Microbial and sensory changes throughout the ripening of Prato cheese made from milk with different levels of somatic cells

The objective of this research was to evaluate the effects of 2 levels of raw milk somatic cell count (SCC) on the composition of Prato cheese and on the microbiological and sensory changes of Prato cheese throughout ripening. Two groups of dairy cows were selected to obtain low-SCC (<200,000 cells/mL) and high-SCC (>700,000 cells/mL) milks, which were used to manufacture 2 vats of cheese. The pasteurized milk was evaluated according to the pH, total solids, fat, total protein, lactose, standard plate count, coliforms at 45 degrees C, and Salmonella spp. The cheese composition was evaluated 2 d after manufacture. Lactic acid bacteria, psychrotrophic bacteria, and yeast and mold counts were carried out after 3, 9, 16, 32, and 51 d of storage. Salmonella spp., Listeria monocytogenes, and coagulase-positive Staphylococcus counts were carried out after 3, 32, and 51 d of storage. A 2 x 5 factorial design with 4 replications was performed. Sensory evaluation of the cheeses from low- and high-SCC milks was carried out for overall acceptance by using a 9-point hedonic scale after 8, 22, 35, 50, and 63 d of storage. The somatic cell levels used did not affect the total protein and salt: moisture contents of the cheeses. The pH and moisture content were higher and the clotting time was longer for cheeses from high-SCC milk. Both cheeses presented the absence of Salmonella spp. and L. monocytogenes, and the coagulase-positive Staphylococcus count was below 1 x 10(2) cfu/g throughout the storage time. The lactic acid bacteria count decreased significantly during the storage time for the cheeses from both low- and high-SCC milks, but at a faster rate for the cheese from high-SCC milk. Cheeses from high-SCC milk presented lower psychrotrophic bacteria counts and higher yeast and mold counts than cheeses from low- SCC milk. Cheeses from low- SCC milk showed better overall acceptance by the consumers. The lower overall acceptance of the cheeses from high-SCC milk may be associated with texture and flavor defects, probably caused by the higher proteolysis of these cheeses.

Intramammary infusion of leptin decreases proliferation of mammary epithelial cells in prepubertal heifers

High energy intake and excessive body fatness impair mammogenesis in prepubertal ruminants. High energy intake and excessive fatness also increase serum leptin. Our objective was to determine if an infusion of leptin decreases proliferation of mammary epithelial cells of prepubertal heifers in vivo. Ovine leptin at 100 mu g/quarter per d with or without 10 mu g of insulin-like growth factor (IGF)-I was infused via the teat canal into mammary glands of prepubertal dairy heifers; contralateral quarters were used as controls. After 7 d of treatment, bromodeoxyuridine was infused intravenously and heifers were slaughtered similar to 2 h later. Tissue from 3 regions of the mammary parenchyma was collected and immunostained for bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (Ki-67), and caspase-3. Leptin decreased the number of mammary epithelial cells in the S-phase of the cell cycle by 48% in IGF-I-treated quarters and by 19% in saline-treated quarters. Leptin did not alter the number of mammary epithelial cells within the cell cycle, as indicated by Ki-67 labeling. Caspase-3 immunostaining within the mammary parenchyma was very low in these heifers, but leptin significantly increased labeling in saline-treated quarters. Leptin enhanced SOCS-3 expression in IGF-I-treated quarters but did not alter SOCS-1 or SOCS-5 expression. We conclude that a high concentration of leptin in the bovine mammary gland reduces proliferation of mammary epithelial cells. The reduced proliferation is accompanied by an increase in SOCS-3 expression, suggesting a possible mechanism for leptin inhibition of IGF-I action. Whether leptin might be a physiological regulator of mammogenesis remains to be determined.

