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Biomedical subjects

D Hardie

Publications and source records attributed to D Hardie.

At least 19 recordsLinked to original sources

Biological responses to the reversal of acidification in surface waters of the English Lake District.

Declining sulphur deposition since the late 1970s has led to increases in pH in lakes and streams of the English Lake District (Cumbria, UK). To determine whether there have been biological responses to the chemical changes, we carried out surveys of stream macroinvertebrates and lake surface sediment diatoms, and compared the results with those from earlier surveys. Macroinvertebrate taxa in five streams (current average pH range 5.1-7.0) sampled in 1999 showed clear changes from those found during 1965/1966 and 1972. For three of the streams, more taxa were present in 1999 than recorded using comparable sampling methods in the 1960s and 1970s, despite lower numbers of individuals being recovered in 1999. Values of the Margalef diversity index could be calculated for both 1965-1972 and 1999 for four of the streams; the index was significantly greater in 1999 (P < 0.001) in three streams, and unchanged in one stream (the most acid). The 1999 survey revealed the presence of acid-sensitive taxa that had been absent in the earlier surveys, notably the three stoneflies Leuctra fusca, L. moselyi and Chloroperla tripunctata. Some taxa that had been present during 1965-1972 were absent in 1999, but few of these were acid-sensitive. Diatoms from the surface sediments of six lakes were classified according to their acid sensitivities. In three of the lakes sampled in 1999 there were more diatoms characteristic of circumneutral waters, and less acidophilic species than had been found in 1983-1985. In the remaining three lakes, no noticeable changes had occurred. Overall, the observed biological changes are qualitatively as expected for the observed increases in pH, and there have been no instances of biological change in the opposite direction. The results support the expectation that changes in freshwater ecological status can be reversed by decreasing the remote emissions of acidifying pollutants.

Acid Rain↗

Novel molecular mechanisms of dendritic cell-induced T cell activation.

In this study we have re-examined the molecular mechanisms involved in activation of T cells by dendritic cells (DC). Human peripheral blood DC (PBDC) were derived by 2 h adhesion followed by 7 day culture in a combination of granulocyte macrophage colony stimulating factor and IL-4, and depletion of residual T and B cells. These PBDC were used to induce autologous T cell proliferation in a CD3-dependent response, and antibodies against CD11a/18 and CD86 were used as control inhibitors of accessory function. Antibodies against five of the cell surface molecules that we have recently identified on the surface of DC, CD13, CD87, CD98, CD147 and CD148, and an antibody which recognizes a molecule that has not as yet been identified, all inhibited the CD3-induced T cell proliferation. These findings were observed not only when antibodies were present throughout the culture, but also when they were prepulsed on to the surface of the DC, suggesting the inhibition was mediated via the antigen-presenting cells rather than the T cell. The same set of antibodies also inhibited an allospecific mixed lymphocyte reaction, confirming that the inhibitory effect was not dependent on the use of a CD3 antibody as the stimulating agent. All the antibodies of known specificity inhibited both CD4 and CD8 T cells equally. Unlike CD87, CD98 and CD147 antibodies, which inhibited activation of both CD45RA (naive) T cells and CD45RO (memory) T cells, CD13 and CD148 appeared to be involved in activation of naive cells only. The molecules identified in this study have not previously been demonstrated to play a role as accessory molecules on DC, the cells that are pivotal for immune induction. Therefore they may provide new potential targets for modulation of the immune response at the APC level.

Antibodies, Monoclonal↗

Inhibition of T cell apoptosis by IFN-beta rapidly reverses nuclear translocation of protein kinase C-delta.

Type I interferons rescue activated human T cells from cytokine deprivation-induced apoptosis. Our data now show that IFN-beta also rapidly inhibits apoptotic signals induced through the Fas receptor (CD95) in human T cells. To identify upstream signaling elements that could be targets of IFN-beta, we have studied protein kinase C (PKC). PKC-delta is actively involved in the regulation of apoptosis and immunofluorescence staining revealed that early in apoptosis PKC-delta accumulated in the nucleus. Addition of IFN-beta to T cells already deprived of survival factors or treated with anti-Fas antibody caused a rapid retranslocation of PKC-delta away from the nucleus. Furthermore, the generation of a constitutively active catalytic fragment by cleavage of PKC-delta by caspase 3 occurred only after translocation of full-length PKC-delta to the nucleus. IFN-beta also inhibited caspase 3 and the proteolytic activation of PKC-delta. We conclude from these studies that nuclear translocation of PKC-delta is an early event in T cell apoptosis and that IFN-beta rapidly reverses this process.

