Biomedical subjects

J F Harris

Publications and source records attributed to J F Harris.

At least 37 records · Page 2Linked to original sources

PubMed1985

A quantitative stability analysis of human monoclonal antibody production by heteromyeloma hybridomas, using an immunofluorescent technique.

We describe a quantitative method of analysis for assessing stability of human monoclonal antibody production by hybridomas. Clones derived from fusion between the SHM-D33 heteromyeloma line and EBV-stimulated human lymphocytes were studied for antibody presence using a fluorescent labelling technique. Frequencies of antibody-negative variants in clonal populations were measured, and measurements on parallel clonal populations were subjected to Luria-Delbrück fluctuation analysis to compute rates of generations of antibody-negative cells. Independent hybridoma clones exhibited a range of stabilities and the corresponding rates varied between 5 X 10(-4) and 6 X 10(-2) cell-1 generation-1. Rates of generation of antibody-negative variants for the more stable heteromyeloma hybridomas compared well with those of 2 established mouse hybridoma lines tested (less than 10(-3) cell-1 generation-1). There was a positive correlation between frequency of antibody-negative variants measured in clonal populations grown to large numbers of cells (greater than 10(7) per culture) and their rate of loss of antibody production. Large variations in frequency of antibody-negative variants were observed in parallel clonal populations, suggesting that loss of ability to produce antibody is due to random, mutation-like events including chromosome loss (Luria and Delbrück, 1943). High frequencies of antibody-negative variants may indicate imminent loss of antibody-producing capacity by a clone growing in suspension culture.

Antibodies, Monoclonal↗

PubMed1985

Quantitative genetic analysis of tumor progression.

Metastasis and resistance to chemotherapy are common features of progressed cancers. With respect to the latter phenotype, it is thought that during tumor growth drug-resistant cells arise spontaneously at rates characteristic of the genetic alterations involved. On application of chemotherapy, such variant tumor cells are more likely to survive, and they may eventually dominate, resulting in a non-responsive malignancy. Aspects of this model have been confirmed in a number of experimental systems and in patients. In contrast to our understanding of drug resistance, steps involved in the progression to metastatic spread of tumor cells are much less well-understood. In this review we describe methodologies of quantitative genetic analysis with reference to development of drug resistance. We then describe attempts by ourselves and others to use a similar approach to investigate metastatic properties. Based on these studies, we have proposed the quantitative 'dynamic heterogeneity' model of tumor metastasis, which is presented here. Using an 'experimental' metastasis assay and Luria-Delbruck fluctuation analysis, we determined that in murine KHT fibrosarcoma and B16 melanoma lines, 'metastatic' variants with a distinct phenotype are generated at high rates. These variants are relatively unstable resulting in a dynamic equilibrium between generation and loss of metastatic variants. The metastatic ability of such a tumor population is thus dependent on the frequency of a subpopulation of metastatic variants which are turning over rapidly. This dynamic heterogeneity model is able to quantitatively provide a unifying explanation for a wide range of observations concerning tumor heterogeneity and clonal instability. Genetic mechanisms involving rapid rates have been characterized in drug-resistant variants. We speculate that similar processes may be involved in different aspects of tumor progression such as those resulting in metastasis.

Animals↗

PubMed1985

Assignment of the human antithrombin III structural gene to chromosome 1q23-25.

The human antithrombin III (ATIII) structural gene was mapped by in situ hybridization and quantitative analysis of ATIII gene dosage in DNA isolated from carriers of chromosome 1 deletions. These studies indicate that the ATIII structural gene maps to human chromosome q23-q25 and so is likely identical to AT3.

Antithrombin III↗

PubMed1985

Hereditary thrombosis in a Utah kindred is caused by a dysfunctional antithrombin III gene.

Maximum likelihood analysis of linkage between antithrombin III (ATIII) DNA polymorphisms and ATIII deficiency in a large Utah kindred suggests that thrombotic disease in this family is caused by a dysfunctional ATIII gene. ATIII-deficient family members were identified on the basis of: (1) reduced anticoagulant activity and (2) the presence of an electrophoretically abnormal inhibitor molecule in their plasmas. Affected individuals have two copies of the ATIII structural gene, and both alleles appear normal at the resolution of whole genome Southern blotting. However, family studies revealed statistically significant cosegregation of ATIII-deficiency trait and a particular ATIII DNA polymorphism haplotype (lod = 3.35; theta = 0.0); this result is consistent with the presence of a dysfunctional ATIII gene on a chromosome of the +, S haplotype.

