SDS-PAGE confirmed that 30.30 cells synthesized and expressed kappa and lambda L chains. Multiparameter flow cytometry was used to demonstrate the existence of kappa+/lambda+ cells in fetal bone marrow and fetal spleen at frequencies of 2-3% of the total surface Ig+ B cell population. The flow cytometry data was confirmed by two-color immunofluorescence microscopy. The existence of normal human B cells expressing cell surface kappa and lambda refutes the widely accepted concept that expression of a single L chain isotype is immutable. The kappa+/lambda+ cells may represent transients undergoing L chain isotype switching.     

Epstein-Barr virus-induced precursor B cell lines from patients with congenital agammaglobulinemia

We have established lymphoid cell lines with diversity of immunoglobulin expression by infection of bone marrow cells of four patients with congenital agammaglobulinemia with Epstein-Barr virus. Immunofluorescent study revealed that the cells of many of these lymphoid cell lines had characteristics of possible precursor B cells, permitting classification as follows: 1) cell lines without any surface or cytoplasmic immunoglobulins (17/28 lines), 2) cell lines which had only cytoplasmic mu heavy chains (line K4 and O2), 3) cell lines with both mu and delta heavy chains in the cytoplasm (line O6 and M5), 4) cell lines which bore surface mu chains and possessed cytoplasmic mu chains but lacked light chain expression (line S5), 5) cell lines which had surface and cytoplasmic delta and lambda chains, and secreted the immunoglobulins (line K5), and 6) cell lines which had surface IgM and cytoplasmic mu and light chains, and secreted the immunoglobulins (5/28 lines). Biosynthetic studies with 14C-leucine confirmed the patterns of immunoglobulin expression found by immunofluorescent analysis. These cell lines may represent some stages of B cell development and provide useful information on the pathophysiology of congenital agammaglobulinemia.     

Regulation of B Lymphocyte Development by the Truncated Immunoglobulin Heavy Chain Protein Dμ

The development of B lymphocytes from progenitor cells is dependent on the expression of a pre–B cell–specific receptor made up by a μ heavy chain associated with the surrogate light chains, immunoglobulin (Ig)α, and Igβ. A variant pre–B cell receptor can be formed in which the μ heavy chain is exchanged for a truncated μ chain denoted Dμ. To investigate the role of this receptor in the development of B cells, we have generated transgenic mice that express the Dμ protein in cells of the B lineage. Analysis of these mice reveal that Dμ expression leads to a partial block in B cell development at the early pre–B cell stage, probably by inhibiting V_H to D_HJ_H rearrangement. Furthermore, we provide evidence that Dμ induces V_L to J_L rearrangements.     

Differential regulation of membrane and secretory mu chain synthesis in human beta cell lines. Regulation of membrane mu or secreted mu

Regulation of membrane and secretory mu synthesis was examined in human lymphoblastoid cell lines representing various stages of differentiation. Immunoglobulin phenotype was determined by surface and cytoplasmic staining with fluorochrome-conjugated antibodies and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of anti-mu precipitable cellular products. The thymidine analogue, 5-bromo-2'-deoxyuridine (BUdR), which inhibits differentiation-specific proteins in a variety of systems, was used to examine regulation of immunoglobulin synthesis. We found that BUdR had a differential effect on membrane (mum) and secretory (mus) type mu heavy chains. Ig production in pre-B and plasma cell-like lines, which make mus, was unaffected by BUdR. However, surface expression of IgM (mum) in B cell lines was drastically inhibited at similar doses of BUdR without diminishing total Ig or protein synthesis. Examination of labeled mu chains from control and BUdR-treated B cell lines by SDS- PAGE revealed the production of two sizes of mu (mum and mus) in control cells and only the smaller size (mus) in BUdR-treated cells. This size difference could not be attributed to alterations in glycosylation of the molecules. These data show that BUdR inhibits the production of membrane mu chains without diminishing secretory mu chain synthesis in the same cell. Our findings suggest that thymidine-rich regions of the genome are involved in the regulation of mum vs. mus during B cell differentiation.     

