Search

Your search keyword '"Werkmeister, J. A."' showing total 192 results
Start Over Author "Werkmeister, J. A."
192 results on '"Werkmeister, J. A."'

7. Composite autologous stem cell and polyamide graft in the ovine prolapse model; Comparing 2 delivery systems, integration and biocompatiblity.

Author

Mukherjee S., Werkmeister J., Gargett C., Rosamilia A., Emmerson S., Karjalainan P., Mukherjee S., Werkmeister J., Gargett C., Rosamilia A., Emmerson S., and Karjalainan P.

Abstract

HYPOTHESIS/AIMS OF STUDY: The aim of this study was to compare two methods for trans-vaginal delivery of autologous endometrial mesenchymal stem cells (eMSC) on a novel polyamide (PA) synthetic mesh with biomechanical properties designed to match human vaginal tissue using an ovine model of vaginal wall weakness. The second aim was to determine the retention of implanted autologous Iodex-labelled eMSC and to assess their effect on the extracellular matrix and infammatory response to the implanted gelatin-coated (PA/G) and non-coated PA mesh. STUDY DESIGN, MATERIALS AND METHODS: Multiparous sheep (n=30) which underwent quantifcation of pelvic organ prolapse using a modifed POP-Q (Pelvic Organ Prolapse Quantifcation) were divided into 4 experimental and 1 incisional control group. Subtotal hysterectomy was performed via ventral midline laparotomy on 12 sheep to collect endometrial tissue. Ovine autologous eMSC were isolated from hysterectomy tissue utilizing cell sorting by fow cytometry of CD271+ ovine eMSCs, cell culture and eMSC labeling with FITC-labelled IODEX paramagnetic nan-oparticles. Polyamide synthetic grafts were fabricated and used alone or with eMSC or were dip-coated in 12% porcine gelatin and stabilized with 0.5% glutaraldehyde for PA/G constructs; autologous labelled ovine eMSC-seeded PA/G constructs were also prepared. Surgery consisted of a 40 mm, full-thickness midline incision on the posterior vaginal wall and eMSC/PA/G (n=6), PA/G (n=6), PA (n=12) were surgically implanted. A 2 step procedure occurred in the fourth group with eMSC in a gelatin hydrogel (eMSC/G) were applied to the 6 already implanted PA meshes (PA + eMSC/G) and the gelatin crosslinked in situ with blue light. No graft was placed for the incisional controls. After suturing the ewes were allowed to recover and were harvested 30 days post implantation. The analyses of explants included histology, collagen analysis using sirius red birefringence, image analysis of immunofuorescence a

Published
2019

8. Pre-clinical evaluation of immune response to stem cell boosted degradable nanostructured surgical constructs for pelvic organ prolapse.

Author

Rosamilia A., Darzi S., Werkmeister J., Gargett C., Mukherjee S., Rosamilia A., Darzi S., Werkmeister J., Gargett C., and Mukherjee S.

Abstract

HYPOTHESIS/AIMS OF STUDY: Until recently, polypropylene meshes were often used as a surgical treatment option for Pelvic Organ Prolapse (POP). However, they have been associated with serious complications such as infammation, pain and erosion. As a result, transvaginal meshes were banned in Australia and New Zealand in 2017. At present, there is no optimal therapy for treatment of chronic POP leaving millions of women in despair. To overcome adverse events associated with trans-vaginal surgery using non degradable synthetic mesh for treating pelvic organ prolapse (POP), we are developing a tissue engineering construct comprising degradable nano-fbrous mesh and endometrial mesenchymal stem/stromal cells (eMSC). The aim of this study was to investigate in vivo foreign body response, angiogenesis and extracellular matrix gene expression to a nano-topographically controlled mesh made of poly (L-lactic acid)-co-poly(-caprolactone) and gelatin incorporating eMSC (PLCL/G/eMSC). Our team discovered a rare population of Mesenchymal stem cells in the endometrium (eMSCs) and has identifed a single marker to isolate these rare perivascular cells. In a phase IV clinical trial, our team has also showed that eMSCs can be collected with ease even from post-menopausal women and kept in an undiferentiated state for clinical use making them an ideal choice for autologous urogynecological application. We further more compared the diferences between the treatment capacity of eMSCs delivered using degradable and non-degradable. STUDY DESIGN, MATERIALS AND METHODS: PLCL polymer and gelatin (1:1, 10%w/w) was electrospun to form uniform nanofbers and assessed for fbre diameter, pore size, hydrophilicity and biomechanical properties. SUSD2+ eMSC were purifed from single cell suspensions obtained from endometrial biopsies from cycling women and by magnetic bead sorting and transfected with mCherry len-tivirus for cell tracking. SEM was used to characterise eMSC incorporation into the nanofber

Published
2019

9. Degradable nanostructured mesh and endometrial stem/stromal cells ASA bioengineered construct for pelvic organ prolapse.

