Herbicide Widespread: The Effects of Pethoxamid on Nonalcoholic Fatty Liver Steatosis In Vitro.

Pethoxamid is a widespread herbicidal product, presenting itself as an extremely flexible active substance and with a high potential for use as an herbicide for preemergence. The emergence of multiple resistance in crops has been addressed using combinations of preemergence and postemergence herbicides in the same seeding-harvest cycle. A winning combination of pethoxamid and glyphosate mainly affected the acidobacteria population. Glyphosate scientific literature has demonstrated an observational link between herbicide exposure and liver disease in human subjects. Identifying and ranking the risk to the public that pethoxamid could exert on target organs has not been evaluated so far. Due to similarities to glyphosate, we did look at the effect of pethoxamid on impaired liver cells HepG2, using a nonalcoholic fatty liver disease (NAFLD) cell model in vitro. Pethoxamid was cytotoxic starting at 1 ppm. Fatty acid accumulation (FA) was enhanced while low doses of pethoxamid slightly decreased LDH protein expression compared to FA-treated HepG2. The same trend was observed for cytochrome c. Based on our data, we can argue that NAFLD hepatic cells react to pethoxamid trying detoxifying strategies, ready to undergo cell death to avoid further degeneration. Downregulation of cytochrome can lead to the hypothesis that pethoxamid should not induce herbicide resistance.

A Semisynthetic Approach to New Immunoadjuvant Candidates: Site-Selective Chemical Manipulation of Escherichia coli Monophosphoryl Lipid†A.

A semisynthetic approach to novel lipid A derivatives from Escherichia coli (E. coli) lipid A is reported. This methodology stands as an alternative to common approaches based exclusively on either total synthesis or extraction from bacterial sources. It relies upon the purification of the lipid A fraction from fed-batch fermentation of E. coli, followed by its structural modification through tailored, site-selective chemical reactions. In particular, modification of the lipid pattern and functionalization of the phosphate group as well as of the sole primary hydroxyl group were accomplished, highlighting the unusual reactivity of the molecule. Preliminary investigations of the immunostimulating activity of the new semisynthetic lipid A derivatives show that some of them stand out as promising, new immunoadjuvant candidates.

Structural characterization of the lipid A from the LPS of the haloalkaliphilic bacterium Halomonas pantelleriensis.

Halomonas pantelleriensis DSM9661(Τ) is a Gram-negative haloalkaliphilic bacterium isolated from the sand of the volcanic Venus mirror lake, closed to seashore in the Pantelleria Island in the south of Italy. It is able to optimally grow in media containing 3-15 % (w/v) total salt and at pH between 9 and 10. To survive in these harsh conditions, the bacterium has developed several strategies that probably concern the bacteria outer membrane, a barrier regulating the exchange with the environment. In such a context, the lipopolysaccharides (LPSs), which are among the major constituent of the Gram-negative outer membrane, are thought to contribute to the restrictive membrane permeability properties. The structure of the lipid A family derived from the LPS of Halomonas pantelleriensis DSM 9661(T) is reported herein. The lipid A was obtained from the purified LPS by mild acid hydrolysis. The lipid A, which contains different numbers of fatty acids residues, and its partially deacylated derivatives were completely characterized by means of ESI FT-ICR mass spectrometry and chemical analysis. Preliminary immunological assays were performed, and a comparison with the lipid A structure of the phylogenetic proximal Halomonas magadiensis is also reported.

A combined fermentative-chemical approach for the scalable production of pure E. coli monophosphoryl lipid A.

Lipid A is the lipophilic region of lipopolysaccharides and lipooligosaccharides, the major components of the outer leaflet of most part of Gram-negative bacteria. Some lipid As are very promising immunoadjuvants. They are obtained by extraction from bacterial cells or through total chemical synthesis. A novel, semisynthetic approach to lipid As is ongoing in our laboratories, relying upon the chemical modification of a natural lipid A scaffold for the fast obtainment of several other lipid As and derivatives thereof. The first requisite for this strategy is to have this scaffold available in large quantities through a scalable process. Here, we present an optimized fed-batch fermentation procedure for the gram-scale production of lipid A from Escherichia coli K4 and a suitable phenol-free protocol for its purification. A study for regioselective de-O-phosphorylation reaction was then performed to afford pure monophosphoryl lipid A with an attenuated endotoxic activity, as evaluated by cytokine production in human monocytic cell line THP-1 in vitro. The reported method for the large-scale obtainment of monophoshoryl lipid A from the fed-batch fermentation broth of a recombinant strain of E. coli may permit the access to novel semisynthetic lipid A immunoadjuvant candidates.

