9-Demethoxy-medicarpin: A potential bone health supplement for the management of protein deficiency-induced bone loss in growing rats.

Human skeleton requires an adequate supply of many different nutritional factors for optimal growth and development. The role of nutrition in bone growth has piqued interest in recent years, especially in relation to maximizing peak bone mass and reducing the risk of osteoporosis. Protein deficiency-induced bone loss was induced in female growing rats. All experimental rodent diets were prepared as per recommendations for growing animals. 9-Demethoxy-medicarpin (DMM) treatment was given to growing Sprague Dawley (SD) rats at 1 mg and 10 mg dose orally for 30 days. Bones were collected for bone mineral density (BMD). Bone marrow cells were isolated from femur for calcium nodule formation. Serum samples were collected for biochemical parameters. We found that DMM treatment speeds up the recovery of musculoskeletal weakness by replenishing nutrients in proven rodent model. DMM supplementation for four weeks showed significantly increased vertebral, femur and tibial BMD compared with the untreated PD group. Albumin levels were significantly enhanced in treatment groups, in which 10 mg dose imparted a better effect. We conclude that DMM treatment led to increased BMD and biochemical parameters in protein deficient condition in growing rats and has potential as a bone growth supplement.

Utility of the pattern-based approach using BIOCHIP-indirect immunofluorescence in the evaluation of subepidermal bullous disorders.

BACKGROUND: Subepidermal bullous disorders (SEBD) are a heterogeneous group of vesiculobullous diseases because of antibody-mediated destruction of proteins of the dermo-epidermal junction. Direct immunofluorescence (DIF) is the gold standard for diagnosis. BIOCHIP-indirect immunofluorescence (IIF) is a novel serological test that combines multiple target antigens in a single field. The present study aimed to evaluate the utility of the pattern-based approach in BIOCHIP-IIF for the diagnosis of SEBD. METHODS: Seventy cases of BIOCHIP-IIF that showed clinical, histopathological, and/or DIF features favoring SEBD were included in the study. The interpretation in the BIOCHIP was categorized into one of the following patterns. Pattern I: basement membrane zone (BMZ) staining in monkey esophagus (ME), primate salt-split skin (SSS)-roof staining, BP180+ and/or BP230+; Pattern II: roof staining in SSS, BP180- and BP230- with or without BMZ staining in ME; Pattern III: floor staining in SSS, BP180- and BP230-; and pattern IV: negative in SSS and other substrates. The findings were correlated with histopathology and/or DIF. RESULTS: Fifty (71.5%) cases showed pattern I or the typical bullous pemphigoid (BP) pattern. Eight (11.4%) cases showed pattern II. Patterns III and IV were observed in seven (10%) and five (7.1%) cases, respectively. BP was the most common diagnosis in patterns I and II, while anti-p200 pemphigoid was most common in pattern III, as confirmed by immunoblotting. The sensitivity of pattern I in the diagnosis of BP was 96%. CONCLUSION: BIOCHIP-IIF showed a good correlation with DIF and histopathology in the diagnosis of SEBD. This can be used as a first-line investigation in case of bullous disorders.

Papillon-Lefèvre syndrome (PLS) with novel compound heterozygous mutation in the exclusion and Peptidase C1A domains of Cathepsin C gene.

Papillon Lefevre syndrome (PLS) manifests with palmoplantar keratoderma, combined with a rapidly progressive periodontitis associated with mutations in Cathepsin C (CTSC) gene. This article reports a 15-year old male proband with typical PLS traits having a novel compound heterozygote with p.Q49X mutation in exon 1 and p.Y259C missense mutation in exon 6 of CTSC gene respectively. The exon 1 mutation, p.Q49X, (found in proband's mother) was located in exclusion domain and exon 6 mutation, p.Y259C (found in proband's father), was present in peptidase C1A, papain C-terminal domain. Interestingly, missense mutation p.Y259C identified in this study was found to be not reported so far. Upon computational analysis, this missense mutation was found to be lethal. Moreover, our protein modelling approach using mutant protein revealed the presence of monomeric structure on contrary to the tetrameric structure of the wild type protein. In addition, in vitro functional characterization of mutant p.Y259C expressed in HEK293 cells showed a significant reduction in CTSC activity (0.015 ± 0.009 mU/ml) when compared with wild type protein (0.21 ± 0.008 mU/ml). Thus, in this study, we have demonstrated that the pathogenic missense mutant p.Y259C might cause PLS by impaired CTSC function.

