Myofibroblast and pro-fibrotic cytokines in fibrosis of IgG4-related disease (IgG4-RD) patients from South Asia: preliminary data.

INTRODUCTION: Fibrosis is a typical pathological characteristic in IgG4-RD patients and often irreversible. There exists a lack of suitable markers for detection of earlier onset of fibrosis in various organs in IgG4-RD patients. Hence, this study aims at analysing ambispectively the myofibroblasts and the pro-fibrotic cytokines, IFN gamma and IL-33 involved in IgG4-RD associated fibrosis in South Asian patients. METHOD: Archived biopsy samples of definite/probable/possible cases of IgG4-RD, classified according to diagnostic criteria, taken from patients who attended the OPD and IPD of our tertiary care centre during January 2015-January 2020 were chosen for this study. The paraffin sections were examined qualitatively for fibrosis and the excessive collagen deposition by Hematoxylin & Eosin and Masson's Trichrome staining. Also, the presence of alpha-Smooth muscle actin (α-SMA) expressing myofibroblasts and the involvement of pro-fibrotic cytokines (IFN-gamma, IL-33) were assessed by Immunohistochemistry and scored semi-quantitatively (+mild, ++moderate, +++ severe). Serum IL-33 levels were analysed by indirect Elisa (R & D Systems). RESULTS: Myofibroblasts were present in 10/12 biopsy samples, in moderate levels in 4 (33%) and very high levels (+++) in 3 (25%) of the patients. IFN-gamma was expressed at low levels in 6 (50%) and absent in 6 (50%). All patients showed IL-33 expression with very high levels in tissue (6, 50%), as well as in serum samples. CONCLUSION: The findings of this study reinforce the role of myofibroblasts and profibrotic cytokines like IL-33 in fibrosis of Ig4-RD patients, pointing to their potential as earlier predictive markers of onset and extent of fibrosis.

Utility of peripheral blood monocyte subsets, circulating immune complexes and serum cytokines in assessment of SLE activity: an observational, cross-sectional study.

INTRODUCTION: SLE disease measurements by current standards are less than perfect. Monocytes and their subsets are part of innate immunity, and one of our objectives was to look at their role in SLE disease activity. We also looked at the common serum cytokines and the role of circulating immune complex (CIC) estimation in the assessment of disease activity. METHODS: We conducted a single-centre observational cross-sectional study of SLE patients with active and inactive disease as the comparison arms. Blood samples were collected for (a) peripheral blood monocyte separation and flowcytometric analysis of monocyte subsets based on CD14 and CD16 surface markers, and (b) ELISA for serum cytokines and CIC estimation. Results were analysed in terms of the difference in medians between the active and inactive disease groups using the Mann-Whitney U test (non-normally distributed data). RESULTS: The absolute monocyte count was lower in the active group than the inactive group (median (IQR) of 329 (228.5) vs. 628 (257)/microliter, p = 0.001). The frequency (%) of the intermediate monocyte subset showed a trend towards an increase in active disease (median (IQR) of 15.10% (9.65) vs. 11.85% (8.00), p = 0.09). It also had a significant positive correlation to the SLEDAI scores (r = 0.33, p = 0.046). The mean fluorescence intensity (MFI) of CD163, expressed primarily by intermediate subsets, was increased, and CD11c MFI was reduced in active disease. Serum TNF-a level was elevated in active disease (median (IQR) of 38 (48.5) pg/ml vs. 9 (48.5) pg/ml, p = 0.042). CIC ELISA at an optimal cut-off of 10 meq/ml provided an area under the curve (AUC) of 0.85 for detecting active SLE. CONCLUSION: Peripheral blood monocytes are depleted in active disease. The intermediate monocyte subset may have a role in disease activity. TNF-alpha correlated modestly with disease activity. CIC estimation by ELISA may be used in addition to or as an alternative to current standards of laboratory tests for the serological assessment of activity.

