Gambling on putative biomarkers of osteoarthritis and osteochondrosis by equine synovial fluid proteomics

Osteoarthritis (OA) and osteochondrosis (OC) are two of the main challenges in orthopedics, whose definitive diagnosis is usually based on radiographic/arthroscopic evidences. Their early diagnosis should allow preventive or timely therapeutic actions, which are generally precluded from the poor relationships occurring between symptomatologic and radiographic evidences. These limitations should be overcome by improving the knowledge on articular tissue metabolism and on molecular factors regulating its normal homeostasis, also identifying novel OA and OC biomarkers suitable for their earlier diagnoses, whenever clinical/pathological inflammatory scenarios between these joint diseases seem somewhat related. To identify proteins involved in their aetiology and progression, we undertook a differential proteomic analysis of equine synovial fluid (SF), which compared the protein pattern of OA or OC patients with that of healthy individuals. Deregulated proteins in OA and OC included components related to inflammatory state, coagulation pathways, oxidative stress and matrix damage, which were suggestive of pathological alterations in articular homeostasis, plasma-SF exchange, joint nutritional status and vessel permeability. Some proteins seemed commonly deregulated in both pathologies indicating that, regardless of the stimulus, common pathways are affected and/or the animal joint uses the same molecular mechanisms to restore its homeostasis. On the other hand, the increased number of deregulated proteins observed in OA with respect to OC, together with their nature, confirmed the high inflammatory character of this disease. Some deregulated proteins in OA found a verification by analyzing the SF of injured arthritic joints following autologous conditioned serum treatment, an emergent therapy that provides positive results for both human and equine OA. Being the horse involved in occupational/sporting activities and considered as an excellent animal model for human joint diseases, our data provide suggestive information for tentative biomedical extrapolations, allowing to overcome the limitations in joint size and workload that are typical of other small animal models.     

Plasma protein changes in horse after prolonged physical exercise: a proteomic study

Physical exercise induces various stress responses and metabolic adaptations that have not yet been completely elucidated. Novel biomarkers are needed in sport veterinary medicine to monitor training levels and to detect subclinical conditions that can develop into exercise-related diseases. In this study, protein modifications in horse plasma induced by prolonged, aerobic physical exercise were investigated by using a proteomic approach based on 2-DE and combined mass spectrometry procedures. Thirty-eight protein spots, associated with expression products of 13 genes, showed significant quantitative changes; spots identified as membrane Cu amine oxidase, α-1 antitrypsin, α-1 antitrypsin-related protein, caeruloplasmin, α-2 macroglobulin and complement factor C4 were augmented in relative abundance after the race, while haptoglobin β chain, apolipoprotein A-I, transthyretin, retinol binding protein 4, fibrinogen γ chain, complement factor B and albumin fragments were reduced. These results indicate that prolonged physical exercise affects plasma proteins involved in pathways related to inflammation, coagulation, immune modulation, oxidant/antioxidant activity and cellular and vascular damage, with consequent effects on whole horse metabolism.     

Characterization of PbPga1, an Antigenic GPI-Protein in the Pathogenic Fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection.     

Proteomic evaluation of sheep serum proteins

Background

Mammary tumours frequently develop in female domestic cats being highly malignant in a large percentage of cases. Chemokines regulate many physiological and pathological processes including organogenesis, chemotaxis of inflammatory cells, as well as tumour progression and metastasization. In particular, the chemokine/receptor pair SDF-1/CXCR4 has been involved in the regulation of metastatic potential of neoplastic cells, including breast cancer. The aim of this study was the immunohistochemical defininition of the expression profile of CXCR4 in primary and metastatic feline mammary carcinomas and the evaluation of the role of SDF-1 in feline mammary tumour cell proliferation.

Results

A total of 45 mammary surgical samples, including 33 primary tumours (31 carcinomas and 2 adenomas), 6 metastases, and 4 normal mammary tissues were anlyzed. Tumor samples were collected from a total number of 26 animals, as in some cases concurrent occurrence of neoplasm in more than one mammary gland was observed. Tissues were processed for standard histological examination, and all lesions were classified according to the World Health Organization criteria. CXCR4 expression in neoplastic cells was evaluated by immunohistochemistry. The level of CXCR4 immunoreactivity was semi-quantitatively estimated as CXCR4 score evaluating both the number of positive cells and the intensity of staining. Six primary, fibroblast-free primary cultures were obtained from fresh feline mammary carcinomas and characterized by immunofluorescence for CXCR4 and malignant mammary cell marker expression. SDF-1-dependent in vitro proliferative effects were also assayed. CXCR4 expression was observed in 29 out of 31 malignant tissues with a higher CXCR4 score observed in 4 out of 6 metastatic lesions than in the respective primary tumours. In 2 benign lesions analyzed, only the single basaloid adenoma showed a mild positive immunostaining against CXCR4. Normal tissue did not show CXCR4 immunoreactivity. CXCR4 score was statistically significantly associated with the histological features of the samples, showing an increase accordingly with the degree of neoplastic transformation (from normal tissue to metastatic lesions). Finally, in the primary cultures obtained from 6 primary feline mammary carcinomas CXCR4 expression was detected in all cells and its activation by SDF-1 in vitro treatment caused a significant increase in the proliferation rate in 5 out of 6 tumours.

