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Biomedical subjects

P Perlmann

Publications and source records attributed to P Perlmann.

At least 181 records · Page 10Linked to original sources
PubMed1983

Surface markers of human natural killer cells as analyzed in a modified single cell cytotoxicity assay on poly-L-lysine coated cover slips.

A modified single cell cytotoxicity assay using poly-L-lysine coated cover slips (PLL-SCCA) was employed to study the frequency and surface marker profile of human peripheral blood lymphocytes (PBL) with NK reactivity against K 562 target cells. When compared with the previously described agarose single cell cytotoxicity assay (A-SCCA) identical results were obtained. For 13 donors tested 18.1 +/- 4.4% of the PBL formed conjugates with K 562 and 2.7 +/- 1.6% displayed NK reactivity. In contrast to the A-SCCA, the PLL-modified assay permits direct identification of both conjugate forming (TBC) and cytolytic PBL (NK) by means of surface markers. Indirect immunofluorescence studies with monoclonal anti-PBL antibodies revealed that neither the plating procedures nor the incubation conditions employed affected the expression of the antigens recognized by these reagents. This method of directly identifying NK cells showed that OKM1+ cells were enriched among the NK cells as compared to PBL and TBC (55% vs. 23% and 43%, respectively). In contrast, the OKT3+ or Leu1+ fraction of the NK cells was reduced as compared to PBL and TBC. However, using this method of identification at the effector cell level, a substantial proportion of the NK cells were OKT3+ or Leu1+ (57% or 58% respectively, 7 donors). Approximately 25% of the NK cells were Leu2a+ and 30% were Leu3a+, respectively. However, the size of the Leu3a+ fraction varied considerably with individual donors and the size of this fraction appeared to be inversely related to that of the donors NK pool.

Antibodies, Monoclonal↗
PubMed1983

Lectins as probes for identification of tumor-associated antigens on urothelial and colonic carcinoma cell lines.

In the search for tumor-associated antigens, seven lectins were used to investigate the cellular distribution of membrane-associated glycoproteins on a panel of human cells derived from tumor or normal tissues. Surface labelled lysates of the different cells were precipitated with the lectins and the precipitates were separated on SDS-PAGE. Comparison of the autoradiographic patterns revealed that a La-reactive 115K glycopeptide (gp 115) was present on transitional-cell carcinoma cells of the urinary bladder, on two spontaneously transformed urothelial cell lines and on a melanoma cell line. Gp 115 was absent from a non-transformed urothelial cell line, a squamous bladder carcinoma line and five unrelated cell lines of miscellaneous tissue origin. When precipitation was performed with a rabbit antiserum raised against the La-reactive components of a TCC cell line the same distribution of gp 115 was observed. From Helix pomatia hemagglutinin (HP) precipitates a 150K glycopolypeptide co-migrating with a previously described HP-reactive differentiation antigen associated with human T cells was present on one of the urothelial cell lines and on a colon carcinoma cell line. When different extracts depleted of ConA binding glycopeptides were compared, a group of three antigens (32K, 35K and 40K) were identified in the extracts of the colon carcinoma cell line, HT29. These antigens were shared by two other colon carcinoma cell lines but were absent from the unrelated cells of our panel. Furthermore, an extensively absorbed rabbit anti-HT29 serum specifically precipitated one of these antigens (35K) from the three colon cell lines.

Antigens, Neoplasm↗
PubMed1983

Humoral and cellular immune reactions against tumor cells in patients with urinary bladder carcinoma. Correlation between direct and antibody-dependent cell-mediated cytotoxicity.