Contact sensitivity in patients with venous leg ulcer: A multi-centric Indian study.

Venous leg ulcers are the most common form of non-healing leg ulcers. They are subjected to treatments such as topical medications, dressings, and compression therapies. This can lead to exposure to a number of allergens with subsequent sensitisation and contact dermatitis of the regional skin. This may contribute to the poor ulcer healing. To detect the various contact sensitisers in patients with venous leg ulcers through patch testing, patients from 6 centres across India with venous leg ulcers of longer than 6 weeks duration were enrolled for the study. They were patch tested using a special parch test kit with 27 antigens. A total of 172 patients were included in the study; 82 (48.2%) tested positive for at least 1 antigen. Among them, polyvalent sensitisation was noted in 71% of patients. Wood tar mix (10.4%) and the framycetin (8.7%) were the most common allergens. There is a high frequency of allergic sensitisation to various ingredients of topical therapies used in the venous ulcer management, which may interfere with wound healing. Avoiding them can help obtain a better therapeutic outcome.

Synthesis and biological evaluation of heterocyclic analogues of pregnenolone as novel anti-osteoporotic agents.

The structural modifications of pregnenolone have been described via the introduction of heterocyclic moieties at C-17 position by limiting the acyl group. Novel heterocyclic analogues of pregnenolone have been synthesized by using Friedlander and Claisen-Schmidt reactions, and the synthesized compounds were evaluated for their osteogenic activity. Among the synthesized derivatives, four compounds showed significantly increased ALP activity. Among all four active compounds, the novel compound 3a has shown significant bone matrix mineralization and mRNA expressions of osteogenic marker genes, BMP2, RUNX-2 and OCN at 1pM concentration.

Transdermal gelation of methacrylated macromers with near-infrared light and gold nanorods.

Injectable hydrogels provide locally controlled tissue bulking and a means to deliver drugs and cells to the body. The formation of hydrogels in vivo may involve the delivery of two solutions that spontaneously crosslink when mixed, with pH or temperature changes, or with light (e.g., visible or ultraviolet). With these approaches, control over the kinetics of gelation, introduction of the initiation trigger (e.g., limited penetration of ultraviolet light through tissues), or alteration of the material physical properties (e.g., mechanics) may be difficult to achieve. To overcome these limitations, we used the interaction of near-infrared (NIR) light with gold nanorods (AuNRs) to generate heat through the photothermal effect. NIR light penetrates tissues to a greater extent than other wavelengths and provides a means to indirectly initiate radical polymerization. Specifically, this heating coupled with a thermal initiator (VA-044) produced radicals that polymerized methacrylated hyaluronic acid (MeHA) and generated hydrogels. A range of VA-044 concentrations changed the gelation time, yielding a system stable at 37 ° C for 22 min that gels quickly (~3 min) when heated to 55 ° C. With a constant irradiation time (10 min) and laser power (0.3 W), different VA-044 and AuNR concentrations tuned the compressive modulus of the hydrogel. By changing the NIR irradiation time we attained a wide range of moduli at a set solution composition. In vivo mouse studies confirmed that NIR laser irradiation through tissue could gel an injected precursor solution transdermally.

A prospective observational study on species differentiation and antifungal susceptibility pattern in patients with genital candidiasis.

Background: Candidial balanitis, balanoposthitis and vulvovaginitis can be diagnosed by direct microscopy, culture and treated with antifungals. Resistance to antifungals is emerging. Hence, we conducted a study to identify the causative species and antifungal susceptibility. Aim: To observe the species differentiation and antifungal susceptibility pattern in patients with genital candidiasis. Materials and Methods: A prospective observational study was carried out that included 54 patients of age group (18-60 years) diagnosed clinically and direct microscopically (KOH) for genital candidiasis. Culture was done using Sabouraud dextrose agar. Species identification and antifungal susceptibility were tested. Descriptive data were expressed in the form of frequency and percentage. Results: Out of 54 patients, 41 had culture positive candidiasis. Among the isolated species, 68.3% were Candida albicans (28/41) and 31.7% were non- albicans Candida spp. Among non-albicans Candida species (13/41), Candida glabrata (19.5%), Candida tropicalis (7.3%), Candida guilliermondii (2.4%), Candida parapsilosis (2.4%) were identified. Antifungal susceptibility was tested for fluconazole (FLU), clotrimazole (CLTZ), itraconazole (ITZ), ketoconazole (KTZ), voriconazole (VOR), amphotericin-B (AMPH-B). Except C. glabrata and C.parapsilosis, all other species were sensitive to all tested antifungals. All isolated species were sensitive to KTZ, VOR, AMPH-B, and CLTZ. Nearly 22% of isolates were resistant to fluconazole. Conclusion: C. glabrata causes complicated, severe recurrent vulvovaginitis which is fluconazole resistant. Drug sensitivity prior prescribing antifungal agent identifies appropriate drug, decreases patient's disease morbidity and cross resistance.