Regulation of B Cell Development by Variable Gene Complexity in Mice Reconstituted with Human Immunoglobulin Yeast Artificial Chromosomes

The relationship between variable (V) gene complexity and the efficiency of B cell development was studied in strains of mice deficient in mouse antibody production and engineered with yeast artificial chromosomes (YACs) containing different sized fragments of the human heavy (H) chain and κ light (L) chain loci. Each of the two H and the two κ chain fragments encompasses, in germline configuration, the same core variable and constant regions but contains different numbers of unique V_H (5 versus 66) or Vκ genes (3 versus 32). Although each of these YACs was able to substitute for its respective inactivated murine counterpart to induce B cell development and to support production of human immunoglobulins (Igs), major differences in the efficiency of B cell development were detected. Whereas the YACs with great V gene complexity restored efficient development throughout all the different recombination and expression stages, the YACs with limited V gene repertoire exhibited inefficient differentiation with significant blocks at critical stages of B cell development in the bone marrow and peripheral lymphoid tissues. Our analysis identified four key checkpoints regulated by V_H and Vκ gene complexity: (a) production of functional μ chains at the transition from the pre B-I to the pre B-II stage; (b) productive VκJκ recombination at the small pre B-II stage; (c) formation of surface Ig molecules through pairing of μ chains with L chains; and (d) maturation of B cells. These findings demonstrate that V gene complexity is essential not only for production of a diverse repertoire of antigen-specific antibodies but also for efficient development of the B cell lineage.     

The Immunoglobulin (Ig)α and Igβ Cytoplasmic Domains Are Independently Sufficient to Signal B Cell Maturation and Activation in Transgenic Mice

The B cell antigen receptor, composed of membrane immunoglobulin (Ig) sheathed by the Igα/Igβ heterodimer plays a critical role in mediating B cell development and responses to antigen. The cytoplasmic tails of Igα and Igβ differ substantially but have been well conserved in evolution. Transfection experiments have revealed that, while these tails share an esssential tyrosine-based activation motif (ITAM), they perform differently in some but not all assays and have been proposed to recruit distinct downstream effectors. We have created transgenic mouse lines expressing chimeric receptors comprising an IgM fused to the cytoplasmic domain of each of the sheath polypeptides. IgM/α and IgM/β chimeras (but not an IgM/β with mutant ITAM) are each independently sufficient to mediate allelic exclusion, rescue B cell development in gene-targeted Igμ⁻ mice that lack endogenous antigen receptors, as well as signal for B7 upregulation. While the (IgM/α) × (IgM/β) double-transgenic mouse revealed somewhat more efficient allelic exclusion, our data indicate that each of the sheath polypeptides is sufficient to mediate many of the essential functions of the B cell antigen receptor, even if the combination gives optimal activity.     

Counterselection against Dμ Is Mediated through Immunoglobulin (Ig)α-Igβ

The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins that lack a complete variable region (Dμ). Positive selection is known to be mediated by membrane immunoglobulin heavy chains through Igα-Igβ, whereas the mechanism for counterselection against Dμ has not been determined. We have examined the role of the Igα-Igβ signal transducers in counterselection against Dμ using mice that lack Igβ. We found that Dμ expression is not selected against in developing B cells in Igβ mutant mice. Thus, the molecular mechanism for counterselection against Dμ in pre-B cells resembles positive selection in that it requires interaction between mDμ and Igα-Igβ.     

