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Biomedical subjects

S M Andrew

Publications and source records attributed to S M Andrew.

At least 37 records · Page 2Linked to original sources
PubMed1994

Redirection of T cell-mediated cytotoxicity by a recombinant single-chain Fv molecule.

We have produced two single-chain Fv (sFv) proteins by bacterial periplasmic secretion, one sFv with specificity for the hapten DNP, and the other for the human transferrin receptor. After solubilization and refolding, we recovered several mg of active sFv per liter of bacterial culture. Each sFv bound to cells bearing the appropriate Ag and could be used to direct targeted cellular cytotoxicity. Targeting relied on a universal bispecific antibody designed to cross-link CD3 on the cytotoxic T cell with a peptide fused to the sFv carboxyl-terminus. The universal bispecific antibody was used in combination with the Ag-specific sFv to redirect human cytotoxic T cells to kill a variety of target cells. Such an approach has a number of advantages that may make it useful for the immunotherapy of cancer and other diseases.

Amino Acid Sequence↗
PubMed1994

Immunohistochemical appearance of corticosteroid contact hypersensitivity reactions.

We have studied, immunohistochemically, hypersensitivity reactions to corticosteroids and compared them with allergic contact dermatitis from nickel and appropriate controls. We could find no qualitative differences between nickel and corticosteroid contact reactions, providing further evidence that hypersensitivity to corticosteroids is an immunologically mediated reaction.

Administration, Topical↗
PubMed1994

Inflammatory cells in normal human fracture healing.

We studied inflammatory cells in specimens of callus taken from normally healing human fractures. Using immunohistochemistry, T-cells, B-cells, macrophages, HLA-DR expression and endothelial proliferation were assessed. Macrophages were present from an early stage but became less numerous later. T-cells were initially specifically recruited into the fracture site at the stage of granulation tissue, but subsequently excluded from areas of bone and cartilage formation. Inflammatory cells may control and coordinate fracture healing as has been proposed for soft tissue wound healing. The most likely mechanism for this is by the cytokines and growth factors which they are known to release.

Adult↗
PubMed1994

Direct analysis of body composition of dairy cows at three physiological stages.

Holstein cows were killed at three physiological stages, prepartum (-7 d, n = 10), early lactation (63 d, n = 7), and late lactation (269 d, n = 8), for determination of chemical composition and prediction of energy changes during lactation. Cows were weighed, slaughtered, and separated into five or six fractions, including carcass, gastrointestinal tract, mammary gland, uterus, and fetus (if present); the remainder was noncarcass. Live BW and weight of all empty body components except fat were unaffected by physiological stage. Empty body fat was reduced 42.3 kg for the early lactation cows compared with that of prepartum cows. Fat-free matter was similar across physiological stages; however, the water content of fat-free matter was greater for the prepartum and early lactation cows than for late lactation cows. In early lactation cows, the percentages of total protein were less in carcass and greater in gastrointestinal tissue than in prepartum and late lactation cows, but fat distribution was not affected. The energy values of 9.2 and 5.57 Mcal/kg for fat and protein in tissue, respectively, were determined by regression and used to apportion energy associated with fat, .925, and protein, .07, during lactation using data adjusted for ash. A maximum loss of 442 Mcal of tissue energy by approximately 77 DIM was determined by regression of empty body energy on DIM).

Animals↗
PubMed1993

An improved method for generating single-chain antibodies from hybridomas.

Cloning the correct VL kappa gene from hybridomas derived from MOPC-21 can be problematic because such cell lines variably express a transcript which is aberrantly rearranged at the VJ recombination site. Cellular levels of the aberrant transcript can exceed that of productive light chain RNA, so a large proportion of the VL gene-derived products obtained on PCR amplification of hybridoma cDNA may not encode a functional protein. We have developed a method in which antibody variable region genes are recovered from hybridoma cDNA using a unique set of V gene family-specific primers; the V region genes are then spliced by PCR, in the form 5'-VL-LINKER-VH-3' (where the linker encodes [GlyGlyGlyGlySer]3), and cloned into an expression vector under control of T7 RNA polymerase. Plasmid DNA is isolated from colonies, and the insert is expressed in an in vitro rabbit reticulocyte lysate-based coupled transcription/translation system, in a microtiter plate format. Since aberrantly rearranged VL kappa genes contain a translation termination codon at amino acid position 105, only constructs containing the correctly rearranged gene produce a protein of the predicted size. We demonstrate the method by producing the single-chain form of OKT9, a murine IgG1 which binds to the human transferrin receptor, and extend the results to show that the protein generated by the in vitro expression system retains the antigen binding properties of the parent antibody. Our method will be generally useful for screening single-chain antibodies for function prior to large scale production in vivo.

