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Biomedical subjects

M A Holmes

Publications and source records attributed to M A Holmes.

At least 19 recordsLinked to original sources
PubMed2005

Controlled clinical trial of the effect of a homoeopathic nosode on the somatic cell counts in the milk of clinically normal dairy cows.

Cows in a 250-cow Holstein-Friesian herd were allocated at random to be treated with either a homoeopathic nosode or a negative control, both treatments being applied by means of an aerosol spray to the vulval mucous membranes. A total of six treatments were given over a period of three days and milk samples were taken for the determination of somatic cell counts (SCC) on days -3, 3, 7, 9, 14, 21 and 28. Individuals applying the treatments or carrying out the SCC determination were unaware of which animals were receiving which treatment. Owing to the wide natural variations in SCC, the trial had only a 71 per cent possibility of detecting a 30 per cent difference in SCC between the two groups. There were no significant differences between the SCC of the two groups on any sample day, but there were significant variations between the SCC on different days (P=0.003) in both groups.

Animals↗
PubMed2001

Structural effects of framework mutations on a humanized anti-lysozyme antibody.

A humanized version of the mouse anti-lysozyme Ab D1.3 was previously constructed as an Fv fragment and its structure was crystallographically determined in the free form and in complex with lysozyme. Here we report five new crystal structures of single-amino acid substitution mutants of the humanized Fv fragment, four of which were determined as Fv-lysozyme complexes. The crystals were isomorphous with the parent forms, and were refined to free R values of 28-31% at resolutions of 2.7-2.9 A. Residue 27 in other Abs has been implicated in stabilizing the conformation of the first complementarity-determining region (CDR) of the H chain, residues 31-35. We find that a Phe-to-Ser mutation at 27 alters the conformation of immediately adjacent residues, but this change is only weakly transmitted to Ag binding residues in the nearby CDR. Residue 71 of the H chain has been proposed to control the relative disposition of H chain CDRs 1 and 2, based on the bulk of its side chain. However, in structures we determined with Val, Ala, or Arg substituted in place of Lys at position 71, no significant change in the conformation of CDRs 1 and 2 was observed.

Amino Acid Substitution↗
PubMed2000

Production and characterisation of two monoclonal antibodies recognising equine IgG Fc receptors.

Despite the importance of IgG Fc receptors in the regulation of various immunological mechanisms, these receptors have not been well characterised in the domesticated animals including equines. This paper describes the production of two monoclonal antibodies (CVS 59 and CVS 61) that recognise equine IgG Fc receptors. Fusions were conducted using BALB/c mice hyperimmunised with equine peripheral blood mononuclear cells. Hybridoma supernatants were screened on the basis of their ability to inhibit the rosetting of equine antibody coated sheep erythrocytes with equine peripheral blood mononuclear cells. The mAbs were then characterised for cellular distribution of the antigen by flow cytometry. Using immunoprecipitation, it was shown that both under reducing and non-reducing conditions, CVS 59 and CVS 61 precipitated a 80 and 40 kD protein, respectively. From the functional study coupled with cellular distribution and molecular weight of the antigen recognised by these mAbs, it would appear that CVS 59 and CVS 61 recognise an epitope on equine equivalent of human and murine FcgammaRIII and FcgammaRII, respectively.

Animals↗
PubMed2000

Immunoglobulin isotypes in sera and nasal mucosal secretions and their neonatal transfer and distribution in horses.

OBJECTIVE: To determine concentrations of IgA and IgG subclasses in serum, colostrum, milk, and nasal wash samples of adult horses and foals. ANIMALS: Seven 2-year-old Welsh ponies, 27 adult mixed-breed horses, and 5 Quarter Horse mares and their foals. PROCEDURE: Serum was obtained from ponies and adult horses. Colostrum and milk were obtained from mares and serum and nasal wash samples from their foals immediately after parturition and on days 1, 7, 14, 28, 42, and 63. Nasal wash samples were also obtained from 23 adult horses. Concentrations of immunoglobulins were determined by use of inhibition ELISA. To determine transfer of maternal isotypes to foals, concentrations in colostrum and milk were compared with those in foal serum. Serum half-lives of isotypes in foals were also determined. RESULTS: IgGb was the most abundant isotype in serum and colostrum from adult horses, whereas IgA was the predominant isotype in milk. The major isotype in nasal secretions of adult horses and foals > or = 28 days old was IgA, but IgGa and IgGb were the major isotypes in nasal secretions of foals < or = 14 days old. Serum half lives of IgGa, IgGb, IgG(T), and IgA in foals were 176, 32, 21, and 3.4 days, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The early immunoglobulin repertoire of neonatal foals comprised IgGa, IgG(T), and IgA; endogenous synthesis of IgGb could not be detected until 63 days after birth. The restricted repertoire of immunoglobulins in foals may influence humoral immune responses to vaccination.

