PubMed Health⌕ Search

Biomedical subjects

S S Alkan

Publications and source records attributed to S S Alkan.

At least 19 recordsLinked to original sources
PubMed1991

Functional heterogeneity of CD4-positive T-cell subsets: the correlation between effector functions and lymphokine secretion is limited.

Several effector functions and the lymphokine secretion pattern of 30 antigen-specific CD4+ T-cell clones have been investigated. The clones were generated directly by limiting dilution cloning of nylon wool-purified T-cells obtained from KLH immunized BALB/c mice and avoiding an initial bulk culture phase. Using this approach the CD4+ T-cell clones were grouped into helper and nonhelper subsets. Among the helper subset, clones which helped B-cells for specific antibody production by either cognate or noncognate recognition were identified. Some but not all of these helper clones fitted into the Th1 and Th2 scheme, if the lymphokine secretion pattern was evaluated. Among the nonhelper subset CD4+ clones which killed activated APC in a MHC class II-restricted and antigen-specific manner were identified. In addition, one clone which suppressed B-cell antibody production mediated by helper clones was found. However, neither the suppression of antibody responses nor the inability of the nonhelper clones to help B-cells is due to the killing of B-cells. Various attempts were made to convert nonhelper into helper clones and helper into killer clones, without success. Thus, the functional properties of these clones are stable traits and not convertible by varying the experimental conditions.

Animals↗
PubMed1990

Three human interferon-alpha 2 subvariants disclose structural and functional differences.

The human interferon-alpha 2 subvariants 2a, 2b and 2c differ by only one or two amino acids at positions 23 and/or 34 of the mature protein. In this study, the coding regions of the three interferon-alpha 2 subvariants were derived from the cDNA of interferon-alpha 2c by site-directed in vitro mutagenesis. The interferon-alpha subvariants were synthesized using the same Escherichia coli strain for production and were subsequently purified. Comparative studies revealed that they differ significantly in their biological and antigenic properties. Therefore, amino acid positions 23 and 34 seem to be crucial for structure/function of human interferon-alpha. Furthermore, the study points to the importance of defining, whether such minor structural variants of naturally occurring polypeptides represent functional variants.

Amino Acids↗
PubMed1989

Structural characterization of antiidiotypic antibodies. Evidence that Ab2s are derived from the germline differently than Ab1s.

We have found that syngeneic Ab2s in the antiarsonate system are serologically and structurally similar to one another. In contrast, the allogeneic Ab2 response is heterogeneous and derives from a large number of unrelated germline gene segments. The Ab2 response of the BALB/c strain to polyclonal A/J Ars A molecules can probably best be compared with a response to a foreign protein and might have been predicted in a strain that completely lacks the H chain V region gene from which the Ab1 derives. Partial variable region sequences of Ab2s from three other systems in addition to previously reported Ab2 structures indicates that this difference in allogeneic vs. syngeneic Ab2s may be a general phenomena. These data support Jerne's hypothesis of complementary V region genes existing in the germline. However, there is good evidence that these antiidiotypic antibodies are not derived directly from the germline, as somatic processes most likely play an important role in their generation. The D segments of Ab2s in the arsonate system as well as in other systems, are novel in structure and cannot easily be explained by previously described germline D segments. D-D fusion may play a role in the generation of the third hypervariable region in these antibodies.

Amino Acid Sequence↗
PubMed1989

Tenascin, an extracellular matrix protein, exerts immunomodulatory activities.

Tenascin is a nonubiquitous extracellular matrix protein mainly expressed during morphogenesis in embryonal life. In adults it reappears in malignant tumors and during inflammation and tissue repair. Extracellular matrix proteins can alter cell morphology, adhesion, motility, differentiation, and growth. Since cells of the immune system can express receptors for extracellular matrix, we investigated the effects of tenascin on human monocytes and T and B lymphocytes. Tenascin inhibited monocyte adhesion to fibronectin and enhanced the LFA-1 (lymphocyte function-associated antigen 1)-dependent clustering of Epstein-Barr virus-transformed B cells. The physiological consequences of the effects of tenascin were studied in several T-cell activation models. Tenascin inhibited T-cell activation induced by a soluble antigen (tetanus toxoid), alloantigens, or the mitogen concanavalin A. However, T-cell activation with phytohemagglutinin, crosslinked anti-CD3 antibody, or a mixture of ionomycin and phorbol ester was not inhibited by tenascin. Tenascin did not prevent interleukin 2-dependent T-cell growth or the cytolytic activity of an antigen-specific CD4+ T-cell clone. These results suggest that tenascin alters the adhesion properties of human monocytes, B cells, and T cells. The in vitro immunosuppressive activity of tenascin might be due to abrogation of an accessory cell function at an early stage of the interaction between antigen-presenting cells and T cells.