Amino Acid Sequence↗
PubMed1993

A bacterially expressed single-chain Fv construct from the 2B4 T-cell receptor.

A single-chain Fv construct of the 2B4 T-cell receptor has been made and expressed in Escherichia coli as bacterial inclusion bodies. After solubilization in 6 M guanidine hydrochloride and formation of mixed disulfides with glutathione, the protein was refolded by diluting out the denaturant and allowing intramolecular disulfide bridges to form by disulfide exchange. Approximately 65-100 mg of refolded protein was obtained from 1 liter of bacterial culture, an appreciable fraction of which was monomeric in nondenaturing solvents. This protein bound to three monoclonal antibodies specific for allotypic or idiotypic determinants on the native 2B4 variable region but did not bind several other anti-T-cell-receptor monoclonal antibodies that lacked such specificity. These experiments show that T-cell-receptor variable regions, like the V regions of antibodies, can form a well-behaved single-chain Fv molecule and provide large amounts of recombinant single-chain Fv T-cell receptor that can be used to study T-cell function.

Amino Acid Sequence↗
PubMed1993

Characterization of single-chain antibody (sFv)-toxin fusion proteins produced in vitro in rabbit reticulocyte lysate.

Chimeric proteins consisting of a fusion between binding-deficient mutants of diphtheria toxin (DT) or Pseudomonas exotoxin A (PE) and a single-chain antibody (E6 sFv) against the human transferrin receptor (TfnR) were expressed in a rabbit reticulocyte lysate system. Molecules utilizing PE40 (the carboxyl terminus 40 kDa of PE, lacking the binding domain) exhibited significant E6 sFv-mediated, cell type-specific cytotoxicity (IC50 1 x 10(-10) M) against a human erythroleukemia-derived cell line, K562. In contrast, a fusion protein between the same sFv and a DT mutant, DTM1 (containing two amino acid substitutions in the binding domain [S(508)F, S(525)F]) was not significantly cytotoxic, despite being enzymatically active. A tripartite protein in the form NH2-DTM1-E6 sFv-PE40-COOH exhibited cytotoxicity comparable to that of the PE40-sFv fusion (IC50 1 x 10(-10) M), suggesting that the deficit in activity of DTM1-sFv is not a function of misfolding of the sFv moiety or of a reduced ability to bind TfnR. In contrast to DTM1-E6 sFv, a fusion protein between a second DT mutant, CRM 107 [S(525)F], and the E6 sFv was specifically cytotoxic (IC50 1 x 10(-9) M), and toxicity could be blocked by addition of excess E6 antibody. The cell-free in vitro expression system we describe is rapid and may be used to express functional toxin-sFv fusion proteins. No protein refolding procedures are required, and the technique may be used to express proteins which, due to restrictions imposed on manipulation of toxin-encoding genes in Escherichia coli, could not be produced by more conventional methods.

ADP Ribose Transferases↗
PubMed1993

Demonstration of TGF-beta 1 mRNA by in situ hybridization in normal human fracture healing.

The role of transforming growth factor beta (TGF-beta) in fracture healing has previously been investigated in a rodent model, but not in human material. We investigated TGF-beta 1 gene expression in specimens of callus from normally healing human fractures, using in situ hybridization to a cDNA TGF-beta 1 probe and an autoradiographic disclosure system. TGF-beta 1 mRNA was present in areas of proliferation of mesenchymal tissue, bone, and cartilage. Levels of expression were lower in cells in the fracture hematoma and in differentiated (hypertrophic) chondrocytes. These results are compatible with those found in various animal models using immunohistochemistry and support the view that locally produced TGF-beta 1 is a regulator of fracture repair in humans from the early (mesenchymal proliferation) stage to the stage of remodeling of woven bone. They also indicate that, for TGF-beta 1, animal models accurately reflect human bone repair.