Animals↗
PubMed1999

Characterisation of equine T helper cells: demonstration of Th1- and Th2-like cells in long-term equine T-cell cultures.

The aim of this study was to characterise CD4+T-cells in equines, as these cells are pivotal in establishing immune responses or regulating established ones. Peripheral blood mononuclear cells from a pony immunised with ovalbumin were cultured in vitro in the presence of the specific antigen and autologous antigen presenting cells. During the antigen starvation phase, cells were maintained on recombinant equine IL-2. After 35 days of culture, most of the cells were CD4+, CD8-and sIg-. Cells proliferated specifically in the presence of antigen, as tested on day 42 of culture. These cells were analysed by in-situ hybridisation to detect m RNA for IL-2 and IL-4, the presence of which suggested the existence of of Th1- and Th2-like cells.

Animals↗
PubMed1999

Validity of saddle pressure measurements using force-sensing array technology--preliminary studies.

Back pain is a common and poorly understood clinical problem. An important factor in this regard is the induction or exacerbation of back pain from badly designed or poorly fitting saddles. This study used a pressure-sensing mat to investigate saddle fit. The aims of the study were to confirm the accuracy and reliability of the force-sensing array technology when used to measure pressure beneath the saddles of horses, and to collect some initial data from normal healthy horses with well-fitting saddles. Experiments were undertaken to establish that a linear relationship existed between the total force (weight) exerted and the pressure measured beneath the saddle, using both a wooden horse and a live horse in the standing position. Further studies were performed to demonstrate that characteristic changes of the centre of pressure occur while horses move at the walk, sitting trot, rising trot, and canter.

Animals↗
PubMed1999

Transurethral needle ablation of the prostate: outcome at 1 year.

BACKGROUND: Urologists continue to search for alternatives to transurethral prostatectomy that carry a lower potential for complications. PATIENTS AND METHODS: Twenty-five patients on the public waiting list for transurethral resection of the prostate, all spontaneously voiding, underwent transurethral needle ablation of the prostate (TUNA). Eight patients had a simultaneous bladder neck incision (BNI). Follow-up with International Prostate Symptom Scores and flow rate measurement was performed at 6 weeks, 7 months, and 1 year. RESULTS: A statistically significant increase in flow rate and fall in symptom scores occurred out to 1 year after TUNA. Despite this result, six patients (24%) were not satisfied with their outcome and underwent a second endoscopic operation. CONCLUSION: In our hands, TUNA produced an unsatisfactory clinical result.

Aged↗
PubMed1999

An exploration of oral health beliefs and attitudes of Chinese in West Yorkshire: a qualitative investigation.

This qualitative study explores oral health beliefs and attitudes among Chinese resident in West Yorkshire using six focus groups differentiated by age and gender. Focus group discussions took place in community settings led by trained Chinese facilitators. All groups believed that they were susceptible to dental disease, and that bleeding gums and total tooth loss were 'normal'; apart from the elderly, tooth loss was seen as undesirable. The elderly and adult groups believed in traditional remedies and claimed that preventive oral health measures were ineffective. These groups lacked faith in dentists, and for them cost, language difficulties and lack of awareness were the main reported barriers to accessing dental services. Traditional Chinese oral health beliefs remain influential for the elderly and adult UK Chinese. In contrast, teenagers thought that toothbrushing and sugar restriction would help to prevent dental diseases. The appropriateness of the focus group and interview methods for exploring oral health beliefs for the Chinese are discussed, as are implications of the reported intergenerational differences for oral health promotion strategy in the UK.

Adolescent↗
PubMed1998

Report of the Second Equine Leucocyte Antigen Workshop, Squaw valley, California, July 1995.

