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.

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.

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.

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.

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 showed a significant correlation with the DIF and can be used as a first line-screening tool in the diagnosis of AIBD.

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.

Efficacy of Topical Timolol versus Saline in Chronic Venous Ulcers: A Randomized Controlled Trial.

Background: Chronic venous ulcers sometimes fail to heal in spite of adequate treatment. Nonhealing is possibly because of chronic activation of ß-2 adrenergic receptor (B2-AR) in the keratinocytes by endogenously generated catecholamines, which inhibits keratinocyte migration. Blocking of B2-AR using ß-blockers has been reported to promote wound healing through keratinocyte migration, angiogenesis, and fibroblast migration. Topical timolol, a B2-AR antagonist, is used to promote wound healing. Aims and Objectives: The aim of this study was to compare the efficacy of topical timolol versus saline in chronic venous leg ulcers and to compare the mean reduction in ulcer area at the end of 4 weeks. Materials and Methods: Twenty patients were randomized into two groups. Patients in Group 1 were treated with one drop of 0.5% topical timolol every alternate days for 4 weeks. Ulcer margins were measured and ulcer area calculated every week for 4 weeks. Similar dressing and measurement were carried out for patients with saline for 4 weeks. Healing rate was assessed by the percentage of reduction of ulcer area of both groups at week 4. Results: The mean reduction in the ulcer size in timolol group was 86.80%, and in saline group 43.82% at the end of 4 weeks. Conclusion: Topical timolol is an easy, noninvasive therapy that can be recommended for chronic ulcer.

Comparison of Efficacy of Autologous Platelet-rich Fibrin versus Saline Dressing in Chronic Venous Leg Ulcers: A Randomised Controlled Trial.

BACKGROUND: Venous leg ulcer is a chronic condition, and various treatment modalities are available. Platelet-rich fibrin (PRF) is one of the newer modalities and it contains fibroblast growth factor (GF), vascular endothelial GF, angiopoitin and platelet-derived GF which enhances the wound healing. Hence, we conducted a randomised controlled trial to compare the efficacy of PRF versus saline dressing in chronic venous leg ulcers. AIM: This study aims to compare the efficacy of autologous PRF with saline dressing in patients with chronic venous leg ulcer and to compare the mean reduction in ulcer area at the end of 4 weeks. MATERIALS AND METHODS: Fifteen patients with chronic venous leg ulcers of >6 months duration having an ulcer area of 1 cm × 1 cm to 5 cm × 5 cm were taken into the study and were randomly divided into two groups. Group 1: Patients received PRF dressing. Ten millilitres of patient's blood was taken and centrifuged at 3000 rpm for 15 min. A fibrin clot obtained in the middle of the tube was removed and used for dressing over the wound surface. It was repeated every week for 4 weeks. Group 2: Patients received saline dressings once a week for 4 weeks. The assessment of the ulcer size was done with the help of photographs, and ulcer area was measured. RESULTS: The mean reduction in the area of the ulcer size in PRF group was 85.51%, and the mean reduction in the area of the ulcer size in Saline group was 42.74% which was statistically significant with a P < 0.001 and t = 4.11. CONCLUSION: We conclude that PRF dressing can be used as it is effective, inexpensive, safe and an outpatient procedure.

Modulating hydrogel crosslink density and degradation to control bone morphogenetic protein delivery and in vivo bone formation.

Bone morphogenetic proteins (BMPs) show promise in therapies for improving bone formation after injury; however, the high supraphysiological concentrations required for desired osteoinductive effects, off-target concerns, costs, and patient variability have limited the use of BMP-based therapeutics. To better understand the role of biomaterial design in BMP delivery, a matrix metalloprotease (MMP)-sensitive hyaluronic acid (HA)-based hydrogel was used for BMP-2 delivery to evaluate the influence of hydrogel degradation rate on bone repair in vivo. Specifically, maleimide-modified HA (MaHA) macromers were crosslinked with difunctional MMP-sensitive peptides to permit protease-mediated hydrogel degradation and growth factor release. The compressive, rheological, and degradation properties of MaHA hydrogels were characterized as a function of crosslink density, which was varied through either MaHA concentration (1-5wt.%) or maleimide functionalization (10-40%f). Generally, the compressive moduli increased, the time to gelation decreased, and the degradation rate decreased with increasing crosslink density. Furthermore, BMP-2 release increased with either a decrease in the initial crosslink density or an increase in collagenase concentration (non-specific MMP degradation). Lastly, two hydrogel formulations with distinct BMP-2 release profiles were evaluated in a critical-sized calvarial defect model in rats. After six weeks, minimal evidence of bone repair was observed within defects left empty or filled with hydrogels alone. For hydrogels that contained BMP-2, similar volumes of new bone tissue were formed; however, the faster degrading hydrogel exhibited improved cellular invasion, bone volume to total volume ratio, and overall defect filling. These results illustrate the importance of coordinating hydrogel degradation with the rate of new tissue formation.

