Fibulin-4 exerts a dual role in LTBP-4L-mediated matrix assembly and function.

Elastogenesis is a hierarchical process by which cells form functional elastic fibers, providing elasticity and the ability to regulate growth factor bioavailability in tissues, including blood vessels, lung, and skin. This process requires accessory proteins, including fibulin-4 and -5, and latent TGF binding protein (LTBP)-4. Our data demonstrate mechanisms in elastogenesis, focusing on the interaction and functional interdependence between fibulin-4 and LTBP-4L and its impact on matrix deposition and function. We show that LTBP-4L is not secreted in the expected extended structure based on its domain composition, but instead adopts a compact conformation. Interaction with fibulin-4 surprisingly induced a conformational switch from the compact to an elongated LTBP-4L structure. This conversion was only induced by fibulin-4 multimers associated with increased avidity for LTBP-4L; fibulin-4 monomers were inactive. The fibulin-4-induced conformational change caused functional consequences in LTBP-4L in terms of binding to other elastogenic proteins, including fibronectin and fibrillin-1, and of LTBP-4L assembly. A transient exposure of LTBP-4L with fibulin-4 was sufficient to stably induce conformational and functional changes; a stable complex was not required. These data define fibulin-4 as a molecular extracellular chaperone for LTBP-4L. The altered LTBP-4L conformation also promoted elastogenesis, but only in the presence of fibulin-4, which is required to escort tropoelastin onto the extended LTBP-4L molecule. Altogether, this study provides a dual mechanism for fibulin-4 in 1) inducing a stable conformational and functional change in LTBP-4L, and 2) promoting deposition of tropoelastin onto the elongated LTBP-4L.

In vitro and in vivo antagonistic activity of new probiotic culture against Clostridium difficile and Clostridium perfringens.

BACKGROUND: Genus Clostridium accompanies more than 200 known species and at least 30 among them are associated with human and animal diseases. At the moment, the treatment of clostridial infections is based on use of antibiotics. However, due to the European ban on the use of antibiotics in livestock production, novel therapeutic strategies for treatment of these hardly curable infections have been evaluated. Hence, in this study the antimicrobial effect of newly designed probiotic culture consisted of natural isolates Lactobacillus helveticus BGRA43, Lactobacillus fermentum BGHI14 and Streptococcus thermophilus BGVLJ1-44 against Clostridium difficile and Clostridium perfringens was analyzed. RESULTS: The probiotic culture showed strong in vitro antimicrobial effect on C. difficile (human clinical isolate). In addition, individual strains and the probiotic combination exhibited immunomodulatory activity. The probiotic combination significantly increased the proliferation of GALT lymphocytes. At the other hand, none of the bacterial treatments (individual strains and the combination) induced the production of proinflammatory cytokines IL-6 and IL-1ß by intestinal epithelial cells, Caco-2. Interestingly, Caco-2 cells exposed to the probiotic combination produced significantly elevated amount of TGFß pointing to potential protecting effect of the probiotic. In addition, the results of field trial on spontaneously infected goats revealed reduction of C. perfringens in goats (below the detection threshold) after the probiotic treatment. CONCLUSIONS: The results of this study indicated that the novel probiotic deserves to be further investigated as a promising antimicrobial agent against C. difficile and C. perfringens.

Proinflammatory cytokine responses in skin and epidermal cells following epicutaneous administration of anticoagulant rodenticide warfarin in rats.

CONTEXT: Dermal toxicity of coumarin anticoagulant rodenticides, such as warfarin, represents potential risk for workers handling these agents and for individuals applying easily available rodenticides in their households as well. OBJECTIVE: In this study, proinflammatory effects of repeated epicutaneous administration of warfarin in rats were explored by examining inflammatory cytokine skin responses. MATERIALS AND METHODS: Ex vivo production of IL-1ß, IL-6, TNF-α and IL-17 by skin explants and by epidermal cells isolated by enzyme (dispase/trypsin) digestion from skin repeatedly (once a day, three consecutive days) exposed to 10 µg of warfarin was measured 24 h and 72 h following the last warfarin application by ELISAs for respective rat cytokines. RESULTS: Warfarin treatment resulted in histological changes, but skin or epidermal cell viability were not compromised, judging by MTT reduction assay. Both skin and epidermal cells responded to administration of this agent by production of all examined inflammatory cytokines (skin explants by TNF-α and IL-17; epidermal cells by IL-1ß and TNF-α) except IL-6. DISCUSSION: Along with histomorphological changes, cytokines indicate functional consequences in treated skin. IL-1ß production, that precede production of TNF-α, might be responsible for production of the latter cytokine. Sustained production of IL-1ß suggests persistence of epidermal cell stimulation or existence of some amplification mechanisms. Requirements for T cells seem to exist concerning epidermal cell IL-17 production. CONCLUSION: Presented data provide additional new information concerning proinflammatory effects of warfarin.

