ORCID Profile
0000-0002-2787-3317
Current Organisations
Monash University
,
Alfred Health
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Publisher: Public Library of Science (PLoS)
Date: 26-03-2008
Publisher: Elsevier
Date: 2001
Publisher: Wiley
Date: 12-2007
Abstract: Fibroblast growth factor receptors (FGFRs) are a family of four transmembrane (TM) receptor tyrosine kinases (RTKs) which bind to a large family of fibroblast growth factor (FGF) ligands with varying affinity and specificity. FGFR signaling regulates many physiological and pathological processes in development and tissue homeostasis. Understanding FGFR signaling processes requires the identification of partner proteins which regulate receptor function and biological outputs. In this study, we employ an epitope-tagged, covalently dimerized, and constitutively activated form of FGFR1 to identify potential protein partners by MS. By this approach, we s le candidate FGFR effectors throughout the life history of the receptor. Functional classification of the partners identified revealed specific subclasses involved in protein biosynthesis and folding structural and regulatory components of the cytoskeleton known signaling effectors and small GTPases implicated in endocytosis and vesicular trafficking. The kinase dependency of the interaction was determined for a subset of previously unrecognized partners by coimmunoprecipitation, Western blotting, and immunocytochemistry. From this group, the small GTPase Rab5 was selected for functional interrogation. We show that short hairpin (sh) RNA-mediated depletion of Rab5 attenuates the activation of the extracellular-regulated kinase (ERK) 1/2 pathway by FGFR signaling. The strategic approach adopted in this study has revealed bona fide novel effectors of the FGFR signaling pathway.
Publisher: American Society of Hematology
Date: 02-2002
Abstract: Granulocyte colony-stimulating factor (G-CSF) is the major regulator of granulopoiesis and acts through binding to its specific receptor (G-CSF-R) on neutrophilic granulocytes. Previous studies of signaling from the 4 G-CSF-R cytoplasmic tyrosine residues used model cell lines that may have idiosyncratic, nonphysiological responses. This study aimed to identify specific signals transmitted by the receptor tyrosine residues in primary myeloid cells. To bypass the presence of endogenous G-CSF-R, a chimeric receptor containing the extracellular domain of the epidermal growth factor receptor in place of the entire extracellular domain of the G-CSF-R was used. A series of chimeric receptors containing tyrosine mutations to phenylalanine, either in idually or collectively, was constructed and expressed in primary bone marrow cells from G-CSF–deficient mice. Proliferation and differentiation responses of receptor-expressing bone marrow cells stimulated by epidermal growth factor were measured. An increased 50% effective concentration to stimulus of the receptor Ynullmutant indicated that specific signals from tyrosine residues were required for cell proliferation, particularly at low concentrations of stimulus. Impaired responses by mutant receptors implicated G-CSF-R Y764 in cell proliferation and Y729 in granulocyte differentiation signaling. In addition, different sensitivities to ligand stimulation between mutant receptors indicated that G-CSF-R Y744 and possibly Y729 have an inhibitory role in cell proliferation. STAT activation was not affected by tyrosine mutations, whereas ERK activation appeared to depend, at least in part, on Y764. These observations have suggested novel roles for the G-CSF-R tyrosine residues in primary cells that were not observed previously in studies in cell lines.
Publisher: Elsevier BV
Date: 12-2021
DOI: 10.1016/J.ACTBIO.2021.09.043
Abstract: Platelets are a reservoir of growth factors, cytokines and chemokines involved in spontaneous wound repair. In this study, a platelet-rich and fibrin-rich hydrogel was generated from expired platelet components that would have otherwise been transfused. The material contained physiological concentrations of transforming growth factor β1 (TGF-β1, platelet-derived growth factor AB (PDGF-AB), PDGF-BB, insulin-like growth factor-1 (IGF-1), fibroblast growth factor 2 (FGF-2), and epidermal growth factor (EGF). The effect of the hydrogel on wound repair was investigated in SKH-1 mice. Full thickness dorsal wounds were created on the mice and treated with the hydrogel at various concentrations. Immunohistochemical staining with CD31 (endothelial cell marker) revealed that wounds treated with the hydrogel showed significantly enhanced vascularisation in the wound bed. Moreover, high levels of interleukin-6 (IL-6) and KC (IL-8 functional homologue) in treated wounds were sustained over a longer period of time, compared to untreated wounds. We postulate that sustained IL-6 is a driver, at least partly, of enhanced vascularisation in full thickness wounds treated with the hydrogel. Future work is needed to explore whether this hydrogel can be utilised as a treatment option when vascularisation is a critical limitation. STATEMENT OF SIGNIFICANCE: The economic cost of wound repair is estimated in billions of dollars each year. In many cases time required to vascularise wounds is a major contributor to slow wound repair. In this study, we developed a blood-derived platelet- and fibrin-rich hydrogel. It contains a number of growth factors actively involved in spontaneous wound healing. This hydrogel significantly improved dermal repair and vascularisation in a full-thickness wound mouse model. This study provides an action mechanism for modulation of localised inflammation.