The final assignment of antibody clusters for leucocyte antigens and immunoglobulins, as described in detail in Sections 3 and 4, is summarized in Table 4. Together with other mAbs developed outside of ELAW II (Table 9) this pool of reagents represent a powerful array of tools for the study of equine immunity. The Second Equine Leucocyte Antigen Workshop made considerable advances in pursuing the objectives of establishing the specificities of mAbs and achieving consensus on the nomenclature for equine leucocyte and immunoglobulin molecules. Of equal importance, several productive collaborations were fostered among the participating laboratories and observers. Overall, enormous advances have been made in the past decade since mAbs specific for equine leucocyte antigens and immunoglobulins were first reported. There remains enormous scope and need for further studies of equine leucocyte antigens and immunoglobulins, both for the purposes of comparative immunology and for the good of the horse. In the future novel techniques will be required to develop reagents for specific target antigens such as the orthologues of the CD25 or CD45 isoforms. In studies of equine immunoglobulins the functional role of the IgG isotypes must be better established, reagents for IgE must be developed, and cloning of the immunoglobulin heavy chain genes will be essential if the complexities of the IgG sub-isotypes are to be elucidated. The tasks still facing the currently small group of equine immunologists throughout the world remain formidable, and will only be tackled successfully in a spirit of collaboration.

Animals↗
PubMed1998

Monoclonal antibodies to subclass-specific antigenic determinants on equine immunoglobulin gamma chains and their characterization.

This paper describes the production of a panel of monoclonal antibodies (mAbs) identifying the four recognised equine IgG subisotypes IgG, IgGa, IgGb, IgGc and IgG(T). Pure preparations of the subisotypes for use in immunisations and testing were produced using a combination of gel filtration, salt precipitation, ion exchange chromatography and protein A and Protein G affinity chromatography. The specificity of mAbs for the IgG subisotypes was confirmed using ELISA assays, by characterisation of affinity purified proteins recognised by the mAbs, and by Western blotting of equine serum proteins. The expression of equine IgG subisotypes by B cells was examined by flow cytometry using the panel of mAbs.

Animals↗
PubMed1998

Conformational correction mechanisms aiding antigen recognition by a humanized antibody.

The crystal structure of the complex between hen egg lysozyme and the Fv fragment of a humanized antilysozyme antibody was determined to 2.7-A resolution. The structure of the antigen combining site in the complex is nearly identical to that of the complexed form of the parent mouse antibody, D1.3. In contrast, the combining sites of the unliganded mouse and humanized antilysozymes show moderate conformational differences. This disparity suggests that a conformational readjustment process linked to antigen binding reverses adverse conformations in the complementarity determining regions that had been introduced by engineering these segments next to human framework regions in the humanized antibody.

Animals↗
PubMed1998

A syndrome of anaemia, immunodeficiency and peripheral ganglionopathy in Fell pony foals.

Fell pony foals developed a syndrome of anaemia, immunodeficiency and peripheral ganglionopathy. They became ill in the second or third week, and died in the second or third month of life. Clinical and pathological investigations revealed severe anaemia associated with small numbers of late erythroid precursors in bone marrow, small thymi, an absence of secondary lymphoid follicles, a lack of plasma cells and neuronal chromatolysis involving trigeminal, cranial mesenteric and dorsal root ganglia. Some of the foals had cryptosporidial enteritis and adenoviral bronchopneumonia and pancreatitis. The clinical and pathological findings were compatible with an intrinsic defect.

Anemia↗
PubMed1998

Organization of the equine immunoglobulin heavy chain constant region genes; III. Alignment of c mu, c gamma, c epsilon and c alpha genes.

Previous restriction analysis of cloned equine DNA and genomic DNA of equine peripheral blood mononuclear cells had indicated the existence of one c epsilon, one c alpha and up to six c gamma genes in the haploid equine genome. The c epsilon and c alpha genes have been aligned on a 30 kb DNA fragment in the order 5' c epsilon-c alpha 3'. Here we describe the alignment of the equine c mu and c gamma genes by deletion analysis of one IgM, four IgG and two equine light chain expressing heterohybridomas. This analysis establishes the existence of six c gamma genes per haploid genome. The genomic alignment of the cH-genes is 5' c mu/(/) c gamma 1/(/) c gamma 2/(/) c gamma 3/(/) c gamma 4/(/) c gamma 5/(/) c gamma 6/(/) c epsilon-c alpha 3', naming the c gamma genes according to their position relative to c mu. For three of the c gamma genes the corresponding IgG isotypes could be identified as IgGa for c gamma 1, IgG(T) for c gamma 3 and IgGb for c gamma 4.

Animals↗
PubMed1997

Structural consequences of humanizing an antibody.