Evaluation of ELISA BP180 and BP230 Autoantibodies in Blister Fluid and Serum in the Diagnosis of Bullous Pemphigoid.

Bullous pemphigoid (BP) is a chronic subepidermal immunobullous disorder. Studies have demonstrated the presence of antibasement membrane zone antibodies (BP180 & BP230) in the blister fluid using enzyme-linked immunosorbent assay (ELISA). To detect and compare BP 180 and BP 230 autoantibodies in the blister fluid and serum of patients with BP by ELISA method. A total of 30 patients diagnosed as BP and not on treatment were included in the study. Blister fluid and serum were subjected to ELISA, and the results were compared. The sensitivity of ELISA BP 180 was found to be 95.8% in the blister fluid and 88.4% in the serum. The sensitivity of ELISA BP 230 in the blister fluid and serum was 20% and 16.6%, respectively. Association between ELISA antibodies done in blister and serum was analysed using Chi-square test and found to be statistically significant with P value <0.05. Blister fluid is an effective alternative to the serum in detecting BP 180 and BP 230 antibodies, especially in uncooperative and elderly patients with poor venous access.

Discontinuation of PTH therapy amplifies bone loss by increasing oxidative stress: An event ameliorated by sequential IL-17 neutralizing antibody therapy.

Osteoporosis leads to excessive bone resorption which is not accompanied by equal amount of bone formation. PTH (1-34) forms the mainstay of bone anabolic therapy. Intermittent PTH (iPTH) has the ability to reconstruct skeleton, a property not shared by other anti-resorptives. In initial phases of PTH treatment, bone formation exceeds bone resorption. However, gradually this phase is replaced by increased bone resorption. Thus, a replacement post PTH discontinuation is much needed. Studies with bisphosphonates and Denosumab post PTH withdrawal have yielded promising but variable results. Thus, there is scope for trying new combinations. Our previous studies have shown the superior skeletal effects of neutralizing IL17 antibody (NIL17) over anti-RANKL antibody. Thus, here we investigated if sequential treatment of NIL17 after PTH withdrawal (SHIFT) could serve as a promising therapeutic approach for osteoporosis treatment. Our results show that PTH withdrawal (PTH-W) led to mitigation of its anabolic effects as evidenced by reduced BMD, bone trabecular and cortical microarchitectural parameters. In the continuous PTH (PTH-C) and the Shift group, all these parameters were preserved as par with the sham group. Shift therapy also significantly increased PINP levels. Most importantly, serum CTX-I levels and osteoclast numbers, which were elevated in PTH groups were significantly suppressed in NIL17 monotherapy and shift group. Also, expression of FOXO1 and ATF-4, the main regulators of redox balance and function in osteoblasts, were found to be enhanced maximally in the sequential therapy group. Our study thus advocates use of NIL17 as a replacement therapeutic option post PTH discontinuation.

Evaluation of the Role of BIOCHIP Mosaic Based Indirect Immunofluorescence and ELISA BP 180 and BP 230 Autoantibodies in the Diagnosis of Bullous Pemphigoid Patients.

