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H Zola

Publications and source records attributed to H Zola.

At least 19 recordsLinked to original sources
PubMed2006

Assessing the potential of immunohistochemistry for systematic gene expression profiling.

Immunohistochemistry (IHC) is a powerful technique for identifying sites of protein expression in tissues at the cellular and sub-cellular level. Here we have investigated the potential of using IHC for genome-wide expression screening by measuring the success rate and specificity of a panel of 35 monoclonal antibodies recognizing 5 well characterised CD antigens. Antibodies were pre-screened on acetone fixed frozen sections of spleen, tonsil and colon tissues. 19/35 antibodies gave staining with a success rate of 0/7 for JAM-2, 1/4 for CD99, 3/6 for CD138, 5/8 for CD45 and 10/10 for MHC-class II. 16/19 of these antibodies also gave staining on formalin fixed paraffin embedded tissue sections of tonsil and colon. All antibodies that had given staining were then profiled on tissues presented in human tissue microarrays. In the frozen microarrays 216 cores from 29 normal tissue types were present and in the formalin fixed paraffin array 344 cores from 35 normal and 4 cancers were represented. Where multiple antibodies were positive, there was evidence of consistent staining of the same tissues with several antibodies. In some cases differences in staining were observed potentially due to differential splice variants, polymorphisms or protein modification. With some antibodies there was evidence of cross-reactivity to inappropriate cells or structures. In addition the staining intensity with formalin fixation was changed quantitatively for some antibodies and in a few cases qualitatively, representing differential sensitivity of specific and non-specific epitopes to fixation. Accordingly, whilst IHC has potential for describing protein expression of unknown genes, these results emphasise a need to systematically address issues of specificity and sensitivity if appropriate profiles are to be described.

12E7 Antigen↗
PubMed2006

Conservation of leukocyte cell surface proteins: implications for the generation of monoclonal antibodies against newly identified leukocyte cell surface proteins.

The availability of mouse monoclonal antibodies has been integral to the classification of human leukocyte cell surface proteins under the "Cluster of Differentiation" or "CD" nomenclature system. The sequencing of the human genome has identified many more proteins that have characteristics similar to the known leukocyte cell surface proteins, but which have not so far been identified using monoclonal antibodies. One factor that may have limited the generation of monoclonal antibodies to some of these proteins is the high level of sequence conservation between the mouse and human proteins, in particular in the extracellular regions that are recognized by most of the widely used antibodies. An alternative approach is to use a more distant species, such as chickens, for the generation of antibody reagents. Here we compare the extent of amino acid differences in the protein CD molecules expressed by human leukocytes and their mouse and chicken homologs. The analysis confirms that the human proteins are more similar to the mouse homologs than the chicken homologs. The results indicate that chicken antibodies have the potential to be used as an alternative to mouse reagents where human-mouse sequence conservation is high.

Amino Acid Sequence↗
PubMed2005

A comparison of Verotoxin B-subunit (Stx1B) and CD77 antibody to define germinal centre populations.

We have directly compared the use of a CD77 antibody with the binding subunit of Shiga-like toxin 1, Verotoxin 1, and (Stx1B) for delineation on human tonsil cells. We determined that the Stx1B produced a greater intensity of staining than the CD77 antibody, allowing three sub-populations of germinal centre cells to be seen. The populations express high, medium, and low levels of globotriaosylceramide as determined by the binding of the Stx1B reagent. The strong staining patterns of Stx1B suggest that it may be useful in defining germinal center B cell populations.

Antibodies, Monoclonal↗
PubMed2005

Detecting antibodies with similar reactivity patterns in the HLDA8 blind panel of flow cytometry data.

The blind panel collected for the 8th Human Leucocyte Differentiation Antigens Workshop (HLDA8; ) included 49 antibodies of known CD specificities and 76 antibodies of unknown specificity. We have identified groups of antibodies showing similar patterns of reactivity that need to be investigated by biochemical methods to evaluate whether the antibodies within these groups are reacting with the same molecule. Our approach to data analysis was based on the work of Salganik et al. (in press) [Salganik, M.P., Milford E.L., Hardie D.L., Shaw, S., Wand, M.P., in press. Classifying antibodies using flow cytometry data: class prediction and class discovery. Biometrical Journal].

Antibodies↗
PubMed2005

Improved antigen binding by a CD20-specific single-chain antibody fragment with a mutation in CDRH1.