Apoptosis↗

Novel monoclonal antibodies to putative selectin carbohydrate ligands that inhibit selectin binding to myeloid cells.

Four newly developed monoclonal antibodies (MAbs) are characterized using flowcytometry, enzyme-linked immunoadsorbent assay (ELISA), immunoprecipitation and Western blots, carbohydrate epitope mapping, glycosidase cleavage, and competition binding assays. Their effects on selectin binding to myeloid cells was tested. These MAbs react only with myeloid cells. MAbs CI-1, BU60, and HIM95 recognize epitopes expressed by CD11/CD18 (beta2) integrins, while HI247 and CSLEX1 do not. The epitopes require Lewis x [Galbeta1-4 (Fucalpha1-3)GlcNAc] based on reactivity with oligosaccharide-polyacrylamide-biotin or oligosaccharide-BSA conjugates. MAb HI247 recognizes a related structure, sialyl-Lewis x, NeuAcalpha2-3GaLbeta1-4(Fucalpha1-3)GlcNAc. The three MAbs against Lewis x show some minor differences in their reactivity such as recognizing their antigens on CD11/CD18 integrins after endo-beta-galactosidase treatment and recognizing free Lewis x. The hydroxyl group on C-3 of the terminal galactose is important for recognition by MAb CI-1, BU60, and HIM95 as its substitution with sulfo group of sialic acid abolishes the binding of these MAbs. The C-3 sialic acid is crucial for the binding of MAb HI247. Its replacement by sulphate or its cleavage by sialidase eliminates recognition by this MAb. MAbs HI247 and CSLEX-1 did not react in ELISA with immobilized CD11/CD18, suggesting that the majority of sialyl Lewis x on CD11/CD18 molecules may have sialic acid 6-linked rather than 3-linked to galactose. Unexpectedly, MAb BU60 inhibited binding of P-selectin mu chain chimera to HL-60 or U937 cells, while CI-1, HIM95 and three other defined anti-Lewis x MAbs (6C7, M6-1 and LeuM1) did not. MAb HI247 inhibited binding of both E- and P-selectin chimeras to these cell lines more effectively than several characterized MAbs (CSLEX-1, FH6, HECA-452) to sialyl Lewis x and related oligosaccharides. Certain combinations of these anticarbohydrate MAbs had additive inhibitory effects on selectin binding, suggesting a potential application of these new MAbs in cell adhesion/migration and tumor metastasis studies.

Animals↗

The prevalence of surface antigen variants of hepatitis B virus in Papua New Guinea, South Africa, and Sardinia.

Three assays, one based on monoclonal antibodies and the others on polyclonal antibodies, were employed to detect hepatitis B surface antigen (HBsAg)-reactive samples in both vaccinated and unvaccinated populations in areas of the world where hepatitis B virus (HBV) is endemic. Any discordant sera were tested by polymerase chain reaction (PCR) to confirm current infection, and sequence data were obtained from the DNA coding for the major hydrophilic region (MHR) of HBsAg of those samples positive for PCR. In all countries studied, samples that reacted in one HBsAg assay but not another were found. In the most extreme case, about 5% of viremic sera in Papua New Guinea were nonreactive in the monoclonal HBsAg assay; 9 of the 13 PCR-positive samples had novel or once-described variants, or a variant out of its usual genotype context. In South Africa, samples with sequences of subtype ayw2 reacted poorly, particularly in the polyclonal assay. Two had novel variants. In Sardinia, antibody to hepatitis B core antigen (anti-HBc) was analyzed as a marker of infection. A significant proportion of anti-HBc-positive, but monoclonal HBsAg-negative, vaccinees and unvaccinated persons were found to be PCR positive, as were some individuals without any markers of hepatitis B virus infection. Five more novel variants were found in these groups. There are implications for the design of HBsAg assays, which may have to be modified according to local sequence variability. Not all discordant samples were explained by variants, indicating that assay sensitivity is fundamental to diagnostic efficacy. Overall, this study defined 16 novel variants and 2 new potential epitope clusters.

