Re-examination of therapeutic management of muscular dystrophies using a vascular smooth muscle-centered approach.

In contrast to the long-standing focus on the pathophysiology of skeletal muscles in the hunt for a cure for Duchenne muscular dystrophy (DMD), we opine that the malfunctioning of dystrophin produced by vascular smooth muscle is a major contributor to the pathology of the illness. We believe that a biological response modifier glucan (BRMG), which has been shown in clinical studies of DMD to boost the expression of vascular smooth muscle dystrophin and provide anti-fibrotic and anti-inflammatory effects, may play a key role in reducing the pathogenesis of DMD. According to the evaluation of biomarkers, this BRMG, which is safe and side-effect-free, reduces the pathogenesis of DMD. We describe the possible mechanisms of action by which this BRMG helps in alleviating the symptoms of DMD by targeting smooth muscle dystrophin, in addition to its advantages over other therapeutic modalities, as well as how it can serve as a valuable adjunct to existing therapies. We suggest that using BRMG adjuncts that target smooth muscle dystrophin would be a potential therapeutic approach that prolongs the lifespan and extends the duration of ambulation from the onset of DMD. Further studies are needed to validate this hypothesis.

Efficacy of N-163 beta-glucan in beneficially improving biomarkers of relevance to muscle function in patients with muscular dystrophies in a pilot clinical study.

Background: Muscular dystrophies other than Duchenne muscular dystrophy (DMD) are genetic diseases characterized by increasing muscle weakness, loss of ambulation, and ultimately cardiac and respiratory failure. There are currently no effective therapeutics available. Having demonstrated the efficacy of a N-163 strain of Aureobasidium Pullulans (Neu-REFIX) produced B-1, 3-1,6-Glucan in pre-clinical and clinical studies of Duchenne muscular dystrophy (DMD) earlier, we assessed the effectiveness of this novel Beta glucan in the other muscular dystrophies in the present study. Methods: In this 60-day study, six patients with muscular dystrophies other than DMD consumed one 8g gel of Neu-REFIX beta-glucan along with their usual standard of care treatment regimen, and their biomarkers of relevance to muscle function such as serum calcium (SC), creatine phosphokinase (CPK), and alkaline phosphatase (ALP) levels along with functional improvement criteria, which is, Medical research council (MRC) scale and North Star Ambulatory assessment (NSAA), assessed at baseline and following the intervention. Results: After the intervention, the SC levels significantly decreased from a mean baseline value of 9.28 mg/dL to 8.31 mg/dL (p-value = 0.02). With a p-value of 0.29, the mean CPK value dropped from 2192.33 IU/L to 1567.5 IU/L. Following the intervention, the ALP levels dropped from 200.33 to 75.5 U/L (p-value = 0.15). MRC scale improved in three out of six patients. NSAA remained stable. There were no adverse effects. Conclusion: This study has proven the safety of Neu REFIX beta-glucan food supplement and its efficacy in improving both plasma biomarkers and functional parameters of muscle in a short duration of 2 months. Further validation by evaluation of muscle function for a longer duration is recommended to confirm the efficacy of Neu-REFIX food supplement as a potential adjuvant DMT in muscular dystrophies.

Improvement of sleep and melatonin in children with autism spectrum disorder after ß-1,3/1,6-glucan consumption: An open-label prospective pilot clinical study.