Publisher: Elsevier BV
Date: 02-2014
DOI: 10.1016/J.BURNS.2013.05.006
Abstract: Cadaveric cutaneous allografts are used in burns surgery both as a temporary bio-dressing and occasionally as definitive management of partial thickness burns. Nonetheless, limitations in the understanding of the biology of these grafts have meant that their role in burns surgery continues to be controversial. A review of all patients suffering 20% or greater total body surface area (TBSA) burns over an eight year period that received cadaveric allografts were identified. To investigate whether tissue viability plays a role in engraftment success, five s les of cryopreserved cadaveric cutaneous allograft processed at the Donor Tissue Bank of Victoria (DTBV) were submitted to our laboratory for viability analysis using two methods of Trypan Blue Exclusion and tetrazolium salt (MTT) assays. During the study period, 36 patients received cadaveric allograft at our institution. The average total burn surface area (TBSA) for this group of patients was 40% and all patients received cadaveric skin as a temporizing measure prior to definitive grafting. Cadaveric allograft was used in complicated cases such as wound contamination, where synthetic dressings had failed. Viability tests showed fewer than 30% viability in processed allografts when compared to fresh skin following the thawing process. However, the skin structure in the frozen allografts was histologically well preserved. Cryopreserved cutaneous cadaveric allograft has a positive and definite role as an adjunct to conventional dressing and grafting where available, particularly in patients with large TBSA burns. The low viability of cryopreserved specimens processed at DTBV suggests that cell viability in cadaveric allograft may not be essential for its clinical function as a wound dressing or even as permanent dermal substitute.
Publisher: MDPI AG
Date: 25-06-2020
DOI: 10.3390/IJMS21124508
Abstract: Engineered dermal templates have revolutionised the repair and reconstruction of skin defects. Their interaction with the wound microenvironment and linked molecular mediators of wound repair is still not clear. This study investigated the wound bed and acellular “off the shelf” dermal template interaction in a mouse model. Full-thickness wounds in nude mice were grafted with allogenic skin, and either collagen-based or fully synthetic dermal templates. Changes in the wound bed showed significantly higher vascularisation and fibroblast infiltration in synthetic grafts when compared to collagen-based grafts (P ≤ 0.05). Greater tissue growth was associated with higher prostaglandin-endoperoxide synthase 2 (Ptgs2) RNA and cyclooxygenase-2 (COX-2) protein levels in fully synthetic grafts. Collagen-based grafts had higher levels of collagen III and matrix metallopeptidase 2. To compare the capacity to form a double layer skin substitute, both templates were seeded with human fibroblasts and keratinocytes (so-called human skin equivalent or HSE). Mice were grafted with HSEs to test permanent wound closure with no further treatment required. We found the synthetic dermal template to have a significantly greater capacity to support human epidermal cells. In conclusion, the synthetic template showed advantages over the collagen-based template in a short-term mouse model of wound repair.
Publisher: Elsevier BV
Date: 09-2002
Publisher: EMBO
Date: 02-11-2007
Abstract: The non‐receptor tyrosine kinase Src is recruited to activated fibroblast growth factor receptor (FGFR) complexes through the adaptor protein factor receptor substrate 2 (FRS2). Here, we show that Src kinase activity has a crucial role in the regulation of FGFR1 signalling dynamics. Following receptor activation by ligand binding, activated Src is colocalized with activated FGFR1 at the plasma membrane. This localization requires both active Src and FGFR1 kinases, which are inter‐dependent. Internalization of activated FGFR1 is associated with release from complexes containing activated Src. Src‐mediated transport and subsequent activation of FGFR1 require both RhoB endosomes and an intact actin cytoskeleton. Chemical and genetic inhibition studies showed strikingly different requirements for Src family kinases in FGFR1‐mediated signalling activation of the phosphoinositide‐3 kinase–Akt pathway is severely attenuated, whereas activation of the extracellular signal‐regulated kinase pathway is delayed in its initial phase and fails to attenuate.