Gene expression profile of patients with Mayer-Rokitansky-Küster-Hauser syndrome: new insights into the potential role of developmental pathways.

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is a rare disease characterized by congenital aplasia of uterus and vagina. Although many studies have investigated several candidate genes, up to now none of them seem to be responsible for the aetiology of the syndrome. In our study, we identified differences in gene expression profile of in vitro cultured vaginal tissue of MRHKS patients using whole-genome microarray analysis. A group of eight out of sixteen MRKHS patients that underwent reconstruction of neovagina with an autologous in vitro cultured vaginal tissue were subjected to microarray analysis and compared with five healthy controls. Results obtained by array were confirmed by qRT-PCR and further extended to other eight MRKHS patients. Gene profiling of MRKHS patients delineated 275 differentially expressed genes, of which 133 downregulated and 142 upregulated. We selected six deregulated genes (MUC1, HOXC8, HOXB2, HOXB5, JAG1 and DLL1) on the basis of their fold change, their differential expression in most patients and their relevant role in embryological development. All patients showed upregulation of MUC1, while HOXB2 and HOXB5 were downregulated, as well as Notch ligands JAG1 and DLL1 in the majority of them. Interestingly, HOXC8 was significantly upregulated in 47% of patients, with a differential expression only in MRKHS type I patients. Taken together, our results highlighted the dysregulation of developmental genes, thus suggesting a potential alteration of networks involved in the formation of the female reproductive tract and providing a useful clue for understanding the pathophysiology of MRKHS.

Enteroglial-derived S100B protein integrates bacteria-induced Toll-like receptor signalling in human enteric glial cells.

OBJECTIVE: Enteric glial cells (EGC) have been suggested to participate in host-bacteria cross-talk, playing a protective role within the gut. The way EGC interact with microorganisms is still poorly understood. We aimed to evaluate whether: EGC participate in host-bacteria interaction; S100B and Toll-like receptor (TLR) signalling converge in a common pathway leading to nitric oxide (NO) production. DESIGN: Primary cultures of human EGC were exposed to pathogenic (enteroinvasive Escherichia coli; EIEC) and probiotic (Lactobacillus paracasei F19) bacteria. Cell activation was assessed by evaluating the expression of cFos and major histocompatibility complex (MHC) class II molecules. TLR expression in EGC was evaluated at both baseline and after exposure to bacteria by real-time PCR, fluorescence microscopy and western blot analysis. S100B expression and NO release from EGC, following exposure to bacteria, were measured in the presence or absence of specific TLR and S100B pathway inhibitors. RESULTS: EIEC activated EGC by inducing the expression of cFos and MHC II. EGC expressed TLR at baseline. Pathogens and probiotics differentially modulated TLR expression in EGC. Pathogens, but not probiotics, significantly induced S100B protein overexpression and NO release from EGC. Pretreatment with specific inhibitors of TLR and S100B pathways abolished bacterial-induced NO release from EGC. CONCLUSIONS: Human EGC interact with bacteria and discriminate between pathogens and probiotics via a different TLR expression and NO production. In EGC, NO release is impaired in the presence of specific inhibitors of the TLR and S100B pathways, suggesting the presence of a novel common pathway involving both TLR stimulation and S100B protein upregulation.

A time-lapse approach to examine chromium and nickel effects on wound healing in vitro.

Chromium and nickel cause allergic contact dermatitis, a common biological skin response to sensitizing agents. This study used a conventional in vitro wounding model to study the impact of sensitizing agents on the innate immune response of human keratinocytes. Experiments were designed to evaluate the involvement of specific Toll-like receptors and metalloproteinases as effectors molecules downstream, at a molecular level. Further, keratinocytes were co-cultured with monocytes (THP-1 cells) to reproduce an inductive stimulus on monocytes made by metals. Human keratinocytes (HaCat) were grown on plates covered with collagen type I, chemically treated, and then mechanically injured with a sterile pipette tip. Restoration of the monolayer integrity was monitored by time-lapse video microscopy. Effector gene expression was evaluated by real-time PCR. The presence of chromium significantly dropped the rate of wound closure, while nickel-induced hyper-proliferation ended in an acceleration of the healing process, an event that does not occur in vivo. This latter outcome led to considering nickel as an unsuitable example for use in the experimental model. Focusing solely on the chromium aspect of this study, RNA profiles of selected molecular markers were generated to ascertain if the detrimental stimulus from chromium was eliminated or persisted both in keratinocytes alone and/or during co-cultures of keratinocytes and monocytes. Monocytes accelerated the process of wound repair. This in vitro experimental model highlighted the involvement of innate immunity in response to chromium and might be useful for test molecules of therapeutic interest for the treatment of skin lesions. However, the experience with nickel reveals that there are limitations to the utility of this wound model system after all.