Adolescent↗
PubMed1993

Dermal dendrocytes in Dupuytren's disease: a link between the skin and pathogenesis?

The skin has previously been implicated in the process of Dupuytren's disease. The dermal dendrocyte is a factor XIIIa positive cell, which has been found in normal skin and some pathological conditions associated with fibrosis. In this study we examined the distribution of factor XIIIa positive cells in and around tissue from Dupuytren's disease. Immunohistochemistry was performed using a panel of antibodies for factor XIIIa, macrophages (CD68 and MAC387) and MHC II. Many factor XIIIa positive dendritic cells were present in and around Dupuytren's tissue; fewer CD68 and MHC II positive cells and very few MAC387 cells were seen. We propose that the factor XIIIa positive cells are dermal dendrocytes. This study may indicate an important link between the skin and pathogenesis of Dupuytren's disease.

Dendritic Cells↗
PubMed1993

Immunochemotherapy of human colon carcinoma xenografts in nude mice using combinations of idarubicin-monoclonal antibody conjugates.

Tumour cell heterogeneity is probably a principal cause of treatment failure and represents a formidable barrier for effective antibody-targeted chemotherapy. Idarubicin (Ida), a more potent and less cardiotoxic analogue of daunomycin, has been demonstrated to specifically target and eradicate homogeneous, cloned, murine tumour cell populations in vitro and in vivo when coupled to monoclonal antibodies (MoAb); however, the antitumour activity of Ida-MoAb conjugates against human tumour xenografts remains to be established. In this study, the value of cotargeting conjugates to different human tumour-associated antigens within a solid tumour has been assessed by comparing the effects of combinations of Ida-anti-colon carcinoma MoAb conjugates with any one Ida-anti-colon carcinoma MoAb conjugate used alone. Individual Ida-MoAb conjugates have previously been evaluated for their specific binding and cytotoxicity to one of two different human colon carcinoma xenografts (Colo 205 or LIM2210) in vitro, although their efficacy alone or in combination required assessment in vivo. Combinations of the most effective Ida-MoAb conjugates were demonstrated to enable a greater number of complete tumour regressions than the most efficacious Ida-MoAb conjugate administered alone in vivo; some combinations inhibited control tumour growth by up to 95%. This study suggests that Ida-MoAb conjugates can be effective against subcutaneous human tumours in nude mice, although it is unlikely that any single conjugate will eradicate all the tumour cells in a solid tumour, and the value of 'cocktails' of drug-MoAb conjugates against some xenografts (i.e. LIM2210) appears to be limited.

Animals↗
PubMed1993

Skeletal mastocytosis.

AIMS: To characterise the condition of skeletal mastocytosis, an uncommon cause of apparently "idiopathic" osteoporosis. METHODS: Transiliac crest biopsy specimens submitted over a period of five years were examined for nodular accumulation of mast cells. The cases were reviewed histologically and clinical follow up was obtained from hospital notes. RESULTS: Six cases of mastocytosis occurring in bone biopsy specimens submitted to our department were identified. Four patients presented initially with vertebral collapse and the other two were known to have extraskeletal mast cell disease at presentation. On clinical review of the four patients with vertebral collapse, one was found to have urticaria pigmentosa. This patient died from his mastocytosis, whereas the three patients without evidence of extraskeletal disease remain alive and well. Histological examination showed that patients with the poorer clinical outcome had severe peritrabecular fibrosis as well as mast cell nodules; those with prolonged disease-free survival had nodules without peritrabecular fibrosis. CONCLUSION: There is a form of mastocytosis which presents clinically as "idiopathic" osteoporosis. Clinically it does not have the same prognostic implications as skeletal disease in "malignant mastocytosis", running a much more benign course.

Aged↗
PubMed1993

Changes in numbers of epidermal cell adhesion molecules caused by oral cyclosporin in psoriasis.

