Human prostate fibroblasts induce growth and confer castration resistance and metastatic potential in LNCaP Cells

The tumor microenvironment is important for progressive and metastatic disease.

Angiotensinergic neurons in sympathetic coeliac ganglia innervating rat and human mesenteric resistance blood vessels

In contrast to the current belief that angiotensin II (Ang II) interacts with the sympathetic nervous system only as a circulating hormone, we document here the existence of endogenous Ang II in the neurons of rat and human sympathetic coeliac ganglia and their angiotensinergic innervation with mesenteric resistance blood vessels. Angiotensinogen - and angiotensin converting enzyme-mRNA were detected by using quantitative real time polymerase chain reaction in total RNA extracts of rat coeliac ganglia, while renin mRNA was untraceable. Cathepsin D, a protease responsible for cleavage beneath other substrates also angiotensinogen to angiotensin I, was successfully detected in rat coeliac ganglia indicating the possibility of existence of alternative pathways. Angiotensinogen mRNA was also detected by in situ hybridization in the cytoplasm of neurons of rat coeliac ganglia. Immunoreactivity for Ang II was demonstrated in rat and human coeliac ganglia as well as with mesenteric resistance blood vessels. By using confocal laser scanning microscopy we were able to demonstrate the presence of angiotensinergic synapses en passant along side of vascular smooth muscle cells. Our findings indicate that Ang II is synthesized inside the neurons of sympathetic coeliac ganglia and may act as an endogenous neurotransmitter locally with the mesenteric resistance blood vessels.

Physiologic cold shock of Moraxella catarrhalis affects the expression of genes involved in the iron acquisition, serum resistance and immune evasion

Background Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. It was previously shown that the prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis are greatest in winter. The aim of this study was to investigate how M. catarrhalis uses the physiologic exposure to cold air to upregulate pivotal survival systems in the pharynx that may contribute to M. catarrhalis virulence. Results A 26°C cold shock induces the expression of genes involved in transferrin and lactoferrin acquisition, and enhances binding of these proteins on the surface of M. catarrhalis. Exposure of M. catarrhalis to 26°C upregulates the expression of UspA2, a major outer membrane protein involved in serum resistance, leading to improved binding of vitronectin which neutralizes the lethal effect of human complement. In contrast, cold shock decreases the expression of Hemagglutinin, a major adhesin, which mediates B cell response, and reduces immunoglobulin D-binding on the surface of M. catarrhalis. Conclusion Cold shock of M. catarrhalis induces the expression of genes involved in iron acquisition, serum resistance and immune evasion. Thus, cold shock at a physiologically relevant temperature of 26°C induces in M. catarrhalis a complex of adaptive mechanisms that enables the bacterium to target their host cellular receptors or soluble effectors and may contribute to enhanced growth, colonization and virulence.

Increased parasite resistance and recurrent airway obstruction in horses of a high-prevalence family

BACKGROUND: Equine recurrent airway obstruction (RAO) shares many characteristics with human asthma. In humans, an inverse relationship between susceptibility to asthma and resistance to parasites is suspected. HYPOTHESIS/OBJECTIVES: Members of a high-incidence RAO half-sibling family (F) shed fewer strongylid eggs compared with RAO-unaffected pasture mates (PM) and that RAO-affected horses shed fewer eggs than RAO-unaffected half-siblings. ANIMALS: Seventy-three F and 73 unrelated, age matched PM. METHODS: Cases and controls kept under the same management and deworming regime were examined. Each individual was classified as RAO affected or RAO unaffected and fecal samples were collected before and 1-3 weeks and 3 months after deworming. Samples were analyzed by combined sedimentation-flotation and modified McMaster methods and classified into 3 categories of 0 eggs per gram of feces (EpG), 1-100 EpG, and > 100 EpG, respectively. RESULTS: PM compared with RAO-affected F had a 16.7 (95% confidence interval [CI]: 2.0-136.3) times higher risk for shedding > 100 EpG compared with 0 EpG and a 5.3 (95% CI: 1.0-27.4) times higher risk for shedding > 100 EpG compared with 0 EpG. There was no significant effect when RAO-unaffected F were compared with their PM. RAO-unaffected compared with RAO-affected offspring had a 5.8 (95% CI: 0.0-1.0) times higher risk for shedding 1-100 EpG. Age, sex, breed, and sharing pastures with other species had no significant confounding effects. CONCLUSION AND CLINICAL IMPORTANCE: RAO is associated with resistance against strongylid parasites in a high-prevalence family.