Fibulin-3, -4, and -5 are highly susceptible to proteolysis, interact with cells and heparin, and form multimers.

Extracellular short fibulins, fibulin-3, -4, and -5, are components of the elastic fiber/microfibril system and are implicated in the formation and homeostasis of elastic tissues. In this study, we report new structural and functional properties of the short fibulins. Full-length human short fibulins were recombinantly expressed in human embryonic kidney cells and purified by immobilized metal ion affinity chromatography. All three fibulins showed various levels of degradation after the purification procedure. N-terminal sequencing revealed that all three fibulins are highly susceptible to proteolysis within the N-terminal linker region of the first calcium-binding epidermal growth factor domain. Proteolytic susceptibility of the linker correlated with its length. Exposure of these fibulins to matrix metalloproteinase (MMP)-1, -2, -3, -7, -9, and -12 resulted in similar proteolytic fragments with MMP-7 and -12 being the most potent proteases. Fibulin-3 proteolysis was almost completely inhibited in cell culture by the addition of 25 µm doxycycline (a broad spectrum MMP inhibitor). Reducible fibulin-4 dimerization and multimerization were consistently observed by SDS-PAGE, Western blotting, and mass spectrometry. Atomic force microscopy identified monomers, dimers, and multimers in purified fibulin-4 preparations with sizes of ∼10-15, ∼20-25, and ∼30-50 nm, respectively. All short fibulins strongly adhered to human fibroblasts and smooth muscle cells. Although only fibulin-5 has an RGD integrin binding site, all short fibulins adhere at a similar level to the respective cells. Solid phase binding assays detected strong calcium-dependent binding of the short fibulins to immobilized heparin, suggesting that these fibulins may bind cell surface-located heparan sulfate.

Complex contributions of fibronectin to initiation and maturation of microfibrils.

Fibrillins constitute the backbone of extracellular multifunctional assemblies present in elastic and non-elastic matrices, termed microfibrils. Assembly of fibrillins into microfibrils and their homoeostasis is poorly understood and is often compromised in connective tissue disorders such as Marfan syndrome and other fibrillinopathies. Using interaction mapping studies, we demonstrate that fibrillins require the complete gelatin-binding region of fibronectin for interaction, which comprises domains FNI6-FNI9. However, the interaction of fibrillin-1 with the gelatin-binding domain of fibronectin is not involved in fibrillin-1 network assembly mediated by human skin fibroblasts. We show further that the fibronectin network is essential for microfibril homoeostasis in early stages. Fibronectin is present in extracted mature microfibrils from tissue and cells as well as in some in situ microfibrils observed at the ultrastructural level, indicating an extended mechanism for the involvement of fibronectin in microfibril assembly and maturation.

Pulmonary immune responses to Aspergillus fumigatus in rats.

OBJECTIVE: To evaluate immunologic mechanisms underlying Aspergillus fumigatus pulmonary infections in immunocompetent Dark Agouti (DA) and Albino Oxford (AO) rats recognized as being susceptible to some inflammatory diseases in different manners. METHODS: Lung fungal burden (quantitative colony forming units, CFU, assay), leukocyte infiltration (histology, cell composition) and their function (phagocytosis, oxidative activity, CD11b adhesion molecule expression) and cytokine interferon-γ (IFN-γ) and interleukin-17 and -4 (IL-17 and IL-4) lung content were evaluated following infection (intratracheally, 1x10(7) conidia). RESULTS: Slower reduction of fungal burden was observed in AO rats in comparison with that in DA rats, which was coincided with less intense histologically evident lung cell infiltration and leukocyte recovery as well as lower level of most of the their activities including intracellular myeloperoxidase activity, the capacity of nitroblue tetrazolium salt reduction and CD11b adhesion molecule expression (except for phagocytosis of conidia) in these rats. Differential patterns of changes in proinflammatory cytokine levels (unchanged levels of IFN-γ and transient increase of IL-17 in AO rats vs continuous increase of both cytokines in DA rats) and unchanged levels of IL-4 were observed. CONCLUSION: Genetically-based differences in the pattern of antifungal lung leukocyte activities and cytokine milieu, associated with differential efficiency of fungal elimination might be useful in the future use of rat models in studies of pulmonary aspergillosis.