An alternative gas-phase in vitro exposure system for toxicity testing: the interaction between nitrous oxide and A549 cells.

An original in vitro approach was adopted to expose cells to volatile agents. The anaesthetic nitrous oxide (N(2)O) was chosen as the model agent, and type II pneumocyte-like cells (A549 cells) were used as the target to represent the lungs. A time-lapse microscopy station was equipped with a manual gas mixer that allowed the generation of a mixture of N(2)O/air/CO(2) in the gas phase, to provide a uniform distribution of the volatile agent. The dissolution of N(2)O in the culture medium was monitored by gas chromatography-electron capture detection. Biochemical alterations, in terms of homocysteine accumulation, demonstrated that intracellular methionine synthase had been inactivated by N(2)O absorbed by the cells, a process that also occurs in vivo. Toll-like receptors, which are key molecules in inflammatory lung diseases, were also investigated at the molecular level. Our experiments indicated that biochemical and molecular alterations occurred in the cells, even under conditions where neither morphologic changes nor consistent alterations in cell proliferation were evident. This in vitro exposure system can be efficiently adopted for looking at the repeat-dose effects of volatile agents on respiratory tissues. Moreover, it could be of further benefit for identifying the wide range of specific cell targets, and for monitoring relevant endpoints in the cellular and molecular processes that occur during exposure to volatile compounds.

In vitro evaluation of Lactobacillus plantarum DSMZ 12028 as a probiotic: emphasis on innate immunity.

In this study, we analyzed the probiotic potential of L. plantarum DSMZ 12028 in vitro using the pathogen E. coli K4 and a certified probiotic, L. paracasei F19, as controls. Adhesion to intestinal epithelial cells was evaluated using two cell lines, CaCo-2 and HT-29, through the plate dilution method. Moreover, the bacteria/epithelial dynamic interaction was continuously monitored using time-lapse microscopy. Expression of the innate immunity receptors, the TLRs, was evaluated by semi-quantitative PCR on an epithelial/bacteria co-culture. Real-time PCR was used to monitor expression of TLRs and cytokines in a monocytic cell line (THP-1) following bacterial exposure. The adherence of the strain to intestinal epithelial cells was comparable to that of the probiotic. Time-lapse experiments showed that E. coli K4 induced cell death while L. plantarum did not affect proliferation at a 10:1 bacteria/cell ratio. L. plantarum down-regulated TLR mRNAs with the exception of TLR2, while L. paracasei F19 and E. coli K4 caused a significant (p<0.05) up-regulation of TLR2 and 4, respectively. To simulate the activation of underlying immune cells in the lamina propria, we analyzed the immunomodulation of L. plantarum on a monocytic cell line, THP-1. Proinflammatory cytokines, such as TNFalpha, were increased by the presence of bacteria. The pathogen E. coli K4 also induced a strong up-regulation of proinflammatory cytokines, such as IL8, IL1beta and IL23. No differences were observed between experimental groups for IFNgamma, IL-10 and IL12p40. Overall, L. plantarum DSMZ 12028 demonstrated probiotic traits, inducing a proinflammatory response just above the "threshold level", which could prevent an inflammatory outcome, while inducing a higher state of alertness in the defense system of the host intestinal epithelial cells.

Human CD34/CD90 ASCs are capable of growing as sphere clusters, producing high levels of VEGF and forming capillaries.