Hepatitis C virus drug resistance and immune-driven adaptations: relevance to new antiviral therapy

The efficacy of specifically targeted anti-viral therapy for hepatitis C virus (HCV) (STAT-C), including HCV protease and polymerase inhibitors, is limited by the presence of drug-specific viral resistance mutations within the targeted proteins. Genetic diversity within these viral proteins also evolves under selective pressures provided by host human leukocyte antigen (HLA)-restricted immune responses, which may therefore influence STAT-C treatment response. Here, the prevalence of drug resistance mutations relevant to 27 developmental STAT-C drugs, and the potential for drug and immune selective pressures to intersect at sites along the HCV genome, is explored. HCV nonstructural (NS) 3 protease or NS5B polymerase sequences and HLA assignment were obtained from study populations from Australia, Switzerland, and the United Kingdom. Four hundred five treatment-naïve individuals with chronic HCV infection were considered (259 genotype 1, 146 genotype 3), of which 38.5% were coinfected with human immunodeficiency virus (HIV). We identified preexisting STAT-C drug resistance mutations in sequences from this large cohort. The frequency of the variations varied according to individual STAT-C drug and HCV genotype/subtype. Of individuals infected with subtype 1a, 21.5% exhibited genetic variation at a known drug resistance site. Furthermore, we identified areas in HCV protease and polymerase that are under both potential HLA-driven pressure and therapy selection and identified six HLA-associated polymorphisms (P resistance sites. CONCLUSION: Drug and host immune responses are likely to provide powerful selection forces that shape HCV genetic diversity and replication dynamics. Consideration of HCV viral adaptation in terms of drug resistance as well as host "immune resistance" in the STAT-C treatment era could provide important information toward an optimized and individualized therapy for chronic hepatitis C.

Antibiotic resistance and genetic diversity in Staphylococcus aureus from slaughter pigs in Switzerland

Nasal carriage of Staphylococcus aureus was evaluated in pigs at slaughterhouse. The nasal cavities of 304 pigs from 54 herds were screened. Eighty-nine percent of the farms harbored pigs that were colonized with S. aureus. Among them, no MRSA were found, indicating a low prevalence. However, pigs were found to harbor S. aureus, which displayed resistance to penicillin (blaZ) (62.5%), tetracycline [tet(M)] (33.3%), streptomycin (strpS194) (27%), clindamycin [erm(B)] (4.1%), erythromycin [erm(B)] (4.1%), kanamycin (4.1%), chloramphenicol (catpC194) (2%) and gentamicin [aac(6')-Ie-aph(2')-Ia] (2%). The S. aureus isolates mainly belong to Ridom spa type t034 (31.3%), t208 (14.6%) and t899 (12.5%). These pig-associated spa types have not yet been detected in hospitalized human patients in Switzerland. Surveillance programs are now necessary at both inland and import levels to rapidly detect and suppress the emergence of MRSA in pigs in Switzerland.

Antibiotic resistance and phylogenetic characterization of Acinetobacter baumannii strains isolated from commercial raw meat in Switzerland.