INTRODUCTION: Poor sleep quality is a major problem in patients with autism spectrum disorder (ASD), and is attributed to low melatonin levels. Melatonin supplementation is recommended; however, its effectiveness varies. ß-Glucans have previously been shown to improve melatonin levels in animal studies. Herein, we examined the effectiveness of Aureobasidium pullulans (Nichi Glucan), a species of black yeast that contains beta-1,3/1,6-glucan, in a pilot study of children with ASD. METHODS: Thirteen children (age, 2.5-13 years) with ASD were recruited for the study. The control group consisted of four patients (Gr. 1), while nine patients were classified into the treatment group (Gr. 2). Gr. 2 received 1 g of Nichi Glucan along with conventional therapy, whereas the Gr. 1 (control) patients received conventional therapy alone for 90 days. Serum melatonin levels and sleep patterns, assessed using a subjective questionnaire, were evaluated before and after treatment. RESULTS: In Gr. 2, the average serum melatonin level increased from 238.85 ng/L preintervention to 394.72 ng/L postintervention. Eight of nine participants (88%) in Gr. 2 showed improvements in sleep pattern and quality, while no improvement was observed in the participants in Gr. 1. CONCLUSION: The consumption of Nichi Glucan for 90 days resulted in visible improvement in sleep quality, sleep pattern, and serum melatonin levels, which was reported for the first time by our study. A larger multicenter study is required to validate our findings.

Improvement of behavioural pattern and alpha-synuclein levels in autism spectrum disorder after consumption of a beta-glucan food supplement in a randomised, parallel-group pilot clinical study.

BACKGROUND: Autism spectrum disorders (ASDs) are a wide range of behavioural disabilities for which there are no definite interventional modalities available. Remedial therapies remain the only option but with varying outcomes. We have evaluated the Childhood Autism Rating Scale (CARS) and alpha-synuclein levels in this parallel-group, multiple-arm pilot clinical study after supplementation with a biological response modifier beta-glucan food supplement (Nichi Glucan). METHODS: Six subjects with ASD (n=6) Gr. 1 underwent conventional treatment comprising remedial behavioural therapies and L-carnosine 500 mg per day, and 12 subjects (n=12) Gr. 2 underwent supplementation with the Nichi Glucan 0.5 g two times per day along with the conventional treatment. RESULTS: There was a significant decrease in the CARS score in all of the children of the Nichi Glucan Gr.2 compared with the control (p=0.034517). Plasma levels of alpha-synuclein were significantly higher in Gr. 2 (Nichi Glucan) than in the control group Gr. 1 (p=0.091701). CONCLUSION: Improvement of the behavioural pattern CARS score and a correlating alpha-synuclein level, followed by a safe beta-glucan food supplement, warrants further research on other parameters, such as gut-microbiota evaluation, and relevant neuronal biomarkers which is likely to cast light on novel solutions.

An efficient polymer cocktail-based transportation method for cartilage tissue, yielding chondrocytes with enhanced hyaline cartilage expression during in vitro culturing.

BACKGROUND: Chondrocytes are used in cell-based therapies such as autologous chondrocyte implantation (ACI) and matrix-associated cartilage implantation (MACI). To transport the cartilage tissue to the laboratory for in vitro culturing, phosphate-buffered saline (PBS), Euro-Collins solution (ECS) and Dulbecco's Modified Eagle's Medium (DMEM) are commonly employed at 4-8 °C. METHODS: In this study, eight samples of human cartilage biopsy tissues from elderly patients with severe osteoarthritis undergoing arthroscopy, which would otherwise have been discarded, were used. The cartilage tissue samples were compared to assess the cell yield between two transportation groups: i) a thermo-reversible gelation polymer (TGP) based method without cool preservation (∼25 °C) and ii) ECS transport at 4 °C. These samples were subjected to in vitro culture in a two-dimensional (2D) monolayer for two weeks and subsequently in a three-dimensional (3D) TGP scaffold for six weeks. RESULTS: The cell count obtained from the tissues transported in TGP was higher (0.2 million cells) than those transported in ECS (0.08 million cells) both after initial processing and after in vitro culturing for 2 weeks in 2D (18 million cells compared with 10 million cells). In addition, mRNA quantification demonstrated significantly higher expression of Col2a1 and SOX-9 in 3D-TGP cultured cells and lower expression of COL1a1 in RT-PCR, characteristic of the hyaline cartilage phenotype, than in 2D culture. CONCLUSION: This study confirms that the TGP cocktail is suitable for both the transport of human cartilage tissue and for in vitro culturing to yield better-quality cells for use in regenerative therapies.

