Potential Influence of Age and Diabetes Mellitus Type 1 on MSH2 (MutS homolog 2) Expression in a Rat Kidney Tissue.

BACKGROUND: Homeostasis of proliferating tissues is strongly dependent on intact DNA. Both neoplastic and non-neoplastic diseases have been associated with MSH2 (MutS homolog 2, a mismatch repair protein) deficiency. In this study, we examined how age and diabetes mellitus influence the expression of MSH2 in the kidney. METHODS: To study the effect of age, three groups of healthy rats were formed: 2 months, 8 months, and 14 months old. Two groups of diabetic rats were formed: 8 months old and 14 months old. Expression of MSH2 in the kidney was studied by quantifying immunofluorescent staining. RESULTS: Age was identified as the main factor that influences MSH2 expression in kidneys. The effect of age followed parabolic dynamics, with peak expression at 8 months of age and similar levels at 2 and 14 months. Diabetes had an age-dependent effect, which manifested as the increase of MSH2 expression in 14-month-old diabetic rats in comparison to healthy animals. CONCLUSIONS: Age influences MSH2 expression in the kidney more than diabetes mellitus. Since ageing is a risk factor for kidney neoplasia, downregulation of MSH2 in older rats might represent one of the pro-oncogenic mechanisms of ageing at a molecular level.

Identification of bone morphogenetic protein 4 in the saliva after the placement of fixed orthodontic appliance.

BACKGROUND: This study was conducted in order to explore the effects of orthodontic tooth movement (OTM) on the changes of salivary proteome. This prospective observational pilot study recruited 12 healthy teenage boys with malocclusion treated with a fixed orthodontic appliance and 6 appropriate control participants. Saliva samples were collected a day before and at 0, 2, 7, and 30 days after initialization of treatment, corresponding to the initial, lag, and post-lag phases of OTM. Pooled samples were analyzed by liquid chromatography-mass spectrometry, ELISA, and Western blotting. To date, there is no published data on the presence of BMP molecules or their antagonists in the saliva or in the gingival cervical fluid related to orthodontic conditions. RESULTS: A total of 198 identified saliva proteins were classified based on their functional characteristics. Proteins involved in bone remodeling were observed exclusively 30 days post appliance placement, including bone morphogenetic protein 4 (BMP4), a BMP antagonist BMP-binding endothelial regulator, insulin-like growth factor-binding protein 3, cytoskeleton-associated protein 4, and fibroblast growth factor 5. Based on the analysis of protein interactions, BMP4 was found to have a central position in this OTM-related protein network. CONCLUSIONS: The placement of a fixed orthodontic appliance induced occurrence of proteins involved in bone remodeling in the saliva at a time corresponding to the post-lag period of OTM. Limitations of this study include a relatively small sample size, limited time of monitoring patients, and the lack of interindividual variability assessment.

Plasma levels and tissue expression of soluble TGFßrIII receptor in women with early-stage breast cancer and in healthy women: a prospective observational study.

BACKGROUND: Mammary carcinogenesis is partly regulated by the transforming growth factor beta (TGFß) signaling pathway. Its function in cancer progression and metastasis is highly dependent on disease stage, and it is likely modulated by the ratio of membrane-bound vs. soluble TGFßrIII (sTGFßrIII). In this prospective observational study, we assessed tissue expression and plasma levels of sTGFßrIII in healthy women, women with benign breast lesions and in early-stage breast cancer patients. METHODS: In a preliminary study, plasma sTGFßrIII levels were determined in 13 healthy women (age 19-40 years) at different phases of the ovarian cycle, and in 15 patients (age 35-75 years) at different times of the day. The main study assessed plasma concentrations of sTGFßrIII in: (i) 158 healthy women in whom breast lesions were excluded; (ii) 65 women with benign breast lesions; (iii) 147 women with newly diagnosed breast cancer classified as American Joint Committee on Cancer (AJCC) stages 0 to IIB. Completers provided blood samples before surgery and at 10-30 and 160-180 days after surgery. Plasma sTGFßrIII concentrations were determined using an indirect ELISA kit. Part of the removed tissues underwent immunohistochemical (IHC) staining and analysis of tissue TGFßrIII expression. RESULTS: There appeared no relevant variations in plasma sTGFßrIII levels at different times of the day or different ovarian cycle phases. Before surgery, breast cancer patients had somewhat higher sTGFßrIII than healthy women, or those with benign breast lesions (by 14.5 and 26 ng/mL, respectively), with a tendency of larger differences at higher age. This correlated with lower expression of TGFßrIII in breast cancer vs. healthy tissue samples. At 160-180 days after surgery, plasma sTGFßrIII levels in breast cancer patients declined by 23-26 ng/mL. CONCLUSIONS: Plasma sTGFßrIII levels do not seem to relevantly vary during the day or the ovarian cycle. The coinciding higher plasma levels in newly diagnosed cancer patients than in healthy subjects and lower TGFßrIII expression in the malignant than in healthy breast tissue suggest ectodomain shedding as a source of circulating sTGFßrIII. Decline in plasma levels after tumor removal supports such a view.

Changes in snail and SRF expression in the kidneys of diabetic rats during ageing.

