Evidence That ß1-Integrin Is Required for the Anti-Viability and Anti-Proliferative Effect of Resveratrol in CRC Cells.

The ß1-integrin receptor is broadly expressed on tumor and other cells in the tumor microenvironment (TME), and is an unfavorable prognostic factor for cancers. Nature-derived resveratrol has preventive and apoptotic effects on tumors, but whether resveratrol can exert its suppressive actions on TME-induced tumorigenesis through ß1-integrin on the surface of CRC cells is still unknown. HCT116 or SW480 cells were exposed to inhibitory antibodies against ß1-integrin, bacitracin (selective ß1-integrin inhibitor), integrin-binding RGD (Arg-Gly-Asp) peptide, and/or resveratrol. We evaluated the anti-tumor actions and signaling impacts of resveratrol in colorectal cancer (CRC)-TME. We found that resveratrol completely altered the ß1-integrin distribution pattern and expression on the surface of CRC cells in TME. Moreover, resveratrol down-regulated CRC cell proliferation, colony formation, viability, and up-regulated apoptosis in a concentration-dependent way. These actions of resveratrol were antagonized mainly by inhibitory antibodies against ß1-integrin but not ß5-integrin, and by an integrin-binding RGD peptide but not by RGE peptide, and by bacitracin in TME. Similarly, resveratrol-blocked TME-induced p65-NF-kB and its promoted gene markers linked to proliferation (cyclin D1), invasion (focal adhesion kinase, FAK), or apoptosis (caspase-3), were largely abrogated by anti-ß1-integrin or RGD peptide, suggesting that ß1-integrin is a potential transmission pathway for resveratrol/integrin down-stream signaling in CRC cells. The current results highlight, for the first time, the important gateway role of ß1-integrins as signal carriers for resveratrol on the surfaces of HCT116 and SW480 cells, and their functional cooperation for the modulatory effects of resveratrol on TME-promoted tumorigenesis.

Curcumin Attenuates Environment-Derived Osteoarthritis by Sox9/NF-kB Signaling Axis.

Inflammation has a fundamental impact on the pathophysiology of osteoarthritis (OA), a common form of degenerative arthritis. It has previously been established that curcumin, a component of turmeric (Curcuma longa), has anti-inflammatory properties. This research evaluates the potentials of curcumin on the pathophysiology of OA in vitro. To explore the anti-inflammatory efficacy of curcumin in an inflamed joint, an osteoarthritic environment (OA-EN) model consisting of fibroblasts, T-lymphocytes, 3D-chondrocytes is constructed and co-incubated with TNF-α, antisense oligonucleotides targeting NF-kB (ASO-NF-kB), or an IkB-kinase (IKK) inhibitor (BMS-345541). Our results show that OA-EN, similar to TNF-α, suppresses chondrocyte viability, which is accompanied by a significant decrease in cartilage-specific proteins (collagen II, CSPG, Sox9) and an increase in NF-kB-driven gene proteins participating in inflammation, apoptosis, and breakdown (NF-kB, MMP-9, Cox-2, Caspase-3). Conversely, similar to knockdown of NF-kB at the mRNA level or at the IKK level, curcumin suppresses NF-kB activation, NF-kB-promotes gene proteins derived from the OA-EN, and stimulates collagen II, CSPG, and Sox9 expression. Furthermore, co-immunoprecipitation assay shows that curcumin reduces OA-EN-mediated inflammation and chondrocyte apoptosis, with concomitant chondroprotective effects, due to modulation of Sox-9/NF-kB signaling axis. Finally, curcumin selectively hinders the interaction of p-NF-kB-p65 directly with DNA-this association is disrupted through DTT. These results suggest that curcumin suppresses inflammation in OA-EN via modulating NF-kB-Sox9 coupling and is essential for maintaining homeostasis in OA by balancing chondrocyte survival and inflammatory responses. This may contribute to the alternative treatment of OA with respect to the efficacy of curcumin.

Intestinal microsporidia infection among cat owners and non-pet owners in Iran: a case-control study.