Beneficial effects of novel aureobasidium pullulans strains produced beta-1,3-1,6 glucans on interleukin-6 and D-dimer levels in COVID-19 patients; results of a randomized multiple-arm pilot clinical study.

OBJECTIVE: In this pilot clinical study, we report the beneficial effects of beta glucans derived from two strains AFO-202 and N-163 of a black yeast Aureobasidium pullulans on the biomarkers for cytokine storm and coagulopathy in COVID-19 patients. METHODS: A total of 24 RT-PCR positive COVID-19 patients were recruited and randomly divided into three groups (Gr): Gr. 1 control (n = 8) - Standard treatment; Gr. 2: Standard treatment + AFO-202 beta glucan (n = 8); and Gr. 3, Standard treatment + combination of AFO-202 and N-163 beta glucans (n = 8) for 30 days. RESULTS: There was no mortality or requirement of ventilation of the subjects in any of the groups. There was a decrease in D-Dimer values (751 ng/ml to 143.89 ng/ml) and IL-6 values (7.395-3.16 pg/ml) in Gr. 1 in 15 days but the levels increased to abnormal levels on day 30 (D-Dimer: 202.5 ng/ml; IL-6 55.37 pg/ml); which steadily decreased up to day 30 in groups 2 (D-dimer: 560.99 ng/dl to 79.615; IL-6: 26.18-3.41 pg/ml) and 3 (D-dimer: 1614 ng/dl to 164.25 ng/dl; IL-6: 6.25-0.5 pg/ml). The same trend was observed with ESR. LCR and LeCR increased while NLR decreased significantly in Gr. 3. CD4 + and CD8 + T cell count showed relatively higher increase in Gr.3. There was no difference in CRP within the groups. CONCLUSION: As these beta glucans are well known food supplements with a track record for safety, larger multi-centric clinical studies are recommended to validate their use as an adjunct in the management of COVID-19 and the ensuing long COVID-19 syndrome.

Successful engraftment of epithelial cells derived from autologous rabbit buccal mucosal tissue, encapsulated in a polymer scaffold in a rabbit model of a urethral stricture, transplanted using the transurethral approach.

BACKGROUND: A pilot study reported an autologous buccal mucosal cell transplant in humans through the trans-urethral route using the buccal epithelium expanded and encapsulated in scaffold-hybrid approach to urethral stricture (BEES-HAUS), a minimally invasive approach to treat urethral stricture. Although successful outcomes were achieved in that study, for further validation, it is essential to prove that the transplanted buccal epithelium was engrafted over the urothelium through histological examination of the urethra, harvested post-transplant, which is infeasible in humans. Herein, we report the successful creation of an animal model of urethral stricture and the engraftment of epithelial cells derived from autologous buccal mucosal tissue, encapsulated in a thermo-reversible gelation polymer (TGP) scaffold, transplanted by trans-urethral route. METHODS: An animal model of urethral stricture was created in Japanese white male rabbits using electro-coagulation. Buccal tissue was harvested from the rabbits and subjected to enzyme digestion, followed by 5-7 days of in vitro culture in conventional two-dimensional (2D) culture and in a 3D platform of thermo-reversible gelation polymer (3D-TGP) culture. The cells harvested from the groups were mixed and encapsulated and transplanted with TGP, by transurethral catheterization. Fourteen days later, the urethra was harvested and subjected to histological examination. The buccal biopsy tissue, cells after digestion and cells post-culture were also subjected to histological examination. Urethrogram and endoscopy images were recorded at different time points. RESULTS: The stricture was successfully created, with the coagulated area markedly stenosed. Histological staining of the cells after in vitro processing showed that the cells grew with native epithelial and rounded cell morphology in 3D-TGP while they differentiated into fibroblast like-cells in 2D culture. Histological staining of the urethral tissue after transplantation revealed the engraftment of the transplanted buccal mucosal cells, with stratified squamous epithelium over the specialized stratified urothelium in the urethrotomy site. CONCLUSION: We used histology to prove the successful engraftment of TGP-encapsulated buccal mucosal epithelial cells in an animal model of urethral injury with healing of the injured tissue. The model of urethral stricture and cell therapy, using a transurethral approach, recapitulates the previously reported BEES-HAUS approach and lays the foundation for larger multi-centric translational clinical studies.

