The electrical pulse application enhances intra-cellular localization and potentiates cytotoxicity of curcumin in breast cancer cells.

Breast cancer is the most common cancer of women, and fifth leading cause of mortality worldwide. Existing breast cancer regimens are costly and produce severe side effects. This highlights a need for the development of efficient novel therapies, which are cost effective and limit side effects. An electrical pulse (EP)-based chemo therapy, known as electrochemotherapy (ECT) using the natural compound curcumin could be an effective alternative. ECT is a non-surgical modality, which produces excellent anti-tumor efficacy at small drug concentrations due to increased uptake of drugs. In clinics, ECT is shown to be effective in treating advanced, recurrent, and metastatic breast cancers, which are refractory to multiple modalities. ECT with curcumin triggers apoptotic cell death in breast cancer cells and could be an effective alternative, due to curcumin's low cost and reduced side-effects. However, there is a lack of studies quantifying the uptake of curcumin in response to EP application. Towards this, we determined the uptake of different curcuminoids (curcumin, desmethoxycurcumin, and bisdemethoxycurcumin) upon EP application and their impact on cell cytotoxicity. Additionally, we studied the combined effect of calcium chloride (CaCl2) and a curcuminoids (Cur) mixture, based on initial studies suggesting calcium electroporation as a potential inexpensive anti-cancer treatment. Our results indicate EP with Cur increases cellular uptake, cell shrinkage, and cytotoxicity. The EP + Cur resulted in the highest uptake of the bisdemethoxycurcumin. Further, EP also potentiated the cytotoxicity of CaCl2 and of the Cur and CaCl2 combination against breast cancer cells and caused apoptosis. Our preliminary data pave the way to further studies on Cur and CaCl2 combination treating breast cancer.

S100B as a new fecal biomarker of inflammatory bowel diseases.

OBJECTIVE: S100 proteins are demonstrated to exert a protective role in the gastrointestinal tract. In the present study, we investigated whether S100B protein, that is typically expressed by enteroglial cells, is detectable in feces and could be a useful noninvasive indicator of gut chronic inflammation. PATIENTS AND METHODS: This clinical prospective study included n=48 patients suffering Crohn's disease (CD) or ulcerative colitis (UC) and non IBD-controls. The clinical disease activity was evaluated using Harvey-Bradshaw or Mayo Score Index while the diagnosis of IBD was defined based on standard endoscopic and histological criteria. S100B and calprotectin were extracted and analyzed using commercial enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: Unlike calprotectin, S100B was significantly decreased in both CD and UC compared to non IBD-patients. The strongest quantitative alterations of S100B were detected concomitantly with signs of active or quiescent disease, including high/normal expression of fecal calprotectin, mucosal damage/cryptitis, mucin depletion and inflammatory infiltrate, as defined by endoscopic evaluation and histological analysis. At the onset of disease and under no Infliximab-based therapy, the lowest was detected suggesting that S100B in feces could have a potential diagnostic value for IBD. CONCLUSIONS: Testing for S100B and calprotectin could be a useful screening tool to better predict IBD activity.

4,6,4'-trimethylangelicin shows high anti-proliferative activity on DU145 cells under both UVA and blue light.

OBJECTIVES: Furocoumarins (psoralens and angelicins) have been already used under ultraviolet A light (UVA) for the treatment of skin diseases and cutaneous T-cell lymphoma. Besides their high anti-proliferative activity, some severe long-term side effects have been observed, for example genotoxicity and mutagenicity, likely strictly related to the formation of crosslinks. It has been demonstrated that blue light (BL) activation of 8-methoxypsoralen, an FDA-approved drug, leads to less mutagenic monoadducts in the DNA. So far, in this work the less toxic and more penetrating BL is proposed to activate 4,6,4'-trimethylangelicin (TMA), an already known UVA photoactivatable compound. MATERIALS AND METHODS: Photocleavage, crosslink formation and oxidative damage were detected in pBR322 plasmid DNA treated with 300.0 µmol/L TMA activated with various exposures of BL. Anti-proliferative activity, reactive oxygen species (ROS) formation and activation status of some signalling pathways involved in cell growth and apoptosis were verified on DU145 cells treated with 5.0 µmol/L TMA plus 2.0 J/cm2 of BL. RESULTS: Under BL-TMA, no mutagenic crosslinks, no photocleavage and neither photooxidative lesions were detected on isolated plasmid DNA. TMA showed high anti-proliferative activity on DU145 cells through induction of apoptosis. Besides ROS generation, the proapoptotic effect seemed to be related to activation of p38 and inhibition of p44/42 phosphorylation. Interestingly, the decrease in nuclear ß-catenin was coupled with a significant dropping of CD44-positive cells. CONCLUSION: Overall, our results indicate that TMA can be activated by BL and may be considered for targeted phototherapy of prostate cancer lesions.