Background: Bullous pemphigoid (BP) is characterized by tissue-bound and circulating Immunoglobulin G (IgG) autoantibodies against BP 180 and BP 230. Diagnosis of BP is a multi-step procedure. Enzyme-linked immunosorbent assay (ELISA) is a quantitative analysis of target antigens, whereas BIOCHIP mosaic-based indirect immunofluorescence (IIF) has a combination of screening and target antigen-specific substrates in a single miniature incubation field. This study is done to compare BIOCHIP mosaic based IIF and ELISA for the diagnosis of BP. Materials and Methods: A total of 42 biopsy and/or direct immunofluorescence (DIF) proven BP patients were included in the study. Serum was subjected to BIOCHIP mosaic-based IIF and ELISA. The results were then compared. Results: Using ELISA, the sensitivity of BP 180 and BP 230 was 92.3% and 54.5%, respectively. The sensitivity of BP 180 and BP 230 by BIOCHIP was 77.4% and 60%, respectively. The association between ELISA and BIOCHIP was analyzed using the Chi-square test and was found to be statistically significant with a P value ≤ 0.05. Conclusion: Our study concluded that both BIOCHIP and ELISA showed comparable sensitivity in diagnosing BP. Both are non-invasive, less time-consuming, and provide fast results. However, BIOCHIP can delineate bullous pemphigoid from other sub-epidermal bullous diseases.

Evaluation of the Role of Oral Mucosal Direct Immunofluorescence and Salivary Desmoglein 1 and 3 Enzyme-Linked Immunosorbent Assay in Patients With Oral Mucosal Pemphigus.

Background: Pemphigus vulgaris (PV) is characterized by antibodies against desmosomal adhesion proteins desmoglein (Dsg) 1 and 3 which can be detected by direct immunofluorescence (DIF) or enzyme-linked immunosorbent assay (ELISA). Oral lesions usually precede cutaneous lesions and an early diagnosis can prevent mortality and morbidity. Dsg antibodies can be detected by ELISA in saliva of patients with oral mucosal pemphigus. This study compares oral mucosal DIF with the salivary Dsg1 and 3 ELISA. Materials and Methods: A total of 26 biopsy and/or DIF-proven PV patients with oral erosions without cutaneous lesions were included in the study. Biopsy of oral mucosa was taken for DIF by standard method. Saliva sample was obtained and processed for ELISA. The results were then compared. Results: Out of 26 patients, 22 (84.6%) had a positive oral mucosal DIF and four patients (15.4%) had negative DIF. Nine patients (34%) had positive salivary Dsg3 ELISA. Seven patients (27%) had positive salivary Dsg1 ELISA. Taking oral DIF as the gold standard, the sensitivity of salivary Dsg1 ELISA was 31.8% and of salivary Dsg3 ELISA was 40.9%. Conclusion: Although DIF is the gold standard for the diagnosis of PV, salivary Dsg1 and 3 ELISA can also be used in the diagnosis of oral pemphigus.

Cladrin alleviates dexamethasone-induced apoptosis of osteoblasts and promotes bone formation through autophagy induction via AMPK/mTOR signaling.

Glucocorticoid-induced osteoporosis (GIOP) is a common clinical consequence that arises due to the extensive usage of glucocorticoids. Cladrin (Clad), a methoxylated isoflavone has been reported to have a bone protecting effect by enhancing osteoblast proliferation and differentiation. However, its consequences on GIOP are not reported yet. This study investigates whether Clad protects against the deleterious effects of Dexamethasone (Dex) on osteoblast and bone. Mice calvarial osteoblasts were treated with Clad and then exposed to Dex to study the effect on osteoblast differentiation, proliferation, and survival. Further, GIOP mice were treated with Clad (5 and 10 mg/kg) doses along with reference standard alendronate (ALN 3 mg/kg) for evaluation of bone protecting effect of Clad. We analyzed bone and vertebral microarchitecture, mechanical strength, and biochemical parameters. We observed that Clad at 10 nM concentration mitigated Dex-induced cytotoxicity and defend osteoblasts against apoptosis. Subsequent results demonstrate that Clad suppressed apoptosis of osteoblast in the presence of Dex by enhancing autophagy in a way that was reliant on the AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathway. Furthermore, micro-CT scanning, eco MRI results, and serum CTX levels revealed that 12 weeks of Clad treatment prevented bone loss and preserved trabecular bone mass in GIOP animals. We also observed that Clad treated osteoblasts had a lower rate of apoptosis and a greater LC3-II/LC3-I ratio than the Dex group. Our findings show that Clad can protect osteoblasts against glucocorticoids by inducing autophagy via the AMPK/mTOR pathway.

Comparative Analysis of BIOCHIP Mosaic-based Indirect Immunofluorescence with Direct Immunofluorescence in Diagnosis of Autoimmune Bullous Diseases: A Cross-Sectional Study.