Alleles↗

PubMed1984

Dynamic heterogeneity: rapid generation of metastatic variants in mouse B16 melanoma cells.

The ability of clonally derived lines of B16F1 and B16F10 melanoma cells to form experimental metastases in C57BL mice after intravenous injection was examined. Luria- Delbruck fluctuation analysis was applied to the results obtained with parallel subclones grown to small population sizes before testing for metastatic ability. The analysis demonstrated that variant cells capable of forming experimental metastases were generated in B16F1 cell populations at an effective rate of about 1.3 X 10(-5) per cell per generation while in B16F10 cell populations the effective rate of production was about 5 X 10(-5) per cell per generation. These results are consistent with a dynamic heterogeneity model of tumor progression. They suggest that the majority of cells in both lines are effectively nonmetastatic and that the higher metastatic ability of the B16F10 population may be due in part to a higher rate of generation of metastatic variants.

Animals↗

PubMed1984

Dynamic heterogeneity and metastasis.

Parallel clonal populations grown to small defined sizes were used to quantitate rates of generation of metastatic cells. In murine KHT fibrosarcoma and B16 melanoma lines, metastatic cells are generated at effective rates of 10(-5) per cell per generation, or greater. These variant cells are unstable, and are apparently lost at very high rates. It thus appears that metastases could arise from unstable variants, and that rapid rates of change in some phenotypes may be an important feature of malignant progression. We have called such rapid variations dynamic heterogeneity. This may be a useful concept for further investigating aspects of tumor heterogeneity.

Animals↗

PubMed1984

Monoclonal antibodies against SSEA-1 antigen: binding properties and inhibition of human natural killer cell activity against target cells bearing SSEA-1 antigen.

We describe the properties of three monoclonal antibodies (Mab) against stage-specific embryonic antigen-1 (SSEA-1) in terms of their binding activity to HL60, K562, OTF9, and SOTF9 tumor target cells and their functional activity in modulating human natural killer (NK) cytotoxicity assays in vitro against these target cells. Indirect binding, competition, and Western blot analyses indicate that the Mab AEC3A1-9 (3A1), ASSEA-1, and AECAB1-32 (AB1) recognize cell-defined SSEA-1 antigen with activity characteristic of the cell source (HL60 greater than OTF9 greater than K562 much greater than SOTF9). The addition of anti-SSEA-1 Mab to the NK cytotoxicity assay resulted in an inhibition of LU per 1 X 10(6) PBL that correlated closely with the expression of SSEA-1 antigen on the target cell. No significant inhibition was seen for seven other Mab. Inhibition of NK activity (greater than 30%) was observed in the presence of anti-SSEA-1 Mab for 18 of 21 and 6 of 7 human donors examined for HL60 and OTF9 target cells, respectively. The pretreatment of fixed competing cells with anti-SSEA-1 Mab reduced the efficacy of those cells to act as cold competitors in a standard NK cytotoxic assay. Taken together these data suggest that SSEA-1 determinants are important at some stage in the cytolysis produced by NK cells.

Animals↗

PubMed1984

Recognition specificities, development and possible biological function of natural killer cells in the mouse. II. Changes in NK recognition during ontogeny and ageing, and examination of role of environment in controlling the expressed recognition repertoire.

We have used a spleen fragment assay to assess subpopulations of NK effector cells in individual mice and to analyse the patterns of inhibition of lytic activity seen in the presence of different sugars (mono-, di- and tri-saccharides). Our data suggest that during ontogeny the heterogeneity (diversity) of the NK effector population increases in a fashion which is somewhat characteristic of the individual strain under investigation. Furthermore, when a similar analysis was performed on NK cells in the spleen of lethally irradiated recipient mice receiving syngeneic or semi-allogeneic bone marrow stem cell precursors, we found that the phenotype of inhibition by different sugars was a characteristic of the bone marrow donor and not of the recipient. In so far as the assay described assesses target recognition by NK cells (and not subsequent parameters involved in the lytic event) these data can be interpreted in terms of a relative independence of the expressed recognition repertoire of NK cells from the environment in which their differentiation occurs.