We have prepared single-chain immunoglobulin Fv fragments from the CD20-specific hybridoma HB13d. One scFv clone demonstrated strong binding to a CD20-derived peptide by ELISA and to CD20-positive cells by flow cytometry, a second had reduced binding, and a third clone did not bind the target antigen. Sequence analysis showed that all three constructs contained shared and unique amino acid changes when compared to the nearest germline match. Molecular modelling of the scFv variants revealed that several of the mutations are located in regions predicted to contact antigen, including a mutation in the heavy chain CDR1 of the strongest binding scFv construct. No similar mutation is present in the highly conserved protein sequences of a number of CD20-specific monoclonal antibodies. BIACORE analysis demonstrated that the mutated scFv had approximately three-fold greater antigen-binding activity than another clone. Competition studies showed that the scFv is able to compete with intact CD20 monoclonal antibody for binding to the target antigen. The improved antigen binding of this scFv will permit the construction of novel CD20-specific reagents for the therapy of lymphomas.

Amino Acid Sequence↗
PubMed2004

In silico evaluation of two mass spectrometry-based approaches for the identification of novel human leukocyte cell-surface proteins.

The identification and quantitation of cell-surface proteins expressed by leukocytes currently use the wide availability of monoclonal antibodies (mAb) in immunohistochemical and flow cytometric assays. Presently, approximately 400 such proteins have been characterized; however, analysis of the completed human genome sequence indicates that it may contain several thousand as-yet unidentified molecules, which may be expressed on the leukocyte cell surface. Recent advances in protein isolation and analysis using mass spectrometry illustrate that it is now feasible to identify the protein composition of a complex sample such as a plasma membrane extract. Such an approach may be useful for the identification of the cell-surface proteins that have not been identified using mAb techniques. Here, we detail the results of an in silico evaluation of the peptides isolated using two methods used to label plasma membrane proteins to determine whether these methods are suitable for the identification of known leukocyte cell-surface proteins by mass spectrometry. The labeling of cell-surface proteins before isolation and characterization is a valuable means of differentiating between plasma membrane and internal membrane proteins The results indicate that although the majority of cell-surface proteins can be identified using either of the approaches, others known to be important diagnostically and/or therapeutically would not be identified using either approach. The implication of this for the use of these techniques in the discovery of new leukocyte cell-surface proteins is discussed.

Amino Acid Sequence↗
PubMed2004

Rapid simultaneous measurement of multiple cytokines using 100 microl sample volumes--association with neonatal sepsis.

Early diagnosis of neonatal infection has proved problematic due to the inadequacy of currently available laboratory tests. Neonatal sepsis is associated with an increase in plasma-derived cytokine levels, but an increase of a single cytokine cannot identify neonatal sepsis specifically and multiple cytokine levels are required. The time constraints and relatively large volume of plasma required to measure multiple cytokines from newborn infants by conventional enzyme-linked immunosorbent assay (ELISA) techniques is prohibitive. We therefore applied cytometric bead array (CBA) technology for simultaneous measurement of multiple cytokines from a group of 18 term neonates with infection confirmed by culture and a control group. 'Normal' ranges were established for each cytokine from 1-7-, 8-14- and 15-21-day-old newborns. There was no significant change in the levels of cytokines from infants in different control age groups, suggesting that basal cytokine levels are unchanged in the first 3 weeks of life. In the patient groups, however, there was a significant difference in several cytokines between the different age groups. Interleukin (IL)-6, IL-10 and IL-12 were increased significantly in the 1-7-day-old patient group compared to either the 8-14 and 15-21 age group, suggesting that infection in utero is associated with increased levels of these cytokines compared to infection acquired following birth. When individual patient cytokine levels were compared to normal control reference ranges, two patients failed to show significant elevation of any cytokine tested. All other patients showed elevated levels of between one and nine cytokines tested (mean of 4.6). There was no correlation between elevated cytokine levels and types of infective organism or patient age. In conclusion, neonatal sepsis is associated with the elevation of multiple plasma cytokines. The use of CBA kits is a rapid, easy, low sample volume and sensitive method to measure multiple plasma cytokines.

Biomarkers↗
PubMed2003

Maternal milk regulation of cell infiltration and interleukin 18 in the intestine of suckling rat pups.