Publisher: Springer Science and Business Media LLC
Date: 21-01-2019
DOI: 10.1007/S00441-018-02986-5
Abstract: Cultured epithelial autograft (CEA) was the birth of skin tissue engineering and encompassed methodologies for the isolation and expansion of autologous basal keratinocytes for burn treatment that are still practiced at some specialised units around the world. One of the limitations of CEA, however, is the reliance on animal-derived material during the manufacturing process and despite all efforts to date, no xeno-free alternative with proven efficacy has been reported. Here, we investigate whether human-derived fibroblast feeder cells and human serum can sufficiently and effectively provide a suitable microenvironment for adult keratinocyte isolation and expansion. Human dermal fibroblasts and epidermal keratinocytes were isolated from discarded skin during abdominoplasty and breast reduction procedures and cultured in xeno-free conditions. We report that these xeno-free adult keratinocytes form similar numbers of colony-forming units as those cultured using the Green's methods however, xeno-free keratinocytes express lower levels of α6 integrin (CD49f a progenitor and stem cell marker). We identified IL-8 as a potential growth factor secreted by adult human fibroblasts that may enhance keratinocyte colony formation in human serum. Finally, we propose a step-by-step xeno-free isolation and cultivation methodology for adult keratinocytes that can be tested further in serial cultivation for clinical application.
Publisher: Springer Science and Business Media LLC
Date: 22-10-2022
DOI: 10.1007/S00441-022-03698-7
Abstract: Safety concerns associated with foetal bovine serum (FBS) have restricted its translation into clinics. We hypothesised that platelet lysate (PL) can be utilised as a safe alternative to produce serum-free 3D-engineered skin. PL supported a short-term expansion of fibroblasts, with negligible replication-induced senescence and directed epidermal stratification. PL-expanded fibroblasts were phenotypically separated into three subpopulations of CD90 + FAP + , CD90 + FAP − and CD90 − FAP + , based on CD90 (reticular marker) and FAP (papillary marker) expression profile. PL drove the expansion of the intermediate CD90 + FAP + subpopulation in expense of reticular CD90 + FAP − , which may be less fibrotic once grafted. The 3D-engineered skin cultured in PL was analysed by immunofluorescence using specific markers. Detection of ColIV and LMN-511 confirmed basement membrane. K10 confirmed near native differentiation pattern of neo-epidermis. CD29- and K5-positive interfollicular stem cells were also sustained. Transmission and scanning electron microscopies detailed the ultrastructure of the neo-dermis and neo-epidermis. To elucidate the underlying mechanism of the effect of PL on skin maturation, growth factor contents in PL were measured, and TGF-β1 was identified as one of the most abundant. TGF-β1 neutralising antibody reduced the number of Ki67-positive proliferative cells, suggesting TGF-β1 plays a role in skin maturation. Moreover, the 3D-engineered skin was exposed to lucifer yellow on days 1, 3 and 5. Penetration of lucifer yellow into the skin was used as a semi-quantitative measure of improved barrier function over time. Our findings support the concept of PL as a safe and effective serum alternative for bioengineering skin for cell therapies.
Publisher: Elsevier BV
Date: 03-2019
DOI: 10.1016/J.BJPS.2018.11.003
Abstract: Last century, our laboratory produced Cultured Epithelial Autograft (CEA) for clinical use by the affiliated adult burn service and other burn units across the country. Production of CEA for clinical use was discontinued after several years because of a low success rate and subsequent low demand. Recently, at our burns unit, a cell culture program was reintroduced as a direct response to the need for improvement in ongoing deficiencies and clinical requirements in burn wound closure. The aim of this study was to validate the laboratory processes and clinical algorithms established and share our recent clinical experiences involving CEA. This observational cohort study recruited patients with burns exceeding 35% TBSA admitted to the Victorian Adult Burns Service at The Alfred (December 2013-December 2016). Autologous keratinocytes were expanded and delivered through sheets of fibrin carrier. Twelve patients were recruited to participate in the study. Thirty-two sites were treated with CEA. CEA applied in combination with widely meshed SSG led to the highest take rate (90.1%) at 7-10 days. Further, debridement and grafting were necessary in sixteen of thirty-two sites treated, all involving wound beds prepared with Cuono method or sites treated with CEA only. It is important to address the problem of wound bed contamination, either through increased resistance on the part of the construct or wound bed sterilization. Improved understanding of the relative importance of vascularization, control of cell behavior, the extracellular matrix, immune function, and intrinsic antimicrobial capacity for graft take would then inform a more targeted approach to skin tissue engineering for wound closure in severe burns.