MOUSE THYMUS-INDEPENDENT AND THYMUS-DERIVED LYMPHOID CELLS : I. IMMUNOFLUORESCENT AND FUNCTIONAL STUDIES

The simultaneous use on mouse lymphoid suspensions of heterologous antisera directed against thymus-derived (T) cell mouse-specific lymphocyte antigen and brain-associated theta antigen (MSLA and BAθ) or thymus-independent (B) cell mouse-specific bone marrow-derived lymphocyte antigen (MBLA) surface antigens allowed direct proof of the different specificity of these antisera by double immunofluorescence (IF) staining with selective visualization of fluorochromes. These antisera and antisera against mouse Ig and its different types of chains were then used with technique of either double IF staining or IF combined with radioautography, allowing the following conclusions: (a) Surface Ig (sIg) was found exclusively on B cells and never on T cells, but not all B cells had sIg. Cells containing detectable amounts of Ig were MBLA+, but had less sIg than other B cells or none at all. There was evidence for the existence of a significant number of MBLA+ lymphocytes, neither bearing nor containing detectable Ig. (b) µ-Chains were the most frequent but not the only heavy chains found on spleen cells; however, it could not be decided with the technique used, if a single cell can bear more than one type of heavy chain. No cell containing γ-chains was found to bear surface µ-chains, although a very few cells containing both µ- and γ-chains were observed. (c) The antigen-binding cells detected after immunization with bacteriophage T4, bovine serum albumin, Maia squinado hemocyanin, and sheep erythrocytes were analyzed for MSLA, MBLA or sIg using double IF, a combination of IF and radioautography, or inhibition of "rosette" formation. Practically all the antigen-binding cells detected were MSLA-, MBLA+, sIg+. (d) More B cells than T cells were found among short-lived lymphoid cells labeled by repeated in vivo injections of tritiated thymidine, but the results did not support a simplified concept equating T cells to long-lived and B cells to short-lived lymphocytes. (e) Cells dividing rapidly in the lymph nodes draining the sites of immunization with various antigens were predominantly T cells 2 days after immunization and in majority B cells a few days later. (f) Incubation of lymphoid cells at 37°C with rabbit anti-mouse Ig or anti-κ chains led to complete disappearance of sIg and to decrease of MBLA ("antigenic modulation"). In the same conditions, anti-MBLA gave partial modulation of MBLA and of sIg; MBLA, however, reappeared much faster than sIg. No modulation of T cell surface antigens by the appropriate antisera was observed. Cell treatment with Pronase could remove MBLA, sIg, MSLA, and BAθ, which reappeared within a few hours. Neuraminidase treatment was without detectable effect on these antigens.     

Chromosomal location of human kappa and lambda immunoglobulin light chain constant region genes

The chromosomal location of human constant region light chain immunoglobulin (Ig) genes has been determined by analyzing a group of human fibroblast/rodent somatic cell hybrids with nucleic acid probes prepared from cloned human kappa and lambda constant region genes. Human chromosomes in each cell line were identified by isoenzyme analysis. The DNA from hybrid cells was digested with restriction endonucleases, size fractionated by gel electrophoresis, transferred to nitrocellulose or DBM paper, and hybridized with (32)P-labeled nucleic acid probes. The C(κ) gene was assigned to human chromosome 2 and the C(λ) genes to chromosome 22, based upon analysis of these hybrid cell lines, and these assignments were confirmed by analysis of subclones. A group of previously unassigned loci can be mapped to chromosome 2 by virtue of their close linkage to C(κ). The λ and κ light chain and heavy chain Ig genes have now been assigned to all three human chromosomes that are involved in translocations with chromosome 8 in human B cell neoplasms. These techniques and probes provide a means to study the detailed arrangement of human Ig genes and their pseudogenes.     

Translational Control of Hemoglobin Synthesis in Thalassemic Bone Marrow

Previous studies of β-thalassemic reticulocytes have implied a decreased amount of functional β-mRNA but unimpaired translation of the β-mRNA present. However, the β/α synthetic ratios in β-thalassemic marrow are higher than those observed in reticulocytes of the same patients. This could imply that marrow cells contain an abnormally functioning β-mRNA no longer active in reticulocytes. To test the function of mRNA found in marrow, intact cells were incubated with [³⁵S]methionine and the relative amounts of nascent α- and β-chains on polysomes of different sizes were measured by tryptic digestion and determination of the specific activities of the respective peptides. Results showed that in normal and β-thalassemic marrow, as well as in reticulocytes, β-chain production, though deficient, occurs predominantly on larger polysomes than the production of α-chains. In one patient with severe thalassemia and very little production of β-chains in marrow or reticulocytes, δ-chain synthesis was found predominantly on larger polysomes than α-chain synthesis. These results indicate that in β-thalassemic as well as in nonthalassemic marrow and reticulocytes, each β- and δ-mRNA initiates protein synthesis at a rate faster than does each α-mRNA, and suggest that the β-mRNA in contact with polyribosomes is normally functioning but quantitatively deficient in β-thalassemic marrow as well as in reticulocytes. No translational defect was detected in a similar study performed in reticulocytes of a patient with hemoglobin H disease, suggesting a normally functioning mRNA in contact with polyribosomes in this condition as well. In both thalassemias, unbalanced synthesis of α- and β-chains was more pronounced on polysomes than in completed chains. This difference possibly reflects a compensatory delay in translation of the nonthalassemic chain, which is present in excess.     

