Isolation and Characterisation of a Recombinant Antibody Fragment That Binds NCAM1-Expressing Intervertebral Disc Cells

Degeneration of the intervertebral discs (IVD) is a leading cause of neck and low back pain. Degeneration begins in the central nucleus pulposus region, leading to loss of IVD osmotic properties. Regeneration approaches include administration of matrix-mimicking scaffolds, cells and/or therapeutic factors. Cell-targeting strategies are likely to improve delivery due to the low cell numbers in the IVD. Single-chain antibody fragments (scFvs) that bind IVD cells were isolated for potential delivery of therapeutics to degenerated IVD. The most cell-distal domain of neural cell adhesion molecule 1 (NCAM1) was cloned and expressed in Escherichia coli. Phage display technology was used to isolate a human scFv against the recombinant domain by panning a scFv library on the immobilised protein. The isolated scFv bound cultured rat astrocytes, as well as bovine nucleus pulposus and annulus fibrosus cells in immunocytochemical studies. The scFv also labelled cells in bovine spinal cord and six-month and two-year old bovine IVD sections by immunohistochemistry. Antibody fragments can provide cell-binding moieties at improved cost, time, yield and functionalisation potential over whole antibodies. The described scFv has potential application in delivery of therapeutics to NCAM1-expressing cells in degenerated IVD.     

Dietary fish oil associated with increased apoptosis and modulated expression of Bax and Bcl-2 during 7,12-dimethylbenz(alpha)anthracene-induced mammary carcinogenesis in rats

The present study investigated the chemopreventive effect of dietary fish oil (Maxepa), rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on induction of apoptosis in mammary carcinogenesis model. Mammary carcinogenesis was initiated by a single, tail vein injection of 7,12-dimethylbenz(alpha)anthracene (DMBA) (0.5mg/0.2ml corn oil/100g body weight) at 7 weeks of animal age. Ninety female Sprague-Dawley rats were divided into two parts: part one was used for histology and immunohistochemical study and part two for morphological analysis. Each part consists of three experimental groups having 15 animals, i.e., Group A (DMBA control), Group B (DMBA+fish oil) and Group C (DMBA+corn oil). Rats were fed either fish oil or corn oil (0.5ml/day/rat) by oral gavage, 2 weeks prior to DMBA injection. Treatment was continued 25 weeks, studying histopathology, expression of Bax and Bcl-2 proteins by immunohistochemistry and apoptosis by TUNEL assay and morphological study at 36 weeks. Results showed that the fish oil-treated group exhibited a substantial increase in Bax (p<0.05) immunolabelling and a reduction of Bcl-2 immunopositivity (p<0.05), and increased TUNEL-positive apoptotic cells (p<0.05); however, corn oil treatment did not show these beneficial effects toward mammary preneoplasia. We conclude that fish oil has the potential to play a significant role in limiting mammary tumourigenesis in vivo.     

Response of the chromatophores in relation to the healing of skin wounds in an Indian Major Carp, Labeo rohita (Hamilton)

Chromatophores show significant changes during healing of skin wounds in Labeo rohita (Common Name - Rohu). Wound area can be divided into regions I, II and III. After infliction of wound, skin colour becomes significantly dark by 2 h that is gradually restored by 2 d. In regions II and III at 5 min, epidermal melanophores appear with beaded dendrites. In these regions at 2 h and in region I at 6 h, epidermal melanophores appear small, rounded or irregular shaped having dendritic processes with aggregated melanosomes. Subsequently, melanophores appear having elongated dendrites with dispersed or aggregated melanosomes. At 24 h, clusters of pigmented bodies appear in regions I and II. These bodies increase up to 2 d, and then diminish gradually and disappear by 8 d. Changes in dermal melanophores in region II at 5 min indicate the onset of degeneration. Degenerating melanophores increase up to 12 h, then gradually decline, and disappear by 4 d. Simultaneously, stellate melanophore reappear, gradually increase and appear like control by 8 d. Dermal melanophores in region III at different intervals appear stellate. In region I stellate dermal melanophores appear at 4 d. Stellate melanophores in all regions show different distribution of dispersed or aggregated melanosomes. With the appearance of dermal melanophores, highly refractive, crystalline structures, possibly the refractive platelets of the iridophores, are visualized around them. At subsequent intervals, these are frequently observed. This study provides interesting insights in injury induced changes in chromatophores in fish. The findings could be considered useful in perception of intriguing features in the development of pigment research in future.     