The spread of antibiotic-resistant bacteria through food has become a major public health concern because some important human pathogens may be transferred via the food chain. Acinetobacter baumannii is one of the most life-threatening gram-negative pathogens; multidrug-resistant (MDR) clones of A. baumannii are spreading worldwide, causing outbreaks in hospitals. However, the role of raw meat as a reservoir of A. baumannii remains unexplored. In this study, we describe for the first time the antibiotic susceptibility and fingerprint (repetitive extragenic palindromic PCR [rep-PCR] profile and sequence types [STs]) of A. baumannii strains found in raw meat retailed in Switzerland. Our results indicate that A. baumannii was present in 62 (25.0%) of 248 (CI 95%: 19.7 to 30.9%) meat samples analyzed between November 2012 and May 2013, with those derived from poultry being the most contaminated (48.0% [CI 95%: 37.8 to 58.3%]). Thirty-nine strains were further tested for antibiotic susceptibility and clonality. Strains were frequently not susceptible (intermediate and/or resistant) to third- and fourth-generation cephalosporins for human use (i.e., ceftriaxone [65%], cefotaxime [32%], ceftazidime [5%], and cefepime [2.5%]). Resistance to piperacillin-tazobactam, ciprofloxacin, colistin, and tetracycline was sporadically observed (2.5, 2.5, 5, and 5%, respectively), whereas resistance to carbapenems was not found. The strains were genetically very diverse from each other and belonged to 29 different STs, forming 12 singletons and 6 clonal complexes (CCs), of which 3 were new (CC277, CC360, and CC347). RepPCR analysis further distinguished some strains of the same ST. Moreover, some A. baumannii strains from meat belonged to the clonal complexes CC32 and CC79, similar to the MDR isolates responsible for human infections. In conclusion, our findings suggest that raw meat represents a reservoir of MDR A. baumannii and may serve as a vector for the spread of these pathogens into both community and hospital settings.

Moraxella catarrhalis AcrAB-OprM efflux pump contributes to antimicrobial resistance and is enhanced during cold shock response

Moraxella catarrhalis is a common pathogen of the human respiratory tract. Multidrug efflux pumps play a major role in antibiotic resistance and virulence in many Gram-negative organisms. In the present study, the role of the AcrAB-OprM efflux pump in antibiotic resistance was investigated by constructing mutants that lack the acrA, acrB, and oprM genes in M. catarrhalis strain O35E. We observed a moderate (1.5-fold) decrease in the MICs of amoxicillin and cefotaxime and a marked (4.7-fold) decrease in the MICs of clarithromycin for acrA, acrB, and oprM mutants in comparison with the wild-type O35E strain. Exposure of the M. catarrhalis strains O35E and 300 to amoxicillin triggered an increased transcription of all AcrAB-OprM pump genes, and exposure of strains O35E, 300, and 415 to clarithromycin enhanced the expression of acrA and oprM mRNA. Inactivation of the AcrAB-OprM efflux pump genes demonstrated a decreased ability to invade epithelial cells compared to the parental strain, suggesting that acrA, acrB, and oprM are required for efficient invasion of human pharyngeal epithelial cells. Cold shock increases the expression of AcrAB-OprM efflux pump genes in all three M. catarrhalis strains tested. Increased expression of AcrAB-OprM pump genes after cold shock leads to a lower accumulation of Hoechst 33342 (H33342), a substrate of AcrAB-OprM efflux pumps, indicating that cold shock results in increased efflux activity. In conclusion, the AcrAB-OprM efflux pump appears to play a role in the antibiotic resistance and virulence of M. catarrhalis and is involved in the cold shock response.

Virulence-associated gene pattern of porcine and human Yersinia enterocolitica biotype 4 isolates.