Aging↗

PubMed1984

Idiotypic analysis of anti-I-Ak monoclonal antibodies. I. Production and characterization of syngeneic anti-idiotypic mAb against an anti-I-Ak mAb.

To analyze the idiotype (Id) of anti-Ia antibodies elicited during alloimmune responses, we produced syngeneic mouse anti-Id monoclonal antibodies (mAb) reactive with the Id of the 11-5.2.1.9 (11-5) mouse anti-I-Ak (BALB/c anti-CKB) mAb. Two such anti-Id mAb, IA2 (IgG2a) and IIID1 (IgG1), detect structurally related idiotopes located within the binding site of 11-5 for I-Ak antigens. A third anti-Id mAb, VC6 (IgG1), detects an idiotope located either inside or outside of, but presumably proximal to, the 11-5 antigen-binding site, because its expression correlates with the antigenic specificity of 11-5. None of the idiotopes detectable by these three anti-Id mAb are accessible when the binding site of 11-5 is occupied by an I-Ak molecule. The association constants of these anti-Id mAb for their cognate Fab-linked Id range from 2 X 10(9) to 1 X 10(10) M-1. The three anti-Id-producing hybridomas were found with a frequency of 0.008% among growing hybrid colonies. Even though these anti-Id mAb detect public idiotopes (IdX) on 11-5, they do not detect the presence of such IdX markers in the sera of five syngeneic BALB/c mice hyperimmunized with C3H (I-Ak) spleen cells. This suggests that 11-5 represents a BALB/c idiotype infrequently expressed by serum immunoglobulins. The 11-5 idiotopes detectable by IA2, IIID1, and VC6 seem to be conformationally determined by the interaction of 11-5 H and L chains and are not confined to one or the other of these subunit polypeptides. Thus, the expression of the 11-5 Id may be regulated by both VH and VL genes.

Animals↗

PubMed1984

Rapid phenotype variation in cells derived from lung metastases of KHT fibrosarcoma.

We have established previously that intravenously derived metastatic variants are generated in KHT fibrosarcoma cells at an effective rate of 10(-5)/cell/generation. To study the properties of these variants further, we examined several lines of KHT fibrosarcoma cells obtained from experimental lung metastases. When tested using an experimental metastasis assay, some of the lines were highly metastatic, relative to parental lines, but these highly metastatic phenotypes were often rapidly lost as the lines were grown in vitro, and both decreases and increases in metastatic ability were observed. In another set of experiments, lines obtained by 10 serial selections of experimental lung metastases without intervening in vitro growth between passages were also analyzed. Again, while highly metastatic phenotypes were observed in some instances, they did not persist beyond 1 or 2 in vivo passages, and the series as a whole failed to reveal a persistent increase in ability to form experimental metastases. We conclude from these experiments that although metastatic variants are generated at high rates in KHT cell lines, the phenotype is lost at even higher rates, and metastatic variants represent only a small proportion of the tumor cell population. Thus, it appears that in this system rapid phenotypic variation may play an important role in the metastatic process.

Animals↗

PubMed1983

Exocrine function of testis with germinal testicular tumour.

Semen collected immediately before or soon after therapeutic unilateral orchidectomy from 86 men with germinal tumours of the testis was analysed. The mean sperm count was 65.6 +/- SEM 10.3 X 10(6) per ejaculate. This was significantly different from that in a control population, in whom the mean was 165.2 +/- 12.9 X 10(6) per ejaculate. The difference appeared to be due to a subpopulation of 32 patients with counts of 20 X 10(6) per ejaculate; the distribution of counts in the remaining patients was similar to that in the controls. The cause of this abnormality of exocrine function is unknown: it may either be due to the tumour or its treatment, or both, or, alternatively, it may predate the events of the malignant transformation, possibly even acting as an inducing or promoting factor.

