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Widespread cerebral deposition of a 40-42 amino acid peptide called amyloid beta peptide (A beta) in the form of amyloid fibrils is one of the most prominent neuropathologic features of Alzheimer's disease (AD). The clinical study provides evidence that accumulation of protofibrils due to the Arctic mutation (E22G) causes early AD onset. Melatonin showed beneficial effects in an AD mouse model. Mice were divided into four different groups (n=8 per group): (i) control group, (ii) scrambled A beta-injected group, (iii) A beta protofibril-injected group and (iv) melatonin-treated group. A single dose of (5 microg) A beta protofibril was administered to the A beta protofibril-injected and melatonin-treated groups via intracerebroventricular injections. The results demonstrate that melatonin treatment significantly reduces A beta protofibril-induced reactive oxygen species (ROS) production, intracellular calcium levels and acetylcholinesterase activity in the neocortex and hippocampus regions. Based on these findings it is suggested that melatonin therapy might be a useful treatment for AD patients.

Oral hepatitis B vaccine formulation was prepared by successful encapsulation of immunogenic peptide representing residues 127–145 of the immunodominant B-cell epitope of hepatitis B surface antigen (HBsAg) in poly( d , l -lactide co-glycolide) (PLG) microparticles. The smooth, spherical PLG microparticles with a diameter of around 10 μm was prepared by using W/O/W double emulsion solvent evaporation method. The entrapment efficiency of B-cell epitope peptide (BCEP) into PLG microparticles was 64%. In vitro studies showed B-cell epitope loaded PLG microparticles (BCEM) released the peptide in sustained profile and reached 64.9% efficiency by Day 25. Single oral immunization of mice with BCEM led to the significant induction of specific serum IgG and IgM anti-HB antibodies. After the termination of antibody induction, the orally immunized mice were infected with HBsAg, which resulted in the rapid production of antibodies against HBsAg as a result of secondary immune response. PLG microparticles formulation approach may have potential in increasing the efficacy of microparticulate systems for the oral administration of hepatitis B vaccine.

Prion protein fragments that are extracted from the brains of patients with Gerstmann-Straussler-Scheinker disease are known to have stimulating action on circulating leukocytes. In particular, the amyloidogenic hydrophobic prion peptide HuPrP (113-127) AGAAAAGAVVGGLGG has been reported to be associated with significant cellular toxicity. In this paper we show that the self assembled form of HuPrP (113-127) and its valine rich domains viz. GAVVGGLG [HuPrP (119-126)] and VVGGLGG [HuPrP (121-127)] are toxic to peripheral lymphocytes. To explore the cytotoxic mechanism of these fragments, we studied 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) reduction, reactive oxygen species (ROS) generation, calcium influx and raft sequestration of' peptide treated lymphocytes. Langmuir monolayer studies on these peptides showed a maximum lipid perturbing property of HuPrP (121-127) as compared to the other two fragments. MTT reduction assays on lymphocytes treated with peptides indicated that the prion peptide fibrils are relatively more toxic than freshly solubilized peptide preparations. Lymphocytes treated with HuPrP (121-127), HuPrP (113-127) and HuPrP (119-126) fibrils underwent 60%, 30% and 40% cell death, respectively. Abeta(1-42), HuPrP (119-126) and HuPrP (121-127) fibrils caused 4 fold increases in intracellular ROS as compared with control cells. However, HuPrP (113-127) fibrils lacked such a significant ROS generating activity, indicating that a subtle difference in sequence leads to a difference in the toxic mechanism in the cell. HuPrP (119-126) and HuPrP (121-127) fibrils also produced maximum raft sequestration and calcium influx. Taken together, these data suggest that the assemblage of prion fragments has significant toxic activity on peripheral lymphocytes, a finding with implications for controlling reactive lymphocytes in prion infected subjects.