Adjuvants, Immunologic↗
PubMed1988

Enhanced antiproliferative action of interferon targeted by bispecific monoclonal antibodies.

It has previously been shown that interferon (IFN) can be coupled covalently to tumor-specific monoclonal antibodies (mAb) and that the in vitro antiviral and antiproliferative action of these IFN-mAb conjugates is superior to that of uncoupled IFN. We now report a different mode of IFN delivery, i.e., via bispecific mAbs, avoiding chemical coupling of IFN. Bispecific mAbs were prepared by cross-linking two mAbs with SPDP, mAb1 being specific for an idiotype of a hybridoma cell-surface immunoglobulin and mAb2 specific for an IFN. Alternatively, Fab' fractions of mAb1 and mAb2 were coupled by disulfide formation to produce F(ab')2. Binding capacity and specificity of both arms of the mAb conjugates were first demonstrated by a solid-phase radioimmunoassay using idiotype-positive mAb as test antigen and 125I-labeled hybrid IFN-alpha B/D. Secondly, hybridomas either idiotype positive or negative were incubated with bispecific mAbs (mAb1-mAb2 or Fab'1-Fab'2) and 125I-labeled IFN at 4 degrees C. After washing away unbound reagents, the uptake of radioactivity into cells was determined. Additionally, the antiproliferative action of cold or labeled IFN targeted via different modes was assessed by an [3H]TdR incorporation method. Results showed that bispecific mAbs could specifically deliver IFN to the target cells and also inhibit their growth in vitro. Furthermore, targeting IFN by any of the three methods, IFN-mAb, mAb1-mAb2, or Fab'1-Fab'2, enhanced its in vitro antiproliferative potency compared to IFN alone.

Animals↗
PubMed1988

Characterization of a monoclonal antibody against infective larvae of Brugia malayi.

Monoclonal antibodies were produced following immunization of mice with live infective larvae of Brugia malayi. One of these, 46.08.76, is an antibody that promotes adherence of mouse peritoneal macrophages and human peripheral blood leucocytes to the infective larvae of B. malayi and Wuchereria bancrofti, respectively, and kills them. Fresh normal serum, as a source of complement, augments this effect. The same monoclonal antibody conferred 89% protection to jirds (Meriones unguiculatus) against challenge infection of B. malayi stage-three larvae. This monoclonal antibody recognizes antigens of 80,000, 67,000, 52,000 and 36,000 MW proteins present among the antigens of larvae, as detected by an immunoblotting technique. The antibody also reacts with antigens of infective larvae of Litomosoides carinii, Dipetalonema viteae and B. pahangi, but to a smaller extent.

Animals↗
PubMed1987

Estimation of heterokaryon formation and hybridoma growth in murine and human cell fusions.

Four mouse myelomas commonly used for cell fusions (X63.Ag8.653, SP2/0, NS1, P3U1), 3 human myeloma-like cell lines (ARH77, U-266, GM1500) and 3 human x mouse hybridomas (SPAZ4, SA2, SA3) were compared for their heterokaryon formation and successful hybridoma growth after cell fusion with polyethylene glycol. The cells were stained with different fluorescent dyes which do not alter hybridoma growth or antibody secretion. After fusion myeloma cells containing at least 1 nucleus from a lymphocyte (heterokaryons) were counted from fluorescence photomicrographs and the heterokaryon frequency was calculated. Mouse myelomas fused at a frequency of 1-7%, whereas human myeloma lines showed a higher heterokaryon frequency ranging from 3-25%. In mouse fusions almost every well contained growing hybridomas showing a minimum hybridoma frequency of 2/10(6) lymphocytes. In human fusions the SPAZ4 and SA2 lines showed a heterokaryon frequency nearly as good as mouse myelomas, whereas U-266 yielded no growing hybridomas despite 20% heterokaryon frequency. Furthermore, human cell lines showed a high tendency of multikaryon formation whereas this phenomenon was rarely observed with murine and murine x human heterohybrids. In individual fusion experiments no correlation was found between heterokaryon formation and the number of growing hybridomas. Thus, our study shows that defects in hybridoma growth may not always result from lack of a successful fusion and human hybridomas might be more sensitive to post-fusion conditions.

