Lymphocytes' intrinsic lymphoproliferative capacity in patients with AIDS treated with zidovudine.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
B J Bast
Publications and source records attributed to B J Bast.
Indicator cell lines for the detection of hidden mycoplasma contamination, using an adenosine phosphorylase screening test.
Mycoplasmas are a major cause of cell culture contamination and are especially troublesome during HAT selection. The enzyme adenosine phosphorylase (adoP) is present in all common mycoplasma species but is considered to have a low activity in mammalian cells. However, using an adoP screening test, we have observed that some cell cultures do possess an intrinsic adoP activity leading to false positive results. Moreover, as a false negative result, we encountered a variant of Mycoplasma orale (identified after cultivation on agar and immunostaining) which was not detectable with the adoP screening in cell culture supernatants and only at low levels in cell lysates. To increase the low signal/noise adoP ratio found there, we used an indicator cell line with low intrinsic activity. Indicator cells were inoculated with the test supernatant and the adoP activity of these infected cells were measured after lysis. The procedure diminished the effect of biological variation in intrinsic enzyme activity between the several cell lines tested. Furthermore, in another mycoplasma infected cell line (with M. fermentans), this infection was only reliably detected using these indicator cells. With this procedure we obtained rapid results which were concordant with those obtained using the time consuming cultivation on agar.
Animals↗
Mitogenic stimulation of malignant B cells CLL: diminished in-vitro stimulation with anti-CR1 antibodies.
Peripheral B lymphocytes of five CLL patients were tested in a radioimmunoassay to determine the density of the C3b receptor (CR1) and the cells were assayed for their ability to mature into IgM secreting cells after in-vitro culture with a combination of Pokeweed Mitogen (PWM) and antibodies directed against CR1. Despite the presence of normal amounts of CR1 on the leukemic B cells, crosslinking of these receptors by anti-CR1 antibodies stimulated only a fraction of the leukemic cell population to differentiate into IgM secreting cells. These results add to the partial functional impairment of CLL-B cells.
Antibodies↗
Enrichment and selection of hybrid hybridomas by Percoll density gradient centrifugation and fluorescent-activated cell sorting.
Hybrid hybridomas, producing bi-specific monoclonal antibodies that react with horseradish peroxidase and human IgA1 were isolated by sorting the double-fluorescent cells on single-cell basis after fusion of two hybridomas, previously labelled green or red by octadecylamine-FITC or -TRITC, respectively. The double-fluorescent fused cells were significantly different in AXL (size) and RAS (internal structure) distribution compared with the (non-fused) mono-fluorescent cells. The percentage of double-fluorescent cells and the viability of these cells could be increased by Percoll density gradient centrifugation. As a result, there was an 8-fold increase of total isolated hybrid hybridomas (up to 30% of all tested clones) compared to isolations without Percoll density gradient centrifugation. All the isolated hybrid hybridoma clones had similar amounts of DNA, equal to the sum of the DNA of both parental hybridomas.
Antibodies, Monoclonal↗
Determination of the growth fraction in monoclonal gammopathy with the monoclonal antibody Ki-67.
The monoclonal antibody Ki-67 reacts with a nuclear antigen that is present only in proliferating cells. The proportion of Ki-67 positive cells may therefore serve as a reliable measurement for the growth fraction in normal and neoplasmic cell populations. We have tested the significance of the MoAb Ki-67 in the classification of monoclonal gammopathy and compared the results with the plasma cell labelling index. In benign monoclonal gammopathy the percentage of Ki-67 positive plasma cells (median 1.6%) was significantly lower than in untreated multiple myeloma (median 9.6). Among the patients with more than 10% Ki-67 positive plasma cells there were some very short survivors. The largest growth fractions (median 41.8%) were found in patients with relapsing multiple myeloma indicating here a different growth pattern more resembling the high-grade lymphomas. A linear correlation between the proportion of Ki-67 positive plasma cells and the labelling index was not found. Determination of the plasma cell growth fraction with the monoclonal antibody Ki-67 in monoclonal gammopathy may help to discriminate benign monoclonal gammopathy from multiple myeloma and will probably identify a subgroup of multiple myeloma patients with a poor prognosis, including those with relapsing multiple myeloma.
Antibodies, Monoclonal↗
Syngeneic leukocytes together with suramin failed to improve immunodeficiency in a case of transfusion-associated AIDS after syngeneic bone marrow transplantation.
