Biomedical subjects

V O Shah

Publications and source records attributed to V O Shah.

At least 19 recordsLinked to original sources

PubMed1998

Z-2 microsatellite allele is linked to increased expression of the aldose reductase gene in diabetic nephropathy.

Epidemiological studies support the hypothesis that genetic factors modulate the risk for diabetic nephropathy (DN). Aldose reductase (ALDR1), the rate-limiting enzyme in the polyol pathway, is a potential candidate gene. The present study explores the hypothesis that polymorphisms of the (A-C)n dinucleotide repeat sequence, located 2.1 kb upstream of the transcription start site, modulate ALDR1 gene expression and the risk for DN. We conducted studies at two different institutions, the University of New Mexico Health Sciences Center (UNMHSC), and the Istituto Scientifico H San Raffaele (HSR). There were four groups of volunteers at UNMHSC: group I, normal subjects; group II, patients with insulin-dependent diabetes mellitus (IDDM) without DN; group III, IDDM with DN; and group IV, nondiabetics with kidney disease. At HSR we studied volunteers in groups I, II, and III. ALDR1 genotype was assessed by PCR and fluorescent sequencing of the (A-C)n repeat locus, and ALDR1 messenger ribonucleic acid (mRNA) was measured by ribonuclease protection assay in peripheral blood mononuclear cells. At UNMHSC we identified 10 alleles ranging from Z-10 to Z+8. The prevalence of the Z-2 allele among IDDM patients was increased in those with DN. Sixty percent of group III and 22% of group II were homozygous for Z-2. Moreover, 90% and 67% of groups III and II, respectively, had 1 or more copy of Z-2. In contrast, among nondiabetics, 19% of group IV and 3% of group I were homozygous for Z-2, and 69% and 32%, respectively, had 1 copy or more of Z-2. Among diabetics, homozygosity for the Z-2 allele was associated with renal disease [odds ratio (OR), 5.25; 95% confidence interval, 1.71-17.98; P = 0.005]. ALDR1 mRNA levels were higher in patients with DN (group III; 0.113 +/- 0.050) than in group I (0.068 +/- 0.025), group II (0.042 +/- 0.020), or group IV (0.015 +/- 0.011; P < 0.01). Among diabetics, ALDR1 mRNA levels were higher in Z-2 homozygotes (0.098 +/- 0.06) and Z-2 heterozygotes (0.080 +/- 0.04) than in patients with no Z-2 allele (0.043 +/- 0.02; P < 0.05). In contrast, among nondiabetics, ALDR1 mRNA levels in Z-2 homozygotes (0.034 +/- 0.04) and Z-2 heterozygotes (0.038 +/- 0.03) were similar to levels in patients without a Z-2 allele (0.047 +/- 0.03; P = NS). At HSR we identified eight alleles ranging from Z- 12 to Z+2. The prevalence of the Z-2 allele was higher in group III than in group II. In group III, 43% of the patients were homozygous for Z-2, and 81% had one copy or more of the Z-2 allele. In contrast, in group II, 4% were homozygous for Z-2, and 36% had one copy or more of the Z-2 allele. IDDM patients homozygous for Z-2 had an increased risk for DN compared with those lacking the Z-2 allele (OR, 18; 95% confidence interval, 2-159). IDDM patients who had one copy or more of Z-2 had increased risk (OR, 7.5; 95% confidence interval, 1.9-29.4) for DN compared with those without the Z-2 allele. These results support our hypothesis that environmental-genetic interactions modulate the risk for DN. Specifically, the Z 2 allele, in the presence of diabetes and/or hyperglycemia, is associated with increased ALDR1 expression. This interaction may explain the observed association between the Z-2 allele and DN.

Adult↗

PubMed1997

Aldose reductase gene expression is increased in diabetic nephropathy.