A 3D Polymer Scaffold Platform for Enhanced in vitro Culture of Human & Rabbit Buccal Epithelial Cells for Cell Therapies.

BACKGROUND: Buccal mucosal epithelial cells show promising application for various regenerative medicine approaches. In this study, we examined the feasibility of culturing rabbit and human buccal mucosal epithelial cells in a novel thermoreversible gelation polymer (TGP) scaffold, without feeder layers or other foreign proteins. METHODS & RESULTS: The results of this 28-day in vitro culture, u sing the conventional technique (2D) and TGP (3D) showed that the epithelial cell morphology could be maintained only in the TGP group while cells in the 2D group de-differentiated to fibroblast morphology in both human and rabbit samples. CK3 expression, a marker for epithelial differentiation was higher in 3D-TGP cultured cells than 2D. CONCLUSION: TGP based in vitro cell culture is a prospective methodology to culture buccal mucosal epithelial cells efficiently without using foreign biological components for tissue engineering applications.

ß-glucans: wide-spectrum immune-balancing food-supplement-based enteric (ß-WIFE) vaccine adjuvant approach to COVID-19.

Conventional vaccines to combat COVID-19 through different approaches are at various stages of development. The complexity of COVID-19 such as the potential mutations of the virus leading to antigenic drift and the uncertainty on the duration of the immunity induced by the vaccine have hampered the efforts to control the COVID-19 pandemic. Thus, we suggest an alternative interim treatment strategy based on biological response modifier glucans such as the Aureobasidium pullulans AFO-202-derived ß-glucan, which has been reported to induce trained immunity, akin to that induced by the Bacille Calmette-Guérin vaccine, by epigenetic modifications at the central level in the bone marrow. These ß-glucans act as pathogen-associated molecular patterns, activating mucosal immunity by binding with specific pathogen recognition receptors such as dectin-1 and inducing both the adaptive and innate immunity by reaching distant lymphoid organs. ß-Glucans have also been used as immune adjuvants for vaccines such as the influenza vaccine. Therefore, until a conventional vaccine is widely available, an orally consumable vaccine adjuvant that acts like biosimilars, termed as the wide-spectrum immune-balancing food-supplement-based enteric (ß-WIFE) vaccine adjuvant approach, with well-reported safety is worth in-depth investigation and can be considered for a clinical trial.

A three-dimensional in vitro culture environment of a novel polymer scaffold, yielding chondroprogenitors and mesenchymal stem cells in human chondrocytes derived from osteoarthritis-affected cartilage tissue.

OBJECTIVE: We evaluated the expression of stem/progenitor biomarkers in osteoarthritic tissue derived chondrocytes cultured using a three-dimensional (3D) thermo-reversible gelation polymer (TGP). METHODS: The chondrocytes from discarded biopsy tissues obtained from human elderly patients with osteoarthritis were cultured using the 3D-TGP up to six weeks. RESULTS: The chondrocytes grew in a tissue-like manner, without de-differentiation into fibroblasts, and the cells thus tissue-engineered were proven positive for CD49e, OCT4, CD-105 and STRO-1 by immunohistochemistry. CONCLUSION: This study establishes the efficacy of this 3D-TGP platform for clinically useable in-vitro tissue-engineered cartilage for improvising the clinical outcome of cell therapy for cartilage repair.