Scaffold Proteins in Autoimmune Disorders.

Cells transmit information to the external environment and within themselves through signaling molecules that modulate cellular activities. Aberrant cell signaling disturbs cellular homeostasis causing a number of different diseases, including autoimmunity. Scaffold proteins, as the name suggests, serve as the anchor for binding and stabilizing signaling proteins at a particular locale, allowing both intra and intercellular signal amplification and effective signal transmission. Scaffold proteins play a critical role in the functioning of tight junctions present at the intersection of two cells. In addition, they also participate in cleavage formation during cytokinesis, and in the organization of neural synapses, and modulate receptor management outcomes. In autoimmune settings such as lupus, scaffold proteins can lower the cell activation threshold resulting in uncontrolled signaling and hyperactivity. Scaffold proteins, through their binding domains, mediate protein- protein interaction and play numerous roles in cellular communication and homeostasis. This review presents an overview of scaffold proteins, their influence on the different signaling pathways, and their role in the pathogenesis of autoimmune and auto inflammatory diseases. Since these proteins participate in many roles and interact with several other signaling pathways, it is necessary to gain a thorough understanding of these proteins and their nuances to facilitate effective target identification and therapeutic design for the treatment of autoimmune disorders.

Natural 3D Extra Cellular Matrix mimicking stem cells seeded decellularized scaffolds as a platform for tendon regeneration.

Achilles tendon, which connects the calf muscles to heel, is the strongest tendon in the body. Despite its strength, it is more prone to injury due to its limited blood supply. Tendon-related injuries are more common in sportspersons, people with labor-intensive work and the aged community. The currently available treatment mode is surgery which is expensive with chances of re-injury. Present study made an attempt to fabricate a tissue-engineered tendon product using decellularized tendon (DT) seeded with stem cells and bioactive components of Tinospora cordifolia extract (TCE). The bare DT tissue scaffold/substitute may also serve as a drug delivery platform for growth factors and cells with a new approach to promote tissue regeneration in clinical applications. DT construct showed good regenerative potential and easily promoted new tissue formation. Decellularization of the tendon was carried out by chemical method using tri (n-butyl) phosphate (TnBP). DT was physicochemically characterized by contact angle measurement, thermal gravimetric analysis (TGA), and mechanical testing. Rabbit adipose derived mesenchymal stem cells (RADMSCs) were isolated and phenotypically characterized by flow cytometry analysis, tri lineage differentiation, and so forth. Further, stem cell seeded DT scaffolds were prepared and found to be non-toxic by cytotoxicity, cell adhesion by scanning electron microscope (SEM) analysis, cell viability by live dead assays, and so forth. The findings of this study yield valid proof for the employability of cell-seeded DT construct as a natural scaffold in repairing injured tendons-the toughest chords of the skeleton. This is a cost effective method for the replacement of injured/damaged tendons for athletes, people in labor-intensive occupations, the elderly population, and so forth-a boon towards the repair of the tendon in damage/injury.

Neural Tissue Engineering with Rat Adipose-Derived Mesenchymal Stem Cells: The Role of an Injectable, Resorbable Hydrogel Scaffold Derived from Oxidized Alginate and Gelatin.