Author

Darzi S., Kadam V., Truong Y., Rosamilia A., Werkmeister J., Gargett C., Mukherjee S., Darzi S., Kadam V., Truong Y., Rosamilia A., Werkmeister J., Gargett C., and Mukherjee S.

Abstract

Introduction: To overcome adverse events associated with transvaginal surgery using non degradable synthetic mesh for treating pelvic organ prolapse (POP), we are developing a tissue engineering construct comprising degradable nanofibrous mesh and endometrial mesenchymal stem/stromal cells (eMSC). The aim of this study was to investigate the in vitro cell-material properties and in vivo foreign body response to a nano-topographically controlled mesh made of poly (L-lactic acid)-copoly( -caprolactone) and gelatin incorporating eMSC (PLCL/G/eMSC). Method(s): PLCL alone and a 1:1 blend with gelatin (w/w) were electrospun from a 10% solution at 1 ml/hr and 18kV over 12.5 cm to form uniform nanofibers and assessed for fibre diameter, pore size, hydrophilicity and biomechanical properties. SUSD2+ eMSC were purified from single cell suspensions obtained from endometrial biopsies from cycling women by magnetic bead sorting and transfected with mCherry lentivirus for cell tracking. SEM was used to characterise eMSC incorporation into the PLCL and PLCL/G nanofiber meshes in vitro and their degradation in NSG mouse skin wound repair model of vaginal repair (2, 6 wks, 4 mice/gp). IF and confocal microscopy was used to assess total (F4/80+), M1 (F4/80+CCR7+) and M2 (F4/80+/CD206+) macrophages. Result(s): Addition of G to PLCL increased fiber size 40% (P<0.05) and pore size 25% (P<0.05) rendering meshes hydrophilic without change in tensile properties. In vitro, eMSC rapidly adhered, penetrated, incorporated and proliferated (P<0.01) in PLCL/G mesh with a 2.5 fold greater coverage than PLCL alone (P<0.05). In vivo, mCherry+ eMSC survived 1 wk in PLCL and PLCL/G implanted mesh, but only a few were retained in PLCL/G and none in PLCL at 6 wk. eMSC prevented PLCL/G nanofiber degradation 2 fold (P <0.0001) and increased cellular infiltration into the mesh 1.5 fold (P<0.05) at 6 wks. eMSC seeded on PLCL/G promoted macrophage switching from M1 to M2 phenotype to a greater extent than s

Published
2019

11. Endometrial Mesenchymal Stem/Stromal Cells Modulate the Macrophage Response to Implanted Polyamide/Gelatin Composite Mesh in Immunocompromised and Immunocompetent Mice

Author

Darzi, S., primary, Deane, J. A., additional, Nold, C. A., additional, Edwards, S. E., additional, Gough, D. J., additional, Mukherjee, S., additional, Gurung, S., additional, Tan, K. S., additional, Vashi, A. V., additional, Werkmeister, J. A., additional, and Gargett, C. E., additional

Published
2018
Full Text
View/download PDF

12. Interaction of preservation methods and radiation sterilization in human skin processing, with particular insight on the impact of the final water content and collagen disruption. Part I: process validation, water activity and collagen changes in tissues cryopreserved or processed using 50, 85 or 98% glycerol solutions

Author

Herson, M. R., primary, Hamilton, K., additional, White, J., additional, Alexander, D., additional, Poniatowski, S., additional, O’Connor, A. J., additional, and Werkmeister, J. A., additional

Published
2018
Full Text
View/download PDF

13. Vaginal wall weakness in parous ewes: a potential preclinical model of pelvic organ prolapse.

Author

Young N., Rosamilia A., Arkwright J., Davies-Tuck M., Melendez J., Werkmeister J., Gargett C.E., Lee J., Young N., Rosamilia A., Arkwright J., Davies-Tuck M., Melendez J., Werkmeister J., Gargett C.E., and Lee J.