Serum IgG fractions from a large and homogeneous group of patients with transitional cell carcinoma of the urinary bladder (TCC) were tested for their capacity to induce antibody-dependent cellular cytotoxicity (ADCC) with lymphocytes from healthy donors against a TCC-derived target cell and one derived from adenocarcinoma of the colon. Both targets have previously been shown to be of comparable susceptibility to cell-mediated lysis in vitro. Some of the IgG preparations showed strong and dose-dependent ADCC against either one or both targets, while others gave weak reactions or none at all. Similar results were obtained with IgG from a matched group of patients with prostatic carcinoma who were used as clinical controls (CC). In parallel experiments, lymphocytes taken from the two donor groups at the same time as the serum samples were tested for their direct cytotoxicity (CMC) against the two targets. CMC gave similar results to ADCC. The differences in cytotoxicity displayed by either IgG or lymphocytes from individual donors were analysed statistically, using nonparametric statistics. To avoid introducing bias due to arbitrary data selection, the entire set of results, comprising both high and low reactors, was included in the statistical assessment. ADCC of the TCC donors' IgG against the TCC target was significantly stronger than against the colon carcinoma and also significantly stronger than that of the control donors. Similarly, the TCC patients' lymphocytes displayed a significantly higher CMC against the TCC target than against the control targets. This was not seen when the lymphocytes from the patients with prostatic carcinoma were tested. When CMC and ADCC of individual donors were compared, a statistically significant correlation between these activities was seen in three of the four donor/target combinations. These results support earlier findings and suggest that a significant fraction of both the disease-related and the 'non-selective' CMC (NK) displayed by cancer patients lymphocytes against allogeneic tumor cells in vitro reflects antibody-dependent reactions.

Aged↗
PubMed1983

Human spontaneous lymphocyte-mediated cytotoxicity (SLMC) against malignant and normal tissue-derived target cell lines tested in autologous and allogeneic combinations by the microcytotoxicity assay.

Effector cell types and effector mechanisms of human spontaneous lymphocyte-mediated cytotoxicity (SLMC) were studied in a 44-h microcytotoxicity titration assay. Peripheral blood lymphocytes from cancer patients and controls were used as effector cells either unfractionated or after fractionation by rosetting techniques or affinity chromatography. The possible immunoglobulin dependency of the reactions was studied by incorporation of specific Fab fragments of rabbit anti-human IgG antibodies in the incubation mixtures. Twelve different target cell lines of either high or low sensitivity to SLMC and with or without easily detectable HLA antigens were used. Most of the target cells were cell lines derived from transitional cell carcinoma of the urinary bladder (TCC). Both allogeneic and autologous lymphocyte target cell combinations were tested. Although high- and low-sensitivity target cells differed significantly in susceptibility to lysis, the predominating SLMC was displayed by Fc-receptor-positive lymphocytes in both allogeneic and autologous combinations. Addition of the Fab anti-immunoglobulin reagent to the incubation mixtures resulted in strong inhibition of cytotoxicity regardless of the type of target cells used and in allogeneic as well as in autologous lymphocyte target cell mixtures. However, in some combinations no inhibition was seen and inhibition was usually not complete, suggesting that both immunoglobulin-dependent (i.e., ADCC-like) and immunoglobulin-independent mechanisms were involved in the cytotoxicity reactions. The results of the microcytotoxicity assay were compared with those obtained with aliquots of the same lymphocytes and target cells in an 18-h 51Cr-release assay. While similar results were obtained with high-sensitivity target cells, with low-sensitivity targets and in some autologous combinations the two assay systems registered lymphocyte/target cell interactions which differed with regard to specificity, effector cell type, and immunoglobulin dependency.

Cell Line↗
PubMed1983

Comparative study of pituitary and bacteria-derived human growth hormone by monoclonal antibodies.

We have established hybridoma lines which secrete mouse monoclonal antibodies (Mabs) to human pituitary growth hormone, hGH. Using indirect competitive ELISA and indirect passive hemagglutination inhibition twelve different Mabs were characterized with regard to cross-reactivity with the hGH-related hormones, human chorionic somatomammotropin, hCS, and human prolactin, hPRL. The reactivity of these Mabs with pituitary hGH was compared to that with either bacterially-produced methionyl-hGH or to that of reduced and S-carboxymethylated hGH, which has an altered conformation. None of the Mabs reacted with hPRL. Four did not react with hCS whereas the others showed varying degree of cross-reactivity with hCS. All Mabs reacted more weakly with reduced and S-carboxymethylated hGH than with the native form of the hormone, which was not seen with conventional rabbit antisera to hGH. Thus in the case of hGH the Mabs are superior to conventional antisera in revealing small conformational differences. However the pituitary and bacterially-derived methionyl-hGH were indistinguishable as determined by the 12 Mabs.