The central nervous system has limited regeneration potential. The multipotency of adipose-derived mesenchymal stem cells (ADMSC) makes them an ideal autologous cell source for the regeneration of neural tissues. However, the likelihood of their differentiation into unwanted cell lineages when transplanted into a hostile injury environment is a serious disadvantage. Transplanting predifferentiated cells via an injectable carrier may aid in site-specific delivery for better survival of cells. Here, we focus on identifying an appropriate injectable hydrogel system that favors stem/progenitor cell attachment and differentiation for neural tissue engineering. An injectable composition of the hydrogel, derived from alginate dialdehyde (ADA) and gelatin, was formulated for this purpose. This hydrogel promoted proliferation/differentiation of ADMSCs to neural progenitors, visualized from the generation of prominent neurospheres and stage-specific expression of a neural progenitor marker (nestin, day 4), an intermittent neuronal marker (ß-III tub, day 5), and a mature neuronal marker (MAP-2, day 8) with neural branching and networking (>85%). The differentiated cells also expressed the functional marker synaptophysin. There was no negative impact on stem/progenitor cell survival (>95%) or differentiation (∼90%) as compared to two-dimensional (2D) culture. Addition of appropriate quantities of asiatic acid specific for neural niche supported cell growth and differentiation without affecting cell survival (>90%) and improved neural branching and elongation. Optimized interconnected porous hydrogel niche exhibited rapid gelation (3 min) and self-healing properties mimicking native neural tissue. Both ADA-gelatin hydrogel by itself and that incorporated with asiatic acid were found to support stem/neural progenitor cell growth and differentiation and have potential applications as antioxidants and growth promoters upon release at the cell transplantation site. In short, the matrix itself or incorporated with phytomoieties could serve as a potential minimally invasive injectable cell delivery vehicle for cell-based therapies of neural diseases.

3D-bulk to nanoforms of modified hydroxyapatite: Characterization and osteogenic potency in an in vitro 3D bone model system.

Synthetic bioceramics are replacing conventional methods of treating bone defects with autografts owing to the high demand of bone substitutes, with their Surface topography and size contributing to favor cytocompatibility in tissue regeneration. This experimental study deals with the comparative evaluation of the physical characterizations of four different in-house synthesized bioceramics from 3D-bulk to nanoforms of hydroxyapatite (HA), Biphasic calcium phosphate (BCP), Strontium doped hydroxyapatite (SrHA) and Silica coated hydroxyapatite (HASi) and also simultaneously evaluates adhesion, proliferation and osteogenic differentiation of rabbit adipose derived mesenchymal stem cells (RADMSCs) on these biomimetic ceramic niches. The osteogenic induced cells grown on 3D scaffolds for a period of 7, 14, 21, and 28 days were analyzed for their viability (MTT, LDH, live-dead assays), morphology (SEM), proliferation (Cytox-Red) and osteogenic differentiation (ALP, osteocalcin expression). Cellular activities and differentiation of RADMSCs were significantly higher on SrHA indicating the role of strontium in the differentiation of mesenchymal stem cells on this ceramic platform to the bone lineage. In order to reinforce the materials for hard tissue implantation and drug delivery, nano-SrHA (nSrHA) became the nanoparticle of choice based on its non-toxicity, cytocompatibility and osteogenic properties (nSrHA > nHASi > nBCP > nHA).

Green synthesis of Cuminum cyminum silver nanoparticles: Characterizations and cytocompatibility with lapine primary tenocytes.

Current treatment systems for tendon injuries are very few and do not ensure complete cure. This is a serious health concern for sports persons and the aged population. It is known that the nano- or microsized particles of natural products such as jeera/cumin seed (Cuminum cyminum) has been used traditionally as a home remedy for the treatment of tendon injuries. Nevertheless, these particles are likely to perform better due to their smaller size, increased absorption and local delivery in conjunction with nanotechnology. In this context, the major objective of this study was to synthesize silver-capped nanoparticles using aqueous extract of Cuminum cyminum (CCE) and to assess their in vitro non-cytotoxic effect with the perspective of clinical application to enhance tendon tissue regeneration. The presence of phytochemicals in CCE was studied by qualitative and quantitative methods. Cuminum cyminum nanoparticles (CCNP) were synthesized by the bioreduction method using silver nitrate and the particles were characterized by X-ray diffraction analysis (XRD), Fourier Transform Infra Red Spectroscopy (FTIR), Zeta potential measurement and scanning electron microscopy (SEM). The antioxidant effect of the particles was studied using total antioxidant activity and reducing power assay. Simultaneously, primary Tenocytes were isolated from rabbit Achilles tendon by collagenase and dispase digestion/treatment and characterized for Type 1 collagen. Further, in vitro non-cytotoxicity of the CCNP in direct contact with L929 mouse fibroblast cells and primary Tenocytes, respectively, was evaluated by MTT assay. Physico-chemical characterizations confirmed the formation and stability of the nanosize of CCNP with antioxidant property. Again, MTT assay confirmed the non-cytotoxicity of CCNP with L929 fibroblasts and primary Tenocytes. CCNP may be attributed as an ideal candidate for therapeutic application towards a faster restoration of worn-out/injured tendon tissue confronted by the geriatric and athlete communities.