Abstract

Introduction and hypothesis: Ewes develop pelvic organ prolapse (POP) and may be a suitable model for preclinical studies evaluating cell-based therapies for POP. The aim of this study was to establish a clinical score of vaginal weakness and to compare POP Quantification System (POP-Q) values in conscious nulliparous and parous ewes and determine whether ewes are a suitable POP model. Method(s): Ewes (n = 114) were examined while conscious, without sedation, and standing in a V conveyer by adapting the human POP-Q measurement. Ovine POP was defined as descent to the introitus from POP-Q points Aa 3 cm above the introitus on the anterior wall, Ap 3 cm above the introitus on the posterior wall, or increased Ba anterior wall descent above the urethra (>=0). A test-retest showed good inter- and intrarater reliability. Result(s): There was no evidence of tissue mobility at Aa, Ap, Ba (all -3 cm) in nulliparous ewes (n = 14). In contrast, multiparous ewes had a median of -1 and interquartile range (IQR) (-2 to 0) for Aa, [0 (-1 to 0)] for Ap and [0 (-2.75 to 0)] for Ba (n = 33; P < 0.0001 in comparison with nulliparous) ewes. Ovine vaginal displacement was seen in 50.9 % of parous ewes and was strongly associated with parity (P = 0.003). Conclusion(s): A modified POP-Q in conscious ewes was established showing that the vaginal wall of parous animals has similar regions of weakness as do women and may be similarly related to parity. Ewes appear to be a representative preclinical model of human vaginal prolapse.Copyright © 2016, The International Urogynecological Association.

Published
2017

14. Harnessing the versatility of bacterial collagen to improve the chondrogenic potential of porous collagen scaffolds

Author

Stevens, MM, Parmar, P, St-Pierre, J, Chow, L, Puetzer, J, Stoichevska, V, Peng, Y, Werkmeister, J, Ramshaw, J, Medical Research Council (MRC), Wellcome Trust, and Commission of the European Communities

Subjects
mesenchymal stem cells, collagen foams, bioactivity, cartilage tissue engineering, biomimetic materials
Abstract

Collagen I foams are used in the clinic as scaffolds to promote articular cartilage repair as they provide a bioactive environment for cells with chondrogenic potential. However, collagen I as a base material does not allow for precise control over bioactivity. Alternatively, recombinant bacterial collagens can be used as blank slate collagen molecules to offer a versatile platform for incorporation of selected bioactive sequences and fabrication into 3D scaffolds. Here, we show the potential of Streptococcal collagen-like 2 (Scl2) protein foams modified with peptides designed to specifically and noncovalently bind hyaluronic acid and chondroitin sulfate to improve chondrogenesis of human mesenchymal stem cells (hMSCs) compared to collagen I foams. Specific compositions of functionalized Scl2 foams lead to improved chondrogenesis compared to both nonfunctionalized Scl2 and collagen I foams, as indicated by gene expression, extracellular matrix accumulation, and compression moduli. hMSCs cultured in functionalized Scl2 foams exhibit decreased collagens I and X gene and protein expression, suggesting an advantage over collagen I foams in promoting a chondrocytic phenotype. These highly modular foams can be further modified to improve specific aspects chondrogenesis. As such, these scaffolds also have the potential to be tailored for other regenerative medicine applications.

Published
2016

17. Novel biomaterials for pelvic organ prolapse (POP) repair.

Author

Supit T., White J., Rosamilia A., Lo L., Edwards S., Ramshaw J., Werkmeister J., Gargett C., Ulrich D., Supit T., White J., Rosamilia A., Lo L., Edwards S., Ramshaw J., Werkmeister J., Gargett C., and Ulrich D.

Abstract

POP is the descent of one or more of the pelvic structures into the vagina and includes uterine, vaginal vault as well as anterior and posterior vaginal wall prolapse. Treatment of POP includes surgical treatment and/or implantation of synthetic mesh. However, the long-term outcome of synthetic mesh surgery is unsatisfactory due to surgical failure and post-surgical complications. We have fabricated 3 novel warpknitted synthetic scaffolds, Polyetheretherketone (PEEK), Polyamide (PA) and gelatine coated PA (PA+G). Our aim was to compare the in vivo biocompatibility of these meshes with a commercial Polypropylene (PP) mesh. A SD rat abdominal hernia model was used to implant PA, PEEK, PA+G, PP meshes (25 . 35 mm, n = 24/gp). After 7, 30, 60, 90 days tissues (n = 6/mesh) were explanted for immunohistochemical assessment of foreign body reaction and tissue integration, using CD31, CD45, CD68, alpha-SMA antibodies. CD68+ macrophages showed a trend toward decreased numbers in PP compared to PA, PA+G and PEEK. The smooth muscle content at 60 days was significantly higher in PA and PA+G meshes compared to PP (p > 0.05), but there were no significant differences at 90 days. The collagen content was similar for all meshes at 90 days(p > 0.05). PA, PA+G and PEEK appear to have similar biocompatibility properties compared to PP, inducing a similar foreign body reaction response. These novel meshes may provide an alternative option for future treatment of POP.