BACKGROUND: Human adult adipose tissue is an abundant source of mesenchymal stem cells (MSCs). Moreover, it is an easily accessible site producing a considerable amount of stem cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have selected and characterized stem cells within the stromal vascular fraction (SVF) of human adult adipose tissue with the aim of understanding their differentiation capabilities and performance. We have found, within the SVF, different cell populations expressing MSC markers--including CD34, CD90, CD29, CD44, CD105, and CD117--and endothelial-progenitor-cell markers--including CD34, CD90, CD44, and CD54. Interestingly, CD34(+)/CD90(+) cells formed sphere clusters, when placed in non-adherent growth conditions. Moreover, they showed a high proliferative capability, a telomerase activity that was significantly higher than that found in differentiated cells, and contained a fraction of cells displaying the phenotype of a side population. When cultured in adipogenic medium, CD34(+)/CD90(+) quickly differentiated into adipocytes. In addition, they differentiated into endothelial cells (CD31(+)/VEGF(+)/Flk-1(+)) and, when placed in methylcellulose, were capable of forming capillary-like structures producing a high level of VEGF, as substantiated with ELISA tests. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate, for the first time, that CD34(+)/CD90(+) cells of human adipose tissue are capable of forming sphere clusters, when grown in free-floating conditions, and differentiate in endothelial cells that form capillary-like structures in methylcellulose. These cells might be suitable for tissue reconstruction in regenerative medicine, especially when patients need treatments for vascular disease.

Effects of low concentrations of benzene on human lung cells in vitro.

Exposure to benzene causes health hazards to humans. The airway epithelium is a physical barrier to inhaled toxicants and particulates. This is an in vitro basic science study to evaluate the effects of benzene on lung cells without the inflammatory responses triggered by inhalation. Dose-response cytotoxicity was assessed using two cell lines: alveolar derived (A549) human epithelial adenocarcinoma and human lung (LL24) fibroblast. A549 cells were more resistant than LL24 fibroblast lung cells to benzene. LL24 cells demonstrated enhanced proliferation with diluted benzene solutions. Moreover, low concentrations of benzene enhanced telomerase activity in LL24 cells while no effects were observed in the adenocarcinoma cells. Proteolysis of lung matrix by matrix metalloproteinases (MMPs) is an early event observed in lung pathologies. Benzene increased MMP-2 and MMP-3 mRNA. Using the ratio (MMP-1+MMP-2+MMP-3)/(TIMP-1+TIMP-2), as an index of prodestructive activity, we observed a dose-dependent increase. The overall higher expression levels of MMPs in benzene treated cells did not appear to be controlled by TIMPs, which are negatively correlated. Comparing different cell lines, we demonstrated how crucial is the target's susceptibility. Our observations may represent early functional alterations that occur in the airways of exposed people.

Matrix metalloproteinases and their inhibitors as biomarkers for metal toxicity in vitro.

Exposure to nickel and chromium, and their compounds, has been associated with adverse health effects. These metals are two human carcinogens whose pathogenesis involves active extracellular matrix degradation and remodelling. In this work we have compared the effects of in vitro exposure to nickel and chromium of a keratinocyte cell line (HaCat). The modulation of matrix metalloproteinase genes was used as biomarker of chemical damage. Confluent cells were constantly exposed to subtoxic chromium and nickel concentrations (10(-5) and 10(-7)M) up to 72 h. Total RNA was extracted and specific matrix metalloproteinase, and inhibitor, gene expression was analyzed by RT-PCR. Moreover, cell cycle alterations were evaluated by flow cytometry. Nickel and chromium showed different results, with an upregulation of MMP-2 mRNA production in nickel-treated cells while chromium exposure down-regulated MMP-2 mRNA production. This result could be correlated to the precocious (6h) over-expression of tissue inhibitor-1 (TIMP-1) mRNA in chromium-treated cells. Cell cycle analysis showed and increase of cells with 4N DNA. These results could be explained as a survival response of cells that escape metal induced apoptosis through the anti-apoptotic effects of TIMP-1. These cells that encompass the genotoxic insult may have a selective proliferation advantage, and therefore represent the precursor pool from which degenerating variants may emerge. To study if the chemical damage was reversible, subconfluent cells were stimulated only for 24 h, then the medium was replaced without metal. Cells were able to recover from nickel exposure, showing only weak alterations in specific mRNA expression and cell cycle alteration respect to control. Chromium-induced damage was irreversible. Our results demonstrated that there is an association between metal toxicity and expression of MMPs and their inhibitors. These biomarkers could be potentially useful to elaborate a prediction model of chemical toxicity.

In vitro evaluation of matrix metalloproteinases as predictive testing for nickel, a model sensitizing agent.