Isolation and partial characterization of Brazilian samples of feline immunodeficiency virus

Feline immunodeficiency virus (FIV) causes a slow progressive degeneration of the immune system which eventually leads to a disease comparable to acquired immune deficiency syndrome (AIDS) in humans. FIV has extensive sequence variation, a typical feature of lentiviruses. Sequence analysis showed that diversity was not evenly distributed throughout the genome, but was greatest in the envelope gene, env. The virus enters host cells via a sequential interaction, initiated by the envelope glycoprotein (env) binding the primary receptor molecule CD134 and followed by a subsequent interaction with chemokine co-receptor CXCR4. The purpose of this study was to isolate and characterize isolates of FIV from an open shelter in Sao Paulo, Brazil. The separated PBMC from 11 positive cats were co-cultured with MYA-1 cells. Full-length viral env glycoprotein genes were amplified and determined. Chimeric feline x human CD134 receptors were used to investigate the receptor utilization of 17 clones from Brazilian isolates of Fly. Analyses of the sequence present of molecular clones showed that all clones grouped within subtype B. In contrast to the virulent primary isolate FIV-GL8, expression of the first cysteine-rich domain (CRD1) of feline CD134 in the context of human CD134 was sufficient for optimal receptor function for all Brazilian FIV isolates tested. (C) 2011 Elsevier B.V. All rights reserved.

Lp95, a novel leptospiral protein that binds extracellular matrix components and activates e-selectin on endothelial cells

Objectives: The study of a predicted outer membrane leptospiral protein encoded by the gene LIC12690 in mediating the adhesion process. Methods: The gene was cloned and expressed in Escherichia coli BL21 (SI) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and used to assess its ability to activate human umbilical vein endothelial cells (HUVECs). Results: The recombinant leptospiral protein of 95 kDa, named Lp95, activated E-selectin in a dose-dependent fashion but not the intercellular adhesion molecule 1 (ICAM-1). In addition, we show that pathogenic and non-pathogenic Leptospira are both capable to stimulate endothelium E-selectin and ICAM-1, but the pathogenic L. interrogans serovar Copenhageni strain promotes a statistically significant higher activation than the non-pathogenic L. biflexa serovar Patoc (P < 0.01). The Lp95 was identified in vivo in the renal tubules of animal during experimental infection with L. interrogans. The whole Lp95 as well as its fragments, the C-terminal containing the domain of unknown function (DUF), the N-terminal and the central overlap regions bind laminin and fibronectin ECM molecules, being the binding stronger with the DUF containing fragment. Conclusion: This is the first leptospiral protein capable to mediate the adhesion to ECM components and the activation of HUVECS, thus suggesting its participation in the pathogenesis of Leptospira. (C) 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Two Atypical Enteropathogenic Escherichia coli Strains Induce the Production of Secreted and Membrane-Bound Mucins To Benefit Their Own Growth at the Apical Surface of Human Mucin-Secreting Intestinal HT29-MTX Cells

In rabbit ligated ileal loops, two atypical enteropathogenic Escherichia coli (aEPEC) strains, 3991-1 and 0421-1, intimately associated with the cell membrane, forming the characteristic EPEC attachment and effacement lesion of the brush border, induced a mucous hypersecretion, whereas typical EPEC (tEPEC) strain E2348/69 did not. Using cultured human mucin-secreting intestinal HT29-MTX cells, we demonstrate that apically aEPEC infection is followed by increased production of secreted MUC2 and MUC5AC mucins and membrane-bound MUC3 and MUC4 mucins. The transcription of the MUC5AC and MUC4 genes was transiently upregulated after aEPEC infection. We provide evidence that the apically adhering aEPEC cells exploit the mucins` increased production since they grew in the presence of membrane-bound mucins, whereas tEPEC did not. The data described herein report a putative new virulence phenomenon in aEPEC.