Published
2012

33. Use of biodegradable urethane-based adhesives to appose meniscal defect edges in an ovine model: a preliminary study.

Author

Field, J. R., Gunatillake, P., Adhikari, R., Ramshaw, J. A. M., and Werkmeister, J. A.

Subjects
STIFLE joint, JOINTS (Anatomy), HEALING, COLLAGEN, VETERINARY medicine
Abstract

Objective To evaluate the biological response to two urethane-based adhesives used to repair full thickness meniscal wounds created in the partially vascularised (red-white) zone. Design An ovine bilateral meniscal defect model was used to evaluate the initial biological response of the meniscal cartilage and synovium over a 1-month period. A 10-mm full-thickness defect was created in the medial meniscus of each femorotibial joint. The defects were either left untreated or repaired using the urethane-based adhesives. Synovial fluid, synovial membrane and the meniscal cartilages were retrieved at necropsy for cytological and histological assessment. Results The ovine model proved to be a suitable system for examining meniscal repair. Untreated defects showed no tissue apposition or cellular healing response, whereas all eight defects repaired with the two urethane-based adhesive formulations showed signs of repair and tissue regeneration with indications of cell infiltration and new collagen deposition in and around the polymer. No adverse cellular response to the adhesives was observed in the meniscal defect or in the synovial membrane and fluid. Conclusion Trauma to the knee commonly results in tears to the meniscal cartilage, with the majority of these occurring in the partially vascularised (red-white) or non-vascularised (white) zones of the meniscus. Repair, and subsequent healing, of these tears is poor because of the reduced vascularity and limited surgical access. The present data indicate that an ovine model is a suitable system for examining meniscal repair, and that development of urethane-based adhesives offers a strategy that may be clinically effective for the treatment of these injuries. [ABSTRACT FROM AUTHOR]

Published
2008
Full Text
View/download PDF

34. Characterization of an inhibitor of cell division released in tumour cell cultures.

Author

Werkmeister, J., Zaunders, J., McCarthy, W., and Hersey, P.

Subjects
*CANCER cells, *CELL division, *TUMORS, *CELL culture, *LYMPHOCYTES, *POLYACRYLAMIDE
Abstract

We have previously shown that tumour cells may release soluble factors which inhibit the division of a variety of cells. These factors had marked immunosuppressive activity in vitro as shown by their ability to Inhibit lymphocyte mitogenic responses to lectins. allogeneic lymphocytes and pokeweed mitogen-induced immunoglobulin production. It was postulated that these factors may play an important role in determining the outcome of tumour host interactions in humans and be the source of immunosuppressive factors in the circulation of patients with cancer. In the present study, characterization of these mitogenic inhibitory factors released into the supernatants of cultured tumour cells was carried out by a number of sequential separation procedures involving affinity chromatography on wheat-germ lectin (WGL). gel filtration on PL-agarose and electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulphate (PAGE-SDS). The factors were identified throughout the separation procedure using inhibition of phytohaemagglutinin (PHA) stimulation of normal lymphocytes as an index of inhibitory function. By these techniques the inhibitory activity was shown to be associated with glycoproteins containing N-acetyl-β-D-glucosamine (NAcGlu) and having molecular weights (mol. wt) of approximately 140.000 and over 200,000 daltons. A further small mol. wt peak of 70,000 daltons was observed on PAGE-SDS. pi values of the fractions were 7.4 and 4.2. The activity was destroyed by heating, low pH and repeated freeze-thawing but was resistant to proteolytic enzymes, deoxyribonuclease, ribonuclease and neuraminidase. The active fractions were not associated with proteolytic activity and the inhibition of mitogenic responses was irreversible in nature. These results form the basis of studies to detect this factor in biological fluids and further evaluate its role in the tumour host relationship. [ABSTRACT FROM AUTHOR]

Published
1980

35. Detection of an inhibitor of cell division in cultures of tumour cells with immunosuppressive activity in vitro.

Author

Werkmeister, J., Zbroja, Rosemary, McCarthy, W., and Hersey, P.