Microsporidia is a group of spore-forming microorganisms with zoonotic potential. This study aimed to compare intestinal microsporidia infections in cat owners and non-pet owners. In total, 210 fecal samples were collected from indoor cats, cat owners, and non-pet owners. DNA extraction was performed and the small subunit ribosomal RNA (SSU rRNA) gene was amplified. To characterize the genotypes, the internal transcribed spacer (ITS) fragment was amplified and sequenced. The phylogenetic trees were drawn to evaluate the relationship among Enterocytozoon bieneusi isolates. Two (2.9%) and one (1.4%) fecal samples from cat owners and one (1.4%) and two (2.9%) fecal samples from non-pet owners were positive for E. bieneusi and Encephalitozoon intestinalis, respectively. E. bieneusi was detected in two cat samples (2.9%). Same infection was not seen between infected cats and their owners. There was no significant difference between the prevalence rate of microsporidia among the cat owners and non-pet owners. Indeed, the genotypes L and type IV were seen in cats, while the genotype D was only detected in human. In this study, E. bieneusi and E. intestinalis were more prevalent among the cat owners and non-pet owners, respectively. Indeed, the higher prevalence of E. bieneusi in cats and their owners might be resulted from the worldwide distribution of this species.

Resveratrol Suppresses Cross-Talk between Colorectal Cancer Cells and Stromal Cells in Multicellular Tumor Microenvironment: A Bridge between In Vitro and In Vivo Tumor Microenvironment Study.

The interaction between tumor cells and the tumor microenvironment (TME) is an important process for the development of tumor malignancy. Modulation of paracrine cross-talk could be a promising strategy for tumor control within the TME. The exact mechanisms of multi-targeted compound resveratrol are not yet fully understood. Whether resveratrol can modulate paracrine signal transduction-induced malignancy in the multicellular-TME of colorectal cancer cells (CRC) was investigated. An in vitro model with 3D-alginate HCT116 cells in multicellular-TME cultures (fibroblast cells, T-lymphocytes) was used to elucidate the role of TNF-ß, Sirt1-ASO and/or resveratrol in the proliferation, invasion and cancer stem cells (CSC) of CRC cells. We found that multicellular-TME, similar to TNF-ß-TME, promoted proliferation, colony formation, invasion of CRC cells and enabled activation of CSCs. However, after co-treatment with resveratrol, the malignancy of multicellular-TME reversed to HCT116. In addition, resveratrol reduced the secretion of T-lymphocyte/fibroblast (TNF-ß, TGF-ß3) proteins, antagonized the T-lymphocyte/fibroblast-promoting NF-κB activation, NF-κB nuclear translocation and thus the expression of NF-κB-promoting biomarkers, associated with proliferation, invasion and survival of CSCs in 3D-alginate cultures of HCT116 cells induced by TNF-ß- or multicellular-TME, but not by Sirt1-ASO, indicating the central role of this enzyme in the anti-tumor function of resveratrol. Our results suggest that in vitro multicellular-TME promotes crosstalk between CRC and stromal cells to increase survival, migration of HCT116 and the resveratrol/Sirt1 axis suppresses this loop by modulating paracrine agent secretion and NF-κB signaling. Fibroblasts and T-lymphocytes are promising targets for resveratrol in the prevention of CRC metastasis.

Investigation of the antimicrobial activity of a short cationic peptide against promastigote and amastigote forms of Leishmania major (MHRO/IR/75/ER): An in vitro study.

Cutaneous leishmaniasis is one of the most endemic global health problems in many countries all around the world. Pentavalent antimonial drugs constitute the first line of leishmaniasis treatment; however, resistance to these drugs is a serious problem. Therefore, new therapies with new modes of action are urgently needed. In the current study, we examined antimicrobial activity of CM11 hybrid peptide (WKLFKKILKVL-NH2) against promastigote and amastigote forms of L. major (MHRO/IR/75/ER). In vitro anti-leishmanial activity was identified against L. major by parasite viability and metabolic activity after exposure to different peptide concentration. In the presentt study, we demostrated that different concentrations of CM11 result in dose dependent growth inhibition of Leishmania promastigotes. Furthermore, we demostrated that CM11 peptide has significant anti-leishmanial activities on amastigotes. Our results demonstrated that CM11 antimicrobial peptide may provide an alternative therapeutic approach for L. major treatment.

The interaction of central nitrergic and GABAergic systems on food intake in neonatal layer-type chicks.