Coordinated regulation of follicle development by germ and somatic cells

The continuum of folliculogenesis begins in the fetal ovary with the differentiation of the oogonia and their isolation within the primordial follicles. Primordial follicle activation is an enigmatic process, whereby some follicles enter the growing pool to become primary follicles, thereby embarking on an irreversible progression towards ovulation or atresia. This process is under the coordinated regulation of factors from the oocyte itself, as well as from the somatic cells of the ovary, in particular the theca and granulosa cells, which are structural components of the follicle. These two influences provide the principal stimuli for the growth of the follicle to the late preantral or early antral stage of development. The endocrine effects of the gonadotrophins FSH and LH are essential to the continued progression of the follicle and most atresia can be attributed to the failure to receive or process the gonadotrophin signals. The peri-ovulatory state has received intensive investigation recently, demonstrating a coordinated role for gonadotrophins, steroids, epidermal growth factor family proteins and prostaglandins. Thus, a complex programme of coordinated interaction of governing elements from both germ and somatic cell sources is required for successful follicle development.

Oestrogen and Progesterone Receptor Gene Expression in Canine Oocytes and Cumulus Cells Throughout the Oestrous Cycle

The aim of this research was to analyze oestrogen receptor-alpha (ER alpha), ER beta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells` total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ER beta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ER beta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ER alpha and ER beta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ER alpha was expressed throughout the oestrous cycle.

Bovine sperm cells viability during incubation with or without exogenous DNA

The aim of this study was to assess the effect of exogenous DNA and incubation time on the viability of bovine sperm. Sperm were incubated at a concentration of 5 x 10(6)/ml with or without plasmid pEYFP-NUC. Fluorescent probes, propidium iodide/Hoechst 33342, FITC-PSA and JC-1, were used to assess plasma membrane integrity (PMI), acrosome membrane integrity (AMI) and mitochondrial membrane potential (MMP) respectively at 0, 1, 2, 3 and 4 h of incubation. Exogenous DNA addition did not affect sperm viability; however, incubation time was related to sperm deterioration. Simultaneous assessment of PMI, AMI and MMP showed a reduction in the number of sperm with higher viability (integrity of plasma and acrosome membranes and high mitochondrial membrane potential) from 58.7% at 0 h to 7.5% after 4 h of incubation. Lower viability sperm (damaged plasma and acrosome membranes and low mitochondrial membrane potential) increased from 4.6% at 0 h to 25.99% after 4 h of incubation. When PMI, AMI and MMP were assessed separately we noticed a reduction in plasma and acrosome membrane integrity and mitochondrial membrane potential throughout the incubation period. Therefore, exogenous DNA addition does not affect sperm viability, but the viability is reduced by incubation time.

Human immature dental pulp stem cells` contribution to developing mouse embryos: production of human/mouse preterm chimaeras

In this study, we aimed at determining whether human immature dental pulp stem cells (hIDPSC) would be able to contribute to different cell types in mouse blastocysts without damaging them. Also, we analysed whether these blastocysts would progress further into embryogenesis when implanted to the uterus of foster mice, and develop human/mouse chimaera with retention of hIDPSC derivates and their differentiation. hIDPSC and mouse blastocysts were used in this study. Fluorescence staining of hIDPSC and injection into mouse blastocysts, was performed. Histology, immunohistochemistry, fluorescence in situ hybridization and confocal microscopy were carried out. hIDPSC showed biological compatibility with the mouse host environment and could survive, proliferate and contribute to the inner cell mass as well as to the trophoblast cell layer after introduction into early mouse embryos (n = 28), which achieved the hatching stage following 24 and 48 h in culture. When transferred to foster mice (n = 5), these blastocysts with hIDPSC (n = 57) yielded embryos (n = 3) and foetuses (n = 6); demonstrating presence of human cells in various organs, such as brain, liver, intestine and hearts, of the human/mouse chimaeras. We verified whether hIDPSC would also be able to differentiate into specific cell types in the mouse environment. Contribution of hIDPSC in at least two types of tissues (muscles and epithelial), was confirmed. We showed that hIDPSC survived, proliferated and differentiated in mouse developing blastocysts and were capable of producing human/mouse chimaeras.