Subjects
*CELL division, *TUMORS, *IMMUNOSUPPRESSION, *PHYTOHEMAGGLUTININS, *LYMPHOCYTES, *IMMUNE response
Abstract

A number of previous reports have described the presence of factors which inhibit the response of lymphoid cells to phytohaemagglutinin. The present study describes an inhibitor of cell division synthesized and released directly from cultured tumour cells which appears to have similar immunosuppressive effects in vitro. This factor(s) was detected in a wide variety of cultured tumour cells and some cultures of normal foetal tissue. It inhibited the mitogenic response of lymphocytes to PHA and also the spontaneous cell division of a variety of cultured cells including the cells producing the factor. Pulse cytophotometry showed that cells were inhibited in the G1 phase. Production of the factor increased with time and was related to the number of cells in culture. Its synthesis was inhibited by cycloheximide. DNA and RNA synthesis was not required for its production. The factor appeared to have a high degree of biological activity m terms of the number of cultured cells required for production and in regard to the number of cells inhibited. The biological significance of this factor in vivo is unknown but in vitro it was shown to inhibit immunoglobulin and mitogen-induced responses which suggests it may play an important role in suppression of the immune response against tumour cells in the host. [ABSTRACT FROM AUTHOR]

Published
1980

36. Lymphocytes from patients with hairy cell leukaemia enable the distinction of two separate subpopulations of activated lymphocyte killer cells generated in mixed lymphocyte culture.

Author

Werkmeister, J. A., Boyd, A. W., Triglia, T., and Burns, G. F.

Subjects
*HAIRY cell leukemia, *LYMPHOPROLIFERATIVE disorders, *KILLER cells, *LEUKEMIA, *CELL culture, *LYMPHOCYTES
Abstract

Blood from patients with hairy cell leukaemia (HCL) was used to investigate subpopulations of the non-specific activated lymphocyte killer (ALK) cells generated in mixed lymphocyte culture (MLC). Mononuclear cells from four of live patients with HCL had decreased natural killer (NK) cell activity and., of these, all showed a decrease in ALK cell activity against K-562 target cells (NK like cells), but three had normal levels of ALK cells able to kill melanoma cells. One patient who had received splenectomy had normal NK cell activity, and in MLC his cells generated normal levels of both subpopulations of ALK cells. The results support the hypothesis of separate subsets of ALK cells: NK like cells with NK cell precursors and which preferentially lyse the K-562 target cell and a second separate population which arises independent of NK cell precursors and which has strong cytolytic activity against an NK insensitive melanoma cell line. [ABSTRACT FROM AUTHOR]

Published
1985

39. Cloned human cytotoxic T lymphocytes develop anomalous killer cell function.

Author

Burns, G. F., Triglia, T., and Werkmeister, J. A.

Subjects
LYMPHOCYTES, CULTURES (Biology), B cells, NEUROENDOCRINE tumors, IMMUNOGLOBULINS, MOLECULAR cloning, MONOCLONAL antibodies
Abstract

Mixed lymphocyte cultures were set up between blood mononuclear cells and irradiated autologous or allogeneic B lymphoblasts infected with Epstein Barr virus. The resulting cytotoxic effector cells were cloned and tested for activity against the stimulating B lymphoblast, K562 and melanoma targets. Specific clones which killed only the stimulating B lymphoblasts (cytotoxic T lymphocytes: CTL) were re-cloned and the subclones tested for cytolysis of B lymphoblasts and melanoma cells. Of seven primary CTL clones generated in allogeneic culture, 308 subclones developed the ability to kill melanoma cells and none retained specific CTL function. In the autologous system, 180 subclones were derived from three specific primary clones: of these. 13 (7%) retained specific function 29(16%) were able to kill both B lymphoblasts and melanoma cells, and 93 (52%) killed only the melanoma target. Testing of random clones demonstrated that whereas both B lymphoblast killing (CTL function) and melanoma cell killing (anomalous killer; AK function) were blocked by a monoclonal antibody to LFA-1, only CTL function was blocked by anti-T3 or anti-T8 antibodies. The factor(s) causing the progression of CTL to AK cells are discussed. These data thus demonstrate that the majority of CTL are capable of mediating AK cell function and are thus potentially suitable for passive immunotherapy. [ABSTRACT FROM AUTHOR]

Published
1986

40. The Chediak-Higashi gene in humans. III. Studies on the mechanisms of NK impairment.

Author

Roder, J. C., Todd, R. F., Rubin, J. P., Haliotis, Tina, Helfand, S. L., Werkmeister, J., Pross, H. F., Boxer, L. A., Schlossman, S. F., and Fauci, A. S.