Aldose reductase gene expression is increased in insulin-dependent diabetes mellitus (IDDM) with nephropathy. Epidemiology studies in patients with IDDM and noninsulin-dependent diabetes mellitus (NIDDM) are consistent with the hypothesis that a genetic factor(s) influences the risk for kidney disease of diabetes mellitus (KDDM). Aldose reductase (AR), the rate-limiting enzyme in the polyol pathway, is a potential candidate gene product. The present study explored the hypothesis that AR gene expression is increased in peripheral blood mononuclear cells obtained from patients with KDDM. We studied four groups of volunteers: group I, normal subjects; group II, IDDM without nephropathy; group III, IDDM with kidney disease; and group IV, nondiabetics with kidney disease. AR messenger ribonucleic acid was measured by a ribonuclease protection assay. The results are expressed as the mean and 95% confidence interval (CI) of the AR/beta-actin messenger ribonucleic acid molar ratios (AR/beta-actin R). Among diabetics, the AR/beta-actin R was higher in group III (0.088; CI, 0.068-0.108) than in group I (0.045; CI, 0.033-0.057; P < 0.01). There were no significant differences in age, hemoglobin A1c, or duration of diabetes between groups II and III (P = NS). The AR/beta-actin R in group III was also higher than that in group II (0.045; CI, 0.030-0.060; P < 0.01) or group IV (0.019; CI, 0.011-0.027; P < 0.001). In contrast, among nondiabetics, AR/beta-actin R values were 2-fold lower in group IV than in group I (P < 0.01). The results of this study are consistent with the hypothesis that the degree of AR gene expression modulates the risk of KDDM.

Actins↗

PubMed1995

Regulation of aldose reductase gene expression in renal cortex and medulla of rats.

A role for aldose reductase-mediated production of polyol in the aetiology of diabetic nephropathy has been supported by both animal and clinical studies. In the renal medulla, the rate of polyol production is influenced in part by regulated changes in the level of aldose reductase gene expression. However, little is known about the expression of aldose reductase in the renal cortex. In this study, we evaluated the regulation of aldose reductase gene expression in the renal cortex and medulla in response to galactose feeding. Four groups of rats (n = 6) were treated for 9 weeks with control or galactose diet in the presence or absence of sorbinil, an aldose reductase inhibitor. In the renal medulla, galactose treatment produced a significant (p < 0.01) decrease in aldose reductase mRNA, to approximately 10% of control levels. Coadministration of sorbinil partially prevented the effect of galactose feeding on medullary aldose reductase mRNA (to 43% of control). Under basal conditions, the concentration of aldose reductase mRNA in the cortex was only 1% that of the renal medulla. Galactose feeding significantly reduced cortical aldose reductase mRNA by 29% relative to control (p < 0.01), and this was completely reversed by addition of sorbinil. Sorbinil administration to rats fed a control diet also decreased aldose reductase expression in the renal medulla and cortex. These results demonstrate that galactose feeding results in dynamic, polyol-dependent regulation of aldose reductase gene expression in the renal cortex as well as the medulla.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1993

Aldose reductase expression and prostaglandin E2 production are coordinately regulated in cultured rat mesangial cells.

There is increasing evidence that a link between the polyol pathway and prostaglandins is important in the pathogenesis of diabetic nephropathy. The presence of the polyol pathway in the kidneys of normal animals, the galactose-fed rat, and animals with experimental diabetes has been established. While aldose reductase (AR) immunoreactive protein (AR-IRP) and AR mRNA are expressed at high levels in renal medulla, the sites of AR synthesis and regulation and metabolic consequences of AR activity in renal cortex are uncertain. The present study was conducted to test the hypothesis that AR expression and PGE2 production are coordinately regulated in glomerular mesangial cells. To test this hypothesis, we measured AR-IRP, AR mRNA, and PGE2 production in mesangial cells isolated from rats maintained on diets containing normal chow (MC-N), 50% galactose (MC-G), and 50% dextrin (MC-D). The rank order for each parameter studied (AR-IRP, AR mRNA, PGE2) was MC-N > MC-G > MC-D. Western blot analysis demonstrated that MC-N (optical density [OD] 1.0), MC-G (OD 0.59), and MC-D (OD 0.25) express AR-IRP. Slot-blot analyses demonstrated that levels of AR mRNA were greatest in MC-N (1.0), intermediate in MC-G (0.49), and lowest in MC-D (0.31). Ribonuclease (RNase) protection analyses demonstrated a similar pattern of AR mRNA expression, with MC-N at 1.0, MC-G at 0.60, and MC-D at 0.33. PGE2 production (pg/5 x 10(4) cells/30 min) was highest in MC-N (278 +/- 29), intermediate in MC-G (110 +/- 9), and lowest in MC-D (37 +/- 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Reductase↗

PubMed1990

In vitro studies with the platelet-reactive antibody 50H.19 and its fragments.