Differences in T-helper polarizing capability between human monocyte-derived dendritic cells and monocyte-derived Langerhans'-like cells.

Langerhans' cells (LCs) represent a specific subset of dendritic cells (DCs) which are important for detecting and processing pathogens that penetrate the skin and epithelial barriers. The aim of our study was to explain what makes their in vitro counterparts - monocyte-derived Langerhans'-like cells (MoLCs) - unique compared with monocyte-derived dendritic cells (MoDCs). Immature MoDCs were generated by incubating peripheral blood monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4. The addition of transforming growth factor-ß (TGF-ß) to this cytokine cocktail resulted in the generation of MoLCs. MoLCs showed a lower expression of CD83, CD86, HLA-DR and CCR7 compared with MoDCs, regardless of their maturational status. Both immature and mature MoLCs secreted higher quantities of IL-23 compared with MoDCs and this finding correlated with a higher secretion of IL-17 in co-culture of MoLCs with allogeneic CD4(+) T cells. Mature MoLCs, which produced higher levels of IL-12 and lower levels of IL-10 compared with mature MoDCs, were more potent at inducing interferon-γ (IFN-γ) production by CD4(+) T cells in the co-culture system. In conclusion, the finding that mature MoLCs stimulate stronger T-helper 1 and T-helper 17 immune responses than mature MoDCs, makes them better candidates for use in the preparation of anti-tumour DC vaccines.

The Influence of Heat-Killed Enterococcus faecium BGPAS1-3 on the Tight Junction Protein Expression and Immune Function in Differentiated Caco-2 Cells Infected With Listeria monocytogenes ATCC 19111.

Listeria monocytogenes, the common foodborne pathogenic bacteria species, compromises the intestinal epithelial barrier, leading to development of the listeriosis, a severe disease especially among immunocompromised individuals. L. monocytogenes infection usually requires antibiotic treatment, however, excessive use of antibiotics promotes emergence of antibiotic resistance and the destruction of gut microbiota. Probiotics, including lactic acid bacteria (LAB), have been repeatedly proven as an alternative approach for the treatment of various infections. We have analyzed the potential of Enterococcus faecium BGPAS1-3, a dairy isolate exhibiting strong direct antilisterial effect, to modulate the response of differentiated Caco-2 intestinal epithelial cells to L. monocytogenes ATCC 19111 infection. We showed that the molecule with antilisterial effect is a bacterial cell-wall protein that is highly resistant to the high-temperature treatment. When we tested the antilisterial potential of heat-killed BGPAS1-3, we found that it could prevent tight junction disruption in differentiated Caco-2 monolayer infected with L. monocytogenes ATCC 19111, induce antilisterial host response mechanisms, and stimulate the production of protective TGF-ß in intestinal epithelial cells. We also showed that the modulation of MyD88 dependent TLR2 and TLR4 pathways by BGPAS1-3 are involved in host response against L. monocytogenes ATCC 19111. Since heat-killed BGPAS1-3 possess strong antilisterial effects, such postbiotic could be used as a controllable and safe therapeutic.

GABA-Producing Natural Dairy Isolate From Artisanal Zlatar Cheese Attenuates Gut Inflammation and Strengthens Gut Epithelial Barrier in vitro.