Most physiological behaviors such as food intake are controlled by the hypothalamus and its nuclei. It has been demonstrated that injection of the paraventricular nucleus of the hypothalamus with nitric oxide (NO) donors elicited changes in the concentration of some amino acids, including GABA. Also, central nitrergic and GABAergic systems are known to provide inputs to the paraventricular nucleus and are involved in food intake control. Therefore, the present study examines the probable interaction of central nitrergic and GABAergic systems on food intake in neonatal layer-type chicks. The results of this study showed that intracerebroventricular (ICV) injection of L-arginine (400 and 800 nmol), as a NO donor, significantly decreased food intake (P < 0.001), but ICV injection of Nω-Nitro-L-arginine methyl ester (L-NAME) (200 and 400 nmol), a NO synthesis inhibitor, increased food intake (P < 0.001). In addition, the orexigenic effect of gaboxadol (0.2 µg), a GABAA agonist, was significantly attenuated in ICV co-injection of L-arginine (200 nmol) and gaboxadol (0.2 µg) (P < 0.001), but it was significantly amplified in ICV co-injection of L-NAME (100 nmol) and gaboxadol (0.2 µg) (P < 0.001). On the other hand, the orexigenic effect of baclofen (0.2 µg), a GABAB agonist, did not change in ICV co-injection of L-arginine (200 nmol) or L-NAME (100 nmol) with baclofen (0.2 µg) (P > 0.05). Also, the hypophagic effect of L-arginine (800 nmol) was significantly amplified in ICV co-injection of picrotoxin (0.5 µg), a GABAA antagonist, or CGP54626 (21 ng), a GABAB antagonist, with L-arginine (800 nmol) (P < 0.001). These results probably suggest an interaction of central nitrergic and GABAergic systems on food intake in neonatal layer-type chicks and GABAA receptors play a major role in this interaction.

Sirtuin-1 (SIRT1) is required for promoting chondrogenic differentiation of mesenchymal stem cells.

Sirtuin-1 (SIRT1), NAD(+)-dependent deacetylase, has been linked to anabolic effects in cartilage, although the mechanisms of SIRT1 signaling during differentiation of mesenchymal stem cells (MSCs) to chondrocytes are poorly understood. Therefore, we investigated the role of SIRT1-mediated signaling during chondrogenic differentiation of MSCs in vitro. High density and alginate cultures of MSCs were treated with chondrogenic induction medium with/without the SIRT1 inhibitor nicotinamide, antisense oligonucleotides against SIRT1 (SIRT1-ASO), IL-1ß, and/or resveratrol. Transient transfection of MSCs with SIRT1-antisense oligonucleotides, nicotinamide, and IL-1ß inhibited chondrogenesis-induced down-regulation of cartilage-specific proteins, cartilage-specific transcription factor Sox9, and enhanced NF-κB-regulated gene products involved in the inflammatory and degradative processes in cartilage (MMP-9, COX-2, and caspase-3), and NF-κB phosphorylation, acetylation, and activation of IκBα kinase. In contrast, the SIRT1 activator resveratrol or BMS-345541 (inhibitor of IKK) inhibited IL-1ß- and NAM-induced suppression of cartilage-specific proteins, Sox9, and up-regulation of NF-κB-regulated gene products. Moreover, SIRT1 was found to interact directly with NF-κB and resveratrol-suppressed IL-1ß and NAM but not SIRT1-ASO-induced NF-κB phosphorylation, acetylation, and activation of IκBα kinase. Knockdown of SIRT1 by mRNA abolished the inhibitory effects of resveratrol on inflammatory and apoptotic signaling and Sox9 expression, suggesting the essential role of this enzyme. Finally, the modulatory effects of resveratrol were found to be mediated at least in part by the association between SIRT1 and Sox9. These results indicate for the first time that SIRT1 supports chondrogenic development of MSCs at least in part through inhibition/deacetylation of NF-κB and activation of Sox9.

Curcumin potentiates antitumor activity of 5-fluorouracil in a 3D alginate tumor microenvironment of colorectal cancer.