Castration↗

PubMed1983

Retinoic acid-induced neural differentiation of embryonal carcinoma cells.

We have previously shown that the P19 line of embryonal carcinoma cells develops into neurons, astroglia, and fibroblasts after aggregation and exposure to retinoic acid. The neurons were initially identified by their morphology and by the presence of neurofilaments within their cytoplasm. We have more fully documented the neuronal nature of these cells by showing that their cell surfaces display tetanus toxin receptors, a neuronal cell marker, and that choline acetyl-transferase and acetyl cholinesterase activities appear coordinately in neuron-containing cultures. Several days before the appearance of neurons, there is a marked decrease in the amount of an embryonal carcinoma surface antigen, and at the same time there is a substantial decrease in the volumes of individual cells. Various retinoids were able to induce the development of neurons in cultures of aggregated P19 cells, but it did not appear that polyamine metabolism was involved in the effect. We have isolated a mutant clone which does not differentiate in the presence of any of the drugs which are normally effective in inducing differentiation of P19 cells. This mutant and others may help to elucidate the chain of events triggered by retinoic acid and other differentiation-inducing drugs.

Animals↗

PubMed1983

Induced muscle differentiation in an embryonal carcinoma cell line.

Cells of the teratocarcinoma-derived line P19S1801A1 (01A1) are pluripotent embryonal carcinoma cells and can be induced to differentiate when aggregated and exposed to dimethyl sulfoxide. Many nonneural cell types appear in dimethyl sulfoxide-treated cultures, cardiac and skeletal muscle being the most easily identified. We have used immunofluorescence procedures with monoclonal antibodies directed against muscle myosin to confirm and quantitate the number of muscle cells formed. A monoclonal antibody reactive with an embryonal carcinoma-specific surface antigen was used to confirm the disappearance of undifferentiated cells after dimethyl sulfoxide treatment. Cardiac muscle cells developed within 4 to 5 days of drug exposure, but skeletal muscle cells did not become evident until 7 to 8 days. We have isolated a mutant cell line (D3) which appears to be incapable of muscle development but which does form neurons and glial cells when exposed to high retinoic acid concentrations. We propose that this system will be useful for investigation of the means by which pluripotent cells become committed to development along the striated muscle lineages.

Animals↗

PubMed1982

Role of Ia antigens in graft vs. host reactions. II. Molecular and functional analysis of T cell alloreactivity by the characterization of host Ia antigens on alloactivated donor T cells.

Graft vs. host response (GVHR)-activated donor T cells bind to stimulatory host cell-derived Ia antigens. Radioimmune cell-binding assays demonstrate that activated donor T cells acquire both host I-A and I-E alloantigens on their surface. Approximately threefold to fivefold less I-E products than I-A products are transferred. Immunoprecipitation and one-dimensional and two-dimensional gel electrophoresis analyses show that radioiodinated alpha and beta polypeptide chains of both I-A and I-E-encoded host Ia molecules may be transferred in an apparently structurally unaltered form from host cells to donor cells. Biosynthetic studies indicate that [35S]methionine-labeled activated donor T cells do not synthesize Ia antigens of the donor haplotype. Functional analyses with fluorescence-activated cell sorter sorted donor T cell subpopulations show that donor T cells that bind host I-A antigens preferentially cooperate with nonimmune host B cells. Donor T cells that do not bind detectable amounts of host I-A antigens preferentially help nonimmune donor B cells. By contrast, donor T cells that either bind or do not bind host I-A antigens display no H-2-restricted interaction and help both donor and host immune B cells. These data reveal that the Ia antigen-binding specificity of distinct functional subpopulations of alloactivated donor T cells regulates their I-region-restricted (self or allo) helper activity for nonimmune B cells but not immune B cells. Furthermore, they suggest that T cell-macrophage and T cell-B cell collaboration is mediated by a complementary anti-Ia:Ia receptor:ligand type of interaction in which the receptor of a T cell binds to the ligand of an antigen-presenting macrophage and/or B cell.

Animals↗

PubMed1982

Metastatic variants are generated spontaneously at a high rate in mouse KHT tumor.