Early Function of Pax5 (BSAP) before the Pre-B Cell Receptor Stage of B Lymphopoiesis

The formation of the pre-B cell receptor (BCR) corresponds to an important checkpoint in B cell development that selects pro-B (pre-BI) cells expressing a functionally rearranged immunoglobulin μ (Igμ) heavy chain protein to undergo the transition to the pre-B (pre-BII) cell stage. The pre-BCR contains, in addition to Igμ, the surrogate light chains λ5 and VpreB and the signal transducing proteins Igα and Igβ. The absence of one of these pre-BCR components is known to arrest B cell development at the pre-BI cell stage. Disruption of the Pax5 gene, which codes for the B cell–specific activator protein (BSAP), also blocks adult B lymphopoiesis at the pre-BI cell stage. Moreover, expression of the mb-1 (Igα) gene and V_H-to-D_HJ_H recombination at the IgH locus are reduced in Pax5-deficient B lymphocytes ∼10- and ∼50-fold, respectively. Here we demonstrate that complementation of these deficiencies in pre-BCR components by expression of functionally rearranged Igμ and chimeric Igμ-Igβ transgenes fails to advance B cell development to the pre-BII cell stage in Pax5 (−/−) mice in contrast to RAG2 (−/−) mice. Furthermore, the pre-BCR is stably expressed on cultured pre-BI cells from Igμ transgenic, Pax5-deficient bone marrow, but is unable to elicit its normal signaling responses. In addition, the early developmental block is unlikely to be caused by the absence of a survival signal, as it could not be rescued by expression of a bcl2 transgene in Pax5-deficient pre-BI cells. Together, these data demonstrate that the absence of Pax5 arrests adult B lymphopoiesis at an early developmental stage that is unresponsive to pre-BCR signaling.     

IMMUNOSUPPRESSION OF MICE INJECTED WITH HETEROLOGOUS ANTI-IMMUNOGLOBULIN HEAVY CHAIN ANTISERA

Neonatal injection of mice with rabbit anti-µ antiserum has been shown to produce complete loss of direct and indirect plaque-forming responses to sheep erythrocytes as well as loss of serum IgM and severe depressions of all other serum immunoglobulins. Similar injection of anti-γ1γ2 or anti-γ1 antibodies effects a loss of the indirect response but induces relatively minor alterations in serum Ig levels. Delaying initiation of anti-µ treatment until young adulthood results in a somewhat diminished effect on plaque-forming responses and serum Ig levels but triggers the release of high serum levels of an aberrant µ-bearing protein. Anti-µ suppression of genetically thymusless mice indicates that at least part of the target cells for suppression are bone marrow derived. A working hypothesis for the maturation of humoral antibody-producing cell lines as it relates to these data is discussed.     

The Normal Counterpart of IgD Myeloma Cells in Germinal Center Displays Extensively Mutated IgVH Gene, Cμ–Cδ Switch, and λ Light Chain Expression

Human myeloma are incurable hematologic cancers of immunoglobulin-secreting plasma cells in bone marrow. Although malignant plasma cells can be almost eradicated from the patient's bone marrow by chemotherapy, drug-resistant myeloma precursor cells persist in an apparently cryptic compartment. Controversy exists as to whether myeloma precursor cells are hematopoietic stem cells, pre–B cells, germinal center (GC) B cells, circulating memory cells, or plasma blasts. This situation reflects what has been a general problem in cancer research for years: how to compare a tumor with its normal counterpart. Although several studies have demonstrated somatically mutated immunoglobulin variable region genes in multiple myeloma, it is unclear if myeloma cells are derived from GCs or post-GC memory B cells. Immunoglobulin (Ig)D-secreting myeloma have two unique immunoglobulin features, including a biased λ light chain expression and a Cμ–Cδ isotype switch. Using surface markers, we have previously isolated a population of surface IgM⁻IgD⁺CD38⁺ GC B cells that carry the most impressive somatic mutation in their IgV genes. Here we show that this population of GC B cells displays the two molecular features of IgD-secreting myeloma cells: a biased λ light chain expression and a Cμ–Cδ isotype switch. The demonstration of these peculiar GC B cells to differentiate into IgD-secreting plasma cells but not memory B cells both in vivo and in vitro suggests that IgD-secreting plasma and myeloma cells are derived from GCs.     