Antibodies, Monoclonal↗
PubMed1983

Sendai-virus-induced cell-mediated cytotoxicity in vitro. The role of viral glycoproteins in cell-mediated cytotoxicity.

Treatment of peripheral blood lymphocytes from normal donors with small amounts of purified Sendai virions results in enhanced cellular cytotoxicity in vitro to uninfected tissue culture target cells (virus-dependent cellular cytotoxicity (VDCC)), without any obvious correlation to the natural cytotoxicity (NK) displayed by the lymphocytes in the absence of virus. Removal from the virions of the two surface components present in the viral envelope, the HN glycoprotein (gp 71), carrying haemagglutinating and neuraminidase activity, and the F glycoprotein (gp 49), carrying fusion activity, by treatment with pronase abrogated their capacity to induce VDCC. Similar results were obtained when virions lacking the HN glycoprotein after treatment with chymotrypsin were added to the lymphocytes. In contrast, treatment of the virus particles with trypsin, which removed the F glycoprotein, did not affect their capacity to induce VDCC. When the solubilized and separated peplomers were used for lymphocyte treatment, either alone or in combination, the purified HN glycoprotein had full capacity to induce VDCC, whereas the F glycoprotein was inactive. These results suggest that the HM peplomer is solely or primarily responsible for the cytolytic activity arising in non-sensitized lymphocytes when confronted with certain viruses.

Adult↗
PubMed1983

Regulation of the immune response in Plasmodium falciparum malaria. II. Antigen specific proliferative responses in vitro.

The antigen-induced DNA synthesis in vitro in lymphocytes from patients with acute Plasmodium falciparum malaria was investigated. The patients and healthy controls from Sweden or Colombia were the same as those studied in the accompanying paper (Troye-Blomberg et al., 1983). The malarial antigens used were sonicated membrane preparations or purified and concentrated supernatants from in vitro cultures of P. falciparum; similar preparations derived from normal human erythrocytes served as control antigen. In the patients' lymphocytes P. falciparum antigens induced a weak or moderate but significant stimulation of DNA synthesis, peaking after 3-4 days of incubation. This early response was specific for P. falciparum since it was not obtained with lymphocytes from healthy donors nor with those from patients with acute P. vivax or P. ovale malaria. No antigen-induced response was seen in about half of the P. falciparum patients. However in a few negative cases, available for consecutive testing, positive reactions were seen with lymphocytes taken 2 weeks after infection when the blood of these patients was free of parasites. The early response induced in patients' lymphocytes to the P. falciparum antigens was not obtained with RBC antigen. However, these preparations frequently induced a response rising to significant levels later during incubation (day 5-6). Similar delayed responses were obtained when either patients' or control donors' lymphocytes were exposed to the P. falciparum antigens. This indicates that both the RBC and the parasite preparations contained mitogenic substances affecting human lymphocytes in general and easily obscuring the P. falciparum specific response seen only in the patients. This latter response was relatively low and short lived, suggesting that it reflected a secondary in vitro stimulation of in vivo primed lymphocytes and that it was regulated by suppressor mechanisms.

Acute Disease↗
PubMed1983

C3 receptors on human lymphocyte subsets and recruitment of ADCC effector cells by C3 fragments.