Subjects
LYMPHOCYTES, TUMORS, KILLER cells, SHEEP as laboratory animals, ERYTHROCYTES, LEUCOCYTES
Abstract

Lymphocytes from six Chediak Higashi (CH) patients were markedly depressed in their ability to lyse tumour cell targets in both 51Cr release and single cell cytotoxicity assays. The frequency of lymphocytes bearing the OKM1 marker and the frequency of T3+, T4+, T8+, la-, Mol+, Mo2+ and BI+ cells was normal among sheep erythrocyte rosetting (E+ ) and non-resetting (E-) peripheral blood leucocytes analysed by flow cytofluorography. Cells expressing the NK shared markers. OKMI mac-1, FcR, and the characteristic large granular lymphocyte (LGL) morphology of NK cells were also present in normal numbers in the highly enriched NK fraction separated on Percoll density gradients. This fraction did not contain detectable numbers of cells expressing the Mo2 marker of human monocytes. Therefore most of the cells stained by monoclonal OKMI and mac-1 in this fraction are likely NK cells, rather than monocytes. and we conclude that the size of the NK pool in CH patients is probably normal. The capacity of CH lymphocytes to recognize anti bind to tumour cells was also normal as was the subsequent burst at oxygen intermediates produced by the NK cells in a chemiluminenscence assay We have shown elsewhere that O2 generation is directly involved in activating subsequent steps in the NK cytolytic pathway. These results suggest that NK cells in CH patients are present in normal frequency but are blocked at some post-recognition, post-activation step in the cytolytic pathway subsequent to the burst of oxygen intermediates but preceding the lethal hit. [ABSTRACT FROM AUTHOR]

Published
1983

41. Inhibition of suppressor T cells in pokeweed mitogen-stimulated cultures of T and B cells by levamisole <em>in vitro</em> and <em>in vivo</em>.

Author

Hersey, P., Ho, K., Werkmeister, J., and Abele, U.

Subjects
T cells, LEVAMISOLE, MITOGENS, IMMUNOGLOBULIN G, LEUCOCYTES, IMMUNE serums
Abstract

The action of levamisole on immunoglobulin production was investigated in pokeweed mitogen-stimulated cultures of B and T cells. Immunoglobulin production was measured by nephelometric methods using specific antisera to IgA, IgG and IgM. Levamisole was shown to have no effect on B lymphocytes but pretreatment of T lymphocytes with levamisole resulted in an increase in immunoglobulin production which was maximal at 10-6 M. The latter increase appeared to be due to an effect on suppressor T cells in that pretreatment of T cells depleted of suppressor cells (by irradiation or removal of T cells with receptors with IgG) did not result in an increase in immunoglobulin production. Similar effects of levamisole were demonstrated against suppressor cells induced by concanavalin A in vitro and against spontaneous suppressor cell activity in vivo. The effect of levamisole and irradiation on suppressor cell activity against particular classes of immunoglobulin varied between individuals. Suppressor cell activity against IgA production alone was demonstrated in six of 17 subjects whereas in the remainder, suppressor cell activity was either not detected or inhibited production of all three immunoglobulin classes. The implications of this action of levamisole for its effects in vivo are unknown, but the results appear consistent with many of the observed effects of the drug. Selection of patients with diseases associated with high suppressor cell activity may lead to more effective use of the drug. [ABSTRACT FROM AUTHOR]

Published
1981

45. Characterization of a Protein-based Adhesive Elastomer Secreted by the Australian Frog Notaden bennetti

Author

Graham, L. D., Glattauer, V., Huson, M. G., Maxwell, J. M., Knott, R. B., White, J. W., Vaughan, P. R., Peng, Y., Tyler, M. J., Werkmeister, J. A., and Ramshaw, J. A.

Abstract

When provoked, Notaden bennetti frogs secrete an exudate which rapidly forms a tacky elastic solid (“frog glue”). This protein-based material acts as a promiscuous pressure-sensitive adhesive that functions even in wet conditions. We conducted macroscopic tests in air to assess the tensile strength of moist glue (up to 78 ± 8 kPa) and the shear strength of dry glue (1.7 ± 0.3 MPa). We also performed nanomechanical measurements in water to determine the adhesion (1.9−7.2 nN or greater), resilience (43−56%), and elastic modulus (170−1035 kPa) of solid glue collected in different ways. Dry glue contains little carbohydrate and consists mainly of protein. The protein complement is rich in Gly (15.8 mol %), Pro (8.8 mol %), and Glu/Gln (14.1 mol %); it also contains some 4-hydroxyproline (4.6 mol %) but no 5-hydroxylysine or 3,4-dihydroxyphenylalanine (l-Dopa). Denaturing gel electrophoresis of the glue reveals a characteristic pattern of proteins spanning 13−400 kDa. The largest protein (Nb-1R, apparent molecular mass 350−500 kDa) is also the most abundant, and this protein appears to be the key structural component. The solid glue can be dissolved in dilute acids; raising the ionic strength causes the glue components to self-assemble spontaneously into a solid which resembles the starting material. We describe scattering studies on dissolved and solid glue and provide microscopy images of glue surfaces and sections, revealing a porous interior that is consistent with the high water content (85−90 wt %) of moist glue. In addition to compositional similarities with other biological adhesives and well-known elastomeric proteins, the circular dichroism spectrum of dissolved glue is almost identical to that for soluble elastin and electron and scanning probe microscopy images invite comparison with silk fibroins. Covalent cross-linking does not seem to be necessary for the glue to set.