Conclusions

These results indicate that malignant feline mammary tumours commonly express CXCR4, with a higher level in malignant tumours, and, in most of the cases analysed, metastatic cells display stronger immunoreactivity for CXCR4 than the corresponding primary tumours. Moreover, CXCR4 activation in primary cultures of feline mammary carcinomas causes increase in the proliferative rate. Thus, SDF-1/CXCR4 system seems to play a tumorigenic in feline mammary gland malignancy and in vitro cultures from these tumour samples may represent an experimental model to investigate the biological and pharmacological role of this chemokinergic axis.     

The FTO gene is associated with adulthood obesity in the Mexican population

Common polymorphisms in the fat mass and obesity-associated gene (FTO) have shown strong association with obesity in several populations. In the present study, we explored the association of FTO gene polymorphisms with obesity and other biochemical parameters in the Mexican population. We also assessed FTO gene expression levels in adipose tissue of obese and nonobese individuals. The study comprised 788 unrelated Mexican-Mestizo individuals and 31 subcutaneous fat tissue biopsies from lean and obese women. FTO single-nucleotide polymorphisms (SNPs) rs9939609, rs1421085, and rs17817449 were associated with obesity, particularly with class III obesity, under both additive and dominant models (P = 0.0000004 and 0.000008, respectively). These associations remained significant after adjusting for admixture (P = 0.000003 and 0.00009, respectively). Moreover, risk alleles showed a nominal association with lower insulin levels and homeostasis model assessment of B-cell function (HOMA-B), and with higher homeostasis model assessment of insulin sensitivity (HOMA-S) only in nonobese individuals (P (dom) = 0.031, 0.023, and 0.049, respectively). FTO mRNA levels were significantly higher in subcutaneous fat tissue of class III obese individuals than in lean individuals (P = 0.043). Risk alleles were significantly associated with higher FTO expression in the class III obesity group (P = 0.047). In conclusion, FTO is a major risk factor for obesity (particularly class III) in the Mexican-Mestizo population, and is upregulated in subcutaneous fat tissue of obese individuals.     

Polymorphisms within the Novel Type 2 Diabetes Risk Locus MTNR1B Determine β-Cell Function

Background

Very recently, a novel type 2 diabetes risk gene, i.e., MTNR1B, was identified and reported to affect fasting glycemia. Using our thoroughly phenotyped cohort of subjects at an increased risk for type 2 diabetes, we assessed the association of common genetic variation within the MTNR1B locus with obesity and prediabetes traits, namely impaired insulin secretion and insulin resistance.

Methodology/Principal Findings

We genotyped 1,578 non-diabetic subjects, metabolically characterized by oral glucose tolerance test, for five tagging single nucleotide polymorphisms (SNPs) covering 100% of common genetic variation (minor allele frequency >0.05) within the MTNR1B locus (rs10830962, rs4753426, rs12804291, rs10830963, rs3781638). In a subgroup (N = 513), insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp, and in a further subgroup (N = 301), glucose-stimulated insulin secretion was determined by intravenous glucose tolerance test. After appropriate adjustment for confounding variables and Bonferroni correction for multiple comparisons, none of the tagging SNPs was reliably associated with measures of adiposity. SNPs rs10830962, rs4753426, and rs10830963 were significantly associated with higher fasting plasma glucose concentrations (p<0.0001) and reduced OGTT- and IVGTT-induced insulin release (p≤0.0007 and p≤0.01, respectively). By contrast, SNP rs3781638 displayed significant association with lower fasting plasma glucose levels and increased OGTT-induced insulin release (p<0.0001 and p≤0.0002, respectively). Moreover, SNP rs3781638 revealed significant association with elevated fasting- and OGTT-derived insulin sensitivity (p≤0.0021). None of the MTNR1B tagging SNPs altered proinsulin-to-insulin conversion.

Conclusions/Significance

In conclusion, common genetic variation within MTNR1B determines glucose-stimulated insulin secretion and plasma glucose concentrations. Their impact on β-cell function might represent the prevailing pathomechanism how MTNR1B variants increase the type 2 diabetes risk.     