The platelet-reactive monoclonal antibody 50H.19, its F(ab')2 fragments, and 99m-technetium (99m-Tc)-labeled fragments used in thrombus imaging were evaluated for their ability to cause platelet aggregation. The intact antibody caused a dose-dependent platelet aggregation in either platelet-rich plasma (PRP) or defined buffer solutions. The F(ab')2 fragments did not cause platelet aggregation except at high calcium concentrations. Neither stannous-ion-treated antibody fragments nor 99m-Tc-labeled antibody fragments caused platelet aggregation. The antibody-induced platelet aggregation was completely inhibited by 8-bromo-cAMP, caffeine, theophylline diltiazem, and staurosporin; partially inhibited by EDTA, EGTA, cytochalasin B, colchicine, aspirin, or indomethacin. Treatment of platelets with the intact 50H.19 antibody resulted in phosphorylation of a 40K dalton platelet protein, similar to that caused by treatment with phorbol myristate acetate (PMA). Phosphorylization of the 40K protein was not observed after treatment with either the 50H.19 F(ab')2 fragments or the calcium ionophore A23187.

Antibodies, Monoclonal↗

PubMed1990

Multiparameter flow cytometric analysis of human fetal bone marrow B cells.

Maturation of adult human bone marrow (BM) B cells is accompanied by the sequential acquisition and loss of characteristic cell surface antigens (Loken et al., Blood 70:1316). Little is known about these changes in fetal BM B cells. In order to compare fetal with adult B cell development, we performed three-color, flow cytometric analyses of cell surface antigens, as well as nuclear TdT staining, on lymphoid cells from fetal BM. Mononuclear cells isolated from fetal BM (18-22 weeks) were stained with combinations of antibodies against CD3, CD10, CD19, CD20, CD21, CD22, CD34, CD45, PCA-1, IgM, and HLA-DR. Analysis of six separate fetal BM specimens indicated that combinations of cell surface antigens were expressed on analogous populations in fetal and adult BM. Consistent with adult BM, greater than 95% of TdT+ cells within the CD10+ population were CD34+, whereas less than 5% were CD34-. This CD10+/CD34+/TdT+ population constituted 30-40% of the total B cell compartment, compared with 10% in adults. Quantitative changes in CD45 expression on fetal BM B cells defined three clear populations, as has been observed in adults. In striking contrast to adult BM, greater than 95% of CD19+ and greater than 95% of surface IgM+ cells were CD10+, indicating that CD10 is a pan-B cell antigen in fetal BM. Virtually no mature B cells expressing CD21, CD22, or PCA-1 were detected in fetal BM. Our results indicate a preponderance of immature phenotypes exist in the fetal BM B cell compartment. These immature cells can be grouped into three distinct populations, and probably correspond to expanded populations found less frequently in adult BM. This striking increase in the earliest identifiable stages of B cell ontogeny is consistent with an active expansion of cells destined to constitute the humoral immune system during fetal development.

Antigens, CD↗

PubMed1988

Flow cytometric analysis of human bone marrow. IV. Differential quantitative expression of T-200 common leukocyte antigen during normal hemopoiesis.

We have correlated the intensity of expression of CD45 Ag (T200 common leukocyte Ag) with mAb reactive with various lineages of hemopoietic cells in normal human bone marrow by using two-color immunofluorescence on a flow cytometer. Mature T lymphocytes (CD3+) and NK cells (CD16+ or CD11b+) expressed CD45 at the highest intensity. B lymphoid cells (CD19+) had three distinct levels of CD45 Ag expression. The bright CD45(3+) cells were mature B cells (CD19+, CD20+), whereas the less intense CD45(2+) cells were less mature B lymphoid cells (CD19+, CD10+). The dim CD45+ cells were very early, B lymphoid precursor cells (CD19+, CD10(2+), CD34+). The intensity of CD45 expression increased as cells matured in the monocytic lineage (CD14+, CD11b+). Among marrow granulocytic cells, CD45 intensity did not change on cells during maturation. Within the erythroid lineage, the most immature cells were CD45+ dim, and CD45 expression decreased during erythroid maturation to become undetectable on mature E. Hemopoietic progenitor cells (CD34+) expressed low levels of CD45 Ag. Expression of CD45R on marrow cells also showed intensity differences on different lineages. All NK cells (CD16+) were positive for CD45R, whereas only about one-half of the T lymphocytes (CD3+) were positive for CD45R. Almost all the cells in the erythroid and myelomonocytic lineages were CD45R-. Quantitative differences in expression of CD45R were observed on marrow B lymphoid cells which were correlated with the expression of CD45. The results show that quantitative changes in CD45 Ag expression accompany the differentiation and maturation of cells in the bone marrow. Comparisons with CD45R showed that this Ag was not always correlated with CD45. Since these Ag are the products of the same gene, these data indicate that the regulation of expression of the T200 molecules during normal hemopoietic development must be both quantitative and qualitative.