the relationship between surface immunoglobulin isotype and immune function of murine B lymphocytes. III. Expression of a single predominant isotype on primed and unprimed B cells

We determined whether primed and unprimed B cells in the spleen of (BALB/c × C57BL/Ka)F(1) mice contain subpopulations that express a predominant surface Ig isotype. Spleen cells were stained for surface isotypes and sorted on the fluorescence-activated cell sorter (FACS) in order to obtain B cells bearing predominantly IgM (μp cells), IgD (δp cells), or IgG (γp cells). Each population was assayed for its capacity to restore the adoptive primary and secondary anti-bovine serum albumin (BSA) antibody response in irradiated syngeneic recipients. In addition, the adoptive response restored by isotype-predominant cells was compared to that restored by isotype- positive cells (B cells bearing a given surface isotype alone or in combination with others). The experimental results show that μp cells restore the adoptive primary and secondary IgM and IgG responses to BSA, and γP cells restore only the primary and secondary IgG response. Δp Cells restored the adoptive secondary IgG response, but failed to restore the adoptive primary response at the cell doses tested. ΓP Cells but not δp cells suppressed the IgM response of the μ(+) and δ(+) cells. The contribution of isotype-predominant cells to both the adoptive primary and secondary anti-BSA response was smaller than that of B cells bearing a combination of surface isotypes. Differences in the Ig isotype pattern expressed on the surface of primed and unprimed B cells are discussed.     

Immunoglobulin production in Epstein-Barr virus-induced human monoclonal lymphoblastoid cell lines

In order to establish clonal human lymphoblastoid cell lines, human tonsillar lymphocytes infected with Epstein-Barr virus were directly seeded in semi-solid agar. After four weeks, resulting colonies were randomly isolated and transferred to suspension culture. At around three months after the initiation of the culture, immunoglobulin (Ig) production of 17 clones thus established was determined. Cytoplasmic and membrane Ig were detected by immunofluorescence staining and secretory Ig in the culture supernatants was detected by double immunodiffusion. The patterns of Ig production by these cell lines were clear-cut. Each clone produced and secreted one class of heavy chains and one type of light chains, i.e., a single Ig. Of 17 clones, 13 clones were IgM producers, 3 clones were IgA producers and one clone was an IgG producer. Membrane Ig of these clones was also identical with cytoplasmic/secretory Ig with the exceptions of three IgM producers in which membrane IgD as well as IgM was detected and the one IgG producer in which no membrane Ig was detected.     

A novel anti-lung cancer agent inhibits proliferation and epithelial–mesenchymal transition

ObjectiveTo synthesize a novel chalcone-1,3,4-thiadiazole hybrid and investigate its anticancer effects against NCI-H460 cells.Methods(E)-3-(4-bromophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one, 1,3-dibromopropane and 1,3,4-thiadiazole-2-thiol were used as chemical materials to synthesize compound ZW97. The NCI-H460 lung cancer cell line was selected to explore the antitumor effects of compound ZW97 in vitro and in vivo.ResultsCompound ZW97 selectively inhibited cell proliferation against lung cancer cell lines NCI-H460, HCC-44 and NCI-H3122 with IC₅₀ values of 0.15 μM, 2.06 μM and 1.17 μM, respectively. ZW97 suppressed migration and the epithelial–mesenchymal transition process in NCI-H460 cells in a concentration-dependent manner. Based on the kinase activity results and docking analysis, compound ZW97 is a novel tyrosine-protein kinase Met (c-Met kinase) inhibitor. It also inhibited NCI-H460 cell growth in xenograft models without obvious toxicity to normal tissues.ConclusionsCompound ZW97 is a potential c-Met inhibitor that might be a promising agent to treat lung cancer by inhibiting the epithelial–mesenchymal transition process.     