Animals↗
PubMed1987

Prostaglandin E2 (PGE2)-dependent suppression of interleukin alpha (IL-2) production in patients with major trauma.

The depression of interleukin-2 synthesis represents a major dysfunction within the cascade of immunologic defects induced by mechanical and thermal trauma. This study was undertaken to elucidate the negative control mechanisms that were responsible for the deficiency of IL-2 production in polytraumatized patients. Peripheral blood mononuclear cells (PBMC's) from 29 patients (average age, 35.8 years; average ISS, 35) were separated on post-trauma days 1, 3, 5, 7, 10, 14, and 21 and cultured as untreated cells (C), cells treated with indomethacin (C + INDO), and cells depleted of adherent cells (C-AC). Cell cultures were assayed for proliferative responses to PHA, IL-2 synthesis, PGE2 production, gamma-interferon levels, and phenotyping studies. On all days post-trauma there was found a marked reduction of IL-2 production compared to controls with a highly significant nadir from day 5 to day 10 with an almost 80% inhibition of IL-2 (p less than 0.005). C + INDO cells showed increases of IL-2 synthesis over untreated cells ranging from 48% (Day 1) to 220% (Day 7). Removal of adherent cells (C-AC) did not reverse the suppression of IL-2 production. gamma-interferon levels were depressed in parallel with IL-2 levels but did not increase with C + INDO. The phenotyping of the PBMC's showed highly significant suppression of OKT3+, OKT4+, and IL-2R+ lymphocytes as well as a highly significant elevation of the monocyte (p less than 0.005) count. There was a highly significant increase of PGE2 synthesis from monocytes, due to the monocytosis and to a higher capacity of synthesis of the individual cells following trauma. PGE2 levels peaked on Day 5 and 7 post-trauma at 400% of control (p less than 0.005). These data suggest that the suppression of IL-2 synthesis post trauma is caused mainly by two factors: the excessive PGE2 output of inhibitory monocytes and inadequate function in immature and/or blocked lymphocytes. The partial restoration of IL-2 synthesis by indomethacin suggests that blockade of the cyclo-oxygenase pathway as an immunomodulating therapy may reverse some of the immunologic abnormalities in multiple trauma patients.

Adult↗
PubMed1987

Monoclonal antibodies to human renin: properties and applications.

A series of 11 different monoclonal antibodies generated against human kidney renin have been characterised. Their binding affinity, inhibition of renin activity, epitope distribution, crossreactivity with related enzymes and finally in vivo pharmacological effects were analysed. All antibodies were found to be specific for primate renin recognising 6 independent antigenic structures on the renin molecule. They expressed different effects on renin activity namely (1) no inhibition, (2) only partial, or (3) complete inhibition. Partially inhibiting antibodies demonstrated specific degrees of inhibition (30, 60 or 80%). One antibody, R-36-16, demonstrated an IC 50 of 1.3 X 10(-11) M/L and, when injected into marmosets, induced complete inhibition of plasma renin activity and reduction of blood pressure. Using a selected pair of antibodies a radioimmunoassay has been established providing a fast and highly reproducible determination of human and marmoset immunoreactive renin, detecting both active and inactive renin down to concentrations of 10 pg/ml (1.25 X 10(-17) moles of renin per 50 microliter sample).

Animals↗
PubMed1986

Epitope mapping of human recombinant interferon alpha molecules by monoclonal antibodies.