Contraceptive steroids levonorgestrel (LNG) and ethinyl estradiol (EE) have been encapsulated with poly(ϵ-caprolactone) (PCL) microspheres using a w/o/w double emulsion method. The microspheres prepared were smooth and spherical, with a mean size from 8–25 μm. In vitro release profiles of microspheres showed a trend of increasing initially at the first week, and thereafter the release was sustained. At the end of the seventh week LNG/EE from 1:5 and 1:10 PCL microspheres were 60 and 48%, 52 and 46%, respectively. An in vitro degradation study shows that at the 20th week the microspheres maintained the surface integrity. The PCL microspheres showed a triphasic in vivo release profile with an initial burst effect due to the release of the steroid adsorbed on the microsphere surface, a second sustained release phase due to the steroid diffusion through the pores or channels formed in the polymer matrix, and third phase due to polymer bioerodible. Histological examination of PCL microspheres injected intramuscularly into thigh muscle of a rat showed a minimal inflammatory reaction demonstrating that contraceptive steroid-loaded microspheres were biocompatible. The level of inflammatory cytokines determined by immunostaining for IL-1α, the tissue response to formulations at the first week was considered mild, whereas at the end of the 20th week the inflammatory response ceased. Thus, this study helped us to evaluate the feasibility of using these microspheres as a long-acting biodegradable drug delivery system for contraceptive steroids. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2006

Vascular amyloidosis in Alzheimer's disease (AD) results in the exposure of red blood cells to beta-amyloid fibrils (A beta). The potential in vivo ramifications of this exposure have been investigated by injecting A beta(1-40) alone or A beta-bound mouse red blood cells into the circulation of C57BL/6 mice. Results indicate that when A beta(1-40) is injected alone, a transient uptake of the fibrils by red blood cells occurs in vivo. When A beta-bound red blood cells were injected, beta-amyloid is rapidly removed from these cells in vivo. Double-labeling experiments indicate that while some of the red blood cells bound to A beta(1-40) are removed from circulation, a major fraction of these cells remain in circulation even after A beta is removed. Immunohistochemistry of murine tissue samples obtained after sacrificing the animals suggests that within 1 h after injection of A beta(1-40) or A beta-bound red blood cells, A beta is found in spleen phagocytes and liver Kupffer cells. Heme staining further indicates that the binding of A beta(1-40) to red blood cells enhances red cell phagocytosis by the spleen.

Collagen is one of the best materials used for nerve guide preparation due to its biocompatibility and desirable tensile strength. In this work, we have compared regeneration and functional reinnervation after sciatic nerve resection with bioresorbable crosslinked collagen guides in 10 mm gap. The crosslinking was carried out either with glutaraldehyde (GTA) or microwave irradiation (MWI). The multilayered collagen membrane used for nerve guides are prepared by lamellar evaporation technique. Functional evaluations of the regenerated nerves were performed by measuring the sciatic functional index (SFI), nerve conduction velocity (NCV), and electromyography (EMG). Transmission electron microscopic studies showed growth of axonal cable with fewer myelinated axons, Schwann cells and more unmyelinated axons present in the case of group treated with uncrosslinked collagen tubes after 1 month of implantation. However, we have observed more myelinated axons in the case of autograft, GTA, and MWI crosslinked collagen tube implants across the gap of 1 cm after the same period of implantation. Smaller myelinated fiber diameter was observed in the case of GTA crosslinked collagen tube group when compared with the autograft and MWI collagen tube groups. There were more myelinated axons during the 3rd and 6th months postoperatively using these conduits as substantiated by light microscopic studies of the regenerated nerve. The conduction velocity and recovery index improved significantly after 5 months reaching the normal values in the autograft and MWI crosslinked collagen groups compared to GTA and uncrosslinked collagen tubes.