The presence of C3 receptors on human peripheral blood lymphocytes (PBL) and on the ADCC-exhibiting subset (K cells) thereof was analyzed by rosetting with bovine erythrocytes (Eb) or chicken erythrocytes (Ec) carrying human C3b, C3bi, or C3d. The indicator cells were coated with 20,000 to 100,000 C3 fragments, obtained by C3 activation with purified proteins of the alternative pathway and trypsin treatment. ADCC was studied at the cellular level by means of a plaque assay, with complement-free or complement-carrying indicator cells as targets. Of the total lymphocytes, 12 to 14% bound EC3b; 6 to 8%, EC3bi; and approximately 2%, EC3d. Surface marker analysis indicated that approximately 75% of the C3b-binding lymphocytes in PBL were either B or null cells and approximately 60% of the C3bi-binding cells were T cells, as characterized by the monoclonal antibodies OKT3 and OKT4 or by presence of receptors for Helix pomatia hemagglutinin. Of the K cells, which constituted from 5 to 10% of the total lymphocytes, approximately 20% bound C3b; 30 to 35%, C3bi; and 7 to 8%, C3d. Here the majority of the C3b binders were null cells, and the majority of the C3bi and C3d binders were T cells. Only one-third of the C3b-binding K cells and one-fifth of the C3bi-binding K cells bound both fragments. The nature of these double binding cells is unknown. In contrast, all C3d-binding K cells bound C3bi as well. C3 fragment-carrying target cells did not induce K cell-mediated lysis in the absence of anti-target antibodies but strongly enhanced ADCC in the presence of sublytic concentrations of such antibodies. The rank order for C3 fragment-induced enhancement was C3bi greater than C3d greater than C3b. It reflected the relative proportions of effector cells binding the different fragments. Enhancement was the expression of effector cell recruitment rather than of increased cytolytic activity of individual K cells. This recruitment was selective in that C3b-carrying target cells primarily recruited effector cells of null type, binding C3b, while C3bi- or C3d-carrying targets primarily recruited C3bi and/or C3d-binding K cells of T gamma type. Thus, these experiments show directly at the effector cell level that cell-bound C3 fragments constitute important recognition structures, which strongly amplify ADCC both by recruiting the proper effector cells into the cytolytic reaction and by very significantly decreasing the antibody concentration needed for its induction.

Animals↗
PubMed1983

Characterization of human K cells by surface antigens and morphology at the single cell level.

Human lymphocytes killing bovine erythrocytes in vitro in antibody-mediated reactions were characterized at the effector cell level in the ADCC plaque assay. Five to 10% of highly purified peripheral blood lymphocytes are active K cells in this system. Forty to 50% of these were T gamma cells expressing the T cell-associated surface antigens T3 and Leu-1. These cells also expressed the T8/Leu-2a antigens (approximately 20%) or the T4/Leu-3a antigens. Although approximately 30% of the K cells were T4+ when examined after completion of the ADCC assay (18 hr), only less than 10% were T4+ (and Leu-3a+) when examined before the assay. The results indicated that exposure to antigen/antibody complexes during the assay induced increased T4 expression, probably linked to Fc gamma R modulation on some initially T4-/T3+ lymphocytes. The expression of the other antigens (including Leu-3a) was not affected by exposure of the lymphocytes to antigen/antibody complexes. Two-color fluorescence experiments further demonstrated that a minor fraction (10 to 20%) of the K cells carrying T cell-associated antigens also expressed the monocyte/null cell-associated antigen M1 as detected with the monoclonal antibody OKM1. A second major category of effector cells, composed of at least 25% of the K cells, were large granular lymphocytes (LGL) that lacked detectable T cell-associated antigens but expressed the HNK-1 (Leu-7) as well as the M1 antigen. As seen from the size of the plaques formed by different effector cells, K cells of the LGL type had a greater recycling capacity and/or cytolytic efficiency than those of T gamma type.

Antibodies, Monoclonal↗
PubMed1983

Monoclonal anti-parasite and anti-RBC antibodies produced by stable EBV-transformed B cell lines from malaria patients.

To produce human monoclonal antibodies associated with infectious disease, peripheral blood lymphocytes (PBL) from patients with Plasmodium falciparum malaria were transformed with EB-virus in vitro. To enrich for malaria-specific B cells, PBL were incubated for 3 days with unsoluble P. falciparum antigen before EBV-transformation. Furthermore, cyclosporin A was added during and after transformation to eliminate T cell suppression of B cell growth. Microcultures were screened for antibodies against blood stage antigens of P. falciparum or of noninfected erythrocytes by ELISA and indirect immunofluorescence. Cultures producing anti-P. falciparum and/or anti-erythrocyte antibodies were developed from the lymphocytes of eight patients, including some individuals with their first infection. Positive cultures were cloned and propagated for several weeks. Seven of 15 clones producing antibody at a stable rate have now been kept in cultures for more than 1 yr. Of six cultures analyzed in detail, all produced IgM antibodies of either K or lambda isotype. Although three clones were monoclonal after one cloning, three were oligoclonal. Of the former, two produced P. falciparum-specific antibodies directed to an antigen associated with the surface of merozoites. One of the oligoclonal cultures produced anti-erythrocyte antibodies, and it was probably reacting with spectrin.