Amino Acid Sequence↗

Preclinical studies with the anti-CD19-saporin immunotoxin BU12-SAPORIN for the treatment of human-B-cell tumours.

The immunotoxin BU12-SAPORIN was constructed by covalently coupling the single-chain ribosome-inactivating protein saporin to the anti-CD19 monoclonal antibody BU12 via a disulphide linker using the heterobifunctional reagent SPDP. The immunoreactivity and specificity of BU12-SAPORIN was identical to that of unmodified native BU12 antibody. BU12-SAPORIN was selectively cytotoxic in vitro in a dose-dependent manner for the CD19+ human common acute lymphoblastic leukaemia (cALL) cell line NALM-6 but exhibited no toxicity for the CD19- T-cell acute lymphoblastic leukaemia (T-ALL) cell line HSB-2. The survival of severe combined immunodeficient (SCID) mice with disseminated NALM-6 leukaemia was significantly prolonged compared with sham-treated control animals by a course of therapy with BU12-SAPORIN but not with the irrelevant anti-CD7 immunotoxin HB2-SAPORIN. BU12-SAPORIN had no therapeutic effect in SCID mice with disseminated CD19- HSB-2 leukaemia. These preclinical studies have clearly demonstrated the selective cytotoxicity of BU12-SAPORIN for CD19+ target cells both in vitro and in vivo. This, taken together with the lack of expression of the CD19 molecule by any normal life-sustaining tissue and its ubiquitous and homogeneous expression by the majority of cALL and B-NHL cells, provides the rationale for undertaking a phase I trial of systemic therapy with BU12-SAPORIN.

Animals↗

Production of immunoglobulins by human sIgD+ and sIgD- human blood B lymphocytes in response to stimulation with activated T cells and agonistic antibodies; effect of IL-10, IL-2 and mode of activation of T cells.

Production of IgM, IgG and IgA was induced from human blood B lymphocytes by culturing with a CD40 MoAb and IL-2 for 9 days. Replacement of IL-2 by IL-10 markedly enhanced production of all three isotypes. High levels of immunoglobulin production also occurred when activated irradiated autologous T cells replaced the CD40 MoAb, and when IL-10 replaced IL-2 in these cultures a spectacular increase in IgG production occurred. The effectiveness of the T cell stimulus depended on the mode of purification of the T cells and the nature of the stimulant used to activate them. Differences in the kinetics and level of expression of CD40L on the various T cell preparations were observed, but did not account for variations in immunoglobulin-inducing efficiency. Immunoglobulin production from sIgD+ and sIgD- B cells was investigated. IgG and IgA were found in sIgD+ cultures, indicating that some isotype switching had occurred, but the major part of the IgG and IgA secreted was from cells already committed to these isotypes. Anti-IgD or anti-IgM MoAbs enhanced the proliferation of B cells induced by anti-CD40 antibody, but immunoglobulin production was not enhanced. Factors affecting the balance of proliferation and differentiation are discussed.

Animals↗

Synergy test for recognition of epitopes on soluble proteins; its application in the study of CD21 and CD23 antigens and their respective antibodies.

Antigens such as CD21 and CD23, which express only one copy of an epitope require two monoclonal antibodies (mAbs) for their detection and estimation. This requirement is exploited in two ways in a technique based on the chromic chloride haemagglutination test. For simple titration of antigen two portions of red cells each coated with one of a pair of synergising mAbs are used in a 1:1 combination. For testing the antigenic specificity of a mAb and assessing its region of epitope binding, the mAb under test is serially diluted in fluid containing a standard amount of antigen and red cells are added to which have been attached a different mAb. If the red cell-bound mAb recognises a determinant topographically distinct from that of the soluble mAb, red cell agglutination to high titre occurs. In titrations of ascitic fluid containing approximately 1 mg/ml mAb, titres of log2(9) to log2(16) were recorded from a starting dilution of 1 in 200. Hence the test is very sensitive and only minute amounts of a mAb are required for testing. The same test system can be used for assessing the relative display of epitopes on antigen obtained from different sources, e.g., culture supernates and body fluids. The method is of general applicability to monomeric antigens and its use is illustrated by analysis of CD21 and CD23 antigens and antibodies.