A 22-year-old man who underwent syngeneic bone marrow transplantation (BMT) for acute lymphoblastic leukemia acquired a human immunodeficiency virus (HIV) infection by transfusion of blood products from a donor at risk. The manifestations were acute encephalopathy together with immune thrombocytopenia in the early posttransplant period, and acquired immunodeficiency syndrome (AIDS) developed 20 months after BMT. Because he had a syngeneic donor, the possibility of reconstituting the immune system was investigated by repeated transfer of healthy syngeneic lymphocytes and by combining repeated transfer of syngeneic lymphocytes with the antiviral agent suramin to protect the infused leukocytes from being attacked by HIV. No improvement was observed clinically or in the patient's immune functions by these efforts.
Acquired Immunodeficiency Syndrome↗
Novel type of proliferating lymphoplasmacytoid cell with a characteristic spotted immunofluorescence pattern.
We examined bone marrow from myeloma patients for the presence of cells with the characteristics of the clonogenic cell in the myeloma stem cell assay. We identified a novel type of cell that contained cytoplasmic immunoglobulin of the relevant idiotype located in a cytoplasmic spot. This "spotted" Ig could be located in the rough endoplasmic reticulum. Spotted cells are highly proliferative, as evidenced by the nuclear staining with the antibody Ki67, and were found in the bone marrow from most of the myeloma patients studied. This type of cell was also present in patients with immunocytomas, in some cases of benign monoclonal gammopathy, and in patients in the state of polyclonal hypergammaglobulinemia. IgG subclass distribution of so-called spotted cells and plasma cells, found in a patient with pseudo biclonal gammopathy, indicates that spotted cells are intermediate between B cells and plasma cells. Spotted cells express the B cell-associated antigens HB4 and HB6 but do not express other B cluster of differentiation antigens or plasmacytoid antigens tested.
Adult↗
Influence of cytomegalovirus infection on the recovery of humoral immunity after autologous bone marrow transplantation.
Recovery of B-cell number and function was studied in 23 patients with hematological malignancies treated with high-dose chemoradiotherapy followed by autologous bone marrow transplantation (auto-BMT) in relation to the presence or absence of cytomegalovirus (CMV) infection. B cells recovered rapidly after auto-BMT and specific antibodies to herpes viruses remained nearly unchanged. Both were independent of the CMV status of the patients. However, the capacity of peripheral blood B cells to differentiate in vitro into cytoplasmic immunoglobulin (Ig)-positive cells (plasma cells) on pokeweed mitogen stimulation in the presence of normal T-cell help was significantly better in CMV-negative patients than in CMV-positive patients after auto-BMT, but was decreased in both groups. Serum Ig levels were, in contrast, higher in CMV-positive patients than in CMV-negative patients after auto-BMT.
Antibodies, Antinuclear↗
Determination of the plasma cell labelling index with bromodeoxyuridine in a double fluorescence technique.
A new technique is described in which plasma cells actively synthesizing DNA can be identified in a heterogeneous cell population such as bone marrow. This method uses bromodeoxyuridine (BrdU) and a fluorescent monoclonal antibody against BrdU in combination with cytoplasmic staining for immunoglobulin (Ig). In 26 patients with monoclonal gammopathy (MG) the plasma cell labelling index (LI) as determined by this immunofluorescent method was compared with the tritiated thymidine (3H-TdR) LI. No difference in sensitivity was found between the two methods. As the determination of the plasma cell LI with the BrdU/anti BrdU method is easy to perform and results can be obtained within 4 h, this immunofluorescent method seems to be an attractive alternative to the laborious time-consuming classic 3H-TdR LI.
Bone Marrow↗
Molecular analysis of a case of IgA2 deficiency.
A family with two members with selective IgA2 deficiency was analysed by direct gene analysis with different probes for the IgCH region. No gross gene deletions or rearrangements were detected. Genetic analysis based on serological and molecular markers did not rule out linkage with the IgCH region. However, a defect of other genes not linked to the Ig heavy chain region and controlling the expression of IgA may be possible as well.
Dysgammaglobulinemia↗
Surface immunoglobulin restriction in B-non Hodgkin's lymphomas in cell suspension and on frozen tissue sections.