Poly-ε-caprolactone composite scaffolds for bone repair.

Synthetic biomaterials combined with cells and osteogenic factors represent a promising approach for the treatment of a number of orthopedic diseases, such as bone trauma and congenital malformations. To guarantee optimal biological properties, bone substitutes are prepared with a 3D structure and porosity grade functional to drive cell migration and proliferation, diffusion of factors, vascularization and cell waste expulsion. In this study, synthetic hydroxyapatite (HA) or rat bone extracellular matrix (BP) were examined in an effort to optimize the mechanical properties and osteogenic activity of poly-ε-caprolactone scaffolds prepared with alginate threads (PCL-AT). Using rabbit bone marrow-derived mesenchymal stem cells (rMSCs), the effects of PCL composite substrates on cell adhesion, growth and osteogenic differentiation were evaluated. Micro-CT analysis and scanning electron microscopy evidenced that porous PCL scaffolds containing HA or BP acquire a trabecular bone-like structure with interconnected pores homogenously distributed and are characterized by a pore diameter of approximately 10 µm (PCL-AT-BP) or ranging from 10 to 100 µm. Although the porosity grade of both PCL-AT-HA and PCL-AT-BP promoted optimal conditions for the cell growth of rMSCs at the early phase, the presence of BP was crucial to prolong the cell viability at the late phase. Moreover, a precocious expression of Runx2 (at 7 days) was observed in PCL-AT-BP in combination with osteogenic soluble factors suggesting that BP controls better than HA the osteogenic maturation process in bone substitutes.

In vitro evaluation of TAT-OP1 osteogenic properties and prospects for in vivo applications.

So far, osteogenic protein 1 (OP1) is biotechnologically produced and approved for the treatment of human lumbar spine fusion and long bone non-union fractures. When combined with the TAT sequence, it has been demonstrated in vitro to be easily taken up by PC12 neuronal cells and to acquire its biological activity after intracellular refolding. In this study, TAT-OP1 was shown to be a useful strategy to efficiently drive denatured OP1 into mouse MC3T3E1 pre-osteoblasts. The correct in vitro protein refolding was verified by the activation of the BMP cascade, while the osteogenic potential of OP1 was demonstrated by increased expression of alkaline phosphatase, osteonectin and osteocalcin.

Growth factors, their receptor expression and markers for proliferation of endothelial and neoplastic cells in human osteosarcoma.

Osteosarcoma is the most common primary malignant tumour of the bone. Although new therapies continue to be reported, osteosarcoma-related morbidity and mortality remain high. Modern medicine has greatly increased knowledge of the physiopathology of this neoplasm. Novel targets for drug development may be identified through an understanding of the normal molecular processes that are deeply modified in pathological conditions. The aim of the present study is to investigate, by immunohistochemistry, the localisation of different growth factors and of the proliferative marker Ki-67 in order to determine whether these factors are involved in the transformation of osteogenic cells and in the development of human osteosarcoma. We observed a general positivity for NGF - TrKA - NT3 - TrKC - VEGF in the cytoplasm of neoplastic cells and a strong expression for NT4 in the nuclear compartment. TGF-beta was strongly expressed in the extracellular matrix and vascular endothelium. BDNF and TrKB showed a strong immunolabeling in the extracellular matrix. Ki-67/MIB-1 was moderately expressed in the nucleus of neoplastic cells. We believe that these growth factors may be considered potential therapeutic targets in the treatment of osteosarcoma, although proof of this hypothesis requires further investigation.

Recombinant human TAT-OP1 to enhance NGF neurogenic potential: preliminary studies on PC12 cells.