BACKGROUND: To overcome the limitations of animal-based experiments, 3D culture models mimicking the tumor microenvironment in vivo are gaining attention. Herein, we investigated an alginate-based 3D scaffold for screening of 5-fluorouracil (5-FU) or/and curcumin on malignancy of colorectal cancer cells (CRC). METHODS: The potentiation effects of curcumin on 5-FU against proliferation and metastasis of HCT116 cell and its corresponding isogenic 5-FU-chemoresistant cells (HCT116R) were examined in a 3D-alginate tumor model. RESULTS: CRC cells encapsulated in alginate were able to proliferate in 3D-colonospheres in a vivo-like phenotype and invaded from alginate. During cultivation of cells in alginate, we could isolate 3 stages of cells, (1) alginate proliferating (2) invasive and (3) adherent cells. Tumor-promoting factors (CXCR4, MMP-9, NF-κB) were significantly increased in the proliferating and invasive compared to the adherent cells, however HCT116R cells overexpressed factors in comparison to the parental HCT116, suggesting an increase in malignancy behavior. In alginate, curcumin potentiated 5-FU-induced decreased capacity for proliferation, invasion and increased more sensitivity to 5-FU of HCT116R compared to the HCT116 cells. IC50 for HCT116 to 5-FU was 8nM, but co-treatment with 5 µM curcumin significantly reduced 5-FU concentrations in HCT116 and HCT116R cells (0.8nM, 0.1nM, respectively) and these effects were accompanied by down-regulation of NF-κB activation and NF-κB-regulated gene products. CONCLUSIONS: Our results demonstrate that the alginate provides an excellent tumor microenvironment and indicate that curcumin potentiates and chemosensitizes HCT116R cells to 5-FU-based chemotherapy that may be useful for the treatment of CRC and to overcome drug resistance.

Application of recombinant Cryptosporidium parvum P23 for isolation and prevention.

Cryptosporidium parvum is a coccidian protozoan that causes diarrhea in immunocompromised humans and newborn animals. Billions of oocysts of C. parvum can be released from the infected calves and can contaminate the environment. The severity of the disease depends on the immunological status of the individual. Oocysts of Cryptosporidium are extremely resistant to many environmental stresses, and no effective disinfectant and curative agent against this organism is available. In our study, recombinant C. parvum P23 was prepared for application in the isolation and prevention of cryptosporidiosis. P23 is a glycoprotein that belongs to a family of protein of 23-27 kDa and is believed to be expressed in the different life stages of the parasite. Immunostaining analysis using the indirect fluorescent antibody test showed that P23 could be recognized on the surface of the oocysts. The antibody prepared in rabbit against P23 was bound to Sepharose 4B and used for the isolation of oocysts. The results showed that the prepared column was able to bind specifically only the oocysts. The effect of specific recombinant C. parvum IgY antibody against infection with C. parvum was examined in a mouse model. For this aim, purified egg yolk antibody prepared from immunized hens was used to analyze the protective effect of recombinant P23 specific antibody in immunosuppressed adult mice. The results showed more than 70% reduction in oocyst shedding after challenge with 1 × 10(4) oocysts. These results support previous studies of other investigators regarding the protective effect of P23 as an antigen against C. parvum infection and showed that it could be possible to design a passive immunization strategy against C. parvum based on the anti-P23 yolk antibody in animals and immunosuppressed humans.

Calebin A modulates inflammatory and autophagy signals for the prevention and treatment of osteoarthritis.

Introduction: Osteoarthritis (OA) is associated with excessive cartilage degradation, inflammation, and decreased autophagy. Insufficient efficacy of conventional monotherapies and poor tissue regeneration due to side effects are just some of the unresolved issues. Our previous research has shown that Calebin A (CA), a component of turmeric (Curcuma longa), has pronounced anti-inflammatory and anti-oxidative effects by modulating various cell signaling pathways. Whether CA protects chondrocytes from degradation and apoptosis in the OA environment (EN), particularly via the autophagy signaling pathway, is however completely unclear. Methods: To study the anti-degradative and anti-apoptotic effects of CA in an inflamed joint, an in vitro model of OA-EN was created and treated with antisense oligonucleotides targeting NF-κB (ASO-NF-κB), and IκB kinase (IKK) inhibitor (BMS-345541) or the autophagy inhibitor 3-methyladenine (3-MA) and/or CA to affect chondrocyte proliferation, degradation, apoptosis, and autophagy. The mechanisms underlying the CA effects were investigated by MTT assays, immunofluorescence, transmission electron microscopy, and Western blot analysis in a 3D-OA high-density culture model. Results: In contrast to OA-EN or TNF-α-EN, a treatment with CA protects chondrocytes from stress-induced defects by inhibiting apoptosis, matrix degradation, and signaling pathways associated with inflammation (NF-κB, MMP9) or autophagy-repression (mTOR/PI3K/Akt), while promoting the expression of matrix compounds (collagen II, cartilage specific proteoglycans), transcription factor Sox9, and autophagy-associated proteins (Beclin-1, LC3). However, the preventive properties of CA in OA-EN could be partially abrogated by the autophagy inhibitor 3-MA. Discussion: The present results reveal for the first time that CA is able to ameliorate the progression of OA by modulating autophagy pathway, inhibiting inflammation and apoptosis in chondrocytes, suggesting that CA may be a novel therapeutic compound for OA.