Neurotrophic factors play an important modulatory role in axonal sprouting during nerve regeneration involving the proliferation of hematogenous and Schwann cells in damaged tissue. We have exposed lesioned sciatic nerves to a collagen prosthesis with covalently bonded small cell adhesive peptides Arg-Gly-Asp-Ser (RGDS), Lys-Arg-Asp-Ser (KRDS), and Gly-His-Lys (GHK) to study local production of growth factors and cytokines in the regenerating tissues. Western/enzyme-linked immunosorbent assay (ELISA) studies were performed after 10 days of regeneration, when the tubular prosthesis is filled with fibrous matrix infiltrated by hematogenous cells and proliferating Schwann cells with growth factors produced locally. Regeneration was also analyzed by morphometrical methods after 30 days. The quantification of growth factors and proteins by ELISA revealed that there was an enhanced expression of the neurotrophic factors nerve growth factor (NGF) and neurotrophins (NT-3 and NT-4) in the regenerating tissues. This was further established by Western blot to qualitatively analyze the presence of the antigens in the regenerating tissues. Schwann cells were localized in the regenerating tissues using antibodies against S-100 protein. Other growth factors including growth-associated protein 43 (GAP-43), apolipoprotein E (Apo E), and pro-inflammatory cytokine like interleukin-1alpha (IL-1alpha) expression in the peptide groups were evaluated by ELISA and confirmed by Western blotting. Cell adhesive integrins in the proliferating cells were localized using integrin-alpha V. The combined results suggest that the early phase of regeneration of peripheral nerves in the presence of peptide-incorporated collagen tubes results in the enhanced production of trophic factors by the recruited hematogenous cells and Schwann cells, which in turn help in the secretion of certain vital trophic and tropic factors essential for early regeneration. Furthermore, hematogenous cells recruited within the first 10 days of regeneration help in the production of inflammatory mediators like interleukins that in turn stimulate Schwann cells to produce NGF for axonal growth.

This paper describes the regeneration of lesioned sciatic nerve with collagen tubes incorporated with RGD cell-adhesive peptide. Collagen implants of 14 mm were grafted to bridge a gap length of 10 mm nerve defect in a rat model. The regenerated tissues were analyzed histomorphologically. The number of myelinated axons in the regenerated mid-graft of the RGD peptide incorporated groups was statistically significant (p

Present study describes the development of a new formulation of levonorgestrel and ethinylestradiol based on double emulsion-solvent evaporation technique using poly(epsilon-caprolactone) (PCL) as biodegradable polymer. The effect of polymer concentration on microspheres and entrapment of drug into microspheres were studied. PCL was selected because of its hydrophobicity and advantages over other biodegradable polymers. Characterization of biodegradable polymer used for controlled drug delivery is essential to ensure reproducibility of in vitro and in vivo performances. The selected characterisation techniques established for PCL microspheres include its loading and entrapment efficiencies, DSC to analyse thermal behaviour, SEM to observe surface morphology, drug content of microspheres and in vitro release of drugs from microspheres. The SEM reports showed that microspheres were with smooth surface and DSC thermograms revealed no interaction between drug and polymer. The entrapment was found to be 58 and 47% for 1:10 and 1:5 batches and in vitro release studies showed that about 69.7% of LNG and 66.7% of EE from 1:10 batch and about 80% of LNG and 75.5% of EE from 1:5 batch for 150 days.

was prepared (0.1, 0.2, 0.3, 0.6, and 0.8 M) and added slowly to the aqueous mannitol solutions (0.1, 0.2, 0.3, 0.6, and 0.8 M, respectively) placed in the thermostat. The specific conductance of the solution was recorded. The abrupt change in the initial slope value at a particular concentration was considered to be the cmc. Fluorescence spectra were recorded on a Hitachi model 65040 fluorimeter. The aggregation number of SDS in mannitol solutions was determined by the fluorescence quenching method 6

AA (amyloid protein A) amyloidosis in mice is markedly accelerated when the animals are given, in addition to an inflammatory stimulus, an intravenous injection of protein extracted from AA-laden mouse tissue. Previous findings affirm that AA fibrils can enhance the in vivo amyloidogenic process by a nucleation seeding mechanism. Accumulating evidence suggests that globular aggregates rather than fibrils are the toxic entities responsible for cell death. In the present study we report on structural and morphological features of AEF (amyloid-enhancing factor), a compound extracted and partially purified from amyloid-laden spleen. Surprisingly, the chief amyloidogenic material identified in the active AEF was diffusible globular oligomers. This partially purified active extract triggered amyloid deposition in vital organs when injected intravenously into mice. This implies that such a phenomenon could have been inflicted through the nucleation seeding potential of toxic oligomers in association with altered cytokine induction. In the present study we report an apparent relationship between altered cytokine expression and AA accumulation in systemically inflamed tissues. The prevalence of serum AA monomers and proteolytic oligomers in spleen AEF is consistent to suggest that extrahepatic serum AA processing might lead to local accumulation of amyloidogenic proteins at the serum AA production site.