Probiotic bacteria are recognized for their health-promoting properties, including maintenance of gut epithelial integrity and host immune system homeostasis. Taking into account the beneficial health-promoting effects of GABA, the presence of the gadB gene, encoding glutamate decarboxylase that converts L-glutamate to GABA, was analyzed in Lactic Acid Bacteria (LAB) natural isolates from Zlatar cheese. The results revealed that 52% of tested Lactobacillus spp. and 8% of Lactococcus spp. isolates harbor the gadB gene. Qualitative and quantitative analysis of GABA production performed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed the highest GABA production by Lactobacillus brevis BGZLS10-17. Since high GABA-producing LAB natural isolates are the most valuable source of naturally produced GABA, the probiotic properties of BGZLS10-17 were characterized. This study demonstrated high adhesion of BGZLS10-17 strain to Caco-2 cells and the ability to decrease the adhesion of Escherichia coli ATCC25922 and Salmonella enterica C29039. Treatment of differentiated Caco-2 cells monolayer with BGZLS10-17 supernatant containing GABA alleviated inflammation (production of IL-8) caused by IL-1ß and significantly stimulated the expression of tight junction proteins (zonulin, occludin, and claudin 4), as well as the expression of TGF-ß cytokine leading to the conclusion that immunosuppression and strengthening the tight junctions can have significant role in the maintenance of intestinal epithelial barrier integrity. Taken together the results obtained in this study support the idea that using of GABA producing BGZLS10-17 probiotic strain could be a good strategy to modulate immunological response in various inflammatory diseases, and at the same time, it could be a good candidate for adjunct starter culture for production of GABA-enriched dairy foods and beverages offering new perspectives in designing the novel functional foods.

New Insight into Biofilm Formation Ability, the Presence of Virulence Genes and Probiotic Potential of Enterococcus sp. Dairy Isolates.

Enterococci have controversial status due to their emerging role in nosocomial infections and transmission of antibiotic resistance genes, while some enterococci strains are used as probiotics for humans and animals and starter cultures in dairy industry. In order to improve our understanding of factors involved in the safe use of enterococci as potential probiotics, the antibiotic susceptibility, virulence and probiotic traits of 75 dairy enterococci isolates belonging to Enterococcus durans (50), En. faecium (15), En. faecalis (6), En. italicus (3), and En. hirae (1) were evaluated. The results revealed that ciprofloxacin resistance and biofilm formation are correlated with isolates originated from Golija mountain (Serbia), while gelatinase activity was more common in isolates from Prigorje region (Croatia), pointing to uncontrolled use of antibiotics and anthropogenic impact on dairy products' microbiota in these regions. The virulence genes were sporadically present in 13 selected dairy enterococci isolates. Interestingly, biofilm formation was correlated with higher ability of strains to reduce the adhesion of E. coli and Salmonella Enteritidis to HT29-MTX cells. To our knowledge this is the first study reporting the presence of the esp gene (previously correlated with pathogenesis) in dairy enterococci isolates, mostly associated with the genes involved in adhesion property. Hence, the results of this study revealed that the virulence genes are sporadically present in dairy isolates and more correlated to adhesion properties and biofilm formation, implicating their role in gut colonization rather than to the virulence traits.

Exopolysaccharide Produced by Probiotic Strain Lactobacillus paraplantarum BGCG11 Reduces Inflammatory Hyperalgesia in Rats.

The aim of this study was to test the potential of high molecular weight exopolysaccharide (EPS) produced by the putative probiotic strain Lactobacillus paraplantarum BGCG11 (EPS CG11) to alleviate inflammatory pain in Wistar rats. The EPS CG11 was isolated from bacterial surface and was subjected to Fourier-transform infrared spectroscopy (FTIR) and thermal analysis. FTIR spectra confirmed the polysaccharide structure of isolated sample, while the thermal methods revealed good thermal properties of the polymer. The antihyperalgesic and antiedematous effects of the EPS CG11 were examined in the rat model of inflammation induced by carrageenan injection in hind paw. The results showed that the intraperitoneal administration of EPS CG11 produced a significant decrease in pain sensations (mechanical hyperalgesia) and a paw swelling in a dose-dependent manner as it was measured using Von Frey anesthesiometer and plethysmometer, respectively. These effects were followed by a decreased expression of IL-1ß and iNOS mRNAs in rat's paw tissue suggesting that the antihyperalgesic and antiedematous effects of the EPS CG11 are related to the suppression of inflammatory response. Additionally, we demonstrated that EPS CG11 exhibits immunosuppressive properties in the peritonitis model induced by carrageenan. Expression levels of pro-inflammatory mediators IL-1ß, TNF-α and iNOS were decreased, together with the enhanced secretion of anti-inflammatory IL-10 and IL-6 cytokines, while neutrophil infiltration was not changed. To the best of our knowledge, this is the first study which reports an antihyperalgesic effect as the novel property of bacterial EPSs. Given the high demands of pharmaceutical industry for the replacement of commonly used analgesics due to numerous side effects, this study describes a promising natural compound for the future pharmacological testing in the area.