BACKGROUND: and aims: In neonates the gastrointestinal tract is exposed to food and bacterial antigens at a time when the gut mucosal immune system has not developed the ability to induce oral tolerance. This increases the risk for an inappropriate immune response to oral antigens. Transforming growth factor beta (TGF-beta) is an immunoregulatory cytokine present in high concentration in maternal milk. Interleukin 18 (IL-18) is a cytokine that mediates early immune events, and drives T cell development. We assessed the role of TGF-beta in mediating mucosal immune development and specifically the effect on endogenous IL-18. METHODS: Rat pups were randomly assigned to the following groups, naturally suckled, maternal milk via cannula, and formula fed with and without physiological levels of TGF-beta2. A comparison of the immune response profile was then carried out. Cytokine profiles, dendritic cell, intestinal mast cell, and eosinophil numbers were assessed. RESULTS: We show that feeding formula deficient in TGF-beta2 resulted in accumulated IL-18 protein release from intestinal epithelial cells and IL-18 mRNA up regulation. A proinflammatory cytokine profile resulted in the gut, along with increased numbers of activated dendritic cells, eosinophils, and mast cells. Supplementation of the formula with TGF-beta2 down regulated the proinflammatory cytokine mRNA as well as the number of activated lymphocytes, eosinophils, mast cells, CD80, and CD86 positive dendritic cells. CONCLUSION: The data suggests an important role for maternal milk, in regulating immune responses after exposure to food antigens, which might otherwise induce deleterious immune responses in the intestine of suckling neonates. This regulation is potentially mediated by milk TGF-beta2, as well as endogenous IL-18.

Animals↗
PubMed2002

Penetration of engineered antibody fragments into the eye.

Antibodies are powerful immunotherapeutic agents but their use for treating ocular disorders is limited by their poor penetration into the eye. We hypothesized that antibody fragments of relatively small size might penetrate the cornea more readily. Monovalent single chain variable region (scFv) antibody fragments and divalent miniantibodies were engineered from existing monoclonal antibodies, expressed in a bacterial expression system, and purified by metal ion affinity chromatography. Corneoscleral preparations from normal pig and cat eyes were mounted in a corneal perfusion chamber. Intact antibodies and antibody fragments were applied topically to the anterior corneal surface over 12-h periods, and samples were collected from the artificial anterior chamber. Similar experiments were performed with whole enucleated pig and human eyes. Penetration of antibodies and fragments was quantified by high-sensitivity flow cytometry on appropriate target cells. Both monovalent scFv and divalent miniantibody fragments (but not whole immunoglobulin molecules) passed through de-epithelialized and intact corneas after topical administration, and could be detected by antigen binding. Addition of 0.5% sodium caprate facilitated penetration through intact corneas. Topically-applied scFv was found to penetrate into the anterior chamber fluid of rabbit eyes in vivo. The engineered fragments were stable and resistant to ocular proteases. Monovalent and divalent antibody constructs of molecular weight 28 kD and 67 kD, respectively, can penetrate through intact corneas into the anterior chamber, with retention of appropriate antigen-binding activity. Such constructs may form novel therapeutic agents for topical ophthalmic use.

Animals↗
PubMed2001

CD antigens 2001.

Explore the source record for details and available documents.

Antigens, CD↗
PubMed2001

Increased yield and activity of soluble single-chain antibody fragments by combining high-level expression and the Skp periplasmic chaperonin.

The success of recombinant antibody fragments as diagnostic reagents and therapeutic agents depends on the availability of sufficient functional material. We have produced a bacterial expression vector that combines high-level expression driven by a modified Shine-Dalgarno sequence with the periplasmic chaperonin Skp. Using this vector, we are able to obtain higher yields of soluble antibody fragments from cultures without the need for supplementation of the culture medium during expression. The fragments produced in the presence of the Skp show improved antigen binding activity compared to when the chaperonin is absent.

Animals↗
PubMed2001

Antibody against CD20 in patients with B cell malignancy.

Cancer patients may make antibodies against antigens on the surface of their malignant cells due either to the expression of unique antigens or to dysregulated responses to self antigens. Patients with B cell malignancy frequently produce autoantibodies and may therefore be a source of immunoglobulin genes for the production of phage display antibody libraries directed against tumour-associated antigens. Patients with autoimmune disease have circulating antibodies against lymphocyte surface antigens, and may also provide a good starting point for the production of a library of lymphocyte-reactive antibody structures. In this study, plasma and serum samples from patients with B cell malignancy or Sjogren's syndrome and from healthy controls were screened for antibodies against the B cell membrane antigens CD20. While the majority of samples showed very low reactivity, some individuals did show significant and reproducible binding to CD20. To identify a good donor for library construction, it would be advisable to screen donors for antibody against the antigens of interest.