Using the Luria-Delbrück fluctuation analysis, we have examined the lung tumor-forming ability of a series of parallel clones derived from the KHT tumor, grown to small defined sizes. From these studies, we conclude that metastatic variants arise spontaneously in the clonal lines during their growth, at an apparent rate of approximately 10(-5) per cell per generation. This rapid rate has implications for our understanding of tumor heterogeneity and the process of tumor progression. Previous results have suggested that heterogeneity observed in cloning experiments reflects stable subpopulations of cells in the original tumor. We propose here an alternative "dynamic heterogeneity" model, in which metastatic variants arise at a high rate (as detected in the cloning experiments) but need not be stable mutations in order to effectively produce metastases.

Animals↗

PubMed1982

Long-term care criteria and standards agreement with professional placement determination.

The decisions made by physician-nurse teams and individual nurse-evaluators following examination of long-term care patients are compared with the results of placement assignment of the same patients obtained by using the New York State Patient Assessment Form (DMS-1), predictor scores, and numerical standard. The descriptors, when applied to long-term care patients and weighted according to intensity, were capable of matching the best judgment of these professionals with a 90 per cent concurrence.

Health↗

PubMed1981

In vitro analysis of allogeneic lymphocyte interaction. V. Identification and characterization of two components of allogeneic effect factor, one of which displays H-2-restricted helper activity and the other, T cell-growth factor activity.

An allogeneic effect factor (AEF) derived from mixed lymphocyte reaction (MLR) cultures of alloactivated A.SW (H-2s) responder T cells and irradiated A/WySn (H-2a) stimulator spleen cells helps an in vitro primary anti-erythrocyte plaque-forming cell PFC response of BALB/c nude spleen cels and also A/WySn but not A.SW T cell-depleted spleen cells. AEF activity is adsorbed by anti-Ik and anti-I-Ak but not by anti-I-Jk, anti-I-ECk, and anti-Is. Gel filtration of ACA 54 resolves AEF into two main components that which appear in the 50,000- to 70,000-mol wt (component I) and 30,000- to 35,000-mol wt (component II) regions, respectively. Component I has a mol wt of 68,000, elutes from DEAE-Sephacel at 0.05-0.1 M NaCl, and has an isoelectric point (pI) of 5.8. It helps A/WySn but not A.SW B cells and, therefore, is H-2 restricted. Component II is not H-2 restricted, because it helps both A.SW and A/WySn B cells. It also stimulates (a) the growth of a long-term cytotoxic cell line in vitro, (b) Con A-induced thymocyte mitogenesis, and (c) the generation of cytotoxic T cells. The latter three properties of component II are not shared by component I. In addition, component II elutes from DEAE-Sephacel at 0.15-0.2 M NaCl and has a pI of 4.3 and 4.9. Ia determinants and Ig VH, CH, L-chain, and idiotypic determinants are not present on either component I or component II. The properties of component II are identical to that of a T cell growth factor produced by Con A-stimulated spleen cells. It is suggested that the H-2-restricted component I of AEF might be an MLR-activated responder T cell-derived Ia alloantigen receptor.

Animals↗

PubMed1980

Derivation of a monoclonal antibody that detects an Ia antigen encoded by 2 complementing I-subregions.

A monoclonal antibody designated A303, which reacts with Ia antigens controlled by the complementation of 2 distinct I-subregion genes, is described. Complementation of both I-A and I-EC subregion gene products is necessary for the cytotoxic, direct and 125I-protein A-amplified indirect cell binding, and immunoprecipitation activities of A303. Positive prototype strains of independent origin include H-2k,p, whereas A303-negative haplotypes include H-2b,d,f,r,s,u. Seven recombinant strains, H-2ap5,as1,h4,i3,i5,t3,t4 representing intra I-region cross-over events between positive and negative haplotypes are A303 negative. Trans complementation occurs between the I-Ak allele and the I-EdCd, I-EkCk and I-EkCd alleles, respectively. Analysis of F1 heterozygotes suggests that A303-binding activity is also controlled by gene-dose effects. Thus, the activity of A303 is formally equivalent to a system of coupled complementation previously described for Ir and Is genes.

Animals↗