Chronically elevated blood glucose levels result in reduced leukocyte function and cell malnutrition, which contribute to a high rate of wound infection and associated healing problems in diabetic patients. In the present study, the role of biotinylated GHK peptide (BioGHK) incorporated collagen biomaterial was tested for wound healing in diabetic rats. The rate of wound contraction and the levels of collagen, uronic acid, protein and DNA in the granulation tissue were determined. Further, the concentration of nitric oxide and other skin antioxidants was also monitored during the study. In diabetic rats treated with BioGHK incorporated collagen (Peptide Incorporated Collagen — PIC), the healing process was hastened with an increased rate of wound contraction. Glutathione (GSH) and ascorbic acid levels in the skin of streptozotocin-induced diabetic rats were higher in the PIC group as compared to control (Untreated) and collagen (Collagen Film — CF) treated groups. Superoxide dismutase (SOD) and catalase (CAT) activity was altered in all the groups. In vitro fibroblast cell culture studies suggest that PIC promotes fibroblast growth. Histological evaluation by haematoxylin–eosin and Masson's trichrome method revealed epithelialization, increased synthesis of collagen and activation of fibroblasts and mast cells in the PIC group. This study provides a rationale for the topical application of BioGHK incorporated collagen as a feasible and productive approach to support diabetic wound healing.

Purpose A controlled-release drug delivery of contraceptive steroids levonorgestrel (LNG) and ethinyl estradiol (EE) has been developed by successful encapsulation of LNG and EE in poly (lactide-co-glycolide) (PLG) microspheres. Materials and Methods Smooth, spherical, steroid-loaded PLG microspheres with a mean size of 10–25 μm were prepared by using the water/oil/water double-emulsion solvent evaporation method. Results In vitro release profiles showed an increased burst release of LNG/EE on Week 1; thereafter, the release was sustained. At the end of Week 7, the release of LNG/EE from 1:5 and 1:10 PLG microspheres was 75.64% and 62.55%. respectively. In vitro degradation studies showed that the PLG microspheres maintained surface integrity up to Week 8 and then eroded completely by Week 20. In an in vivo study, the serum concentration of LNG/EE in rats showed a triphasic release response, with an initial burst release of 8 ng/mL LNG and 14 pg/mL EE on Day 1; thereafter, a controlled release of the drugs to the systemic circulation was maintained until Week 15, maintaining constant drug levels of 2 ng/mL LNG and 3–4 pg/mL EE in the blood. Histological examination of steroid-loaded PLG microspheres injected intramuscularly into the thigh muscle of Wistar rats showed minimal inflammatory reaction, demonstrating that contraceptive-steroid-loaded microspheres were biocompatible. Conclusion This controlled-release and biocompatible nature of the PLG microspheres may have potential application in contraceptive therapy.

The structure of insulin in amyloid fibrillar form has been recently shown as a well folded conformation using cryoelectron microscopy [Jimenez, J.L., Nettleton, E.J., Bouchard, M., Robinson, C.V., Dobson, C.M., Saibil H.R., 2002. The protofilament structure of insulin amyloid fibrils. Proc. Natl. Acad. Sci. USA. 99 9196–9201.]. Most of the amyloid aggregates elicit maximum toxicity in the protofibrillar (PF) intermediate state. Here, we describe PF intermediates of insulin are made-up monomers with flexible conformers. We also show protofibrils have three-dimensionally extended hydrophobic cavity to bind with 1-anilinonaphthalene-8-sulphonate (ANS) molecules. Energy transfer measurement revealed that ANS dye binding site of PF is within the range of FRET distance of insulin tyrosine residues. Significant proportion of β-sheet, helical, and turn structures in the PF form indicate conformational dynamics in the folded chain of insulin in the PF assembled form. Though the conformational flexibility is noticeably present in the assembly, addition of GdnHCl could completely unfold PF into disordered structure suggesting structural “zipping” in the PF form. We have also shown that helical conformer inducing solvent 2,2,2-trifluoroethanol (TFE) could dissociate the PF aggregate indicating possible involvement of β-sheets in contributing to PF stability.