Expression of Extracellular Matrix Proteins in Human Dental Pulp Stem Cells Depends on the Donor Tooth Conditions

Introduction: Stem cells are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. An important source of adult stem cells is the dental pulp. In dentistry, regenerative strategies are of importance because of hard dental tissue damage especially as result of caries lesions, trauma, or iatrogenic procedures. The regeneration of dental tissues relies on the ability of stem cells to produce extracellular (ECM) proteins encountered in the dental pulp tissue. Thus, the aim of this study was to analyze the expression and distribution of proteins encountered in dental pulp ECM (type I collagen, fibronectin, and tenascin) in stem cells. Methods: Human immature dental pulp stem cells (hIDPSCs) from deciduous (DL-1 and DL-4 cell lines) and permanent (DL-2) teeth were used. The distribution of ECM proteins was observed using the immunofluorescence technique. The gene expression profile was evaluated using reverse transcription polymerase chain reaction (RT-PCR) analysis. Results: Positive reactions for all ECM proteins were observed independently of the hIDPSCs analyzed. Type I collagen appeared less evident in DL-2 than in other hIDPSCs. Fibronectin and tenascin were less clear in DL-4. The RT-PCR reactions showed that type I collagen was lesser expressed in the DL-2 cells, whereas fibronectin and tenascin were similarly expressed in all hIDPSCs. Conclusions: The distribution and expression of ECM proteins differ among the hIDPSCs. These differences seemed to be related to the donor tooth conditions (deciduous or permanent, retained or erupted, and degree of root reabsorption). (J Endod 2010;36:826-831)

The neuroprotective effect of dental pulp cells in models of Alzheimer`s and Parkinson`s disease

Aim of the present study was to investigate the neuroprotective effect of dental pulp cells (DPCs) in in vitro models of Alzheimer and Parkinson disease. Primary cultures of hippocampal and ventral mesencephalic neurons were treated for 24 h with amyloid beta (A beta(1-42)) peptide 1-42 and 6-OHDA, respectively. DPCs isolated from adult rat incisors were previously cultured in tissue culture inserts and added to the neuron cultures 2 days prior to neurotoxin treatment. Cell viability was assessed by the MTT assay. The co-culture with DPCs significantly attenuated 6-OHDA and A beta(1-42)-induced toxicity in primary cultures of mesencephalic and hippocampal neurons, and lead to an increase in neuronal viability in untreated cultures, suggesting a neurotrophic effect in both models. Furthermore, human dental pulp cells expressed a neuronal phenotype and produced the neurotrophic factors NGF, GDNF, BDNF, and BMP2 shown by microarray screening and antibody staining for the representative proteins. DPCs protected primary neurons in in vitro models of Alzheimer`s and Parkinson`s disease and can be viewed as possible candidates for studies on cell-based therapy.

Tooth Tissue Engineering: Optimal Dental Stem Cell Harvest Based on Tooth Development

Our long-term objective is to devise reliable methods to generate biological replacement teeth exhibiting the physical properties and functions of naturally formed human teeth. Previously, we demonstrated the successful use of tissue engineering approaches to generate small, bioengineered tooth crowns from harvested pig and rat postnatal dental stem cells (DSCs). To facilitate characterizations of human DSCs, we have developed a novel radiographic staging system to accurately correlate human third molar tooth developmental stage with anticipated harvested DSC yield. Our results demonstrated that DSC yields were higher in less developed teeth (Stages 1 and 2), and lower in more developed teeth (Stages 3, 4, and 5). The greatest cell yields and colony-forming units (CFUs) capability was obtained from Stages 1 and 2 tooth dental pulp. We conclude that radiographic developmental staging can be used to accurately assess the utility of harvested human teeth for future dental tissue engineering applications.