Osteogenic protein 1 (OP1), also known as bone morphogenic protein-7 (BMP7), is a multifunctional cytokine with demonstrated neurogenic potential. As the recombinant OP1 (rhOP1) was shown to provide axonal guidance cues and to prevent the reduction of dendritic growth in the injury-induced cortical cultures, it was suggested that an in vivo efficient rhOP1 delivery could enhance neurite growth and functional reconnectivity in the damaged brain. In the present work, we engineered a chimeric molecule in which rhBMP7 was fused to a protein transduction domain derived from HIV-1 TAT protein to deliver the denatured recombinant BMP7 into cells and obtain its chaperone-mediated folding, circumventing the expensive and not much efficient in vitro refolding procedures. When tested on rat PC12 cells, a widely used in vitro neurogenic differentiation model, the resulting fusion protein (rhTAT-OP1) demonstrated to enter fastly into the cells, lose HIV-TAT sequence and interact with membrane receptors activating BMP pathway by SMAD 1/5/8 phosphorylation. In comparison with nerve growth factor (NGF) and BMP7, it proved itself effective to induce the formation of more organized H and M neurofilaments. Moreover, if used in combination with NGF, it stimulated a significant (P < 0.05) and more precocious dendritic outgrowth with respect to NGF alone. These results indicate that rhTAT-OP1 fused with TAT transduction domain shows neurogenic activity and may be a promising enhancer factor in NGF-based therapies.

Pancreatic acellular matrix supports islet survival and function in a synthetic tubular device: in vitro and in vivo studies.

Increasing pancreatic islet survival and function is a starting point for obtaining a valuable bioartificial pancreas for the treatment of type 1 diabetes. In this context, decellularized matrices, obtained after the removal of tissue cellular part, are known to support in vitro adhesion, growth, and function of several cell types. We demonstrate that a homologous acellular pancreatic matrix is a suitable scaffold for rat islet cultures maintaining their long-term viability and function. Islets adhered to the pancreatic matrix showed a constant glucose-induced insulin release during long-term in vitro incubation, while islets cultured without a matrix or on the liver matrix showed a progressive reduction. In order to obtain implantable devices, acellular matrix/islet cultures were entrapped into poly(vinyl alcohol) (PVA)/ poly(ethylene glycol) (PEG) tubes obtained by the freezing/thawing procedure. Under this condition, an in vitro constant insulin release was detected. The devices were then implanted into diabetic rats where reduced insulin requirement was noted suggesting insulin secretory activity of islets contained in the device. Indeed, immunofluorescence confirmed the presence of insulin- and glucagon-producing cells into the explanted devices. These data show that PVA/PEG semi-permeable membrane can obtain devices that restore, at least in part, insulin secretion.

In vitro biological activity of bovine milk ribonuclease-4.

Several members of the ribonuclease superfamily possess a variety of interesting biological properties, including ribonucleolytic, angiogenic, antiproliferative, cytotoxic, embryotoxic, aspermatogenic and antitumoral activity. In this study, we report the purification from bovine milk of a protein with structural and enzymatic properties very similar to those of ribonuclease-4 (RNase-4), which is normally present in the liver and lungs, and examined its functional properties, biological activity and cytotoxic effects. RNase-4, at physiological concentrations, had a positive effect on the vitality and proliferation of human umbilical vein endothelial cells. Moreover, it induced an increase in cellular migration and the formation of in vitro capillary-like structures. We also evaluated the effect of RNase-4 in vitro on human breast, colorectal and cervical carcinoma cell lines. The protein was revealed to have a cytotoxic effect similar to that of RNase-A. We suggest that the positive effects of RNase-4 on normal cells were due to its particularly close interaction with RNase inhibitor, while good conformational stability and resistance to proteolytic degradation potentially favour ribonuclease cytotoxicity.

Middle-term expansion of hematopoietic cord blood cells with new human stromal cell line feeder-layers and different cytokine cocktails.