Nickel and Ultraviolet-B Stresses Induce Differential Growth and Photosynthetic Responses in Pisum sativum L. Seedlings

Enhanced level of UV-B radiation and heavy metals in irrigated soils due to anthropogenic activities are deteriorating the environmental conditions necessary for growth and development of plants. The present study was undertaken to study the individual and interactive effects of heavy metal nickel (NiCl(2)·6H(2)O; 0.01, 0.1, 1.0?mM) and UV-B exposure (0.4 W m(-2); 45?min corresponds to 1.08 KJ m(-2)) on growth performance and photosynthetic activity of pea (Pisum sativum L.) seedlings. Ni treatment at high doses (0.1 and 1.0?mM Ni) and UV-B alone reduced chlorophyll content and photosynthetic activity (oxygen yield, carbon fixation, photorespiration, and PSI, PSII, and whole chain electron transport activities), and declining trends continued with combined doses. In contrast to this, Ni at 0.01?mM appeared to be stimulatory for photosynthetic pigments and photosynthetic activity, thereby enhanced biomass was observed at this concentration. However, combined dose (UV-B + 0.01?mM Ni) caused inhibitory effects. Carotenoids showed different responses to each stress. Nickel at high doses strongly inhibited PSII activity and the inhibition was further intensified when chloroplasts were simultaneously exposed to UV-B radiation. PSI activity appeared to be more resistant to each stress. High doses of Ni (0.1and 1.0?mM) and UV-B alone interrupted electron flow at the oxygen evolving complex. Similar damaging effects were caused by 0.01 and 0.1?mM Ni together with UV-B, but the damage extended to PSII reaction center in case of 1.0?mM Ni in combination with UV-B. In conclusion, the results demonstrate that low dose of Ni stimulated the growth performance of pea seedlings in contrast to its inhibitory role at high doses. However, UV-B alone and together with low as well as high doses of Ni proved to be toxic for P. sativum L.     

Uveal melanoma cell-based vaccines express MHC II molecules that traffic via the endocytic and secretory pathways and activate CD8+ cytotoxic, tumor-specific T cells

We are exploring cell-based vaccines as a treatment for the 50% of patients with large primary uveal melanomas who develop lethal metastatic disease. MHC II uveal melanoma vaccines are MHC class I⁺ uveal melanoma cells transduced with CD80 genes and MHC II genes syngeneic to the recipient. Previous studies demonstrated that the vaccines activate tumor-specific CD4⁺ T cells from patients with metastatic uveal melanoma. We have hypothesized that vaccine potency is due to the absence of the MHC II-associated invariant chain (Ii). In the absence of Ii, newly synthesized MHC II molecules traffic intracellularly via a non-traditional pathway where they encounter and bind novel tumor peptides. Using confocal microscopy, we now confirm this hypothesis and demonstrate that MHC II molecules are present in both the endosomal and secretory pathways in vaccine cells. We also demonstrate that uveal melanoma MHC II vaccines activate uveal melanoma-specific, cytolytic CD8⁺ T cells that do not lyse normal fibroblasts or other tumor cells. Surprisingly, the CD8⁺ T cells are cytolytic for HLA-A syngeneic and MHC I-mismatched uveal melanomas. Collectively, these studies demonstrate that MHC II uveal melanoma vaccines are potent activators of tumor-specific CD4⁺ and CD8⁺ T cells and suggest that the non-conventional intracellular trafficking pattern of MHC II may contribute to their enhanced immunogenicity. Since MHC I compatibility is unnecessary for the activation of cytolytic CD8⁺ T cells, the vaccines could be used in uveal melanoma patients without regard to MHC I genotype.     

Antidiabetic Property of Symplocos cochinchinensis Is Mediated by Inhibition of Alpha Glucosidase and Enhanced Insulin Sensitivity

The study is designed to find out the biochemical basis of antidiabetic property of Symplocos cochinchinensis (SC), the main ingredient of ‘Nisakathakadi’ an Ayurvedic decoction for diabetes. Since diabetes is a multifactorial disease, ethanolic extract of the bark (SCE) and its fractions (hexane, dichloromethane, ethyl acetate and 90% ethanol) were evaluated by in vitro methods against multiple targets relevant to diabetes such as the alpha glucosidase inhibition, glucose uptake, adipogenic potential, oxidative stress, pancreatic beta cell proliferation, inhibition of protein glycation, protein tyrosine phosphatase-1B (PTP-1B) and dipeptidyl peptidase-IV (DPP-IV). Among the extracts, SCE exhibited comparatively better activity like alpha glucosidase inhibition (IC₅₀ value-82.07±2.10 µg/mL), insulin dependent glucose uptake (3 fold increase) in L6 myotubes, pancreatic beta cell regeneration in RIN-m5F (3.5 fold increase) and reduced triglyceride accumulation (22% decrease) in 3T3L1 cells, protection from hyperglycemia induced generation of reactive oxygen species in HepG2 cells (59.57% decrease) with moderate antiglycation and PTP-1B inhibition. Chemical characterization by HPLC revealed the superiority of SCE over other extracts due to presence and quantity of bioactives (beta-sitosterol, phloretin 2′glucoside, oleanolic acid) in addition to minerals like magnesium, calcium, potassium, sodium, zinc and manganese. So SCE has been subjected to oral sucrose tolerance test to evaluate its antihyperglycemic property in mild diabetic and diabetic animal models. SCE showed significant antihyperglycemic activity in in vivo diabetic models. We conclude that SC mediates the antidiabetic activity mainly via alpha glucosidase inhibition, improved insulin sensitivity, with moderate antiglycation and antioxidant activity.