Published
2005

46. TLiSA1, a human T lineage-specific activation antigen involved in the differentiation of cytotoxic T lymphocytes and anomalous killer cells from their precursors.

Author

Burns, G F, Triglia, T, Werkmeister, J A, Begley, C G, and Boyd, A W

Abstract

The characteristics of a novel T lineage-specific activation antigen, termed TLiSA1, are described. The antigen was detected with a mouse monoclonal antibody, LeoA1, that was raised against activated human T cells generated in mixed lymphocyte culture (MLC). The antigen became strongly expressed on T cells 48-72 h after stimulation with phytohemagglutinin, and retained expression on MLC-activated T cells after 10 d of culture. The antigen was absent from a range of human T, B, myeloid, fibroblast, and tumour cell lines, but was present on the surface of the interleukin 2 (IL-2)-dependent gibbon cell line MLA-144. Analysis of the antigen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained from activated human T cells demonstrated a broad band in the region of 70 kD, whereas precipitates obtained from MLA-144 revealed a single narrow band of 95 kD. The molecule was expressed with a maximum density of 66,000 copies per cell on the surface of MLC-activated T cell blasts, as assessed by Scatchard analysis. TLiSA1 was distinguished from the IL-2 receptor bound by the anti-Tac monoclonal antibody by demonstrating that the antigens did not comodulate or coprecipitate, and by constructing an IL-2-independent human T X T hybrid that expressed the TLiSA1 but not the Tac antigen. MLC with B lymphoblasts was used to generate cytotoxic T lymphocytes (CTL) specific for the stimulating cell, and anomalous killer (AK) cells able to kill melanoma target cells. The presence of LeoA1 or F(ab')2 fragments of the antibody from the beginning of coculture did not affect proliferation in these cultures, but did inhibit the induction of both CTL and AK cells from their precursors. This inhibition of differentiation by LeoA1 was confirmed under conditions of limiting dilution, where it was shown that the antibody reduced the frequency of CTL produced, and greatly (fourfold) reduced the frequency of AK cells generated from their precursors. We discuss the possibility that human CTL may express a differentiation factor receptor that is distinct from the receptor for IL-2.

Published
1985
Full Text
View/download PDF

47. Chemiluminescence response of human natural killer cells. I. The relationship between target cell binding, chemiluminescence, and cytolysis.

Author

Helfand, S L, Werkmeister, J, and Roder, J C

Abstract

The binding of tumor cells or fetal fibroblasts to human natural killer (NK) cells led to a rapid chemiluminescence response within seconds of target-effector interaction. The degree of chemiluminescence was dependent on the concentration of NK-enriched lymphocytes or target cells, and plasma membrane vesicles from K562 also induced a chemiluminescence response. Mild glutaraldehyde treatment of effector cells abrogated their ability to generate chemiluminescence, whereas K562 target cells treated in the same way were almost fully able to induce a chemiluminescence response to NK-enriched lymphocytes. These results show a directionality of response with NK as the responders and tumor cells as the stimulators. A survey of eight different tumor cell lines and fetal fibroblast lines revealed a striking correlation (r greater than 0.93, P less than 0.001) between the ability of a given line to bind to NK-enriched lymphocytes, induce chemiluminescence, and to be lysed. Three differentiated sublines of K562 grown in butyrate and cloned induced little chemiluminescence compared with the K562 parent, and they were selectively resistant to NK-mediated binding and cytolysis. In addition, treatment of K562 cells with higher concentrations of glutaraldehyde for longer periods led to varying degrees of target antigen preservation, as measured in cold target competition assays and in conjugate formation. The degree of NK target antigen preservation correlated directly with the ability of the cells to induce chemiluminescence (r greater than 0.95). The degree of NK activation was also important because interferon-pretreated effectors generated more chemiluminescence upon stimulation with K562 or MeWo targets. Monocytes or granulocytes did not contribute to the chemiluminescence induced by NK-sensitive targets. Some NK-resistant tumor cell lines were sensitive to monocyte-mediated cytolysis and also induced chemiluminescence in monocytes but not NK cells. These results show that the target structures recognized by the NK cell may play a role in NK activation because the degree of chemiluminescence was directly proportional to the ability of a given target cell line to bind to the NK cell and to be lysed.