Animals↗

A subset of anti-CD21 antibodies promote the rescue of germinal center B cells from apoptosis.

Germinal center cells (GCC) are programmed to die by apoptosis unless they receive a positive signal for rescue. The primary signal in vivo is believed to be dependent on interaction with antigen held as immune complexes on follicular dendritic cells (FDC), a subset of which express large amounts of CD23, a low-affinity receptor for IgE. Recombinant soluble CD23 (sCD23) and interleukin-1 have been found to potentiate the survival of GCC in vitro. Recently, CD23 was shown to interact specifically with a ligand other than IgE, namely CD21 (CR2/Epstein-Barr virus receptor). In the present study, we show that a subset of anti-CD21 monoclonal antibodies behave similarly to soluble CD23 in their effect on GCC inasmuch as they: (i) diminish the occurrence of apoptosis; (ii) promote a plasmacytoid appearance in rescued cells; (iii) up-regulate expression of the Bcl-2 proto-oncogene. These findings indicate that FDC-derived CD23 exerts its effects on GCC via CD21.

Antibodies, Monoclonal↗

Association of hypomagnesemia and mortality in acutely ill medical patients.

OBJECTIVE: To test the hypothesis that the mortality rate of acutely ill patients admitted to a medical ward or medical ICU is higher for those patients who present with hypomagnesemia than for those patients who do not present with hypomagnesemia. DESIGN: Prospective, observational study. SETTING: Emergency Department admissions to the medical ward and medical ICU of a tertiary care teaching hospital serving an inner city patient population. SUBJECTS: A total of 381 consecutive acutely ill patients. MEASUREMENTS: Serum magnesium concentrations and other metabolic variables were measured on admission from the Emergency Department. Acute Physiology and Chronic Health Evaluation (APACHE II) scores were computed for all patients, and mortality rates were determined for hypomagnesemic and normomagnesemic groups. MAIN RESULTS: Hypomagnesemic and normomagnesemic groups had comparable APACHE II scores and other variables. However, the mortality rates of the hypomagnesemic ward and medical ICU groups were approximately twice (p < .01) the rate of the normomagnesemic groups. Additionally, the duration of hospital survival in those patients who died was approximately 8 days less for hypomagnesemia than normomagnesemia, but not for ward admissions. Other associated metabolic abnormalities were frequently observed in both hypomagnesemic and normomagnesemic groups, including hypokalemia and hypocalcemia. CONCLUSIONS: Hypomagnesemia detected at the time of admission of acutely ill medical patients is associated with an increased mortality rate for both ward and medical ICU patients.

Adolescent↗

Soluble forms of CD21 and CD23 antigens in the serum in B cell chronic lymphocytic leukaemia.

By using pairs of monoclonal antibodies (MAbs) to different epitopes on CD21 and CD23 antigens, it has been shown that both antigens are readily detectable in cell-free supernates of cultures of B cells expressing these antigens on the cell surface. The antigens remained in the soluble fraction after high speed centrifugation. Sera from normal individuals contained significant amounts of CD21 antigen, whereas little CD23 antigen was detectable. By contrast CD23 but not CD21 antigen was present in urine. Sera from patients with B cell chronic lymphocytic leukaemia (B-CLL) contained increased amounts of both antigens. The levels were related to the surface expression of antigen on the leukaemic cells and the number of cells in the blood. The possible functional role of soluble forms of B cell antigens and the diagnostic potential of their detection in body fluids are discussed.

Antibodies, Monoclonal↗

A phenotypic study of cells from Burkitt lymphoma and EBV-B-lymphoblastoid lines and their relationship to cells in normal lymphoid tissues.

Cells of 7 EBV-B-LCL, 10 Burkitt lines and 13 EBV-B-LCL/Burkitt line hybrids have been phenotyped for antigens of the major B cell clusters and for some other antigens. High levels of CD23 and CD39 and low levels of CD38 (T10) were characteristic of EBV-B-LCL; the converse was true for Burkitt lines. In hybrids the EBV-LCL phenotype was dominant. The phenotype of Burkitt-line cells correlated strongly with that of germinal centre B cells in tonsil sections, but differed markedly from that of marginal zone B cells or follicular mantle cells. The results are discussed in relation to the origin of Burkitt tumours of "sporadic" and "endemic" type, in particular to histopathological evidence that Burkitt lymphomas develop in germinal centres. Recent studies on the location of the breakpoints of Burkitt chromosomal translocations are also considered to be compatible with this concept, even though different regions of the immunoglobulin heavy chain locus are involved in the two types of BL.