The epitopes of six recombinant human interferon alpha (IFN alpha) subtypes have been analysed using 22 monoclonal antibodies (MAbs) obtained from different sources. The IFN alpha subtype specificity of each MAb was determined by a combined immunoprecipitation-bioassay. Eight different epitopes were identified; the number of epitopes on a given IFN alpha subtype varied between four and eight. Each subtype possessed a unique combination of epitopes. Using the best pair of monoclonal antibodies, predicted from epitope mapping studies, subtype-specific two-site (tandem) assays were developed. It was observed that some non-cross-reactive MAbs influenced each other's binding, indicating the flexible nature of IFN molecules. Competitive radioimmunoassay and the combined immunoprecipitation-bioassay were used to identify a common epitope, present on IFN alpha-A, B, C, F and J but not on IFN alpha-D. In neutralization studies, all MAbs that identified this common epitope inhibited the antiviral activity of all IFN alpha molecules tested. It was concluded that the epitope is located within the receptor-binding region of IFN molecules and is important for biological activity. A tentative localization of the common epitope and the other identified epitopes is proposed.

Antibodies, Monoclonal↗
PubMed1985

Thrombin specificity. Selective cleavage of antibody light chains at the joints of variable with joining regions and joining with constant regions.

Selective cleavage of polypeptides by alpha-thrombin can be reasonably predicted [Chang, J.Y. (1985) Eur. J. Biochem. 151,217-224]. This knowledge was applied to the selective cleavage of antibody light chains with the aim of producing intact fragments of both variable region and constant region. (a) Mouse kappa light chains 10K26 and 10K44 from anti-(azobenzene arsonate) antibodies contain 20 Arg/Lys-Xaa bonds. Only two of them, one ProArg-Thr bond located at the joint of the variable region with the joining peptide and one ValLys-Ser bond located near the carboxyl-terminal end of the constant region, were selectively cleaved by alpha-thrombin. The ProArg-Thr bond has a 50% cleavage time of about 10 min under the designated conditions, whereas the ValLys-Ser has a 50% cleavage time approx. 9-10 h. A single selective cleavage at the joining position of the variable region and joining peptide can be achieved by short-time thrombin digestion. Fragments containing intact variable region (1-96) and intact joining peptide-constant region (97-214) obtained from both denatured and native light chains of 10K26 can be separated by gel filtration. (b) lambda light chains from both human and mouse all begin with the GlnProLys-(Ala/Ser) structure (positions 108-111) at their constant regions. This ProLys-Ala/Ser bond is also susceptible to specific thrombin cleavage. Four human lambda chain (KERN, NEI, NEW, VOR) and one mouse lambda chain (RPC20) were shown to be selectively cleaved by thrombin at these ProLys-Ala/Ser bonds. For human lambda chains, the 50% cleavage time at this ProLys-Ala bond was approx. 3-4 h under the designated conditions. Six additional thrombin specific cleavages were also detected within the variable regions of NEI, VOR and RPC-20. (c) Heparin inhibits thrombin cleavage of Arg/Lys-Xaa bonds located near the center of the antibody light chain, but slightly activates thrombin cleavage of those located near the amino or carboxyl-terminal ends of the protein. The significance of these findings is threefold. (a) It demonstrates that selective cleavage of large polypeptides by alpha-thrombin can also be reasonably predicted. (b) It provides a useful method for light chain fragmentation which can greatly facilitate amino acid sequencing of antibodies. (c) It serves to generate fragments containing intact variable regions and constant regions from antibody light chains of human and mouse. Such fragments may be useful for chemical semisynthesis of a human-mouse light chain chimeras.

Amino Acid Sequence↗
PubMed1985

Biological activities of human recombinant interferon alpha/beta targeted by anti-Epstein-Barr virus monoclonal antibodies.

The requirement of high doses of interferon (IFN) during therapy severely restrict its application. Thus a model using an Epstein-Barr virus (EBV) membrane antigen (MA) specific monoclonal antibody (MAb) was developed to assess the feasibility of coupling minimal amounts of IFN to a MAb and specifically delivering the IFN to the target cells. Coupled IFN was first shown to retain fully both its anti-viral and anti-proliferative properties when tested on human tumor cell lines QIMR-WIL (EBV-MA+) and the U-266 (EBV-MA-). A series of in vitro pulsing experiments demonstrated the specific targeting of both the anti-viral and anti-proliferative properties of IFN to the EBV-MA+ QIMR-WIL cells and not EBV-MA- cell lines.