Evaluation of Nanonanoliposomal Curcumin on Cutaneous Leishmaniasis Skin Lesions Caused by Leishmania major in BALB/c Mice.

Background: Curcumin is an extract of rhizome turmeric (diferuloylmethane), with antioxidant, anti-inflammatory, antimicrobial, and anti-parasitic properties, which making it a potential candidate for the treatment of leishmaniasis. The aim of the presented study was to evaluate curcumin as possible candidate for treatment of cutaneous leishmaniasis. Methods: We investigated the physicochemical properties and anti-leishmanial effects of nanoliposomal curcumin (40, 80, and 120 µM) in Leishmania major (MRHO/IR/75/ER) infected BALB/c mice at the faculty of Veterinary Medicinem University of Tehran, Iran. For this aim, L. major promastigotes (MHROM/IR/75/ER) at stationary phase (2×106) were inoculated sub-cutaneously into the upper area of the tail in BALB/c mice (six groups, n= 10 per group). For evaluation of nanoliposomal curcumin, the zeta potential, particle size and stability of nanoliposomal curcumin was determined. Furthermore, the anti-leishmanial effects of nanoliposomal curcumin formulation on the lesion sizes was determined and the parasite burden in the leishmania induced lesion was performed using semi quantitative PCR. Results: Treatment of L. major infected BALB/c mice with nanoliposomal curcumin led to a reduction in the kinetic of the skin lesion size development. The semi quantitative PCR analysis of DNA extracted from the lesions showed reduction of parasite burden. The most effective treatment could be found in 80 µM nanoliposomal curcumin. Treatment with Glucantime, as a positive control, also showed a nearly similar effect compared to the effect of 80 µM nanoliposomal curcumin. Conclusion: Nanoliposomal curcumin could be considered as a potential drug against cutaneous leishmaniasis caused by L. major in susceptible animal models.

Sirt-1 is required for the inhibition of apoptosis and inflammatory responses in human tenocytes.

Tendon overuse injuries and tendinitis are accompanied by catabolic processes and apoptosis of tenocytes. However, the precise molecular mechanisms of the destructive processes in tendon are not fully understood. Sirt-1, a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase, has been linked to transcriptional silencing and appears to play a key role in inflammation. The purpose of this study was to examine whether down-regulation of Sirt-1 using antisense oligonucleotides (ASO) affects inflammatory and apoptotic signaling in tenocytes. Transient transfection of tenocytes with ASO against Sirt-1 induced expression of Bax and other proteins involved in apoptosis (cleaved caspase-3 and poly(ADP-ribose)polymerase), acetylation of tumor suppressor p53, and mitochondrial degradation. Interestingly, Sirt-1 was found to interact directly with p53. In contrast, Sirt-1 activator resveratrol inhibited interleukin-1ß (IL-1ß)- and nicotinamide-induced NF-κB activation and p65 acetylation and suppressed the activation of IκB-α kinase. Resveratrol also reversed the IL-1ß- or nicotinamide-induced up-regulation of various gene products that mediate inflammation (cyclooxygenase-2) and matrix degradation (matrix metalloproteinase-9) that are known to be regulated by NF-κB. Knockdown of Sirt-1 by using ASO abolished the inhibitory effects of resveratrol on inflammatory and apoptotic signaling including Akt activation and SCAX suppression. Down-regulation of histone deacetylase Sirt-1 by mRNA interference abrogated the effect of resveratrol on NF-κB suppression, thus highlighting the crucial homeostatic role of this enzyme. Overall, these results suggest for the first time that Sirt-1 can regulate p53 and NF-κB signaling via deacetylation, demonstrating a novel role for resveratrol in the treatment of tendinitis/tendinopathy.