AIM: To determine the effects of a three month course of low dose cyclosporin on the expression of epidermal cell adhesion molecules. METHODS: Eighteen patients with psoriasis were treated for 12 weeks with either 2.5 or 5 mg/kg/day of oral cyclosporin. Biopsy specimens taken from skin before, during, and after cyclosporin treatment were stained immunohistochemically for CD 54 (ICAM-1), CD 29 (beta-1 integrins), and CD18 (beta-2 integrins). RESULTS: There was a highly significant (p < 0.01) clinical response after 12 weeks of cyclosporin as assessed by the Psoriasis Area and Severity Index (PASI) score. The staining of CD 29 on keratinocytes of affected and unaffected psoriatic skin was not affected by cyclosporin. Epidermal CD54 was variably expressed in active psoriatic plaques and changed unpredictably after cyclosporin (p = NS). Staining for CD18 on large epidermal dendritic cells was reduced after cyclosporin (p < 0.02). The expression of CD18 by large epidermal dendritic cells during treatment correlated strongly with the PASI score at that time and one month after stopping cyclosporin (p < 0.02). CONCLUSIONS: Persistence of epidermal staining for CD 54 in psoriasis is compatible with a good clinical response to cyclosporin. Residual staining for CD 18 on large epidermal dendritic cells may be a useful marker for early clinical relapse.

Antigens, CD↗
PubMed1992

Bispecific antibodies and retargeted cellular cytotoxicity: novel approaches to cancer therapy.

We have used a relatively new technology to increase the number of human lymphocytes that will react with human ovarian carcinoma cells. This technology, often called "retargeting of the immune system," can temporarily redirect the activity of immune cells that were originally committed to react with foreign substances other than cancer cells. In the example presented here, the antitumor effects of retargeted human T lymphocytes, collected from normal donors, were tested in immunodeficient mice with a human ovarian carcinoma line growing intraperitoneally. We retargeted T cells in vitro with a bispecific antibody that reacted with the T cell receptor complex and with a cell-surface antigen expressed by the ovarian carcinoma cells. Retargeted lymphocytes, injected intraperitoneally into mice 4 days after intraperitoneal injection of the tumor cells, impeded tumor growth and doubled the host survival time. These findings provide support for the concept that treatment of ovarian cancer patients with retargeted T cells could prove beneficial.

Animals↗
PubMed1992

Targeting of anti-tumor responses with bispecific antibodies.

T cells can be induced to specifically lyse tumor cells with bispecific antibodies containing anti-T cell receptor mAbs crosslinked to anti-tumor mAbs. Such "targeted cytolysis" requires that the target cell be bound directly to the cytotoxic cell. In addition, targeted T cells mediate a second activity, the secretion of factors that can block the growth of both tumor target cells and bystander tumor cells. When given to nude mice bearing intraperitoneal human ovarian carcinoma, targeted human T cells cause the rapid removal of most tumor cells from the peritoneum, and markedly prolong the times of survival of treated mice. The efficacy of targeted T cells for treating human cancer is currently being tested in clinical trials.

Animals↗
PubMed1992

A quantitative immunohistochemical study of the expression of integrins by nerves in psoriatic and normal skin.

Qualitative and quantitative assessment of integrin expression by dermal nerves was made by an avidin-biotin immunoperoxidase method on snap-frozen biopsies from affected psoriatic skin, and skin from normal control subjects with no history of skin disease. Nerves expressed alpha 1, alpha 2, alpha 3, alpha 6, beta 1 and beta 4 integrin subunits, and perineural sheaths in the mid-dermis also expressed these subunits, with the exception of alpha 2. There were more upper dermal nerve segments expressing alpha 1 integrin compared with other integrins both in controls and in psoriatic skin. The greater number of nerves expressing alpha 1 integrin compared with other integrins may be due to anatomical or functional differences between groups of nerves. There were significantly more nerves expressing alpha 1, alpha 2, alpha 3, alpha 6 and beta 4 integrins in psoriatic skin compared with control skin. This generalized increase may indicate a secondary trophic effect on all nerves rather than a specific increase in one type of nerve. However, the expression of alpha 2 integrin may be significant in the pathogenesis of the psoriatic plaque, in that it was barely detectable in the normal site-matched biopsies, but much greater in psoriatic plaques. The study of the expression of adhesion molecules by neurones in psoriasis offers a new avenue for investigation of the role of neuronal hypertrophy in the initiation and maintenance of psoriatic plaques.

Adult↗