Isolation and characterization of cytotoxic and insulin-releasing components from the venom of the black-necked spitting cobra Naja nigricollis (Elapidae)

Four peptides with cytotoxic activity against BRIN-BD11 rat clonal β-cells were purified from the venom of the black-necked spitting cobra Naja nigricollis using reversed-phase HPLC. The peptides were identified as members of the three-finger superfamily of snake toxins by ESI-MS/MS sequencing of tryptic peptides. The most potent peptide (cytotoxin-1N) showed strong cytotoxic activity against three human tumour-derived cell lines (LC50 = 0.8 ± 0. 2 µM for A549 non-small cell lung adenocarcinoma cells; LC50 = 7 ± 1 µM for MDA-MB-231 breast adenocarcinoma cells; and LC50 = 9 ± 1 µM for HT-29 colorectal adenocarcinoma cells). However, all the peptides were to varying degrees cytotoxic against HUVEC human umbilical vein endothelial cells (LC50 in the range 2-22 µM) and cytotoxin-2N was moderately hemolytic (LC50 = 45 ± 3 µM against mouse erythrocytes). The lack of differential activity against cells derived from non-neoplastic tissue limits their potential for development into anti-cancer agents. In addition, two proteins in the venom, identified as isoforms of phospholipase A2, effectively stimulated insulin release from BRIN-BD11 cells (an approximately 6-fold increase in rate compared with 5.6 mM glucose alone) at a concentration (1 µM) that was not cytotoxic to the cells suggesting possible application in therapy for Type 2 diabetes

Conformational analysis and in vitro immunomodulatory and insulinotropic properties of the frog skin host-defense peptide rhinophrynin-27 and selected analogs

The study investigates conformational analysis and the in vitro cytokine-mediated immunomodulatory and insulin-releasing activities of rhinophrynin-27 (ELRLPEIARPVPEVLPARLPLPALPRN; RP-27), a proline-arginine-rich peptide first isolated from skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae). In both water and 50% trifluoroethanol-water, the peptide adopts a polyproline type II helical conformation with a high degree of deviation from the canonical collagen-like folding and a pronounced bend in the molecule at the Glu13 residue. Incubation of mouse peritoneal cells with RP-27 significantly (P < 0.05) inhibited production of the pro-inflammatory cytokines TNF-α and IL-1β and stimulated production of the anti-inflammatory cytokine IL-10. The peptide significantly (P < 0.01) stimulated release of insulin from BRIN-BD11 rat clonal β-cells at concentrations ≥ 1 nM while maintaining the integrity of the plasma membrane and also stimulated insulin release from isolated mouse islets at a concentration of 10−6 M. Increasing the cationicity of RP-27 by substituting glutamic acid residues in the peptide by arginine and increasing hydrophobicity by substituting alanine residues by tryptophan did not result in analogues with increased activity with respect to cytokine production and insulin release. The combination of immunosuppressive and insulinotropic activities together with very low cytotoxicity suggests that RP-27 may represent a template for the development of an agent for use in anti-inflammatory and Type 2 diabetes therapies

Evaluation of neck masses by CT or MRI

Aim: To evaluate the role of CT or MRI in neck masses for pre-operative characterization based on location, extent, morphological characteristics and enhancement pattern. Materials & methods: The present study was conducted in the department of   Radiodiagnosis in co-ordination with the departments of E.N.T and Pathology at K.E.M. Hospital, Mumbai. A total of 50 (25 each of CT and MRI) patients with   palpable neck masses were included. Results: The study comprised of nodal and non-nodal masses. Out of 50 cases studied, 23 cases (46%) had benign lesions and 27 (54%) cases had malignant lesions. The overall male to female ratio was 0.85:1. MRI and CT made a correct diagnosis in 48 out of 50 cases, having a diagnostic accuracy of 96%. For evaluation of neck masses by CT or MRI, the sensitivity, specificity, positive predictive value and negative predictive value are 96.30%, 95.65%, 96.30% and 95.65% respectively. Conclusion: CT and MRI ensure accurate anatomical localization and lesion characterization in benign lesions. MRI has obvious advantages over CT in neck imaging like better soft tissue resolution, lack of ionizing radiation and safer contrast agents.&nbsp