The highly conserved ubiquitin-proteasome system (UPS) controls the stability of most nuclear and cytoplasmic proteins and is therefore essential for virtually all aspects of cellular function. We have previously shown that the UPS is impaired in the presence of aggregated proteins that become deposited into cytoplasmic inclusion bodies (IBs). Here, we report that production of protein aggregates specifically targeted to either the nucleus or cytosol leads to global impairment of UPS function in both cellular compartments and is independent of sequestration of aggregates into IBs. The observation of severe UPS impairment in compartments lacking detectable aggregates or aggregation-prone protein, together with the lack of interference of protein aggregates on 26S proteasome function in vitro, suggests that UPS impairment is unlikely to be a consequence of direct choking of proteasomes by protein aggregates. These data suggest a common proteotoxic mechanism for nuclear and cytoplasmic protein aggregates in the pathogenesis of neurodegenerative disease.

Alzheimer's disease is associated with vascular amyloidosis. As blood flows through the microcirculation, red blood cells (RBCs) come in contact with the vasculature. RBCs as well as endothelial cells (ECs) are known to bind beta amyloid fibrils. This suggests that a potential effect of amyloidosis may involve the interactions of RBCs with ECs lining the wall of the blood vessels mediated by amyloid fibrils. We have studied the effect of beta-amyloid peptide[1-40] (Abeta1-40) fibrils on the interactions of murine RBCs with ECs derived from bovine lung microvascular endothelium (BLMVEC) as well as bovine pulmonary arterial endothelium (BPAEC) in culture. We show that the initial incorporation of Abeta fibrils onto either RBCs or ECs cause RBCs to adhere to the ECs with greater affinity for the microvascular cells than the arterial cells. In addition, there is a transfer of Abeta fibrils between the RBCs and the ECs. Both the transfer and adhesion occurs when the amyloid fibrils are on the ECs or on the RBCs. However, with the amyloid fibrils on the RBCs, the adhesion and the transfer are greater than with the fibrils on the ECs. These results suggest that amyloidosis may affect the flow of RBCs through the microcirculation and that RBCs may play a role in propagating amyloidosis through the vasculature.

The wound healing process involves extensive oxidative stress to the system, which generally inhibits tissue remodeling. In the present study, an improvement in the quality of wound healing was attempted by slow delivery of antioxidants like curcumin from collagen, which also acts as a supportive matrix for the regenerative tissue. Curcumin incorporated collagen matrix (CICM) treated groups were compared with control and collagen treated rats. Biochemical parameters and histological analysis revealed that increased wound reduction, enhanced cell proliferation and efficient free radical scavenging in CICM group. The higher shrinkage temperature of CICM films suggests increased hydrothermal stability when compared to normal collagen films. Spectroscopic studies revealed that curcumin was bound to the collagen without affecting its triple helicity. Further we adopted the antioxidant assay using 2,2'-azobisisobutyronitrile to assess in vitro antioxidant activity of CICM. The antioxidant studies indicated that CICM quenches free radicals more efficiently. This study provides a rationale for the topical application of CICM as a feasible and productive approach to support dermal wound healing.

Increased neuronal nitric oxide synthase (nNOS) activity was observed during the prodromal period of seizures in various rat brain regions following administration of GABAA receptor antagonist, picrotoxin (PCT). Pretreatment with the selective nNOS inhibitor 7-nitroindazole (7-NI), dose- and time-dependently delayed the onset of clonus with a corresponding decrease in nNOS activity. The threshold dose of antiepileptic drugs (AEDs; diazepam, phenobarbitone and gabapentin) have potentiated the anticonvulsant action by pretreatment with graded doses of 7-NI. The increase in efficacy of anticonvulsant action correlated with a corresponding decrease of PCT-evoked increase in nNOS activity. The present data support a role for abnormal nNOS activity in mechanisms that trigger seizures and suggest a possible NO-mediated interplay between GABAA and glutamate receptors. The results of the present study provide evidence for a trigger role of neuronally produced NO in epileptogenesis induced by PCT and the participation of nNOS inhibitory mechanisms in the action of AEDs.