Cord blood (CB) is a source of hematopoietic stem cells (HSCs) and is an alternative to bone marrow for allogenic transplantation in patients with hematological disorders. The improvement of HSC in vitro expansion is one of the main challenges in cell therapy. Stromal components and soluble factors, such as cytokines, can be useful to induce in vitro cell expansion. Hence, we investigated whether feeder-layers from new stromal cell lines and different exogenous cytokine cocktails induce HSC expansion in middle-term cultures. CB HSC middle-term expansion was carried out in co-cultures on different feeder-layers exposed to three different cytokine cocktails. CB HSC expansion was also carried out in stroma-free cultures in the presence of different cytokine cocktails. Clonogenic tests were performed, and cell growth levels were evaluated. Moreover, the presence of VCAM-1 mRNA was assessed, and the mesenchymal cell-like phenotype expression was detected. All feeder-layers were able to induce a significant clonogenic growth with respect to the control culture, and all of the cytokine cocktails induced a significant increase in CB cell expansion indexes, even though no potential variation dependent on their composition was noted. The modulative effects of the different cocktails, exerted on each cell line used, was dependent on their composition. Finally, all cell lines were positive for CD73, CD117 and CD309, similar to mesenchymal stem cells present in adult bone marrow and in other human tissues, and negative for the hematopoietic markers. These data indicate that our cell lines have, not only a stromal cell-like phenotype, but also a mesenchymal cell-like phenotype, and they have the potential to support in vitro expansion of CB HSCs. Moreover, exogenous cytokines can be used in synergism with feeder-layers to improve the expansion levels of CB HSCs in preparation for their clinical use in allogenic transplantation.

Experimental hepatology applied to stem cells.

Transplantation is an accepted treatment today for many people suffering from organ failure. More and more patients are referred for transplant surgery, and the waiting lists are growing longer because not enough organs and tissues are donated for transplantation. This has led to several potentially viable alternatives being considered, including bio-artificial support devices, the transplantation of mature cells or stem/progenitor cells and the potential transplantation of xenogenic organs and cells [Burra P, Samuel D, Wendon J, Pietrangelo A, Gupta S. Strategies for liver support: from stem cells to xenotransplantation. J Hepatol 2004;41:1050-9]. Numerous investigators around the world are engaged in these investigations and the pace of discovery has begun to accelerate in recent years. To take stock of the achievements of recent years, the AISF sponsored a Single-Topic Conference, held in Padua on 26-27 May, 2006, with the participation of many leading investigators from various parts of Italy and Europe. This present paper summarizes the content of the Conference. Different issues were analysed, from the biology of stem cells to the possible use of gene therapy. The speakers were clinicians and scientists interested in diseases not only of the liver but also of other organs such as the kidney or heart. The fact that numerous specialties were represented helped the audience to understand the stem cell research area from different standpoints, and what research has achieved so far.

Effects of some vanadyl coordination compounds on the in vitro insulin release from rat pancreatic islets.

Many lines of evidence indicate that vanadium inorganic salts possess insulin-mimetic and insulinotropic properties. However, they are poorly absorbed, so high oral doses are required to achieve effective plasma concentrations with possible undesirable toxic side-effects ensuing. Various organically-chelated vanadium compounds have been synthesized that are more potent than inorganic vanadium salts in their insulin-like effects due to their greater bioavailability. Unfortunately, little is known about the possible insulin secretagogue action of organic vanadyl coordination compounds. Hence, we investigated the effect of [VO(metformin)2]H2O, [VO(salicylidene-ethylenedimmine)2] and [VO(pyrrolidine-N-dithiocarbamate)2](VODTC) on insulin release from isolated rat pancreatic islets, and compared it to that of vanadyl sulfate (VOSO4). Of the three coordination compounds, only VODTC was found to exert insulin secretagogue action. VODTC, within concentrations ranging from 0.1 to 1.0 mM, enhanced both basal and glucose (11 mM)-stimulated insulin release. The effect involves calcium channels, since it was not appreciable in Ca2+-free medium. The stimulating action of VODTC required the presence of the whole metal-chelator complex inasmuch as the chelator DTC alone was ineffective. VOSO4 was unable to bring about any significant rise in insulin release from isolated islets. Taken together, our findings indicate that VODTC may be considered a potential elective pharmaceutical tool in the therapy of diabetes, especially of type 2, through its concomitant stimulatory effect on insulin secretion and insulin-mimetic action.

Experimental abdominal wall defect repaired with acellular matrix.