Yersinia enterocolitica 4/O:3 is the most important human pathogenic bioserotype in Europe and the predominant pathogenic bioserotype in slaughter pigs. Although many studies on the virulence of Y. enterocolitica strains have showed a broad spectrum of detectable factors in pigs and humans, an analysis based on a strict comparative approach and serving to verify the virulence capability of porcine Y. enterocolitica as a source for human yersiniosis is lacking. Therefore, in the present study, strains of biotype (BT) 4 isolated from Swiss slaughter pig tonsils and feces and isolates from human clinical cases were compared in terms of their spectrum of virulence-associated genes (yadA, virF, ail, inv, rovA, ymoA, ystA, ystB and myfA). An analysis of the associated antimicrobial susceptibility pattern completed the characterization. All analyzed BT 4 strains showed a nearly similar pattern, comprising the known fundamental virulence-associated genes yadA, virF, ail, inv, rovA, ymoA, ystA and myfA. Only ystB was not detectable among all analyzed isolates. Importantly, neither the source of the isolates (porcine tonsils and feces, humans) nor the serotype (ST) had any influence on the gene pattern. From these findings, it can be concluded that the presence of the full complement of virulence genes necessary for human infection is common among porcine BT 4 strains. Swiss porcine BT 4 strains not only showed antimicrobial susceptibility to chloramphenicol, cefotaxime, ceftazidime, ciprofloxacin, colistin, florfenicol, gentamicin, kanamycin, nalidixic acid, sulfamethoxazole, streptomycin, tetracycline and trimethoprim but also showed 100% antibiotic resistance to ampicillin. The human BT 4 strains revealed comparable results. However, in addition to 100% antibiotic resistance to ampicillin, 2 strains were resistant to chloramphenicol and nalidixic acid. Additionally, 1 of these strains was resistant to sulfamethoxazole. The results demonstrated that Y. enterocolitica BT 4 isolates from porcine tonsils, as well as from feces, show the same virulence-associated gene pattern and antibiotic resistance properties as human isolates from clinical cases, consistent with the etiological role of porcine BT 4 in human yersiniosis. Thus, cross-contamination of carcasses and organs at slaughter with porcine Y. enterocolitica BT 4 strains, either from tonsils or feces, must be prevented to reduce human yersiniosis.

The association of feeding behavior with the resistance and tolerance to parasites in recently diverged sticklebacks

Divergent natural selection regimes can contribute to adaptive population divergence, but can be sensitive to human-mediated environmental change. Nutrient loading of aquatic ecosystems, for example, might modify selection pressures by altering the abundance and distribution of resources and the prevalence and infectivity of parasites. Here, we used a mesocosm experiment to test for interactive effects of nutrient loading and parasitism on host condition and feeding ecology. Specifically, we investigated whether the common fish parasite Gyrodactylus sp. differentially affected recently diverged lake and stream ecotypes of three-spined stickleback (Gasterosteus aculeatus). We found that the stream ecotype had a higher resistance to Gyrodactylus sp. infections than the lake ecotype, and that both ecotypes experienced a cost of parasitism, indicated by negative relationships between parasite load and both stomach fullness and body condition. Overall, our results suggest that in the early stages of adaptive population divergence of hosts, parasites can affect host resistance, body condition, and diet.

Expression, localization, and functional model of cholesterol transporters in lactating and nonlactating mammary tissues of murine, bovine, and human origin

Members of the ATP-binding cassette (ABC) transporters play a pivotal role in cellular lipid efflux. To identify candidate cholesterol transporters implicated in lipid homeostasis and mammary gland (MG) physiology, we compared expression and localization of ABCA1, ABCG1, and ABCA7 and their regulatory genes in mammary tissues of different species during the pregnancy-lactation cycle. Murine and bovine mammary glands (MGs) were investigated during different functional stages. The abundance of mRNAs was determined by quantitative RT-PCR. Furthermore, transporter proteins were localized in murine, bovine, and human MGs by immunohistochemistry. In the murine MG, ABCA1 mRNA abundance was elevated during nonlactating compared with lactating stages, whereas ABCA7 and ABCA1 mRNA profiles were not altered. In the bovine MG, ABCA1, ABCG1, and ABCA7 mRNAs abundances were increased during nonlactating stages compared with lactation. Furthermore, associations between mRNA levels of transporters and their regulatory genes LXRalpha, PPARgamma, and SREBPs were found. ABCA1, ABCG1, and ABCA7 proteins were localized in glandular MG epithelial cells (MEC) during lactation, whereas during nonlactating stages, depending on species, the proteins showed distinct localization patterns in MEC and adipocytes. Our results demonstrate that ABCA1, ABCG1, and ABCA7 are differentially expressed between lactation and nonlactating stages and in association with regulatory genes. Combined expression and localization data suggest that the selected cholesterol transporters are universal MG transporters involved in transport and storage of cholesterol and in lipid homeostasis of MEC. Because of the species-specific expression patterns of transporters in mammary tissue, mechanisms of cholesterol homeostasis seem to be differentially regulated between species.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