Antigens, CD20↗
PubMed2001

The Fc receptor for IgG (Fc gamma RII; CD32) on human neonatal B lymphocytes.

B cells express an Fc receptor for IgG (FcgammaRII; CD32) which is involved in feedback inhibition of antibody production. Engagement of FcgammaRII during ligation of the antigen receptor provides an inhibitory signal. FcgammaRII exists as several isoforms, with FcgammaRIIb (which carries an immunoreceptor tyrosine-based inhibition motif; ITIM) being predominant form on adult B cells. The inhibitory role of FcgammaRIIb may be unhelpful to the infant, since primary exposure to infectious agents is likely to be in the presence of maternal IgG. We hypothesized that neonatal B cells would be less susceptible to feedback inhibition by antibody, either through the expression of activation-competent FcgammaRII isoforms (FcgammaRIIa and FcgammaRIIc) or through reduced expression of the inhibitory FcgammaRIIb isoforms. Cord and adult B cells were examined for expression of FcgammaRII isoforms using monoclonal antibodies and RT-PCR. In vitro assays were performed to assess susceptibility of cord and adult cells to FcgammaRII-mediated suppression. Although there is no phenotypic difference in FcgammaRII expression (FcgammaRIIb predominating on both adult and cord B cells), FcgammaRIIb is expressed at lower levels on cord cells. This quantitative difference in FcgammaRIIb expression may explain the reduced susceptibility of cord B cells to antibody-mediated inhibition observed in these experiments.

Adult↗
PubMed2001

Immune modulation in suckling rat pups by a growth factor extract derived from milk whey.

Oral tolerance to foreign enteral antigens is not fully developed in early neonatal life. Epidemiological evidence supports a role for maternal milk in the development of immune responses, including oral tolerance. Formula fed infants have an increased susceptibility to food allergy and the later development of autoimmune disease. This may relate to the lack in infant formula of growth factors found in maternal milk. Bovine milk contains proteins, growth factors and cytokines. Various studies have outlined the immune modulating potential of bovine milk-derived products. Fractionated whey extracts have therapeutic potential in disease states where there is an excessive inflammatory reaction, and disease preventive potential for infants who are not breast-fed. We have shown that daily oral administration of a growth factor-enriched fraction from milk whey to naturally suckling rat pups between days 4-9 postnatal can down-regulate immune activation to a specific orally administered food antigen, ovalbumin, assessed by lymphocyte proliferation. In addition, non-specific down-regulation in the intestine was observed as assessed by the expression of MHC I. Treatment of rat pups with whey extract at the time of oral sensitisation to ovalbumin also resulted in an increased secretion of TGF-beta into the culture supernatant of spleen cells incubated with specific antigen. TGF-beta is an immuno-down-regulatory cytokine involved in tolerance induction. Immune modulation by extracts derived from milk whey could be of potential benefit for formula-fed and pre-term infants in reducing susceptibility to inappropriate activation to food antigens.

Animals↗
PubMed2001

CD antigens 2001.

The most recent Human Leucocyte Differentiation Antigen Workshop ("HLDA7") took place in 2000 in Harrogate, UK and the proceedings are about to be published (Leucocyte Typing VII). New Sections were introduced in this Workship (Dendritic cells, Stem/progenitor cells, Erythroid cells and Carbohydrate Structures) and monoclonal antibodies were selected for which at least some molecular data were already available (to avoid "blind" screening of reagents against known specificities). A total of more than 80 new CD specificities were established (previously the average was less than 30 new CD specificities per Workshop) and these are listed in this article. There is already evidence for the existence of many new leucocyte surface molecules for study at the next HLDA Workshop (in Adelaide in 2004), and we have listed in this article a number of such potential CD candidates (identified following the production of monoclonal antibodies or via gene cloning). There are also today an increasing number of lineage- and/or stage-restricted leucocyte-associated molecules localised within the cell cytoplasm (or nucleus): they will certainly prove of intense in the future for many laboratories studying human haematopoietic cells (regardless of whether a new "intracellular CD" categorisation scheme is devised for such molecules).

Antigens, CD↗
PubMed2001

CD antigens 2001.

Explore the source record for details and available documents.

Antibodies, Monoclonal↗