Amyloid beta-protein (A beta) accumulation in brain is thought to be important in causing the neuropathology of Alzheimer's disease (AD). A beta interactions with both neurons and microglial cells play key roles in AD. Since vascular deposition of A beta is also implicated in AD, the interaction of red cells with these toxic aggregates gains importance. However, the effects of A beta interactions with red blood cells are less well understood. Synthetic amyloid beta-protein (1-40) was labeled with biotin and preincubated at 37 degrees C for 4, 14 and 72 h to produce fibrils. Flow cytometry was used to study the binding of these fibrils to red cells. The amyloid fibrils had a high affinity for the red cell with increased binding for the larger fibrils produced by longer preincubation. Bovine serum albumin (BSA) did not reverse the binding, but actually resulted in a more efficient binding of the A beta fibrils to the red cells. The interaction of A beta with red cells increased the mean cell volume and caused the cells to become more spherical. This effect was greater for the longer fibrils. At the same time the interaction of A beta with red cells produced an increase in their fluorescence measured after 16-h incubation at 37 degrees C. This increase in fluorescence is attributed to the formation of fluorescent heme degradation products. The effect of prior hemoglobin oxidation, catalase inhibition and glutathione peroxidase inhibition indicated that the amyloid-induced oxidative damage to the red cell involved hydrogen peroxide-induced heme degradation. These results suggest that amyloid interactions with the red cell may contribute to the pathology of AD.

We have been developing antioxidants incorporated collagen matrix as a novel biomaterial for various biomedical applications. In this study we made use of quercetin incorporated collagenous matrix for dermal wound healing in rat. Quercetin incorporated collagen (QIC) treated groups were compared with control and collagen (CS) treated animals. QIC treated animal showed a better healing when compared to control and CS treated wound. The biochemical parameters like hydroxyproline, protein, uronic acid content in the healing wound, revealed that there is an increase in proliferation of cells in quercetin treated groups when compared to CS group and there is considerable increase in wound contraction when compared to CS treated group. In addition we adapted the antioxidant assay using 2,2'-azobisisobutryonitrile (AIBN) to assess in vitro antioxidant activity of QIC. The antioxidant studies indicate QIC quench the radicals more efficiently. These results suggested that quercetin incorporated collagen matrix could be a novel dressing material for dermal wound healing.

Collagen, a unique connective tissue protein finds extensive application as biocompatible biomaterial in wound healing, as drug carriers, cosmetics, etc. A study has been undertaken to stabilise Type-I collagen of rat-tail tendon using plant polyphenol (Acacia Mollissima) in the presence of an acrylic polymer. It has been found that collagen fibres pre-treated with acrylic polymer followed by the treatment with Acacia Mollissima exhibited an increase in hydrothermal stability by 25°C. Infrared spectroscopic studies display the changes in the spectral characteristics of native and treated collagen films. Transmission electron microscopic and circular dichroic studies provide an insight into the understanding of the improved stabilisation of collagen, due to treatment with acrylic polymer and plant polyphenols. The study is expected to enhance the biomaterial applications of collagen tissues.

A novel peptide, tert-butyloxycarbonyl-L-arginine-L-proline lauryl ester laurate, synthesised by a solution-phase method, formed micelles in aqueous solutions and was observed to exhibit anticoagulant activity as shown by clotting assays such as the thrombin time (TT) test, the prothrombin time (PT) test and the activated partial thromboplastin time (APTT) test. TT and PT were found to be normal up to a particular concentration of peptide, and above that level they increased with increasing concentration of peptide, while APTT did not exhibit much significance. Conductometric and potentiometric studies showed that the peptide formed a stable micelle, and the anticoagulant activity of this peptide was also compared with a non-arginine-containing peptide (control) known to form micelles. The anticoagulant action in the micellar form could be due to the inhibition of thrombin, as seen from the decrease in amidolytic activity. The inhibitory activity of the peptide was explained in the light of micelle formation.