The diagnostic relevance of different tests for detection of surface immunoglobulin on tumour cells of B-type non-Hodgkin's lymphomas (B-NHL) was investigated by comparison of the direct antiglobulin rosetting reaction (DARR) in suspension with two-colour direct immunofluorescence (DIF) on frozen tissue sections. In benign lymph nodes (n = 27) the kappa/lambda ratio by DARR test ranged from 0.9 to 2.8. Tested by suspension and frozen tissue analysis, light chain restriction was found in 24 and 27 of 31 cases of B-NHL, respectively. Heavy chain restriction was found in half of the cases (14 of 26) studied in suspension and in almost all (28 of 31) tested on sections. In 9 cases DARR tests showed restriction of more than one Ig class on tumour cells, which was infrequent (2 of 28) in frozen section analysis. Although both tests appeared valuable for routine diagnostic purposes, we found the DIF analysis on tissue sections somewhat more discriminative, especially in detection of heavy chain restriction in B-NHL.
Cells, Cultured↗
T cells in B cell chronic lymphocytic leukemia. II. Lack of antigen-specific T suppressor cells and their progenitors.
Nylon wool-purified T cells (Tn) of two patients with chronic lymphocytic leukemia of the B cell type were phenotyped and tested in various assays for antigen-specific T helper (Th), T suppressor effector (Tse), T suppressor precursor (Tsp), and T suppressor inducer (Tsi) function. Antigen-specific Th as well as Tsi activity could be effectively generated. Although phenotypically CD8+ T cells, carrying the receptor for the Fc part of IgG, were present in mononuclear blood cells and Tn fractions, no antigen-specific Tse cell activity could be induced. In addition, Tsp cells were found to be functionally absent. These findings are discussed in relation to a tumor-induced limited heterogeneity within the T suppressor (Ts) cell compartment.
B-Lymphocytes↗
Recovery of T cell subsets after autologous bone marrow transplantation is mainly due to proliferation of mature T cells in the graft.
In 22 patients with malignancies, treated with high-dose chemoradiotherapy and autologous bone marrow transplantation (BMT), peripheral blood T cell subsets and functions were studied. In ten cytomegalovirus (CMV)-negative patients, CD4+ and CD8+ T cells (representing T cells of the helper/inducer phenotype and T cells of the suppressor/cytotoxic phenotype, respectively), recovered slowly and simultaneously. In 12 CMV-positive patients, however, CD8+ T cells recovered more rapidly than CD4+ T cells and rose to increased counts. No T cells with an immature phenotype (CD1+, OKT6+) were observed. Lymphocyte stimulation by herpes simplex virus infected fibroblasts (and by CMV-infected fibroblasts in CMV-positive patients) in contrast remained high and even increased after BMT in both groups. These data indicate that T cell recovery after autologous BMT is mainly due to proliferation of mature T cells present in the BM graft and not to generation of new T cells from T cell precursors.
Antibodies, Viral↗
Cellular expression of idiotopes defined by monoclonal antibodies.
Monoclonal anti-idiotype (Id) antibodies with specificities for determinants related to the antigen-binding sites of 3 BALB/c myeloma proteins, MOPC-460, HOPC-8 and J558, were used to study Id expression on murine lymphocytes. The monoclonal antibodies were shown to react only with Id structures associated with immunoglobulin on B cells. None of these 3 Id nor a VH Id, detected by a monoclonal antibody made against HOPC-8 heavy chain, were found on T cells. These Id were detected on splenic B cells in neonatal mice; the frequencies in normal, nude and germ-free mice were similar: MOPC-460 Id+: 1.05 +/- 1.7/10(4) spleen cells, HOPC-8 Id+: 1.45 +/- 1.2/10(4) and J558: 0.35 +/- 0.6/10(4). Almost all Id+ cells bore surface IgM, a few expressed surface IgG. MOPC-460 Id+ IgG+ cells were mainly gamma 2a+ or gamma 2b+, whereas J558 and HOPC-8 Id+ IgG+ cells were gamma 3+.
Aging↗
Characterization of the colony-forming cell in monoclonal gammopathies.
The nature of the colony-forming cell in the bone marrow of patients with monoclonal gammopathy, as defined in the stem cell assay described by Hamburger and Salmon, has been studied. It could be shown that the colony-forming cells produce immunoglobulins of the same idiotype, heavy chain and light chain, as the monoclonal bone marrow cells in the patient. Data regarding the presence or absence of J chain in the colonies, the failure to observe isotype-switch in the growing colonies, as well as the lack of inhibition of colony formation using antiidiotypic antibodies, strongly suggest that colony formation in vitro reflects proliferation of the clonogenic stem cell in the bone marrow without apparent differentiation. The stem cell may be of plasma cell nature.
Antibody-Producing Cells↗
Fc receptors on rabbit lymphocytes. Existence of receptors for IgG antibody complexed with antigen; conditions for its detection.