Immunotherapy with injectable hydrogels to treat obstructive nephropathy.

Hydrogels are gaining attention as injectable vehicles for delivery of therapeutics for a range of applications. We describe self-assembling and injectable Dock-and-Lock hydrogels for local delivery of interleukin-10 (IL-10) to abate the progression of inflammation and fibrosis that leads to chronic kidney disease. As monitored with a fluorescent tag, hydrogels degraded within a few days in vitro and matched IL-10 release profiles; however, hydrogels remained in the kidney for up to 30 days in vivo. A unilateral ureteral obstruction (UUO) mouse model was used to investigate in vivo outcomes after hydrogel injection and IL-10 delivery. Eight groups were investigated (7, 21, 35 days, n = 4): healthy, sham, healthy injected with mouse serum albumin (MSA), healthy + hydrogel, UUO, UUO + IL-10, UUO + hydrogel, UUO + hydrogel/IL-10. 15 µL of IL-10, hydrogel, or hydrogel/IL-10 was injected under the renal capsule 3 days after the UUO. Immunohistochemistry (IHC) was performed on paraffin sections to identify macrophages and apoptotic cells and trichrome staining was used to evaluate fibrosis. There were no significant differences in inflammatory markers between all control groups. With hydrogel delivery, macrophage infiltration and apoptosis were significantly reduced at days 21 and 35 compared to untreated animals. By day 35, IL-10 delivery via hydrogel reduced macrophage infiltration and apoptosis more than IL-10 injection alone. Fibrosis was decreased by day 35 in all treatment groups. This work supports the use of hydrogel delivery of IL-10 to treat chronic kidney disease.

Amyloidosis cutis dyschromica.

Amyloidosis cutis dyschromica is a very rare form of primary cutaneous amyloidosis characterized by prepubertal onset of hyper and hypopigmented spots and amyloid deposits in the papillary dermis. We report a case of a 26 year old female with amyloidosis cutis dyschromica who presented with dyschromic skin since birth.

Laser and light based treatments of acne.

Medical treatments for acne vulgaris include a variety of topical and oral medications. Poor compliance, lack of durable remission, and potential side effects are common drawbacks to these treatments. Therefore, there is a growing demand for a fast, safe, and side-effect-free novel therapy. Acne often improves after exposure to sunlight, and this has led to the development of laser and other light therapies resulting in the overall ease of treatment, with minimal adverse effects. A variety of light and laser devices has been used for the treatment of acne, including the potassium titanyl phosphate laser, the 585- and 595-nm pulsed dye lasers, the 1450-nm diode laser, radiofrequency devices, intense pulsed light sources, and photodynamic therapy using 5-aminolevulinic acid and indocyanine green. These devices are thought to target the underlying pathogenic factors such as propionibacterium acnes colonization, increased sebaceous gland activity, and the cutaneous inflammatory response. In this article, we review the current status of light- and laser-based treatment of acne.

The influence of hyaluronic acid hydrogel crosslinking density and macromolecular diffusivity on human MSC chondrogenesis and hypertrophy.