BACKGROUND: Diabetic nephropathy is a progressive condition which develops for many years. We analyzed expression of Snail and serum response factor (SRF), epithelial-mesenchymal transition (EMT) regulatory transcription factors with a key role in renal fibrosis, in different renal areas of diabetic rats during ageing. METHODS: Male Sprague-Dawley rats were treated with 55 mg/kg streptozotocin (model of type 1 diabetes mellitus; DM group) or citrate buffer (control). DM group received insulin weekly to prevent ketoacidosis. After 2 weeks, 2, 6 and 12 months kidney samples were collected and analysed in different renal areas. RESULTS: Snail expression was located within cortex in proximal convoluted tubules, in control and DM groups, in the cytoplasm. Percentage of Snail-positive cells in control groups was high and decreased with time, whereas in DM groups the highest percentage was after 2 weeks. In all time points, smaller percentage of Snail expression was seen in DM groups compared to controls. SRF expression was mostly located in the proximal convoluted tubules, always in the cytoplasm. In control groups SRF was expressed in all time periods in proximal convoluted tubules, with decrement after 12 months. Percentage of SRF-positive cells was higher in control groups compared to DM in all time points, with the exception of 12 months. To a smaller degree, SRF expression was seen in the glomeruli and distal convoluted tubules, with more SRF positive cells in DM compared to their control groups. CONCLUSIONS: While Snail expression remained lower in diabetic tissues, compared to controls, expression of SRF increased in diabetic tissues in the second part of the year. These changes may need long time to develop, and, in line with earlier reports, it is possible that insulin treatment of DM rats once a week reduces possibility of EMT and development of renal fibrosis even in the long term.

Elevated plasma RANTES in fibrodysplasia ossificans progressiva - A novel therapeutic target?

Fibrodysplasia ossificans progressiva (FOP) is a rare hereditary disease caused by a mutation in the intracellular domain of the activin A receptor type I and is characterized by episodes (flare-ups) of progressive heterotopic endochondral ossification (HO) in the soft tissues. The mutation alone is not sufficient for the occurrence of HO since flare-ups are triggered by inflammation and activation of the innate immune system. A number of cellular and humoral mediators have been implicated in animal and in vitro models. Observations in humans support the inflammatory nature of the condition, but data on the involved mediators are variable. We hypothesize that for induction of flare-ups in patients with FOP increase in at least one of the pro-inflammatory cytokines is both essential and sufficient to trigger the entire process of the inflammatory cells influx resulting in the novel ectopic bone formation and we suggest that C-C motif ligand 5 (CCL5), a pro-inflammatory chemokine also known as Regulated on activation, normal T-cell expressed and secreted (RANTES), might be the key candidate. CCL5 is a chemoattractant for all cellular types implicated in HO and is produced by the cells of the tissue microenvironment at the sites of HO as well as by the pro-inflammatory cellular mediators. CCL5 induces ossification in cultured human pluripotent mesenchymal cells (hMSCs) and in the primary culture of monocytes from FOP patients (but not from their healthy relatives), stimulation with lipopolysaccharide induces CCL5 expression. Finally, in a pilot study we used a panel of 23 cytokines and chemokines to screen the plasma samples of three subjects: a female patient with FOP during a flare-up; a female patient with hyperostosis corticalis generalisata (van Buchem disease), another rare disease characterized by excessive bone formation at the sites where it regularly occurs that does not include inflammatory events; and a healthy woman without bone disorders. There appeared a rather clear-cut signal of a 2-fold higher level of CCL5 in the FOP patient vs. the healthy subject and the van Buchem patient. Evaluation of the hypothesis would require an international prospective study, with main motivation being the lack of a conclusive treatment as the major unmet need in FOP. A treatment targeting CCL5 receptor already exists and is used in HIV-infected patients.

Long-term streptozotocin diabetes impairs arachidonic and docosahexaenoic acid metabolism and ∆5 desaturation indices in aged rats.

We have investigated the long term effects of insulin dependent diabetes mellitus (IDDM) on the fatty acid profile of tissues in aging rats. For this purpose, a rat model for IDDM was established by streptozotocin application. The rats were randomly divided into four groups of 8 animals each: CON 6 (control group sacrificed after 6 months of the experiment), CON 12 (control group sacrificed after 12 months of the experiment), DM 6 (streptozotocin treated and sacrificed after 6 months of diabetes) and DM 12 (streptozotocin treated and sacrificed after 12 months of diabetes). The periods of 6 and 12 months were taken to observe the changes in lipid metabolism for chronic, long-term diabetes. Fatty acid profiles of the liver and skeletal muscle total lipids and phospholipids as well as desaturation indices for ∆6 desaturase (D6D), ∆5 desaturase (∆6D), ∆9 desaturase (∆9D) and de novo lipogenesis index (DNL) were estimated. Additionally the long-term effects (12 months) were tested in the brain, perirenal fat and bone marrow. The fatty acid composition of lipids was altered in IDDM rats in all tested tissues. The desaturation indices revealed the expected significant decrease in ∆9D and ∆5D indices in tested tissues, while indices for ∆6D were not influenced by diabetes. DNL revealed the strong inhibition of de novo lipogenesis in the liver tissue. Values for arachidonic C20:4n6 (arachidonic acid) significantly decreased in liver total lipids in DM 6 and DM 12 groups and in phospholipids in the DM 12 group. Surprisingly, values for C20:4n6 were also significantly lower in the brain tissue in the DM 12 group. Accumulation of C20:4n6 precursors (C18:2n6 and C20:3n6) was visible in all tissues. Docosahexaenoic acid (C22:6n3) significantly decreased in liver total lipids, liver phospholipids and in the brain phospholipids of the DM 12 group. The present results show that age could exacerbate the expected decrease in the liver synthesis of C20:4n6 in IDDM. Moreover, long-term diabetes could impair C22:6n3 synthesis in the liver and muscle, and incorporation of both important fatty acids into brain phospholipids. In conclusion, numerous changes in fatty acid composition are caused by long-term diabetes in aged rats. These changes could be involved in the pathogenesis of senile and diabetes-induced damage. The results could have clinical significance due to the increasing age of diabetic patients.