The effect of potassium channel opener pinacidil on the non-pregnant rat uterus.

The effects of the K(+) channel opener, pinacidil on the spontaneous rhythmic contractions and contractions provoked by electrical field stimulation (50 Hz) or by oxytocin were investigated in the isolated uterus of the non-pregnant rat in oestrus. Pinacidil produced more potent inhibition of oxytocin-elicited contractions than of spontaneous rhythmic contractions or electrical field stimulation-induced contractions. Glibenclamide, a selective blocker of adenosine triphosphate (ATP)-sensitive K(+) (K(ATP)) channels, antagonized the pinacidil-induced inhibition of contractions elicited by oxytocin in a competitive manner. However, the pinacidil-induced inhibition of electrical field stimulation-elicited contractions and spontaneous rhythmic contractions was antagonized non-competitively by glibenclamide. In the uterine strips pre-contracted with 80 mM K(+), the pinacidil-induced maximal relaxation was not affected. The present data show that pinacidil exhibits potent relaxant properties in the rat non-pregnant uterus in oestrus and therefore should be taken into account as a possible agent for treatment of dysmenorrhoea. Based on glibenclamide affinity, it appears that the inhibitory response to pinacidil involves K(ATP )channels. We need further investigations to explain why the interaction between glibenclamide and pinacidil in this experimental model depends on the nature of contractions. The ability of pinacidil to completely relax the rat non-pregnant uterus pre-contracted with K(+)-rich solution suggests that K(+) channel-independent mechanism(s) also play a part in its relaxant effect.

Lactobacillus fermentum Postbiotic-induced Autophagy as Potential Approach for Treatment of Acetaminophen Hepatotoxicity.

The aim of this study was to investigate the potential of postbiotics originated from Lactobacillus fermentum BGHV110 strain (HV110) to counteract acetaminophen (APAP)-induced hepatotoxicity in HepG2 cells. This strain was selected according to its autophagy inducing potential, based on previous studies reporting protective role of autophagy in APAP caused cellular damage. Cell viability was assessed using MTT and LDH assays, while autophagy was monitored by qPCR analysis of BECN1, Atg5, p62/SQSTM1, and PINK1 mRNA expression and by Western blot analysis of p62/SQSTM1 and lipidated LC3 accumulation. Our results showed that detrimental effect of APAP on cell viability was suppressed in the presence of HV110 which was linked with increased conversion of LC3 protein and p62/SQSTM1 protein degradation. Additionally, higher p62/SQSTM1 and PINK1 mRNA transcription were noticed in cells co-treated with APAP/HV110, simultaneously. In conclusion, this study suggests that HV110 enhances activation of PINK1-dependent autophagy in HepG2 cells and its eventual co-supplementation with APAP could be potentially used for alleviation of hepatotoxic side effects caused by APAP overdose.

The effect of gender and alcohol placement in the processing of sexual intent.

INTRODUCTION AND AIMS: Alcohol consumption in women is known to be perceived by men as signalling sexual intent. However, it is unclear whether such assumptions extend to the simple presence of alcohol. The present study investigated the association between gender and alcohol placement on processing of sexual intent. DESIGN AND METHODS: One hundred and forty-seven sexually experienced male and female participants were shown a brief video of a social interaction between a man and woman depicted with a bottle of water or alcohol. Participants were then asked to rate the female target on sexual intent. RESULTS: Men inferred greater sexual intent compared to women in the female target when she was depicted with alcohol as compared to water. Contrary to previous research, personality traits did not contribute to perceptions of sexual intent. However, state (sexual-related) variables such as likely sexual relationship between targets and attractiveness of the female target, did increase the level of sexual intent processed. DISCUSSION AND CONCLUSIONS: These findings suggest that alcohol may be a cue used by men in their social environment to process sexual intent. The association of a woman with alcohol suggesting sexual intent could have potential implications for advertising practice which influences sexual beliefs toward women.

Cadmium administration affects circulatory mononuclear cells in rats.