In the surgical repair of congenital abdominal-wall defects (AWD), the ready availability of a non-immunogenic and non-prosthetic biomaterial that could guide the regeneration of normal tissue is a fascinating possibility. Biomaterials are already in use, but in our experience, an acellular matrix (ACM) can stimulate exact regeneration of the absent tissue. We explored the possibility of using an ACM to repair a muscular AWD in an animal model. Male New Zealand white rabbits (3-4 kg, n = 18) were anesthetized and the abdominal wall was shaved and scrubbed; a vertical incision was made in the left lower quadrant and a large patch of external-oblique muscle was resected (3 x 3 cm). The animals underwent reconstruction with homologous diaphragm acellular matrix (HDAM) grafts that were previously prepared using a detergent enzymatic method. The patches were evaluated histologically at 9 (n = 6), 40 (n = 6), and 90 (n = 6) days post-surgery in each group; moreover, 90 days post-surgery an electromyogram (EMG) (n = 6) of the implanted matrix was recorded. Histologic analysis demonstrated that the HDAM supported fibroblast migration, deposition of newly-formed collagen, and neovascularization. No signs of necrosis, or evidence of skeletal-muscle-cell ingrowth were detected. The EMG revealed minimum muscular electrophysiologic activity, probably due to muscle underlying the patch. The HDAM we employed was thus not able to produce reconstruction of the skeletal muscle, and was progressively remodeled into fibrous tissue. Since the ultimate reason for failure of muscle regeneration is a lack of myogenesis, future studies will use ACMs preconditioned by various regulators of myoblast proliferation and differentiation.

Essential amino acids increase the growth and alkaline phosphatase activity in osteoblasts cultured in vitro.

An inadequate protein intake seems to be involved in the pathogenesis of osteoporosis. Moreover, protein from animal sources appears to protect against hip fracture, while protein from vegetable sources, which present low levels of essential amino acids, has no effect. In this preliminary work, the growth, the alkaline phosphatase activity and the collagen synthesis were evaluated in osteoblast cultures obtained from calvaria of newborn Sprague-Dawley rats and incubated with lysine, threonine, methionine, triptophan and arginine. Our results have shown that the essential amino acids can modulate the growth and the differentiation of osteoblasts cultured in vitro, confirming the relationship between osteoporotic hip fracture and inadequate protein intake. The compounds have mainly enhanced cell growth and alkaline phosphatase activity, and, to a lower degree, collagen synthesis. In summary, the essential amino acids can stimulate bone formation and could represents useful agents for the prevention and therapy of osteoporosis.

Antioxidative and antiatherosclerotic effects of human apolipoprotein A-IV in apolipoprotein E-deficient mice.

Mice expressing human apolipoprotein A-IV (apoA-IV) mainly in the intestine were obtained in an apolipoprotein E-deficient (apoE(0)) background (apoA-IV/E(0) mice). Quantification of aortic lesions and plasma lipid determination showed that compared with their control apoE(0) counterparts, the apoA-IV/E(0) mice are protected against atherosclerosis without an increase in HDL cholesterol. Because oxidized lipoproteins play an important role in atherogenesis, we tested whether the protection observed in these animals is accompanied by an in vivo reduction of the oxidation parameters. The lag time in the formation of conjugated dienes during copper-mediated oxidation, the aggregation state of LDL, and the presence of anti-oxidized LDL antibodies were measured. The presence of oxidized proteins in tissues and the presence of oxidation-specific epitopes in heart sections of atherosclerotic lesions were also analyzed. Except for lag time, the results showed that the oxidation parameters were reduced in the apoA-IV/E(0) mice compared with the apoE(0) mice. This suggests that human apoA-IV acts in vivo as an antioxidant. In addition, human apoA-IV accumulation was detected in the atherosclerotic lesions of apoA-IV/E(0) mice, suggesting that apoA-IV may inhibit oxidative damage to local tissues, thus decreasing the progression of atherosclerosis.

Prostaglandin F2 alpha can modulate the growth and the differentiation of bovine corneal epithelial cells cultured in vitro.