PEGPH20 Attenuates Hyaluronan-CD44 Mediated Renal Injury in Obesity Related Glomerulopathy

Background and aims: Obesity is considered as one of the biggest public health threats worldwide and increases the risk of incident chronic kidney disease. Hyaluronan (HA) is a major component of the extracellular matrix (ECM). We hypothesised that HA content was increased in the ECM of Obesity-Related Glomerulopathy (ORG) and PEGPH20, human recombinant PEGylated hyaluronidase PH-20, reversed high-fat (HF) diet-induced HA accumulation and renal dysfunction. We further investigated whether its renal protective effect was dependent upon the main HA receptor, CD44. Materials and methods: C57BL/6 mice, global CD44-deficient (cd44-/-) mice, and their wildtype littermate controls (cd44+/+) were fed a chow diet or HF diet for 16 weeks. HF diet-fed mice received injections of either vehicle or PEGPH20 once every 3 days for 24 days. After treatment, mice were euthanized and tissue and blood samples were collected. CD44 expression was detected by Western Blotting. PAS staining was used to observe renal pathology. HA accumulation and α-SMA expression were assessed by histochemistry. Collagen deposition was measured by picrosirius red staining and renal function was assessed by a creatinine assay kit. Results: HF diet feeding in C57BL/6 mice led to an increase in CD44 expression, HA accumulation, glomerular area and tubular injury score in the renal tissue, as well as an increase in serum creatinine levels. PEGPH20 treatment ameliorated some of these adverse effects including reducing renal CD44 expression (3.02 ± 0.33 (Vehicle HF) vs 1.49 ± 0.16 (PEGPH20 HF) fold increase relative to chow-fed mice, p =0.0092), HA accumulation (31.39 ± 3.48 vs 13.58 ± 1.81 %, p =0.0078), tubular injury score (3.28 ± 0.15 vs 1.39 ± 0.06, p =0.0003) and serum creatinine levels (1.40 ± 0.09 vs 0.79 ± 0.07 mg/dl, p =0.0002),without affecting glomerular area. Furthermore, PEGPH20-treated obese mice had reduced renal fibrosis evidenced by decreased α-SMA expression (5.18 ± 0.17 vs 1.21 ± 0.11 %, p =0.0001) and collagen deposition (9.76 ± 0.76 vs 6.30 ± 0.23 %, p =0.0164) compared with obese mice receiving vehicle. Global deletion of cd44 gene in HF-fed mice significantly reduced renal CD44 expression (1.00 ± 0.05 (cd44+/+ HF) vs 0.13 ± 0.02 (cd44-/- HF) fold change, p =0.0001), HA accumulation (24.44 ± 3.33 vs 6.60 ± 0.58 %, p =0.0001) and tubular injury score (3.28 ± 0.20 vs 1.89 ± 0.06, p =0.0001), as well as reduced α-SMA expression (5.02 ± 0.16 vs 0.43 ± 0.06 %, p =0.0001) and collagen deposition (9.48 ± 0.29 vs 6.08 ± 0.73 %, p =0.0063). PEGPH20 caused further reductions in serum creatinine levels, in HF-fed cd44-/- mice (0.67 ± 0.08 mg/dl) when compared with HF-fed cd44 +/+ mice (1.63 ± 0.05 mg/dl, p =0.0001). Conclusion: PEGPH20 treatment and global cd44 deletion suppressed HA accumulation during ORG, which prevented obesity-associated renal fibrosis and dysfunction. Our results demonstrate that HA plays an important role in the pathogenesis of ORG, perhaps in part through binding to receptor CD44. Supported by: BHF Project Grant PG/18/56/33935, BX Qi is supported by a scholarship from CSC Disclosure: B. Qi: None