Antibodies, Monoclonal↗
PubMed1984

Antiviral and antiproliferative effects of interferons delivered via monoclonal antibodies.

We present in vitro experiments demonstrating the feasibility of specifically targeting IFNs by coupling to monoclonal antibodies (MAbs). Human interferon alpha (HuIFN-alpha), both natural and recombinant, were coupled to Epstein-Barr virus (EBV) membrane antigen (MA) specific MAbs and shown to retain full antiviral and antiproliferative activities when tested on several human cell lines. A series of in vitro pulsing experiments demonstrated that a minimum amount of MAb-delivered IFN exerts pronounced antiviral and antiproliferative effects on human QIMR-WIL target cells which carry EBV (viral) membrane antigens (EBV-MA+) and not on U-266 or Hep2 cell lines (EBV-MA-). Additionally the antiproliferative action of MAb-coupled rIFN-alpha appeared to be enhanced compared with that of uncoupled.

Antibodies, Monoclonal↗
PubMed1984

Monoclonal antibodies recognizing structural components of murine retroviruses including an FMR antigen on protein p12.

Monoclonal antibodies were prepared from mice and rats immunized with Friend leukaemia virus and BALB/c xenotropic virus. By immunoprecipitation of 125I-labelled and [35S]methionine-labelled viruses and by protein blotting, ten antibodies were found to react with the viral components p12, p15, p30, gp70 and p15E/p12E. A dot-immunobinding assay was found to be a reliable method to type the antibody reactivity with different murine leukaemia viruses (MuLVs). When tested on a panel of ecotropic and xenotropic MuLVs the antibodies revealed the following antigenic specificities: ecotrop-specific on p15E/p12E; xenotrop-specific on p15E; group-specific on p30 and p15E; FM-specific on gp70; FR-specific on gp70 and p15. Of particular interest is a cytotoxic antibody recognizing an FMR determinant localized on p12.

Animals↗
PubMed1983

Heterogeneity of cross-reactive idiotypes. Serological and structural analysis of monoclonal anti-p-azobenzene-arsonate antibodies expressing major and minor idiotypes.

Hybridoma-derived monoclonal anti-p-azobenzene-arsonate (ABA) antibodies were obtained from fusions of ABA-KLH primed A/J spleen cells with three different myeloma cell lines. Of the 156 antibody secreting hybridomas 24% carried the cross-reactive idiotype (CRI), which is known to be shared by 20-70% of anti-ABA serum antibodies in A/J mice. The isotypes, SDS-PAGE patterns and the partial amino acid sequences of the V-regions of one CRI negative and six CRI positive hybridoma proteins were determined. These antibodies were IgGl, kappa and IgG2b, kappa. Some idiotype carrying monoclonal antibodies appeared to be serologically identical. Although the partial VH amino acid sequences of these monoclonal antibodies showed great homology with each other and with serum antibody, several sequence variations in framework residues as well as in the first and second complementarily determining regions (CDRs) were found. The cross-reactive idiotype of the anti-ABA antibodies, therefore, exhibits structural microheterogeneity, i.e. it consists of a family of non-identical but closely related molecules as previously reported (Alkan et al., 1980; Estess et al., 1980; Marshak-Rothstein et al. 1980). Here the N-terminal sequence of the VH regions from 14 CRI+ and 8 CRI- antibodies as well as the VL regions from 11 CRI+ and 8 CRI- monoclonal antibodies are compared. Analysis of the available data demonstrated that there are pairs of hybridoma proteins (both CRI+ and CRI-) which have identical sequences for VH or VL. This suggests that there exist a minimum of 4 germ line genes coding for CRI+ VH, CRI+ VL, CRI- VH and CRI- VL respectively. In addition, CRI+ VL has always been found in association with a CRI+ VH.

Amino Acid Sequence↗