Published
1982
Full Text
View/download PDF

48. Synergism between membrane gangliosides and Arg-Gly-Asp-directed glycoprotein receptors in attachment to matrix proteins by melanoma cells.

Author

Burns, G F, Lucas, C M, Krissansen, G W, Werkmeister, J A, Scanlon, D B, Simpson, R J, and Vadas, M A

Abstract

The identification of specific cell surface glycoprotein receptors for Arg-Gly-Asp-containing extracellular matrix proteins such as fibronectin has focused attention on the role of gangliosides in this process. Is their involvement dependent or independent of the protein receptors? In attachment assays with cells from a human melanoma cell line, titration experiments with an antibody (Mel 3) with specificity for the disialogangliosides GD2 and GD3, used together with a synthetic peptide containing the cell binding sequence Arg-Gly-Asp, show that their joint effect is synergistic. Both the Mel 3 antibody and the synthetic peptide individually cause rapid detachment of melanoma cells from fibronectin substrate but, when used together, much smaller concentrations of both are required to achieve the same effect. The Mel 3 antibody was not nonspecifically reducing receptor binding to the Arg-Gly-Asp sequence since, in binding assays with radiolabeled peptide performed with cells in suspension, very little peptide is bound by the melanoma cells under these conditions but addition of Mel 3, an antibody of IgM isotype, causes a two- to threefold increase in specific binding. The simplest interpretation of these data is that the Mel 3 antibody is causing sufficient clustering of membrane gangliosides in local areas and producing a favorably charged environment to facilitate peptide binding by specific glycoprotein receptors.

Published
1988
Full Text
View/download PDF

49. Frequent deletion and duplication of the steroid 21-hydroxylase genes

Author

Werkmeister, J W, New, M I, Dupont, B, and White, P C

Subjects
Genetic Markers, Polymorphism, Genetic, Adrenal Hyperplasia, Congenital, Genetic Linkage, HLA Antigens, Steroid Hydroxylases, Humans, Nucleic Acid Hybridization, Complement C4, DNA, Steroid 21-Hydroxylase, Chromosome Deletion, Research Article
Abstract

Congenital adrenal hyperplasia due to 21-hydroxylase (21-OHase) deficiency is an HLA-linked disorder resulting from a mutation in the 21-OHase B gene encoding the adrenal cytochrome P450 specific for steroid 21-hydroxylation. To identify polymorphisms associated with 21-OHase deficiency, DNA samples from 22 unrelated patients with this disorder were examined with a human cDNA clone encoding the enzyme. Deletions of the active 21-OHase gene were found in almost one-fourth of classical 21-OHase deficiency alleles. In contrast, mild, "nonclassical" 21-OHase deficiency is associated with a duplicated 21-OHase gene.

Published
1986

50. Cloned human cytotoxic T lymphocytes develop anomalous killer cell function

Author

Burns, G F, Triglia, T, and Werkmeister, J A

Subjects
Cytotoxicity, Immunologic, B-Lymphocytes, Melanoma, Experimental, hemic and immune systems, Cell Transformation, Viral, Binding, Competitive, Clone Cells, Killer Cells, Natural, hemic and lymphatic diseases, Humans, Interleukin-2, Antigens, Research Article, T-Lymphocytes, Cytotoxic
Abstract

Mixed lymphocyte cultures were set up between blood mononuclear cells and irradiated autologous or allogeneic B lymphoblasts infected with Epstein-Barr virus. The resulting cytotoxic effector cells were cloned and tested for activity against the stimulating B lymphoblast, K562 and melanoma targets. Specific clones which killed only the stimulating B lymphoblasts (cytotoxic T lymphocytes; CTL) were re-cloned and the subclones tested for cytolysis of B lymphoblasts and melanoma cells. Of seven primary CTL clones generated in allogeneic culture, 308 subclones developed the ability to kill melanoma cells and none retained specific CTL function. In the autologous system, 180 subclones were derived from three specific primary clones: of these, 13 (7%) retained specific function, 29 (16%) were able to kill both B lymphoblasts and melanoma cells, and 93 (52%) killed only the melanoma target. Testing of random clones demonstrated that whereas both B lymphoblast killing (CTL function) and melanoma cell killing (anomalous killer; AK function) were blocked by a monoclonal antibody to LFA-1, only CTL function was blocked by anti-T3 or anti-T8 antibodies. The factor(s) causing the progression of CTL to AK cells are discussed. These data thus demonstrate that the majority of CTL are capable of mediating AK cell function and are thus potentially suitable for passive immunotherapy.

Published
1986

Catalog

Books, media, physical & digital resources
See catalog results