Resveratrol modulates interleukin-1ß-induced phosphatidylinositol 3-kinase and nuclear factor κB signaling pathways in human tenocytes.

Resveratrol, an activator of histone deacetylase Sirt-1, has been proposed to have beneficial health effects due to its antioxidant and anti-inflammatory properties. However, the mechanisms underlying the anti-inflammatory effects of resveratrol and the intracellular signaling pathways involved are poorly understood. An in vitro model of human tenocytes was used to examine the mechanism of resveratrol action on IL-1ß-mediated inflammatory signaling. Resveratrol suppressed IL-1ß-induced activation of NF-κB and PI3K in a dose- and time-dependent manner. Treatment with resveratrol enhanced the production of matrix components collagen types I and III, tenomodulin, and tenogenic transcription factor scleraxis, whereas it inhibited gene products involved in inflammation and apoptosis. IL-1ß-induced NF-κB and PI3K activation was inhibited by resveratrol or the inhibitors of PI3K (wortmannin), c-Src (PP1), and Akt (SH-5) through inhibition of IκB kinase, IκBα phosphorylation, and inhibition of nuclear translocation of NF-κB, suggesting that PI3K signaling pathway may be one of the signaling pathways inhibited by resveratrol to abrogate NF-κB activation. Inhibition of PI3K by wortmannin attenuated IL-1ß-induced Akt and p65 acetylation, suggesting that p65 is a downstream component of PI3K/Akt in these responses. The modulatory effects of resveratrol on IL-1ß-induced activation of NF-κB and PI3K were found to be mediated at least in part by the association between Sirt-1 and scleraxis and deacetylation of NF-κB and PI3K. Overall, these results demonstrate that activated Sirt-1 plays an essential role in the anti-inflammatory effects of resveratrol and this may be mediated at least in part through inhibition/deacetylation of PI3K and NF-κB.

Resveratrol induces apoptosis by modulating the reciprocal crosstalk between p53 and Sirt-1 in the CRC tumor microenvironment.

Introduction: P53 represents a key player in apoptosis-induction in cancers including colorectal cancer (CRC) that ranks third worldwide in cancer prevalence as well as mortality statistics. Although a pro-apoptotic effect of resveratrol has been repeatedly proven in CRC cells, its pathway mechanisms are not completely understood, as there are controversial statements in the literature regarding its activation or inhibition of the counteracting proteins Sirt-1 and p53. Methods: CRC cells as wild-type (HCT-116 WT) or p53-deficient (HCT-116 p53-/-) were cultured using multicellular tumor microenvironment (TME) cultures containing T-lymphocytes and fibroblasts to elucidate the role of p53/Sirt-1 modulation in resveratrol's concentration-dependent, pro-apoptotic, and thus anti-cancer effects. Results: Resveratrol dose-dependently inhibited viability, proliferation, plasticity as well as migration, and induced apoptosis in HCT-116 WT more effectively than in HCT-116 p53-/- cells. Moreover, resveratrol stimulated Sirt-1 expression when administered at low concentrations (<5µM) but suppressed it when added at high concentrations (>10µM) to CRC-TME. In parallel, similar to the knockdown of Sirt-1 at the mRNA level, treatment with high-concentration resveratrol boosted the acetylation of p53, the expression of p21, Bax, cytochrome C, caspase-3, and ultimately induced apoptosis in CRC WT but not in CRC p53-/- cells. Notably, increasing concentrations of resveratrol were found to promote hyperacetylation of p53 and FOXO3a as post-translational substrates of Sirt-1, indicating a negative regulatory loop between Sirt-1 and p53. Discussion: These results demonstrate for the first time, a negative reciprocal crosstalk between the regulatory circuits of p53 and Sirt-1, consequently, apoptosis induction by higher resveratrol concentrations in CRC-TME.

RFLP Analysis of Fragments of the 18S rRNA and Cox1 Genes to Identify Sarcocystis cruzi in Water Buffalo (Bubalus bubalis) In Guilan Province, North of Iran.