BACKGROUND: Autoimmune bullous diseases (AIBD) are a heterogeneous group of diseases characterized by autoantibodies against desmosomal proteins in the pemphigus group of disorders and adhesion molecules of the dermal-epidermal junction in pemphigoid group of diseases. Direct immunofluorescence (DIF) establishes the diagnosis of AIBD by demonstrating intercellular deposits of IgG and C3 in case of pemphigus and linear deposits of IgG and C3 along the basement membrane zone (BMZ) in bullous pemphigoid (BP). BIOCHIP mosaic-based indirect immunofluorescence (IIF), a novel diagnostic approach employs detection of characteristic staining pattern and target antigens in a single miniature incubation field. AIM: To compare the BIOCHIP mosaic-based IIF with DIF in the diagnosis of AIBD. MATERIALS AND METHODS: A total of 40 patients of AIBD in the active phase of the disease were included in the study. Skin biopsy was done in these patients for DIF study and serum was subjected to BIOCHIP mosaic-based IIF assay. The results were then compared. RESULTS: DIF revealed a diagnosis of Pemphigus in 18 patients and BP in 22 patients. BIOCHIP showed a diagnosis of pemphigus in 18 patients, BP in 18 patients and floor pattern staining in four patients, which could be attributed to any of the floor pattern staining subepidermal blistering disease. LIMITATIONS: Small sample size, lack of control group and no comparison made with ELISA. CONCLUSION: This study concludes that the result of BIOCHIP shows correlation with the DIF and can be used as a first line-screening tool in the diagnosis of AIBD.

Octanoic acid a major component of widely consumed medium-chain triglyceride ketogenic diet is detrimental to bone.

Octanoic acid is a medium-chained saturated fatty acid found abundantly in the ketogenic dietary supplements containing medium chained triglycerides (MCT) along with decanoic acid. The MCT ketogenic diet is commonly consumed for weight loss but has also showcased neuroprotective potential against neurodegenerative disorders. However, recent clinical findings have reported a critical disadvantage with the long-term consumption of ketogenic diet i.e. bone loss. The following study was employed to investigate whether the two major components of MCT diet also possess bone loss potential as observed with classical ketogenic diet. Swiss albino mice aged between 10 and 12 weeks, were divided into 3 treatment groups that were administered with oral suspensions of octanoic acid, decanoic acid and a combination of both for 4 weeks. Bone specific markers, microarchitectural parameters, using micro computed tomography, and biomechanical strength were analyzed. Remarkably deleterious alterations in the trabecular bone microarchitecture, and on bone markers were observed in the octanoic acid treated groups. Our results suggest significant negative effects on bone health by octanoic acid. These findings require further investigation and validation in order to provide significant clinically relevant data to possibly modify dietary composition of the MCT ketogenic diet.

Unusual Presentation of Trichoblastoma.

We report a 39-year-old man who presented with skin-colored plaque over the glabella and root of the nose. Histopathology revealed the diagnosis of trichoblastoma. This case is reported to emphasize the rare presentation of trichoblastoma as it usually presents as an isolated nodule.

Anti-P 200 pemphigoid - The most common floor binding subepidermal autoimmune bullous disease in a tertiary care center in south India.

BACKGROUND: The pemphigoid group of diseases may present clinically and immunologically in a very similar fashion. Indirect immunofluorescence microscopy with readily available salt-split human skin in a BIOCHIP™ helps to classify these conditions as those with either with roof binding or floor binding of immunoreactants. Epidermolysis bullosa acquisita, anti-laminin 332 pemphigoid and anti-p200 pemphigoid show floor binding, while in the most frequent type of pemphigoid disease, bullous pemphigoid, epidermal side staining pattern is seen on salt-split skin Aims: The aim of the study was to detect the target antigens in sub-epidermal bullous diseases. METHODS: Forty patients with bullous pemphigoid diagnosed by lesional histopathology and direct immunofluorescence microscopy were re-evaluated by a BIOCHIP™ mosaic containing both tissue substrates and recombinant target antigens. Sera with floor pattern staining on salt-split skin were further evaluated by immunoblotting with dermal extract. RESULTS: Five patients with floor staining had anti-p200 pemphigoid. LIMITATIONS: We could not perform serration pattern analysis of direct immunofluorescence in our patients. CONCLUSION: Histopathology and direct immunofluorescence microscopy cannot differentiate between various entities of pemphigoid diseases. A multivariant approach using a BIOCHIP™ mosaic including salt-split skin followed by immunoblotting with dermal extract helps to identify the target antigen.