Although numerous investigations have demonstrated a direct effect of cadmium (Cd) on peripheral blood mononuclear cell (PBMC) activity in humans, there is virtually no data concerning the in vivo impact of this metal on circulatory mononuclear cells. In this study, the effects of a sub-lethal Cd (1 mg/kg) dose were examined in rats 48 h following a single intraperitoneal injection. Cd treatment resulted in increased total peripheral blood leukocyte levels; however, decreases in PBMC numbers were seen. These changes coincided with an accumulation of mononuclear cells in the lungs and an increase in mononuclear cells expressing CD11b. A lack of effect of Cd on spontaneous nitric oxide (NO) production and on iNOS mRNA levels in the PBMC was also noted. Differential effects of Cd on PBMC inflammatory cytokine (IL-1ß, TNFα, IL-6, IFNγ, and IL-17) gene expression and production were also seen. Specifically, except for IL-1ß (levels increased), there were decreases (relative to controls) in mRNA levels for all the other cytokines examined. While there were no Cd treatment-related changes in spontaneous production of the cytokines assessed, there seemed to be a trend (p = 0.06) toward a decrease in spontaneous IL-6 release. When these harvested cells were stimulated ex vivo, there was no effect from Cd exposure on LPS-stimulated IL-1ß and TNFα or on ConA-stimulated IFNγ or IL-17 production, but a decrease in IL-6 production in response to LPS was, again, noted. A preliminary study with a lower Cd dose (0.5 mg/kg) revealed some of the same outcomes noted here (mononuclear cell infiltration into lungs, increases in PBMC IL-1ß mRNA levels), but differential (increased IL-17 mRNA levels) or newly detected outcomes (increased levels of IL-1α mRNA) as well. The described effects of the single in vivo exposure to Cd on PBMC might contribute to a better overall understanding of the immunomodulatory potential of this environmental contaminant.

Heparin/heparan sulfate controls fibrillin-1, -2 and -3 self-interactions in microfibril assembly.

Fibrillins form multifunctional microfibrils in most connective tissues. Deficiencies in fibrillin assembly can result in fibrillinopathies, such as Marfan syndrome. We demonstrate the presence of heparin/heparan sulfate binding sites in fibrillin-2 and -3. Multimerization of all three fibrillins drastically increased the apparent affinity of their interaction with heparin/heparan sulfate. Surprisingly, contrary to other reports heparin/heparan sulfate strongly inhibited homo- and heterotypic N-to-C-terminal fibrillin interactions. These data suggest that heparin/heparan sulfate controls the formation of microfibrils at the bead interaction stage.

Regional cytokine responses to pulmonary aspergillosis in immunocompetent rats.

Rat models of pulmonary aspergillosis are used widely in diagnostic studies and in exploring antifungal therapeutic modalities, but there is lack of data concerning antifungal immunity in rats. In this study, cytokine response to pulmonary infection to Aspergillus fumigatus in non-immunosuppressed rats is explored. Temporal display (from the start of infection up to its eradication) of proinflammatory cytokines (IFN-γ and IL-17) as well as Th2/anti-inflammatory ones (IL-4 and IL-10) was explored by measuring their presence in the environment in which elimination of infection occur (lung homogenates), by production of these mediators by lung cells (recovered by enzyme digestion or by bronchoalveolar lavage) as well as by cells of draining lymph nodes (as sites of generation of cytokine-producing cells). Reduction of infection (1 × 107conidia) was associated with an increase of IFN-γ and IL-17 content in lung homogenates, but with unchanged IL-4 and IL-10 content. Lung cells produced proinflammatory cytokines with differential dynamics (IFN-γ earlier than IL-17). Differential pattern of Th2/anti-inflammatory cytokine production by lung cells was observed (unchanged IL-4 and increased IL-10), with the levels of the latter higher than proinflammatory cytokines. Upregulation of IFN-γ, IL-17 and IL-10 production and gene expression, but downregulation of IL-4, by draining lymph node cells (dLN cells) accounted essentially for the observed ex vivo cytokine response in lungs. Similar pattern of cytokine production by dLN cells following restimulation with A. fumigatus conidia confirmed the specificity of cytokine response to the fungus. Draining lymph node CD4⁺ cells seems to be the main source of proinflammatory cytokines, significant contributors to IL-10 production and the target for down regulation of IL-4. The knowledge of immune-based mechanisms of defense against A. fumigatus in rats might be helpful in the future use of rat models of pulmonary aspergillosis particularly those that develop immune-based therapeutic interventions as an adjunct treatment of fungal diseases.