Publisher: S. Karger AG
Date: 2021
DOI: 10.1159/000514223
Abstract: b i Background: /i /b Platelet-rich plasma (PRP) and its derivatives are an emerging biotechnology whereby concentrated platelets provide damaged tissue with growth factors, cytokines, and other mediators to improve healing outcomes. Although there is strong evidence in the benefits of autologous PRP for both acute and chronic wounds, allogeneic PRP has been studied far less in comparison. b i Summary: /i /b In this mini-review, we discuss critical steps of allogenic PRP (and its derivatives) preparation. We performed a non-systematic review of the literature to identify animal and human subject studies testing allogenic PRP for wound treatment. We searched OVID Medline and PubMed for articles using the keywords “wound, ulcer, lesion, skin, and cutaneous” and “PRP, or platelet-rich plasma, or platelet-rich fibrin, or PRF, or platelet releasate” and “homologous, allogeneic or allogenic,” which were limited to non-review articles and English language. Two studies in animal models and 8 studies in patients were reviewed. There were inconsistencies in preparation methods, treatment regimens, and some lacked a control group in their studies. Despite these variations, none of the studies identified any major side effects or adverse events. The treatment resulted in a reduced time to heal and/or reduced wound size in most cases. b i Key Messages: /i /b In situations where autologous PRP is not available or suitable, allogeneic PRP appears to provide a safe alternative. Its efficacy, however, requires larger-scale studies with appropriate controls. Standardization in PRP preparation and treatment regime are also needed to be able to interpret allogenic PRP efficacy.
Publisher: Wiley
Date: 26-07-2019
DOI: 10.1111/WRR.12748
Abstract: Several issues persist in clinical translation and application of cultured epithelial autografts during treatment of patients with massive burn injuries. The aim of this systematic review is to determine (1) current practice and trends in clinical application and (2) clinical efficacy of cultured epithelial autografts. A structured literature search was performed in Ovid MEDLINE from 1946 and Ovid EMBASE from 1974 until present. All published peer-reviewed randomized or non-randomized clinical studies, cohort studies, prospective, or retrospective series involving human application of cultured epithelial autografts in the setting of burn injury were included. From 7,267 studies initially identified, 77 studies were included in the analysis. In 96% (74/77) of these series, the s le size was less than 100 patients. In 76.6% (59/77) publications, average burn treated exceeded 40% total body surface area. Overall, cultured epithelial autograft take rates reported in the literature were inconsistent and varied significantly from 0 to 100%. There was a recent trend for co-application of cultured grafts with autologous skin grafts, achieving relatively high and consistent take rates of 73-96%. Results from cultured epithelial autograft application remained unpredictable. This technology remains an adjunct or biological dressing, and not an alternative to conventional split skin graft. However, it has contributed to wound closure and it has been life saving in selected circumstances. Skin tissue engineering should continue as the clinical need for skin replacement is foreseeable into the future.
Publisher: American Chemical Society (ACS)
Date: 12-08-2005
DOI: 10.1021/PR050090C
Abstract: Electron capture dissociation (ECD) offers many benefits for the analysis of peptides and proteins, and consequently shows great potential for the field of proteomics. Recent developments have reduced the time scale required for ECD to milliseconds resulting in the technique's compatibility with on-line separation techniques, e.g., HPLC. Here, we demonstrate incorporation of ECD into a high-throughput data-dependent LC-MS/MS approach for the analysis of proteomic s les. The approach is applied to analysis of the protein Fc-ROR2 isolated from chondrocytes and is the first ex le of LC-ECD-MS/MS of such a s le. Protein sequence coverage was 29%. Within that coverage, fifteen peptides were isolated and subjected to ECD. In most cases, the sequence tag generated by ECD was over 70% (in terms of the number of peptide backbone cleavages). The ECD data were searched against the nonredundant human NCBI database using the SEQUEST algorithm. Protein ROR2 was assigned, as was IgG (Fc domain). The results demonstrate the suitability of ECD as an integral technique in high-throughput proteomic strategies.
No related grants have been discovered for shivaa akbarzadeh.