Dual origin of mesenchymal stem cells contributing to organ growth and repair

In many adult tissues, mesenchymal stem cells (MSCs) are closely associated with perivascular niches and coexpress many markers in common with pericytes. The ability of pericytes to act as MSCs, however, remains controversial. By using genetic lineage tracing, we show that some pericytes differentiate into specialized tooth mesenchyme-derived cells-odontoblasts-during tooth growth and in response to damage in vivo. As the pericyte-derived mesenchymal cell contribution to odontoblast differentiation does not account for all cell differentiation, we identify an additional source of cells with MSC-like properties that are stimulated to migrate toward areas of tissue damage and differentiate into odontoblasts. Thus, although pericytes are capable of acting as a source of MSCs and differentiating into cells of mesenchymal origin, they do so alongside other MSCs of a nonpericyte origin. This study identifies a dual origin of MSCs in a single tissue and suggests that the pericyte contribution to MSC-derived mesenchymal cells in any given tissue is variable and possibly dependent on the extent of the vascularity.

Expression of Homeobox Genes in Oral Squamous Cell Carcinoma Cell Lines Treated With All-Trans Retinoic Acid

Oral squamous cell carcinoma (OSCC) may arise from potentially malignant oral lesions. All-trans retinoic acid (atRA), which plays a role in cell growth and differentiation, has been studied as a possible chemotherapeutic agent in the prevention of this progression. While the mechanism by which atRA suppresses cell growth has not been completely elucidated, it is known that homeobox genes are atRA targets. To determine if these genes are involved in the atRA-mediated OSCC growth inhibition, PCR array was performed to evaluate the expression of 84 homeobox genes in atRA-sensitive SCC-25 cells compared to atRA-resistant SCC-9 cells following 7 days with atRA treatment. Results showed that the expression of 8 homeobox genes was downregulated and expression of 4 was upregulated in SCC-25 cells but not in SCC-9 cells. Gene expression levels were confirmed for seven of these genes by RT-qPCR. Expression of three genes that showed threefold downregulation was evaluated in SCC-25 cells treated with atRA for 3, 5, and 7 days. Three different patterns of atRA-dependent gene expression were observed. ALX1 showed downregulation only on day 7. DLX3 showed reduced expression on day 3 and further reduced on clay 7. TLX1 showed downregulation only on days 5 and 7. Clearly the expression of homeobox genes is modulated by atRA in OSCC cell lines. However, the time course of this modulation suggests that these genes are not direct targets of atRA mediating OSCC growth suppression. Instead they appear to act as downstream effectors of atRA signaling. J. Cell. Biochem. 111: 1437-1444, 2010. (C) 2010 Wiley-Liss, Inc.

Release of cytokines by stimulated peripheral blood mononuclear cells in chronic periodontitis

Objective: The emergence of periodontal medicine increased interest in defining the behaviour of peripheral blood cells in periodontitis subjects in comparison with healthy group. The aim of this study was to evaluate the levels of interleukin (IL)-8, tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-10 released by Escherichia coli lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC) obtained from the peripheral blood of chronic periodontitis subjects. Design: PBMC samples were isolated from 19 systemically healthy donors, divided into generalized chronic periodontitis (n = 10) and healthy (n = 9) subjects. Cells were incubated for 24-48 h in 500 mu L wells containing RPM! 1640 and stimulated with 1.0 ng/mL of E. coli LPS. Supernatants were used to quantify the amounts of IL-8, TNF-alpha, IL-6 and IL-10 released using enzyme-linked immunosorbent assay (ELISA). Results: PBMC cells from periodontitis subjects released higher levels of TNF-alpha and IL-6 than those from healthy subjects (P < 0.05). Conversely, the supernatants of the stimulated PBMC cells obtained from healthy subjects presented higher amounts of IL-8 than those from periodontitis (P < 0.05). No differences were observed in the levels of IL-10 (P > 0.05) between groups. Conclusion: In conclusion, the results of the present study showed that E. coli LPS-stimulated PBMC from subjects with periodontitis present a different pattern of cytokine release when compared to PBMC from healthy subjects. This phenomenon could have implications locally, in periodontitis, as well as in systemic diseases. (C) 2010 Elsevier Ltd. All rights reserved.