Systemic immunomodulatory effects of topical dinitrochlorobenzene (DNCB) in rats. Activity of peripheral blood polymorphonuclear cells.

Topical application of dinitrochlorobenzene (DNCB) is employed in the immunotherapy of skin diseases. Activation of T-cell mediated immune responses (Th1/type1) is the supposed mechanism of the clinical effect of DNCB, but there are no data concerning innate/inflammatory mechanisms. In this study, the effect of repeated topical DNCB application on peripheral blood polymorphonuclear (PMN) leukocytes has been examined in two rat strains which differ in the propensity to mount Th1/type1 or Th2/type2 responses. The dynamics of changes in PMN numbers and effector activities (respiratory burst, nitric oxide production and myeloperoxidase content), as well as in adhesion and TNF-α production following the rat skin sensitization with low (0.4%) and high (4%) DNCB doses were measured. Both priming and activation of PMNs were observed following skin sensitization with DNCB, with dose-dependent as well as time-dependent differences in some PMN activities. Obtained data might be relevant for understanding the immune mechanisms of topical DNCB therapy.

Immunomodulatory properties of mesenchymal stem cells derived from dental pulp and dental follicle are susceptible to activation by toll-like receptor agonists.

Adult mesenchymal stem cells (MSCs) have recently become a potent tool in regenerative medicine. Due to certain shortcomings of obtaining bone marrow MSCs, alternate sources of MSCs have been sought. In this work, we studied MSCs from dental pulp (DP-MSCs) and dental follicle (DF-MSCs), isolated from the same tooth/donor, to define differences in their phenotypic properties, differentiation potential, and immunomodulatory activities. Both cell types showed colony-forming ability and expressed typical MSCs markers, but differed in the levels of their expression. DF-MSCs proliferated faster, contained cells larger in diameter, exhibited a higher potential to form adipocytes and a lower potential to form chondrocytes and osteoblasts, compared with DP-MSCs. In contrast to DF-MSCs, DP-MSCs produced the transforming growth factor (TGF)-ß and suppressed proliferation of peripheral blood mononuclear cells, which could be neutralized with anti-TGF-ß antibody. The treatment with toll-like receptor 3 (TLR3) agonist augmented the suppressive potential of both cell types and potentiated TGF-ß and interleukin-6 secretions by these cells. TLR4 agonist augmented the suppressive potential of DF-MSCs and increased TGF-ß production, but abrogated the immunosuppressive activity of DP-MSCs by inhibiting TGF-ß production and the expression of indolamine-2,3-dioxygenase-1. Some of these effects correlated with the higher expression of TLR3 and TLR4 by DP-MSCs compared with DF-MSCs. When transplanted in imunocompetent xenogenic host, both cell types induced formation of granulomatous tissue. In conclusion, our results suggest that dental MSCs are functionally different and each of these functions should be further explored in vivo before their specific biomedical applications.

Analysis of the vasorelaxant action of angiotensin II in the isolated rat renal artery.

Taking into consideration that mechanisms involved in the vasodilatator actions of angiotensin II have not yet been completely elucidated, the present study was undertaken in order to examine the mechanisms underlying the angiotensin II-induced relaxation of rat renal artery (RRA). Angiotensin II produced concentration-dependent and endothelium-independent relaxation of isolated RRA. Angiotensin II-induced relaxation was partially reduced by inhibitors of nitric oxide synthase and guanylyl cyclase. The remaining dilatation was inhibited by a potassium channel blocker, charybdotoxin. Precontraction of RRA with high concentration of K(+) partially reduced angiotensin II-evoked relaxation, while indomethacin, glibenclamide, apamin and barium did not alter the angiotensin II concentration-response curve. Losartan had no effect on angiotensin II effect. Oppositely, HOE 140 and PD123319, separately or in combination, partially antagonized vasorelaxation induced by angiotensin II. Complete blockade of RRA response was obtained after simultaneous incubation of all three receptor antagonists HOE-140, PD123319, and losartan; L-NOARG plus HOE-140; or PD123319 plus charybdotoxin. These results indicate that angiotensin II produces endothelium-independent relaxation of RRA, which is most probably mediated by the interaction of the NO-cGMP pathway and K(+) channels. Moreover, we can assume that AT(1), AT(2), and B(2) receptors are involved in the vasorelaxant effect of angiotensin II.