Mesenchymal Stem Cells Seeded Decellularized Tendon Scaffold for Tissue Engineering.

Tendon is a collagenous tissue to connect bone and muscle. Healing of damaged/injured tendon is the primary clinical challenge in musculoskeletal regeneration because they often react poorly to treatment. Tissue engineering (a triad strategy of scaffolds, cells and growth factors) may have the potential to improve the quality of tendon tissue healing under such impaired situations. Tendon tissue engineering aims to synthesize graft alternatives to repair the injured tendon. Biological scaffolds derived from decellularized tissue may be a better option as their biomechanical properties are similar to the native tissue. This review is designed to provide background information on the current challenges in curing torn/worn out the tendon and the clinical relevance of decellularized scaffolds for such applications.

Novel flourescent spiroborate esters: potential therapeutic agents in in vitro cancer models.

The current treatment system in cancer therapy, which includes chemotherapy/radiotherapy is expensive and often deleterious to surrounding healthy tissue. Presently, several medicinal plants and their constituents are in use to manage the development and progression of these diseases.They have been found effective, safe, and less expensive. In the present study, we are proposing the utility of a new class of curcumin derivative, Rubrocurcumin, the spiroborate ester of curcumin with boric acid and oxalic acid (1:1:1), which have enhanced biostability for therapeutic applications. In vitro cytocompatibility of this drug complex was analysed using MTT assay, neutral red assay, lactate dehydrogenase assay in 3T3L1 adipocytes. Anti tumour activity of this drug complex on MCF7 and A431 human cancer cell line was studied by morphological analysis using phase contrast microscopy, Hoechst staining and cell cycle analysis by FACS. To explore the chemotherapeutic effect, the cytotoxic effect of this compound was also carried out. Rubrocurcumin is more biostable than natural curcumin in physiological medium. Our results prove that this curcumin derivative drug complex possess more efficacy and anti-cancer activity compared with curcumin. The findings out of this study suggests this novel compound as potential candidate for site targeted drug delivery.

Influence of curing agent on fibrosis around silicone implants.

Severe capsular contracture around silicone expander breast implants leading to pain and failure is a major clinical problem. Even though earlier studies have implicated the immunogenicity of silicone, the role of physical and chemical properties of the silicone material in excessive collagen deposition and fibrosis has been less addressed. The present study investigates whether there is any correlation between the type of curing systems i.e. addition and free radical curing and the fibrosis around silicone elastomer. The experiment carried out uses commercially available silicone ventriculo-peritoneal shunt material elastomer cured by platinum and the results are compared with results obtained in a similar study carried out by the authors using commercially available silicone tissue expander material cured by peroxide. Ultra-high molecular weight poly-ethylene (UHMWPE), the standard reference for biocompatibility evaluation, was used as the control material. The materials were implanted in rat skeletal muscle for 30 and 90 days. Inflammatory cells, myofibroblasts, cytokines, and collagen deposition at the material-tissue interface were identified by haematoxylin-eosin and Masson's Trichrome stains and semi-quantitated based on immunohistochemical studies. Results indicate that even though the cellular response in the initial phase of wound healing was similar in both platinum and peroxide-cured materials, the collagen deposition in the proliferative phase was more around peroxide-cured material in comparison to the platinum-cured silicone elastomer. There is a need to look into the molecular mechanisms of this interaction and the possibility of using curing systems other than free radical peroxide in the manufacture of silicone elastomer expanders for breast prosthesis.