CPT1c is localized in endoplasmic reticulum of neurons and has carnitine palmitoyltransferase activity

CPT1c is a carnitine palmitoyltransferase 1 (CPT1) isoform that is expressed only in the brain. The enzyme has recently been localized in neuron mitochondria. Although it has high sequence identity with the other two CPT1 isoenzymes (a and b), no CPT activity has been detected to date. Our results indicate that CPT1c is expressed in neurons but not in astrocytes of mouse brain sections. Overexpression of CPT1c fused to the green fluorescent protein in cultured cells demonstrates that CPT1c is localized in the endoplasmic reticulum rather than mitochondria and that the N-terminal region of CPT1c is responsible for endoplasmic reticulum protein localization. Western blot experiments with cell fractions from adult mouse brain corroborate these results. In addition, overexpression studies demonstrate that CPT1c does not participate in mitochondrial fatty acid oxidation, as would be expected from its subcellular localization. To identify the substrate of CPT1c enzyme, rat cDNA was overexpressed in neuronal PC-12 cells, and the levels of acylcarnitines were measured by high-performance liquid chromatography-mass spectrometry. Palmitoylcarnitine was the only acylcarnitine to increase in transfected cells, which indicates that palmitoyl-CoA is the enzyme substrate and that CPT1c has CPT1 activity. Microsomal fractions of PC-12 and HEK293T cells overexpressing CPT1c protein showed a significant increase in CPT1 activity of 0.57 and 0.13 nmol.mg(-1).min(-1), respectively, which is approximately 50% higher than endogenous CPT1 activity. Kinetic studies demonstrate that CPT1c has similar affinity to CPT1a for both substrates but 20-300 times lower catalytic efficiency.     

Toll-like receptor 3 activation induces antiviral immune responses in mouse sertoli cells

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and elicit antimicrobial immune responses. In the testis, viruses can induce pathological conditions, such as orchitis, and may participate in the etiology of testicular cancer; however, the molecular mechanisms involved remain under investigation. It has been suggested that because they constitutively express interferon (IFN)-inducible antiviral proteins, Sertoli cells participate in the testicular antiviral defense system. Previously, we demonstrated a key function of mouse Sertoli cells in the bactericidal testicular defense mechanism mediated by a panel of TLRs. To better characterize the potential role of Sertoli cells in the response against testicular viral infections, we investigated the TLR3 expression and function in these cells. Sertoli cells express TLR3, and under stimulation with the synthetic double-stranded RNA analogue poly (I:C), they produce the proinflammatory molecule ICAM1 and secrete functionally active CCL2 chemokine. Using both pharmacological and genetic approaches, we found that these effects are TLR3-dependent. Moreover, using ELISA, we found that IFNA is constitutively produced and not further inducible, whereas IFNB1 is absent and dramatically induced only by transfected poly (I:C), indicating different control mechanisms underlying IFNA and IFNB1 production. To conclude, poly (I:C) elicits both inflammatory and antiviral responses in Sertoli cells.     

C825T Polymorphism of the G Protein β3 Subunit Is Associated with Obesity but Not with Insulin Sensitivity

Objective: The common C825T polymorphism of the gene that encodes the G protein β₃ subunit has been shown to influence lipolysis in human adipocytes and to be associated with hypertension, body fat distribution, and obesity. In addition, it has been shown to be associated with insulin resistance in a small group of hypertensive subjects. We investigated whether this polymorphism contributed to the variability in obesity in our population from southern Germany and whether it was associated with insulin sensitivity of lipolysis and/or glucose disposal. Research Methods and Procedures: We determined percentage body fat, body fat distribution, glucose tolerance [oral glucose‐tolerance test (OGTT)], insulin sensitivity, and serum free fatty acids using data from OGTTs (N = 774) and clamp (euglycemic hyperinsulinemic clamp, N = 216) in normal and impaired glucose tolerant subjects who were genotyped for this polymorphism. Results: Compared with noncarriers of the C825T mutation, subjects with the C825T variant (prevalence ～32%) had higher percentage body fat (p = 0.02) and higher BMI (p = 0.03). No conclusive effect was seen on serum free fatty acids measured either during fasting or at the end of a 2‐hour OGTT. Insulin sensitivity determined during the OGTT and during the clamp, both adjusted for age, gender, and percentage body fat, was not different between the genotypes (p = 0.33 and p = 0.48, respectively). Discussion: We have concluded that the C825T polymorphism in the G protein β₃ subunit played an important role in the determination of obesity in this German population. However, it probably had no direct effects on insulin sensitivity of lipolysis and glucose disposal.