Cross-Protection Induced by Encephalitis Vaccines against COVID-19 Might be a Reason for Relatively Lower Mortality Rate in Some Countries.

COronaVIrus Disease 2019 (COVID-19) is an on-going pandemic attributed to a novel virus named SARS-CoV-2. Comparing the statistics of incidence and death rates between nations reveals that there is discrepancy amongst countries in these regards, even between countries that share borders. We herein present information from the literature indicating how cross-protection against COVID-19 conferred by the encephalitis vaccine could be the reason for lower fatality rate in the countries where immunization against encephalitis is widespread or included in national programs. This may pave the way for arriving at efficient prevention strategies as well as vaccine development.

Articular chondrocytes from osteoarthritic knee joints of elderly, in vitro expanded in thermo-reversible gelation polymer (TGP), exhibiting higher UEA-1 expression in lectin microarray.

Autologous chondrocytes in vitro expanded, are used as tools of regenerative therapies for cartilage injuries. However, inability to maintain the hyaline phenotype both in vitro and post in vivo transplantation, remains one of the major hurdles for long term efficacy under clinical settings. We have reported earlier, hyaline phenotype maintenance of both human and rabbit chondrocytes for a long duration both in vitro when cultured conditions using a Thermo-reversible Gelation Polymer (TGP) scaffold-based methodology and in vivo post-transplantation animal model of cartilage damage. Having intrigued by such encouraging outcome, we in this study, analysed the similar TGP culture environment whether would be able to allow in vitro expansion of severe osteoarthritis affected cartilage tissue from elderly patients and evaluated the cells using lectin microarray characterization for pluripotency. Cartilage tissue were obtained from patients (n = 7; age: 60-85 years) undergoing total knee arthroplasty for severe osteoarthritis. Chondrocytes were isolated and cultured in two groups: i. conventional culture without scaffold (2D) and ii. using a TGP scaffold-based culture (3D) up to 18 weeks. In addition to earlier reported findings such as maintenance of hyaline phenotype having been confirmed in this study as well, surface glycoprotein analysis by lectin microarray demonstrated that the α1-2 Fuc recognition lectin (UEA-1) (marker reported in literature for pluripotent stem cells) was found to be more highly expressed in 3D culture compared to 2D culture and even increased over time in 3D culture. We have developed an environment where osteoarthritis affected chondrocytes from the elderly could be cultured up to 18 weeks in vitro using TGP scaffold which express pluripotent cell associated surface glycoproteins compared to the conventional methodology.

Buccal epithelium Expanded and Encapsulated in Scaffold-Hybrid Approach to Urethral Stricture (BEES-HAUS) procedure: A novel cell therapy-based pilot study.

OBJECTIVES: To describe the feasibility of a novel cell-based endoscopic technique using buccal epithelium, expanded and encapsulated in a thermoreversible gelation polymer scaffold for the treatment of urethral stricture. METHODS: Six male patients with bulbar urethral stricture ranging from 2.0 to 3.5 cm in length were included in this pilot study. Autologous buccal epithelial cells from a small buccal mucosal biopsy were isolated, cultured and encapsulated in thermoreversible gelation polymer scaffold, and were implanted at the stricture site after a wide endoscopic urethrotomy. RESULTS: All the patients voided well, with a mean peak flow rate of 24 mL/s. Urethroscopy carried out at 6 months showed healthy mucosa at the urethrotomy site. However, two of the six patients had recurrence at 18 and 24 months, respectively. CONCLUSIONS: This endoscopic-based Buccal epithelium Expanded and Encapsulated in Scaffold-Hybrid Approach to Urethral Stricture (BEES-HAUS) technique is a promising alternative for the open substitution buccal graft urethroplasty. It is possible to achieve the benefits of open substitution buccal urethroplasty with this endoscopic technique.

Human corneal endothelial cell transplantation using nanocomposite gel sheet in bullous keratopathy.