We have determined the crystal structure of the Fv fragment of a humanized anti-hen eggwhite lysozyme Ab (HuLys). This molecule is a composite of known structures: complementarity-determining regions (CDRs) were from the mouse anti-lysozyme Ab D1.3, heavy chain framework regions were from the human myeloma protein NEW, and the light chain framework regions were consensus sequences similar to the Bence Jones protein REI. HuLys crystallized in space group P4(3)2(1)2, with two Fv molecules in the crystallographic asymmetric unit. The structure was solved by molecular replacement, using a composite of the parent structures as a search model. The resolution of the structure was 2.87 A, with an R factor of 22%. There were several unanticipated structural similarities and differences between HuLys and the mouse and human structures. The framework regions of HuLys were as close or closer in conformation to D1.3 than to the NEW or REI protein, despite overwhelming sequence identity with the human framework regions. The effect was most pronounced at the CDR-framework junction, showing that the CDRs can induce structural changes in framework residues, having the net effect in HuLys of reconforming the framework into a conformation more like D1.3. In the combining site, the grafted CDRs retained conformational equilibria seen in D1.3, demonstrating that humanizing can be applied to Abs that bind Ags through isomeric equilibrium or induced fit mechanisms. In addition, HuLys showed systematic differences from D1.3 that resembled domain rotations reported for other Abs. However, the V(H)-V(L) interface itself was unaffected, and the apparent movement was actually caused by rearrangements distant from this surface.

Amino Acid Sequence↗
PubMed1997

Cytokine RNA expression in an equine CD4+ subset differentiated by expression of a novel 46-kDa surface protein.

Two monoclonal antibodies (MAb), HB65A (IgG2a) and HB86A (IgGI), recognize a unique cell surface molecule on equine T-lymphocytes. The molecule, designated EqWC4, identified by these MAbs is present on a subpopulation of CD4+ equine lymphocytes (6.3-10.2% of Arabian lymphocytes CD4+ WC4+) and a smaller population of CD8+ lymphocytes (0.5% to 1.2% of Arabian lymphocytes CD8+ WC4+). EqWC4 is absent from B-lymphocytes, granulocytes, and macrophages. Both MAbs bound to a 46-kDa protein following immunoprecipitation reactions with lysates of surface labeled thymocytes. Immunoaffinity purification using HB65A yielded two molecules of 46 kDa and 52 kDa under reducing conditions and a third 92-kDa molecule was present in nonreduced conditions. Activation by mitogen did not increase expression of EqWC4 on equine lymphocytes. Lymphocytes from Arabian, Pony, and Thoroughbred breeds showed a common distribution of EqWC4 among leukocytes. However, there were significantly fewer Pony lymphocytes bound to HB65A and HB86A when compared to Arabian and Thoroughbred breeds. Using reverse transcriptase-polymerase chain reaction (RT-PCR), magnetically enriched populations (to 80% of cells isolated) of EqWC4+ lymphocytes expressed a cytokine RNA profile dominated by -interleukin2 (IL-2) and interferon-gamma (IFN-gamma) for unstimulated cells. Upon mitogen stimulation, IL-4 was also expressed at low levels while the IL-2 levels decreased and the IFN-gamma levels increased relative to unstimulated cells. EqWC4 is similar to CD28 in molecular weight and its formation of dimers and could therefore be the equine orthologue. However, because of the differences in CD28 expression, EqWC4 probably represents a previously uncharacterized equine lymphocyte marker.

Animals↗
PubMed1997

Serum and mucosal antibody isotype responses to M-like protein (SeM) of Streptococcus equi in convalescent and vaccinated horses.

Equine strangles, caused by the clonal pathogen Streptococcus equi, is a source of serious economic loss despite the widespread use of commercial vaccines. The anti-phagocytic 58 kDa M-like protein (SeM) is an important protective antigen. The objective of this study was to define differences, if any, between SeM-specific convalescent serum and mucosal IgA and IgG subisotypes and those induced by vaccination with commercial strangles vaccine. SeM-specific opsonophagocytic IgGb was the predominant serum antibody in horses intramuscularly vaccinated or recently recovered from infection. Infection also induced high levels of specific opsonophagocytic serum IgGa during and shortly after S. equi infection whereas vaccination stimulated only low levels of serum IgGa. Specific serum IgGc and opsonophagocytic IgA were present at very low levels following infection or vaccination. A strong specific mucosal antibody response occurred during the acute and convalescent phases of infection whereas vaccinated horses made no mucosal response. Specific IgGb was generally predominant in nasopharyngeal washings during the acute phase but was replaced by specific IgA during convalescence. SeM-specific mucosal IgGa and IgG(T) but not IgGc were detected only during the acute and early convalescent phase. The results therefore indicate that vaccination, although inducing SeM-specific serum isotype responses qualitatively and quantitatively similar to those seen in convalescence, did not induce mucosal responses. This suggests that mucosal immunity may be important in acquired resistance to strangles.

Animals↗