Background: Sarcocystosis is a zoonotic disease worldwide caused by Sarcocystis spp., some of these species can show clinical and subclinical manifestations, resulting in financial losses. Our study was performed for identifying Sarcocystis sp., in slaughtered buffalo by PCR-RFLP based strategy with sequencing in Guilan, North of Iran. Methods: Overall, 400 fresh muscle samples were prepared via naked-eye observation from 100 buffaloes (esophagus, diaphragm, shoulder, and thigh), followed by the digestion of samples. The PCR was done to amplify partial parts of the 18S rRNA and mitochondrial cytochrome c oxidase subunit I (Cox1) genes. Then, the PCR products were digested by endonuclease SspI, DraI, and FokI. Sequencing of all species was done to confirm the RFLP results. Results: Five macroscopic cysts (1.25%) were visible in the sample by naked-eye examination. Furthermore, 293 samples (73.25%) were found to be Sarcocystis sp. positive through tissue digestion and microscopic observation, whereas 376 samples (94%) were positive by PCR. In addition, the findings of PCR-RFLP and nucleotide sequence samples exhibited the infection of buffaloes with S. cruzi. Conclusion: Based on the data presented herein, Bovine sarcocystosis caused by S. cruzi is very common in buffalo in the Guilan region. Regarding the high prevalence of sarcocystosis, developing disease control and prevention policies for buffaloes is necessary, and a change of attitude in traditional farming is recommended.

Morphological characteristics of Echinococcus granulosus derived from buffalo in Iran.

Cystic echinococcosis is a significant parasitic disease in Iran, where a variety of animals act as intermediate hosts. In this study, 25 isolates of Echinococcus granulosus obtained from water buffalo from various parts of Iran were characterized on the basis of the morphology of the metacestode and the adult worm. The characteristics of protoscoleces from the different studied areas were nearly similar. They showed 2 rows of alternating large and small hooks and their shapes were smooth in outline. In contrast to the protoscoleces, the adult rostellar hooks showed a rough outline. The results showed that the total length, the blade lengths of the large and small hooks and the number of hooks are almost similar to those isolated from sheep but significantly different from those isolated from camels. The growth rates of adult E. granulosus (total worm length, segmentation and maturation) of buffalo origin, at 35 and 41 days post-infection of dogs, were nearly comparable to the common sheep strain. The form of the strobila and the morphology of the reproductive system were also similar to those of sheep origin. This suggests that the common sheep strain (G1) of E. granulosus may also use buffaloes as its intermediate host.

Detection of Theileria orientalis in Iran by semi-nested PCR.

In order to identify and differentiate Theileria orientalis in cattle which may be infected with Theileria annulata simultaneously, a semi-nested PCR was performed. Thus, 160 blood samples were collected from apparently healthy native cattle in Golestan province of northern Iran, during 2009 to 2011. The Tbs-S/Tbs-A primer set derived from the 18S rRNA encoding gene was used for first PCR amplification, and the amplified sequence weight by this primer set for Theileria sp. was 426-430 bp. Then, DNA solution from purified PCR product was used for the semi-nested PCR analysis. The first PCR product amplified using T. orientalis primer set (To-S/Tbs-A) derived from the 18SrRNA encoding gene, and this specific primer weight was 235 bp. Also, the first PCR product amplified using T. annulata primer set (Ta-S/Tbs-A) derived from the 18SrRNA encoding gene and this specific primer weight was 193 bp. Having extracted DNA of each sample, using Tbs-S/Tbs-A primer set for PCR and analyzing the PCR products on the 2% agarose gel electrophoresis, 13 out of 160 blood samples (8.12%) were positive for Theileria sp. Meanwhile, performing semi-nested PCR with T. orientalis-specific primers, 9 out of 13 blood samples (5.62%) were positive and performing semi-nested PCR with T. annulata-specific primers, 12 out of 13 blood samples (7.5%) were also positive. This molecular assay approves the presence of T. orientalis in the native cattle of northern parts of Iran for the first time. In addition, this procedure will detect the concurrent infection of T. orientalis and T. annulata in the cattle too.

Babesia ovis as the main causative agent of sheep babesiosis in Iran.