Adult↗

PubMed1988

Discriminating between damaged and intact cells in fixed flow cytometric samples.

A vital, nucleic acid stain (LDS-751) was used to discriminate intact from damaged cells in a flow cytometer even after the samples had been fixed with paraformaldehyde. Three major cell populations with different fluorescence properties with LDS-751 were found in the fixed samples. Cells not staining or only dimly staining with LDS-751 were identified as erythrocytes and platelets, respectively. Cells staining with intermediate amounts of LDS-751 were found to be intact cells, while cells intensively stained were identified as damaged cells. Confirmation of the identity of the populations was obtained by light microscopic examination of the sorted populations and by correlating the fluorescent signals of FDA and LDS-751 in nonfixed cell preparations. Agglutinated cells could also be identified by the increased fluorescent signal in the LDS-751 channel as compared with single cells. The spectral properties of this dye permit excitation at 488 nm with emission in the far red portion of the spectrum. This allowed two-color immunofluorescence to be combined with the intact/damaged cell discrimination on fixed samples. Therefore, intact single cells could be distinguished during flow cytometric analysis, increasing the accuracy of the immunofluorescence measurements. The visualization of the multidimensional data was facilitated using color to discriminate cell populations depicted in multiple perspectives.

Antibodies, Monoclonal↗

PubMed1988

Technetium-99m labeled 50H.19 antibody fragments: interaction of the antibody with platelets.

The monoclonal antibody 50H.19 recognized three antigens (Mr = 31-, 40-, 45-K) on normal and thromboasthenic platelets, but only one (Mr = 31-K) on Bernard-Soulier platelets. The intact antibody and its F(ab')2 fragments, had direct platelet-aggregating activity, and induced the platelet release reaction. The intact antibody potentiated platelet aggregation induced by platelet-activating factor or thrombin. Additions of indomethacin did not inhibit aggregation: addition of PGI2, or a calcium channel blocker completely inhibited aggregation. A reduced amount of platelet-aggregating activity was observed with antibody fragments prepared for labeling with 99mTc by pre-exposure to stannous ions, and herein used in biodistribution studies and elsewhere in thrombus imagining studies (J. Nucl. Med. 27: 1315; 1986). Antibody fragments radiolabeled with 99mTc bound to isolated platelets and to clots containing platelets.

Antibodies, Monoclonal↗

PubMed1988

Anti-colon/ovarian tumor antigen: localization of colon cancer xenografts in athymic rats.

Tumor localization studies in athymic rats bearing human colon tumors were performed using the radioiodinated monoclonal antibody SP-21 and its F(ab')2. Antibody preparations isolated from ascitic fluid and antibody from bioreactor effluent were used in these studies with similar radioimmunolocalization results. The intact antibody had an optimal localization time of 4-8 days after injection, while the F(ab')2 fragments had an optimal localization time of 3-4 days. Whole-body autoradiography, whole-body immunohistochemistry, and scintigraphy confirmed that the intact antibody and the antibody fragments localized preferentially in the tumor. The antibody distribution within the tumor was uniform, and not confined to the periphery, nor to focal areas within the tumor. Dose-response studies were performed with the intact antibody over a range of 10-100 micrograms/kg of total body weight with no clear-cut relationships observed. Comparisons of different radio-iodination methods indicated that the chloramine-T-based methods resulted in preparations with higher tumor uptake.

Animals↗

PubMed1988

Flow cytometric analysis of normal B lymphoid development.