Antibodies, Monoclonal↗
PubMed1983

Regulation of the immune response in Plasmodium falciparum malaria. I. Non-specific proliferative responses in vitro and characterization of lymphocytes.

The mitogen-induced DNA synthesis in vitro in lymphocytes from 20 patients acutely ill with Plasmodium falciparum malaria was compared with that of 16 healthy donors. Within both groups part of the donors were individuals who had only experienced short exposure or none at all to the parasite (Sweden) while the other part were donors living in a malaria endemic area (Colombia). The proliferative response to the T cell mitogen La (leucoagglutinin from PHA) of the patients was significantly reduced as compared with that of the controls. With pokeweed mitogen which stimulates T cells and induces a T cell-dependent activation of B cells, no difference between patients or controls was seen. The results were similar for the donors of different geographical origin and malaria background. Lymphocytes and monocytes from the peripheral blood of these donors were also studied for surface marker distribution by means of monoclonal antibodies. Both the absolute and the relative frequencies of T cells in the blood of the malaria patients were significantly reduced as compared with the controls. Furthermore, in almost all eight patients tested, the ratio between T4+ T cells (including the helper/inducer subsets) and T8+ T cells (including the suppressor and cytotoxic subsets) were below 1:1 while they were close to 2:1 in the controls. The results indicate that the relative frequency of T8+ T cells, expressed as percentage total T cells (T3+) was significantly elevated in the P. falciparum patients. The possible relationship between this imbalance and the irregular La response of the patients lymphocytes requires further investigation of lymphocyte function.

Acute Disease↗
PubMed1983

Characterization of the humoral immune response in Plasmodium falciparum malaria. I. Estimation of antibodies to P. falciparum or human erythrocytes by means of microELISA.

An enzyme linked immunosorbent assay (ELISA) has been developed to estimate disease related antibodies in sera from malaria patients or individuals living in malaria endemic areas. As antigen, Percoll enriched fractions (mainly late trophozoites, schizonts) from Plasmodium falciparum in vitro cultures were used. An ELISA with ghosts from normal human red blood cells (RBC) was performed in parallel. One hundred and seventy-five sera were tested for their reactivity with either one of the two antigens. Seven sera from patients with acute P. falciparum infection were negative. Most of these had been taken very early in infection and consecutive samples taken later usually were positive. The antibodies reacting with the P. falciparum antigen had a high parasite specificity as indicated by inhibition and absorption experiments. Many sera also had elevated levels of antibodies specific for RBC antigens. A correlation, most pronounced in the IgM system, was also seen between the anti-RBC and the anti-P. falciparum antibody levels.

Animals↗
PubMed1983

Characterization of the humoral immune response in Plasmodium falciparum malaria. II. IgG subclass levels of anti-P. falciparum antibodies in different sera.

The IgG subclass levels of anti-Plasmodium falciparum antibodies in human sera were determined in ELISA with monoclonal mouse antibodies specific for the human IgG subclasses as analytical reagents. The parasite antigen was a trophozoite/schizont enriched preparation of in vitro cultivated P. falciparum. Serum samples were from Swedish malaria patients and adult Liberians. Parasite specific antibodies were found in all four subclasses. Relatively elevated levels of IgG1 antibodies were found both in Swedish patients and in the Liberians. Relative to the Liberians, high IgG2 antibody levels were seen in most Swedish patients. In Liberian sera but not in those from Swedish patients elevated IgG3 levels were found. In two Swedish patients followed consecutively for a period of 15 weeks, elevated levels of IgG1 and IgG3 antibodies were seen after relapse. No differences between the groups in regard to the levels of IgG4 antibodies were noticed. The differences between the Swedish patients and the Liberians were also in part reflected by differences in the total amounts of the four IgG subclasses.

Animals↗
PubMed1983

Studies on the specificity of anti-erythrocyte antibodies in the serum of patients with malaria.