PEGPH20 Attenuates Hyaluronan-CD44 Mediated Renal Injury in Obesity Related Glomerulopathy

Background and aims: Obesity is considered as one of the biggest public health threats worldwide and increases the risk of incident chronic kidney disease. Hyaluronan (HA) is a major component of the extracellular matrix (ECM). We hypothesised that HA content was increased in the ECM of Obesity-Related Glomerulopathy (ORG) and PEGPH20, human recombinant PEGylated hyaluronidase PH-20, reversed high-fat (HF) diet-induced HA accumulation and renal dysfunction. We further investigated whether its renal protective effect was dependent upon the main HA receptor, CD44. Materials and methods: C57BL/6 mice, global CD44-deficient (cd44-/-) mice, and their wildtype littermate controls (cd44+/+) were fed a chow diet or HF diet for 16 weeks. HF diet-fed mice received injections of either vehicle or PEGPH20 once every 3 days for 24 days. After treatment, mice were euthanized and tissue and blood samples were collected. CD44 expression was detected by Western Blotting. PAS staining was used to observe renal pathology. HA accumulation and α-SMA expression were assessed by histochemistry. Collagen deposition was measured by picrosirius red staining and renal function was assessed by a creatinine assay kit. Results: HF diet feeding in C57BL/6 mice led to an increase in CD44 expression, HA accumulation, glomerular area and tubular injury score in the renal tissue, as well as an increase in serum creatinine levels. PEGPH20 treatment ameliorated some of these adverse effects including reducing renal CD44 expression (3.02 ± 0.33 (Vehicle HF) vs 1.49 ± 0.16 (PEGPH20 HF) fold increase relative to chow-fed mice, p =0.0092), HA accumulation (31.39 ± 3.48 vs 13.58 ± 1.81 %, p =0.0078), tubular injury score (3.28 ± 0.15 vs 1.39 ± 0.06, p =0.0003) and serum creatinine levels (1.40 ± 0.09 vs 0.79 ± 0.07 mg/dl, p =0.0002),without affecting glomerular area. Furthermore, PEGPH20-treated obese mice had reduced renal fibrosis evidenced by decreased α-SMA expression (5.18 ± 0.17 vs 1.21 ± 0.11 %, p =0.0001) and collagen deposition (9.76 ± 0.76 vs 6.30 ± 0.23 %, p =0.0164) compared with obese mice receiving vehicle. Global deletion of cd44 gene in HF-fed mice significantly reduced renal CD44 expression (1.00 ± 0.05 (cd44+/+ HF) vs 0.13 ± 0.02 (cd44-/- HF) fold change, p =0.0001), HA accumulation (24.44 ± 3.33 vs 6.60 ± 0.58 %, p =0.0001) and tubular injury score (3.28 ± 0.20 vs 1.89 ± 0.06, p =0.0001), as well as reduced α-SMA expression (5.02 ± 0.16 vs 0.43 ± 0.06 %, p =0.0001) and collagen deposition (9.48 ± 0.29 vs 6.08 ± 0.73 %, p =0.0063). PEGPH20 caused further reductions in serum creatinine levels, in HF-fed cd44-/- mice (0.67 ± 0.08 mg/dl) when compared with HF-fed cd44 +/+ mice (1.63 ± 0.05 mg/dl, p =0.0001). Conclusion: PEGPH20 treatment and global cd44 deletion suppressed HA accumulation during ORG, which prevented obesity-associated renal fibrosis and dysfunction. Our results demonstrate that HA plays an important role in the pathogenesis of ORG, perhaps in part through binding to receptor CD44. Supported by: BHF Project Grant PG/18/56/33935, BX Qi is supported by a scholarship from CSC Disclosure: B. Qi: None