Identification of the antigens expressed on marrow B lineage cells can be used to develop a model for the sequential acquisition of cell surface antigens during B lymphocyte development. The data suggest that the surface antigen expression is highly controlled during the development of B cells with the coordinated acquisition of multiple cell surface antigens during the maturational process. The developmental scheme in figure 6 is inferred from the expression of cell surface antigens on single samples. Confirmation of the progression from one stage to the next requires the isolation of a particular stage with subsequent induction to the next stage in-vitro. These data suggest that the development of B lymphoid cells may be discrete rather than continuous. The most immature cells identifiable in the bone marrow express CD34+ as well as HLA-DR. The earliest recognizable B lineage cells (CD19+, bright CD10+) also express CD34+. These cells are smaller by forward light scattering when compared to the cells which express only CD34+ (precursor of myeloid cells). Cells within stage I also express TdT in the nucleus and are proliferating. As the cells progress from stage I to stage II, the B lineage cells lose cell surface CD34 and nuclear TdT. At this time the density of HLA-DR and CD45 increases while the amount of CD10 decreases. These changes occur with no detectable change in cell size as assessed by forward light scattering. HLA-DP is first detected on the cells at this time. The progression of cells from stage II to stage III is marked by the acquisition of CD20, HLA-DQ, and sIgM. The amount of CD45 increases further in the transition between stage II and stage III. The acquisition CD21 and CD22 as well as the loss of CD10 distinguishes stage IV from stage III. Once the cellular composition of normal marrow has been defined, perturbations from homeostasis can be identified. Since marrow is the tissue most sensitive to injury by most antineoplastic chemotherapy and radiotherapy regimens, a means of quantifying the changes from the normal state can provide an assessment of the cytotoxic injury produced in individual patients. By monitoring the return to normal, it may be possible to more precisely individualize therapy for each patient. With a clear understanding of normal hematopoiesis, it should also be possible to identify maturational blocks which occur in hypoplastic marrow states. This may provide a means of identifying the regulatory points for each lineage and provide strategies for overcoming the inhibition of development.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗

PubMed1988

Assessment of proliferation during maturation of the B lymphoid lineage in normal human bone marrow.

Different stages of B lymphoid maturation were identified in normal bone marrow using multiple cell surface markers. The proliferation status of each of these maturational stages was determined by simultaneous quantitative DNA analysis on a flow cytometer. The technique used to quantify these parameters preserved the cell surface immunofluorescence, the light scattering properties and the stoichiometric binding to DNA. The proliferating cells were confined to a distinct population of cells expressing CD10. The number of proliferating cells in these populations was relatively constant among 12 separate bone marrow samples. The data suggest that the timing and rate of proliferation of cells within a single lineage may be a preprogrammed aspect of normal maturation.

Antibodies, Monoclonal↗

PubMed1987

A comparison of monoclonal antibodies against distinct colon tumor-associated antigens in immunohistochemistry and in tumor localization.

Monoclonal antibodies to colon/ovary tumor antigen (COTA), carcinoembryonic antigen (CEA), colon-specific antigen (CSA), and colon-specific antigen "protein" (CSAp) were evaluated for specificity, reactivity with normal tissues, and tumor localizations using athymic rats bearing xenografted human colon tumors. Radioiodine labeled anti-CSA and anti-COTA retained immunoreactivity and effectively localized the tumors; anti-CSAp retained immunoreactivity, but localized less effectively; and anti-CEA lost most of its immunoreactivity and localized poorly. Of the antibodies tested, anti-COTA showed potential for human colorectal tumor radiolocalization.

Animals↗

PubMed1987

Flow cytometric analysis of human bone marrow: I. Normal erythroid development.

Flow cytometry was used to identify maturational differences of erythroid lineage cells in normal human bone marrow by combining physical characteristics, the expression of multiple cell surface antigens, and nucleic acid content. Normal low-density bone marrow cells could be divided into four populations, based on forward and right-angle light scattering. Erythroid cells, at different maturational stages, were found in three of these four marrow subpopulations. The sequentially correlated expression of three cell surface markers--HLe-1, transferrin receptor, and glycophorin--allowed us to study erythroid maturation from the colony forming cell to the mature erythrocyte. HLe-1 was expressed on the earliest identifiable erythroid cells and was progressively lost as the cells matured. Transferrin receptor began to be expressed at the BFU-E stage and disappeared at the late reticulocyte stage. Transferrin receptor expression preceded glycophorin expression, the latter beginning on morphologically recognizable erythroid precursors just after the CFU-E stage. In contrast to both HLe-1 and transferrin receptor, which were progressively lost during the maturational process, once glycophorin had been maximally expressed on the cell surface, it remained at constant quantities to the mature erythrocyte stage. Although developing nucleated erythroid cells at approximately the normoblast stage had light-scattering properties similar to those of lymphoid cells, these two cell types could be resolved by cell surface antigen expression. Normoblasts were glycophorin positive and HLe negative, whereas lymphoid cells expressed HLe and either Leu 4, Leu 11, or Leu 12. Decreases in cellular nucleic acid content, corresponding first to the extrusion of the nucleus and second to the loss of reticulum, characterized the later stages of erythroid development. These characteristics and instrumentation can be used to purify erythroid cells at various developmental stages.

Antibodies, Monoclonal↗

PubMed1987

Flow cytometric analysis of human bone marrow. II. Normal B lymphocyte development.