Hyaluronic acid (HA) hydrogels formed via photocrosslinking provide stable 3D hydrogel environments that support the chondrogenesis of mesenchymal stem cells (MSCs). Crosslinking density has a significant impact on the physical properties of hydrogels, including their mechanical stiffness and macromolecular diffusivity. Variations in the HA hydrogel crosslinking density can be obtained by either changes in the HA macromer concentration (1, 3, or 5% w/v at 15 min exposure) or the extent of reaction through light exposure time (5% w/v at 5, 10, or 15 min). In this work, increased crosslinking by either method resulted in an overall decrease in cartilage matrix content and more restricted matrix distribution. Increased crosslinking also promoted hypertrophic differentiation of the chondrogenically induced MSCs, resulting in more matrix calcification in vitro. For example, type X collagen expression in the high crosslinking density 5% 15 min group was ~156 and 285% higher when compared to the low crosslinking density 1% 15 min and 5% 5 min groups on day 42, respectively. Supplementation with inhibitors of the small GTPase pathway involved in cytoskeletal tension or myosin II had no effect on hypertrophic differentiation and matrix calcification, indicating that the differential response is unlikely to be related to force-sensing mechanotransduction mechanisms. When implanted subcutaneously in nude mice, higher crosslinking density again resulted in reduced cartilage matrix content, restricted matrix distribution, and increased matrix calcification. This study demonstrates that hydrogel properties mediated through alterations in crosslinking density must be considered in the context of the hypertrophic differentiation of chondrogenically induced MSCs.

Enhanced MSC chondrogenesis following delivery of TGF-ß3 from alginate microspheres within hyaluronic acid hydrogels in vitro and in vivo.

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair and members of the transforming growth factor-beta (TGF-ß) superfamily are a key mediator of MSC chondrogenesis. While TGF-ß mediated MSC chondrogenesis is well established in in vitro pellet or hydrogel cultures, clinical translation will require effective delivery of TGF-ßs in vivo. Here, we investigated the co-encapsulation of TGF-ß3 containing alginate microspheres with human MSCs in hyaluronic acid (HA) hydrogels towards the development of implantable constructs for cartilage repair. TGF-ß3 encapsulated in alginate microspheres with nanofilm coatings showed significantly reduced initial burst release compared to uncoated microspheres, with release times extending up to 6 days. HA hydrogel constructs seeded with MSCs and TGF-ß3 containing microspheres developed comparable mechanical properties and cartilage matrix content compared to constructs supplemented with TGF-ß3 continuously in culture media, whereas constructs with TGF-ß3 directly encapsulated in the gels without microspheres had inferior properties. When implanted subcutaneously in nude mice, constructs containing TGF-ß3 microspheres resulted in superior cartilage matrix formation when compared to groups without TGF-ß3 or with TGF-ß3 added directly to the gel. However, calcification was observed in implanted constructs after 8 weeks of subcutaneous implantation. To prevent this, the co-delivery of parathyroid hormone-related protein (PTHrP) with TGF-ß3 in alginate microspheres was pursued, resulting in partially reduced calcification. This study demonstrates that the controlled local delivery of TGF-ß3 is essential to neocartilage formation by MSCs and that further optimization is needed to avert the differentiation of chondrogenically induced MSCs towards a hypertrophic phenotype.

Arginyltransferase is an ATP-independent self-regulating enzyme that forms distinct functional complexes in vivo.

Posttranslational arginylation mediated by arginyl transferase (ATE1) plays an important role in cardiovascular development, cell motility, and regulation of cytoskeleton and metabolic enzymes. This protein modification was discovered decades ago, however, the arginylation reaction and the functioning of ATE1 remained poorly understood because of the lack of good biochemical models. Here, we report the development of an in vitro arginylation system, in which ATE1 function and molecular requirements can be tested using purified recombinant ATE1 isoforms supplemented with a controlled number of components. Our results show that arginylation reaction is a self-sufficient, ATP-independent process that can affect different sites in a polypeptide and that arginyl transferases form different molecular complexes in vivo, associate with components of the translation machinery, and have distinct, partially overlapping subsets of substrates, suggesting that these enzymes play different physiological functions.

Extra ocular sebaceous carcinoma: A rare case report.

Extra ocular Sebaceous Carcinoma is a rare malignancy when compared to Peri ocular variant and these are derived from sebaceous gland epithelium. The aggressive types of extra ocular sebaceous neoplasm are reported with lymph node and visceral metastasis associated with poor prognosis. Here we report a case of extensive cutaneous extra ocular sebaceous cell carcinoma confined to large area of scalp proven by Immunohistochemistry without intra cranial involvement, distant metastases or evidence of Muir-Torre syndrome.