Mediatory role of interleukin-6 in α smooth muscle actin induction and myofibroblast formation around silicone tissue expander.

Materials used for medical devices are usually tested for their biocompatibility, before use. However, it is known that long-term implantation in the body may lead to degradation of the material leading to an adverse tissue response. The failure of silicone breast implants due to excessive fibrosis and contracture has led to studies to delineate the cause of fibrosis around this material. To detect the biological moieties involved, conditioned media from RAW 264.7 macrophages seeded over commercially available silicone tissue expander material was added to L929 fibroblasts. Ultrahigh-molecular-weight polyethylene and tissue culture grade polystyrene served as the control materials. The gene expression of fibrogenic cytokines, interleukin-6 (IL-6), and transforming growth factor beta (TGFß) in the RAW macrophages and myofibroblast marker alpha smooth muscle actin (αSMA) in L929 cells were quantitated by real time polymerase chain reaction. Protein expression analysis of αSMA was carried out by immunocytochemical staining and confocal microscopy. An in vitro degradation study of silicone expander material in pseudoextracellular fluid (PECF) and the αSMA expression in fibroblasts incubated with the silicone extract containing PECF has revealed the role of silicone leachants in induction of myofibroblasts. This in vitro expression study revealed the additional profibrotic role of IL-6 in fibroblast to myofibroblast transition and the synergy between material aspects and biomolecules in regulating fibrosis around Silicone implants. These findings may help in targeting newer biological moieties in the profibrotic pathway and in devising better manufacturing processes aiding the life of millions of patients.

Investigative study of myofibroblasts and cytokines in peri-implant tissue of silicone breast expander by rt-PCR in a rat model.

The excessive collagen deposition around silicone breast implants followed by contracture and development of severe pain is a major clinical problem. This study was conducted to investigate the profibrotic and antifibrotic cytokines secreted by inflammatory cells and development of myofibroblasts at the tissue material interface around silicone breast expander and ultra-high-molecular-weight polyethylene (UHMWPE). Both materials were implanted in rats for 30, 90 and 180 days. Inflammatory cells and collagen deposition at the material-tissue interface were assessed with Haematoxylin-Eosin and Masson's Trichrome stain. Gene expression of TGFbeta, IL-1beta, IFNgamma, IL10 and alpha-SMA was quantitated by real-time (RT)-PCR in the peri-implant tissue. Results indicate a difference in collagen deposition and myofibroblast development around both materials with involvement of TGFbeta, IFNgamma and IL10. The results emphasise the need for further investigation into the molecular mechanisms of protomyofibroblast and myofibroblast formation around silicone implants, which would provide information on these target cells for inhibitory therapy in the clinical situation.

Role of immune cells and inflammatory cytokines in regulation of fibrosis around silicone expander implants.

This study aimed to investigate the progress of wound healing around silicone expander with particular emphasis on fibroblasts, myofibroblasts and collagen in the repair phase. Semi-quantitative evaluation of inflammatory cells and their cytokines, fibroblasts and myofibroblasts at the tissue-material interface was carried out. Commercially available silicone expander was implanted in gluteus muscle of young female Wistar rats for 3, 7, 14, 30, 90 and 180 days. Ultra high molecular weight polyethylene served as control. The cellular response was studied by immunohistochemistry and Transmission Electron Microscopy. A thick collagenous fibrous capsule was observed around the silicone expander at 180 days, with persistent myofibroblasts, lymphocytes and macrophages as compared to the thin fibrous encapsulation around the UHMWPE implants. The regulatory role of cytokines and immune cells in myofibroblast persistence in tissue-implant interface around silicone expander has been extensively studied. Results of this study indicate the need to elucidate the signaling molecules in the transition of fibroblast to myofibroblast around silicone expander implants.