A panel of B lymphoid-reactive monoclonal antibodies was used to analyze the phenotypic changes that accompany B lymphocyte development in normal human bone marrow. The B lymphoid cells were identified using light scattering and the expression of CD19 on a flow cytometer. Quantitative three-color immunofluorescence was then used to correlate other cell surface antigens on these cells identified as B lymphoid in normal marrow. CD10 and CD20 identified almost exclusive populations and provided a convenient means of discriminating between the less and more mature B lineage cells. The CD10+ cells could be further subdivided using CD34. The population of CD19+, CD10+, CD34+ cells comprised only 0.6% of marrow cells, but these contained the majority of terminal deoxynucleotidyl transferase (TdT+) cells. In the assessment of class II antigens, HLA-DR was expressed on all B lineage cells whereas HLA-DP preceded HLA-DQ in appearance during the developmental process. Among the later antigens expressed on B lineage cells, cell surface IgM, CD20, and HLA-DQ were expressed at essentially the same time. Cell surface CD10 was lost at the time when CD21 and CD22 were acquired on the cell surface. These data illustrate that multiparameter flow cytometry can be used to define a continuous progression of stages of B lymphocyte development based on cell surface antigen expression even though these cells represent a minor fraction of normal marrow cells.

Antibodies, Monoclonal↗

PubMed1984

Antibody-dependent cell-mediated cytotoxicity of cryopreserved human monocytes.

Human peripheral blood monocytes (PBM) modulate and participate in a variety of host defences. Cryopreservation of PBM has facilitated studies of their function. Peripheral blood samples cleared of red cells and granulocytes by centrifugation over Ficoll-Hypaque were cryopreserved at 1 degree C/min in 10% Me2SO and stored at -150 degrees C. Cryopreserved cells were thawed rapidly, diluted at a constant rate over 10 min with 9 vol of media, and washed twice prior to study. Antibody-dependent cell-mediated cytotoxicity (ADCC) activity against anti-D-coated Rh-positive erythrocytes of both fresh and cryopreserved PBM was tested and found to be equal (52.5 vs 51%). The myeloperoxidase positive, EA-rosette-positive population in cryopreserved cells was 39% compared with 17% for fresh cells (P less than 0.0001). This difference is due to preferential recovery of cryopreserved monocytes among mononuclear cells. The proportion of cells expressing Fc receptors among the myeloperoxidase-positive mononuclear cell population increased after freezing, suggesting an alteration in membrane structure induced by cryopreservation. It is concluded that PBM can be cryopreserved in Me2SO and that ADCC function is fully retained in the cryopreserved cells. This study along with a previous study (R.S. Weiner and S.J. Norman, J. Natl. Cancer Inst. 66, 255-260, 1981) demonstrate the feasibility of using cryopreserved human PBM for functional studies.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1983

Purified human monocyte subsets as effector cells in antibody-dependent cellular cytotoxicity (ADCC).

Human peripheral blood monocytes are heterogeneous with respect to their size and function. Two monocyte subsets were isolated by countercurrent centrifugal elutriation and were studied with respect to their ability to effect antibody-dependent cellular cytotoxicity (ADCC) and for the presence of Fc receptors on their surface. Both monocyte subsets display Fc surface receptors and are effectors of ADCC against sensitized human erythrocyte target cells. The demonstration of ADCC by monocyte effectors is dependent on their concentration in the incubation mixture. Dilution of monocytes below 10% by unlabeled and unsensitized erythrocytes or lymphocytes significantly suppresses ADCC, presumably by steric inhibition of effector and target contact.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1980

Purification of human monocytes: isolation and collection of large numbers of peripheral blood monocytes.

Monocytes from human peripheral blood were purified by elutriation centrifugation. Up to 1.5 x 10(9) peripheral blood mononuclear cells could be separated in isotonic media to yield 6--8 x 10(8) lymphocytes with 95% purity and 1.5--2.5 x 10(8) monocytes with greater than 90% purity. The temperature at which elutriation was performed determined the purity of the monocyte fraction. Both the lymphocyte and monocyte fractions were characterized by cell sizing and histochemical staining. Most of the myeloperoxidase positive mononuclear cells (monocytes) had a modal volume of 378 micrometer 3 while 10% were within the modal volume of the lymphocytes, 131 micrometer 3. Thus, there are two populations of cells with histochemical properties of monocytes which are separable by size. The collection and isolation of human peripheral blood monocyte populations in large numbers will facilitate studies of their functional characteristics.

Cell Separation↗