ADP-ribosyl Cyclase↗

Immunogenic and antigenic epitopes of immunoglobulins. XVIII. Subpopulations of human lambda chains defined with a panel of monoclonal antibodies.

Following immunization with various lambda Bence-Jones proteins, over 100 hybridomas were established. A majority of the monoclonal antibodies (McAb) produced were demonstrated to recognize epitopes common to all lambda chains or unique to the immunogen. Twenty-nine McAb exhibited profiles of 'restricted' reactivity. Analysis with a panel of serologically defined antigens allowed McAb recognizing the lambda II subgroup to be identified. Other McAb recognized epitopes selectively expressed by lambda II and lambda III subgroup proteins. A further twenty-one McAb exhibited individual specificity profiles, some of which detect minor lambda chain subpopulations. Several of these McAb have potential for analysis of the clonality of specific antibody responses and for detecting monoclonal B-cell proliferations.

Antibodies, Monoclonal↗

An antigenic study of human plasma cells in normal tissue and in myeloma: identification of a novel plasma cell associated antigen.

A mouse monoclonal antibody named BU11 which detects an antigen strongly expressed on human plasma cells is described. The antibody stains plasma cells in tonsil sections, fresh and cultured plasmacytoid cells from the bone marrow of patients with multiple myeloma and cells of the plasmacytoid cell line RPMI 8226 used as the immunogen. In vitro studies of pokeweed mitogen (PWM) stimulated peripheral blood B cells and Epstein-Barr virus (EBV) stimulated tonsil B cells show that the antigen is present mainly on cells coexpressing the OKT10 antigen and containing cytoplasmic immunoglobulin (cIg). The BU11 antigen is expressed weakly on some normal B cells and is not present on T cells, monocytes or granulocytes. The antigen is of molecular weight 58kD under reducing conditions and is biochemically distinct from previously described plasma cell antigens.

Animals↗

Immunogenic and antigenic epitopes of immunoglobulins. X: Monoclonal antibodies specific for human IgA, the IgA1 and IgA2 subclasses and an nA2m(2) iso-allotypic epitope.

Monoclonal antibodies (McAb) specific for human IgA, the IgA1 and IgA2 subclasses and the iso-allotypic epitope nA2m(2) have been produced. Three distinct McAb recognize an IgA1-specific epitope expressed in the C alpha 2 domain or the hinge region whilst a further McAb is directed possibly to an IgA1 hinge region epitope. The McAb having nA2m(2) specificity recognizes an iso-allotypic epitope expressed within the Fab region. IgA1 and IgA2 epitopes were detected in gorilla but not rhesus or baboon sera suggesting that the IgA subclasses represent a recent gene duplication even. However, these epitopes were also detected in some non-primate sera.

Animals↗

Immunogenicity and antigenicity of immunoglobulins. XII. Intact light chain and heavy chain isotype-restricted Vk-associated epitopes.

Immunization with intact IgG has allowed the isolation of four hybridomas producing antibodies recognizing epitopes expressed within subpopulations of human kappa light chains unrelated to known polymorphisms (Km) and previously defined V-region subgroups. The V-region-associated epitopes recognized are conformation-dependent, being expressed on intact light chain but not on isolated VK or CK fragments. The frequency of expression within paraprotein panels of different heavy chain isotypes varied between individual antibodies. An epitope recognized by B2A6, expressed by greater than 85% IgGK paraproteins, was not represented in 16 IgM paraproteins tested, suggesting that association of VK with mu chains does not result in display of the epitope recognized, or alternatively, that selective association between VK and CH gene products occurs. These data contrast with the reactivity of other McAb for CK epitopes which were reactive with isolated CK fragments, and for all kappa-bearing paraproteins, regardless of heavy chain isotypes.

Antibodies, Monoclonal↗