Angiotensinergic and noradrenergic neurons in the rat and human heart

Although the physiological and pharmacological evidences suggest a role for angiotensin II (Ang II) with the mammalian heart, the source and precise location of Ang II are unknown. To visualize and quantitate Ang II in atria, ventricular walls and interventricular septum of the rat and human heart and to explore the feasibility of local Ang II production and function, we investigated by different methods the expression of proteins involved in the generation and function of Ang II. We found mRNA of angiotensinogen (Ang-N), of angiotensin converting enzyme, of the angiotensin type receptors AT(1A) and AT(2) (AT(1B) not detected) as well as of cathepsin D in any part of the hearts. No renin mRNA was traceable. Ang-N mRNA was visualized by in situ hybridization in atrial ganglial neurons. Ang II and dopamine- -hydroxylase (D H) were either colocalized inside the same neuronal cell or the neurons were specialized for Ang II or D H. Within these neurons, the vesicular acetylcholine transporter (VAChT) was neither colocalized with Ang II nor D H, but VAChT-staining was found with synapses en passant encircle these neuronal cells. The fibers containing Ang II exhibited with blood vessels and with cardiomyocytes supposedly angiotensinergic synapses en passant. In rat heart, right atrial median Ang II concentration appeared higher than septal and ventricular Ang II. The distinct colocalization of neuronal Ang II with D H in the heart may indicate that Ang II participates together with norepinephrine in the regulation of cardiac functions: Produced as a cardiac neurotransmitter Ang II may have inotropic, chronotropic or dromotropic effects in atria and ventricles and contributes to blood pressure regulation.

Intraneuronal angiotensinergic system in rat and human dorsal root ganglia

To elucidate the local formation of angiotensin II (Ang II) in the neurons of sensory dorsal root ganglia (DRG), we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of protein renin, Ang II, Substance P and calcitonin gene-related peptide (CGRP) in the rat and human thoracic DRG. Quantitative real time PCR (qRT-PCR) studies revealed that rat DRG expressed substantial amounts of Ang-N- and ACE mRNA, while renin mRNA as well as the protein renin were untraceable. Cathepsin D-mRNA and cathepsin D-protein were detected in the rat DRG indicating the possibility of existence of pathways alternative to renin for Ang I formation. Angiotensin peptides were successfully detected with high performance liquid chromatography and radioimmunoassay in human DRG extracts. In situ hybridization in rat DRG confirmed additionally expression of Ang-N mRNA in the cytoplasm of numerous neurons. Intracellular Ang II staining could be shown in number of neurons and their processes in both the rat and human DRG. Interestingly we observed neuronal processes with angiotensinergic synapses en passant, colocalized with synaptophysin, within the DRG. In the DRG, we also identified by qRT-PCR, expression of Ang II receptor AT(1A) and AT(2)-mRNA while AT(1B)-mRNA was not traceable. In some neurons Substance P and CGRP were found colocalized with Ang II. The intracellular localization and colocalization of Ang II with Substance P and CGRP in the DRG neurons may indicate a participation and function of Ang II in the regulation of nociception. In conclusion, these results suggest that Ang II may be produced locally in the neurons of rat and human DRG and act as a neurotransmitter.