Transplantation of in vitro expanded human corneal endothelial precursors (HCEP) cells using a nanocomposite (D25-NC) gel sheet as supporting material in bovine's cornea has been earlier reported. Herein we report the transplantation of HCEP cells derived from a cadaver donor cornea to three patients using the NC gel sheet. In three patients with bullous keratopathy, one after cataract surgery, one after trauma and another in the corneal graft, earlier performed for congenital corneal dystrophy, not amenable to medical management HCEP cells isolated from a human cadaver donor cornea in vitro expanded using a thermoreversible gelation polymer (TGP) for 26 days were divided into three equal portions and 1.6 × 105 HCEP cells were injected on to the endothelium of the affected eye in each patient using the D25-NC gel sheet as a supporting material. The sheets were removed after three days. The bullae in the cornea disappeared by the 3rd-11th post-operative day in all the three patients. Visual acuity improved from Perception of light (PL)+/Projection of rays (PR)+ to Hand movements (HM)+ in one of the patients by post-operative day 3 which was maintained at 18 months follow-up. At 18 months follow-up, in another patient the visual acuity had improved from HM+ to 6/60 while in the third patient, visual acuity remained HM+ as it was prior to HCEP transplantation. There were no adverse effects during the follow-up in any of the patients.

Combining autologous peripheral blood mononuclear cells with fibroblast growth factor therapy along with stringent infection control leading to successful limb salvage in diabetic patient with chronic renal failure and severe toe gangrene.

Peripheral arterial disease (PAD) is a common complication of Diabetes Mellitus (DM) and often culminates in amputation of the affected foot. Pseudomonas aeruginosa infections associated with PAD are difficult to treat due to their multi-drug resistance. Herein we report a 38 year old male who reported with DM, chronic kidney disease (CKD) and rest pain of the right second toe in October 2011. He underwent percutaneous transluminal angioplasty (PTA) which was unsuccessful. The gangrene of the toes worsened and amputation of the right second toe was done. Bacteriological examination showed presence of P. aeruginosa which during the course of antibiotic therapy became multi-drug resistant. Gangrene and abscess of the foot worsened and amputation of the right third toe was performed. Then autologous peripheral blood mononuclear cell (PBMNC) therapy was performed but as infection control could not still be achieved, the fourth toe was amputated. A protocol of foot bath using carbonic water, local usage of antibiotics (Polymyxin-B), and basic fibroblast growth factor (b-FGF) spray was then employed after which the infection could be controlled and improvement in vascularity of the right foot could be observed in angiography. This combined approach after proper validation could be considered for similar cases.

In vitro culture and characterization of enteric neural precursor cells from human gut biopsy specimens using polymer scaffold.

In vitro expansion and characterization of neural precursor cells from human gut biopsy specimens with or without Hirschsprung's disease using a novel thermoreversible gelation polymer (TGP) is reported aiming at a possible future treatment. Gut biopsy samples were obtained from five patients undergoing gut resection for Hirschsprung's disease (n = 1) or gastrointestinal disorders (n = 4). Cells isolated from the smooth muscle layer and the myenteric plexus were cultured in two groups for 18 to 28 days; Group I: conventional culture as earlier reported and Group II: using TGP scaffold. Neurosphere like bodies (NLBs) were observed in the cultures between 8th to 12th day and H & E staining was positive for neural cells in both groups including aganglionic gut portion from the Hirschsprung's disease patient. Immunohistochemistry using S-100 and neuron specific enolase (NSE) was positive in both groups but the TGP group (Group II) showed more number of cells with intense cytoplasmic granular positivity for both NSE and S-100 compared to Group I. TGP supports the in vitro expansion of human gut derived neuronal cells with seemingly better quality NLBs. Animal Studies can be tried to validate their functional outcome by transplanting the NLBs with TGP scaffolds to see whether this can enhance the outcome of cell based therapies for Hirschsprung's disease.