The presence of Fc receptors (FcR) on rabbit peripheral blood leukocytes is demonstrated using rosette formation with an ox erythrocyte-antibody (EoxA) complex. The receptor is specific for the Fc fragment of IgG (neither IgM nor F (ab')2 anti-Eox mediates rosette formation) that is antigen-bound (aggregated rabbit IgG inhibits the rosette formation only transiently). The receptor is species-specific: guinea pig IgG/Eox, goat IgG/Eox and sheep IgG/Eox complexes do not show rosette formation, and goat IgG aggregates do not inhibit rosette formation. The origin of the target erythrocytes is of importance. Sheep erythrocytes are not useful, and within Eox large differences between donors were found. Rosette formation was only inhibited by pretreatment of the rosette-forming cells with homologous immune complexes, whereas the size of the antigen greatly influenced the degree of inhibition. The rabbit FcR is pronase-resistant, unlike the human and murine RcR. The interaction of IgG and the FcR is not inhibited by isolated C gamma 3 domains. Further evidence for the requirement of the whole Fc region was obtained in experiments where inhibition of the rosette formation was observed using antisera directed to the C gamma 3 and the C gamma 2 domain, respectively. Anti-Fab antiserum did not inhibit rosette formation. Results are discussed in relation to the mechanism of allotypic suppression.
Animals↗
Fc receptors on rabbit lymphocytes. Identification and organ distribution of rosette-forming cells; cocapping with surface immunoglobulin.
Fc receptor (FcR)-bearing cells were demonstrated using ox erythrocytes coated with homologous IgG-type antibodies (EA gamma) in rabbit peripheral blood leukocytes (PBL) and in various lymphoid organs. Discrimination of the rosette-forming cells (RFC) is carried out after prior ingestion of tetramethylrhodamine isothiocyanate-labeled latex particles and in transmission electron microscopic studies. Most of the nonlymphoid cells (5-10%) in PBL and spleen cell suspensions expose FcR. These nonlymphoid cells are almost absent in other lymphoid organs, except in bone marrow. The average percentage of cells rosetting with IgG-sensitized erythrocytes (EA gamma RFC) in lymphoid cell preparations of the various tissues was as follows: PBL 25%, bone marrow 65%, appendix 37%, spleen 40%, Peyer's patches 44%, thymus 2% and peripheral lymph node 27%. The nature of FcR-bearing PBL was further studied using F (ab')2 anti-IgM, anti-IgA or anti-T cell conjugates. About half of the population of B cells, bearing IgM or IgA express FcR. Moreover, about 80% of the RFC are found within the B cell population. Only a few T cells were found rosetting with EA gamma suggesting that most of the non-B lymphoid RFC are "null" cells. In different lymphoid organs, the percentages of EA gamma RFC and B cells are comparable but not identical A greater part of the EA gamma RFC also expresses the receptor for the third component of complement. After capping of membrane IgM determinants, FcR is located in the same cap on the majority (60%) of the FcR-positive IgM-capped cells.
Animals↗
Surface Ig on rabbit lymphocytes. Rabbit B and T cells are distinct populations.
Rabbit peripheral blood lymphocytes (PBL) were analyzed by immunofluorescence using anti-T cell conjugates and anti-Fab, anti-a1 allotype, anti-IgM and anti-IgA conjugates. In addition, T cells were demonstrated by rosetting with papain-treated homologous erythrocytes. Control experiments, using acid treatment and incubation at 37 degrees C for 18 h after or without pronase treatment, revealed the endogenous origin of all surface determinants tested. A good correlation was found between results obtained with the two anti-T cell conjugates used and the T rosette test on PBL and on lymphoid cells isolated from various organs. In lymphocytes isolated from peripheral blood and from various lymphoid organs, the percentages of T and B cells were respectively 45 and 38 for PBL, 10 and 46 for bone marrow, 27 and 31 for appendix, 40 and 45 for spleen, 42 and 46 for Peyer's patches, 96 and 0.3 for thymus and 70 and 16 for peripheral lymph nodes. The percentage of "null" cells in lymphocyte populations derived from bone marrow and appendix is rather high. The final percentages of T and B cells in rabbit PBL depend to a significant extent on the method of isolation, especially isolation by Ficoll-Hypaque centrifugation results in a depletion of T cells. Moreover, a rather impure lymphoid cell suspension is obtained. In double incubation experiments, T cells (as defined by T cell antigen(s) or rosette formation) and B cells (Fab-bearing cells) were entirely different subpopulations. Allotypes of the a locus could not be detected on the surface of T cells. The results are discussed with respect to genetic coding of antigen receptors on B and T cells.
Animals↗