Soluble and Membrane-bound Forms of Signaling Lymphocytic Activation Molecule (SLAM) Induce Proliferation and Ig Synthesis by Activated Human B Lymphocytes

In this study it is shown that both membrane-bound and soluble forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by activated human B cells. Activated B cells express the membrane-bound form of SLAM (mSLAM), the soluble (s) and the cytoplasmic (c) isoforms of SLAM, and the expression levels of mSLAM on B cells are rapidly upregulated after activation in vitro. Importantly, recombinant sSLAM and L cells transfected with mSLAM efficiently enhance B cell proliferation induced by anti-μ mAbs, anti-CD40 mAbs or Staphylococcus aureus Cowan I (SAC) in the presence or absence of IL-2, IL-4, IL-10, IL-12, or IL-15. sSLAM strongly enhances proliferation of both freshly isolated B cells and B cells derived from long-term in vitro cultures, indicating that SLAM acts not only during the initial phase of B cell activation but also during the expansion of preactivated B cells. In addition, sSLAM enhances production of IgM, IgG, and IgA by B cells activated by antiCD40 mAbs. SLAM has recently been shown to be a high affinity self-ligand, and the present data suggest that signaling through homophilic SLAM–SLAM binding during B–B and B–T cell interactions enhances the expansion and differentiation of activated B cells.     

Suppressive Role of B Cells in Chronic Colitis of T Cell Receptor α Mutant Mice

The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-α^−/− mice was explored by creating double mutant mice (TCR-α^−/− × immunoglobulin (Ig)μ^−/−), which lack B cells. TCR-α^−/− × Igμ^−/− mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-α^−/− mice. Colitis was induced in recombination-activating gene-1 (RAG-1^−/−) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-α^−/− × Igμ^−/− mice. When purified B cells from TCR-α^−/− mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1^−/− mice. Administration of the purified Ig from TCR-α^−/− mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-α^−/− × Igμ^−/− mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-α^−/− × Igμ^−/− mice as compared to Igμ^−/− mice and TCR-α^−/− mice. Administration of the purified Ig from TCR-α^−/− mice into TCR-α^−/− × Igμ^−/− mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.     

Studies of T- and B-Cell Interactions in Adult Patients with Combined Immunodeficiency

Cellular interactions involved in the pathogenesis of hypogammaglobulinemia were studied in six patients with common variable immunodeficiency. Amounts of immunoglobulin (Ig)G and IgM in the supernate of pokeweed mitogen-stimulated cocultures of normal and immunodeficient mononuclear cells were measured by radioimmunoassays. Mononuclear cells from three of six patients inhibited Ig production of normal B cells (P < 0.005). When purified patient and normal T cells were added to B cells in various autologous or allogeneic combinations, it was observed that immunodeficient T cells (AT) from four patients suppressed normal IgM synthesis. Allogeneic normal T cells did not provide help for B cells from these same immunodeficient patients. In two patients, autologous T cells were able to help autologous B-cell IgM synthesis in vitro. In five patients, AT cells inhibited normal B-cell IgG synthesis. Removal of T cells bearing Ia determinants or T cells with Fc-IgG receptors did not diminish the suppressive effect of AT cells on normal B-cell Ig synthesis. Addition of indomethacin, a prostaglandin synthetase inhibitor, did not abrogate the suppressive effect of immunodeficient mononuclear cells. Addition of hydrocortisone succinate (10 μM) did reverse the suppressive effect of AT cells on IgM production in one patient; however, no in vitro reversal of suppressor cell effect was recorded in five. Suppression by immune-deficient T cells was eliminated by 2,000 rad of x-ray irradiation in three patients. After x-ray irradiation immunedeficient T cells could function as helpers of normal B cells.     

Novel dermacozine-1-carboxamides as promising anticancer agents with tubulin polymerization inhibitory activity

In the present study, novel dermacozine-1-carboxamides (8a–8n) were synthesized and screened for their in vitro cytotoxic activity against three different cancer cell lines: MCF-7 (breast cancer), A-549 (lung cancer) and DU145 (prostate cancer). All the compounds showed more efficiency against the DU145 cell line than against the MCF-7 and A-549 cell lines. Furthermore, 8a (CTC₅₀: 7.02 μM) and 8l (CTC₅₀: 6.32 μM) have been found to be more effective against the DU145 cells as they arrest the cell cycle at the G2/M phase by interfering with tubulin polymerization; these results indicate that these compounds act as potential anti-cancer agents by inhibiting tubulin polymerization.

In the present study, novel dermacozine-1-carboxamides (8a–8n) were synthesized and screened for their in vitro cytotoxic activity against three different cancer cell lines: MCF-7 (breast cancer), A-549 (lung cancer) and DU145 (prostate cancer).