Sera from patients with Plasmodium falciparum, P. vivax or P. ovale malaria were selected according to their high levels of antibodies against human erythrocyte membranes as measured in a microELISA. The specificity of the anti-erythrocyte antibodies in these sera and two normal sera was investigated by means of an immunoblotting technique in combination with SDS-polyacrylamide gel electrophoresis. All the patients' sera as well as the control sera contained antibodies against several erythrocyte polypeptides. As compared with normal sera, most malaria sera showed elevated levels of antibodies against polypeptides of 80K, 70K, 40K and 28K molecular weights. Two sera reacted strongly against a polypeptide with an electrophoretic mobility similar to the alpha subunit of spectrin. One serum showed strong reaction and several other sera, including normal sera, showed weak reaction against a 45K molecular weight polypeptide corresponding to actin. No pervading differences were seen in the pattern of specificities of the anti-erythrocyte ghost antibodies between sera from patients with P. falciparum, P. vivax or P. ovale infections.

Antibody Specificity↗
PubMed1982

The lipid nature of a tumour-associated autoantigen from a chemically induced rat hepatoma.

The target antigens for the complement-dependent cytotoxic antibodies in D23 tumour-bearer serum from rats carrying the chemically induced D23 hepatoma were extractable from extranuclear membranes of tumours with chloroform/methanol. The antigenic activity, measured by inhibition of D23 TBS cytotoxicity against D23 cells, was recovered in the phospholipid-containing fraction after silica gel chromatography. Preparative thin-layer chromatography (in chloroform/methanol/0.25% CaCl2) revealed that the antigenic activity migrated similarly to phosphatidylethanolamine and cardiolipin, although these phospholipids did not cause the inhibition seen with the antigenic fractions. Phospholipase A2 digestion of the phospholipid fraction did not affect the antigenic activity nor did it after the mobility of the antigen when analysed with thin-layer chromatography. From these results it was concluded that the antigen extracted from D23 hepatoma may be of phospholipid nature, although its molecular identity remains to be established.

Animals↗
PubMed1982

Cytotoxicity of tumour-bearer serum to D23 hepatoma cells derived from solid tumours, ascites or in vitro cultures.

The complement-dependent cytotoxicity of antibodies in tumour-bearer serum (TBS) from rats carrying the chemically induced D23 hepatoma was investigated. Target cells were D23 cells from solid tumours (D23sol), from ascites tumours (D23asc), or from in vitro growing cell cultures (D23cc). The D23asc and D23cc cells were not lysed when used as target cells in the assay, although they evoke cytotoxic antibodies when growing in vivo. The D23 ascites cells became susceptible to complement-dependent lysis after trypsin treatment. This was, however, not due to unmasking of target antigens, since untreated D23 ascites cells absorbed cytotoxic antibodies as efficiently as trypsinized cells. No increase in susceptibility to complement-dependent lysis was observed after trypsin treatment of D23cc cells. Absorption of cytotoxic antibodies with D23cc cells showed no or very low antigen expression on the surface of these cells. They did, however, contain the antigen(s), since 3 M KCl extracts of the D23cc cells could inhibit the complement-dependent cytotoxicity of D23sol TBS against D23sol cells. From these data it was concluded that the susceptibility of hepatoma cells for antibody-mediated complement lysis is not only correlated with antigen expression, as was the case with the in-vitro-cultivated cells, but is also dependent on increased lytic susceptibility after trypsin treatment of the cells.

Animals↗
PubMed1982

Role of IgM in human monocyte-mediated target cell destruction in vitro.

The presence on human monocytes of surface receptors binding IgM molecules (Fc mu receptors) and the role of IgM antibodies in monocytic effector functions were investigated. IgM antibodies were first purified by one passage through anti-Fc gamma immunosorbent. Rosette techniques in which either the indicator erythrocytes or the monocytes had been pretreated with IgM antibodies did not reveal the presence of Fc mu receptors on monocytes. Phagocytosis and cytolysis of bovine erythrocytes were, however, regularly observed in the presence of high concentrations of one IgM preparation. Inhibition experiments with human IgG or IgM indicated that the phagocytic and cytolytic effects obtained with this IgM preparation were due to contaminating IgG antibodies. This was confirmed by further immunosorbent purification of the IgM antibody preparation. Lymphocytes carrying Fc micro receptors did not transmit any effector cell activity to co-cultivated autologous monocytes as a result of interaction with IgM. It was concluded that IgM does not mediate antibody-dependent target cell destruction by human monocytes that seem to lack Fc micro receptors.

Animals↗