Babesiosis is a haemoparasitic disease with high economical losses in livestock industry worldwide. The early diagnosis and successful therapy of babesiosis belong to the key steps of control and health management of livestock. Ethanol-fixed blood samples of 400 sheep were analyzed for Babesia infection. Reverse line blot (RLB) was established specifically for Theileria lestoquardi, Theileria (China 1), Theileria (China 2), Theileria ovis, Theileria separata, Babesia ovis, Babesia motasi, Babesia crassa, and Babesia (Lintan). The DNA was extracted from the ethanol-fixed blood samples and amplified with a common primer pair derived from 18S rRNA gene, amplifying both Theileria spp. as well as Babesia spp. Regarding the differences in the length of nucleotide sequences of the polymerase chain reaction (PCR) products obtained from Theileria spp. and Babesia spp., the PCR products derived from Babesia spp. were out screened and analyzed by RLB. The RLB analysis showed that 28 samples within the 400 blood samples were B. ovis positive. No B. motasi, B. crassa, or Babesia (Lintan) could be detected. The sequence analysis of one PCR product as a representative for other B. ovis-positive PCR products confirmed the results of RLB. Our results and the results of other investigators showed that B. ovis could be considered as a main causative agent of sheep babesiosis in Iran. Furthermore, our results also showed that RLB can be used as a reliable method for a simultaneous differentiation of Theileria and Babesia species from each other.

ß1-Integrin plays a major role in resveratrol-mediated anti-invasion effects in the CRC microenvironment.

Background: Tumor microenvironment (TME) is one of the most important factors in tumor aggressiveness, with an active exchange between tumor and other TME-associated cells that promotes metastasis. The tumor-inhibitory effect of resveratrol on colorectal cancer (CRC) cells has been frequently reported. However, whether resveratrol can specifically suppress TME-induced CRC invasion via ß1-integrin receptors has not been fully elucidated yet. Methods: Two CRC cell lines (HCT116, RKO) were cultured in multicellular, pro-inflammatory 3D-alginate TME cultures (containing fibroblasts, T-lymphocytes) to investigate the role of ß1-integrin receptors in the anti-invasive and anti-metastatic effect of resveratrol by antisense oligonucleotides (ASO). Results: Our results show that resveratrol dose-dependently suppressed the migration-promoting adhesion adapter protein paxillin and simultaneously enhanced the expression of E-cadherin associated with the phenotype change of CRC cells, and their invasion. Moreover, resveratrol blocked TME-induced phosphorylation and nuclear translocation of p65-NF-κB, which was associated with changes in the expression pattern of epithelial-mesenchymal-transition-related biomarkers (slug, vimentin, E-cadherin), metastasis-related factors (CXCR4, MMP-9, FAK), and apoptosis (caspase-3). Finally, transient transfection of ß1-integrin, in contrast to knockdown of NF-κB, abrogated most anti-invasive, anti-metastatic effects as well as downstream signaling of resveratrol, resulting in a concomitant increase in CRC cell invasion, indicating a central role of ß1-integrin receptors in the anti-invasive function of resveratrol. Conclusion: These results demonstrate for the first time that silencing ß1-integrins may suppress, at least in part the inhibitory effects of resveratrol on invasion and migration of CRC cells, underscoring the crucial homeostatic role of ß1-integrin receptors.

Recombinant C-type lectin protein of Toxocara canis increases the population of spleen Foxp3+ regulatory T cells in BALB/c mice.

Toxocara canis (T. canis) is a common parasitic nematode in dogs and cats. Parasitic worms can cause chronic and long-term infections in their host, due to their ability to neutralize the host's defense mechanisms. They can stimulate immune response-mediated regulatory T (T-reg) cells. The aim of this study is to evaluate the effect of recombinant T. canis C-type lectin protein (TCTL-1) on cell infiltration in the brains of BALB /c mice as well as the number of regulatory T cells. Six-week-old female BALB/c mice received the recombinant C-type lectin protein of T. canis six times intravenously and intraperitoneally. Twenty-eight days after the first injection, the spleen and brain of mice were removed under sterile conditions. The brains of mice were examined by histopathological staining methods. The FOXP3+ regulatory T cell population was determined by flow cytometry. The cell populations of regulatory T cells in spleen mononuclear cell culture of 3 female BALB/c mice injected with recombinant TCTL-1 (group I) were 2.59%, 1.64%, and 1.78 and in spleen mononuclear cell culture of three female BALB/c mice injected with sterile PBS (group II) as a control group were 1.14%, 1.13%, and 1.15%. Also, no cell infiltration was seen around the cerebral arteries of mice receiving this protein. This recombinant protein would increase the population of FOXP3+ regulatory T cells. These results suggest that recombinant C-type lectin protein of T. canis can modulate immune responses, reduce severe inflammatory responses, and induce FOXP3+ regulatory T cells.