The effects of PGF2 alpha on the growth, morphology, morphometry and keratinization pattern of bovine corneal epithelial cells cultured in vitro were studied. The cells were grown with a basal medium or, in the presence of keratocytes and/or their products, using a keratocyte-conditioning medium. Cell growth was evaluated by MTT assay. Daily treatments with exogenous PGF2 alpha at concentrations equal to or lower than 10(-6) M induced significant increases in cell proliferation when the epithelial cells were cultured on a keratocyte feeder-layer or with the conditioning medium. No variations were observed in cultures grown with the basal medium. 10(-5) M PGF2 alpha induced a decrease in cell growth under all culturing conditions. PGF2 alpha did not affect cell morphology and modified only nuclear dimensions among the cells grown under different culturing conditions. No variations of any parameters were observed between cells cultured on feeder-layer, with conditioning or basal medium and the corresponding cultures supplemented with the autacoid. Moreover, PGF2 alpha induced only the disappearance of 43 kDa keratin in cells grown on basal medium, while the keratin pattern of epithelial cells cultured on feeder-layer or with the conditioning medium was not modified by the autacoid. From these data we can suppose that a cooperation could exist between PGF2 alpha and fibroblasts and their products for the modulation of cell growth. Finally, it was observed that the autacoid had no effect on cell morphology and morphometry, except for nuclear dimensions, despite the presence of other prostaglandins, such as PGE2.

Short bowel syndrome: experimental approach to increase intestinal surface in rats by gastric homologous acellular matrix.

BACKGROUND/PURPOSE: In this preliminary work the authors used homologous acellular matrix obtained by the gastric wall to increase the small bowel surface in Sprague-Downey rats; through this experimental model the authors verified that homologous acellular matrix can support cell migration and the reconstruction of the intestinal wall. METHODS: A tract of about 2 cm of tubular gastric acellular matrix was inserted with bilateral anastomosis in an isolated ileal loop, which was located in endoabdominal position through a short subcutaneous tunnel. Twelve animals were analyzed at each of the time-points ranging from 1 to 6 weeks after surgery. RESULTS: Histologic evaluation showed that the implanted matrix can be reintegrated in the normal small bowel in a period ranging between 3 and 6 weeks from surgery. The implanted matrix was organized with 4 different tonacae from the third week after the surgery, without interruption at the site of the anastomosis. CONCLUSIONS: To date, the authors do not have a demonstration of the function of the ileal loop reconstructed with this technique; based on these results the authors are engaged in an experimental trial of restoration of intestinal viability with the ileal prosthesis after 3 weeks to study its function.

Protein degradation by the proteasome and its implications in aging.

Free radical damage to cellular components is believed to contribute to the aging process. Studies on proteins have shown both an age-related decline in several enzyme activities and an age-related accumulation of oxidized forms of protein. Oxidized forms of protein are generally degraded more rapidly than their native counterparts. Indeed, the normal functions of the cell involve the regular elimination of these altered molecules. The proteasome, a multienzymatic proteolytic complex, is the major enzymatic system in charge of cellular "cleansing" and plays a key role in the degradation of damaged proteins. Consequently, proteasome function is very important in controlling the level of altered proteins in eukaryotic cells. Because the steady-state level of oxidized protein reflects the balance between the rate of protein oxidation and the rate of protein degradation, age-related accumulation of altered protein can be due to an increase of free radical-mediated damage, a loss of protease activity, or the combination of both mechanisms. One of the hypotheses put forward to explain the accumulation of altered proteins is the decrease of proteasome activity with age. In this paper, the importance of oxidative damage to proteins and that of their elimination by the proteasome are first described. Then, evidence for a decline of proteasome activity upon aging and upon oxidative stress is provided by studies from our and other laboratories.

Increase of oxidatively modified protein is associated with a decrease of proteasome activity and content in aging epidermal cells.

For the process of aging in epidermal cells to be characterized, the status of oxidized and damaged protein accumulation and removal by the proteasome has been investigated. Modified protein content and proteasome activity were assayed in lysates of epidermal cells from donors of different ages. Increased levels of oxidized proteins, glycated proteins, and proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were observed in cells from old donors. At the same time, a decline of chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities of the proteasome was found in aging keratinocytes. This age-related decline of the proteasome peptidase activities can be explained, at least in part, by a decreased proteasome content as observed by immunoblotting and enzyme-linked immunosorbent assay. In keratinocyte cultures, a decrease of proteasome activity and content was observed upon serial passaging. In cultures, as well as in skin, an inverse relationship was found between the aging marker 1-galactosidase and the proteasome content. These results suggest that proteasome is downregulated during replicative senescence as well as in aged cells in vivo, possibly resulting in the accumulation of modified proteins.