The identification of potential damage due to chemical exposure in the workplace is a major health and regulatory concern. Traditional tests that measure both sensitization and elicitation responses require the use of animals. An alternative to this widespread use of experimental animals could have a crucial impact on risk assessment, especially for the preliminary screening of new molecules. We developed an in vitro model for the screening of potential toxic compounds. Human keratinocytes (HaCat) were used as target cells while matrix metalloproteinases (MMP) were selected as responders because they are key enzymes involved in extracellular matrix (ECM) degradation in physiological and pathological conditions. Chemical exposure was performed using nickel sulphate as a positive tester. Nickel contact induced upregulation of MMP-2 and IL-8 mRNA production. Molecular activation occurred even at very low nickel concentrations even though no phenotypic changes were observed. MMP-9 accumulation was found in the medium of treated cells with respect to controls. These observations led to the hypothesis that even minimal exposure can accumulate transcriptional activity resulting in long-term clinical signs after contact. Our simple in vitro model can be applied as a useful preliminary complement to the animal studies to screen the effects of new potential toxic compounds.

Evaluation of a high temperature immobilised enzyme reactor for production of non-reducing oligosaccharides.

There is interest in the production of non-reducing carbohydrates due to their potential application in various industrial fields, particularly the food industry. In this paper, we describe the development of an immobilised cell bioprocess for the synthesis of non-reducing maltodextrins at high temperatures. The trehalosyl-dextrins-forming enzyme (TDFE) isolated from the thermoacidophilic archaeon Sulfolobus solfataricus (strain MT4), was recently expressed at high yields in Escherichia coli (strain Rb-791). Here, we evaluate different matrices, such as polyacrylamide gel, crude egg white, chitosan and calcium alginate for their effectiveness in immobilising whole recombinant E. coli cells subjected to prior thermal permeabilisation. Calcium-alginate based gels formed a solid biocatalyst with a good activity yield and the best enzymatic stability at the operating temperature (75 degrees C). Therefore, these beads were used to pack a glass column reactor to perform the bioconversion of interest. Optimal operating parameters were defined in relation to the substrate stream flow-rate and the substrate-to-biocatalyst ratio. The production of trehalosylmaltotetraose from maltohexaose reached equilibrium with a constant of about 2.6 at 75 degrees C. The bioreactor was exploited for production of trehalosylmaltodextrins from a commercial mixture of maltodextrins, achieving a productivity of 106.5 mg ml(-1) h(-1) (g biocatalyst)(-1) with ~40% conversion when using a 30% (w/v) solution.

High cell density cultivation of probiotics and lactic acid production.

The commercial interest in functional foods that contain live microorganisms, also named probiotics, is paralleled by the increasing scientific attention to their functionality in the digestive tract. This is especially true of yogurts that contain strains of lactic-acid bacteria of intestinal origin, among these, Lactobacillus delbrueckii ssp. bulgaricus is extensively used in the dairy industry and it has been demonstrated to be a probiotic strain. In this work we describe high cell density cultivations of this microorganism also focusing on the stereospecific production of lactic acid. Key parameters such as medium composition (bactocasitone concentration) and diverse aeration conditions were explored. The results showed that the final concentration of biomass in anaerobic fermentation was lower than the one obtained in microaerophilic conditions, while it gave a very high productivity of lactic acid which was present as a racemic mixture in the permeate. Fermentation experiments carried out with air sparging, even at very low flow-rate, led to the production of the sole L(+) lactic acid giving sevenfold increase in biomass yield in respect to the batch cultivation. Finally, a mathematical model was developed to describe the microfiltration bioprocess applied in this research considering an inhibition kinetic and enucleating a suitable mathematical description for the decrease of the transmembrane flux.

Grafting of "abbreviated" complementarity-determining regions containing specificity-determining residues essential for ligand contact to engineer a less immunogenic humanized monoclonal antibody.

Murine mAb COL-1 reacts with carcinoembryonic Ag (CEA), expressed on a wide range of human carcinomas. In preclinical studies in animals and clinical trials in patients, murine COL-1 showed excellent tumor localization. To circumvent the problem of immunogenicity of the murine Ab in patients, a humanized COL-1 (HuCOL-1) was generated by grafting the complementarity-determining regions (CDRs) of COL-1 onto the frameworks of the variable light and variable heavy regions of human mAbs. To minimize anti-V region responses, a variant of HuCOL-1 was generated by grafting onto the human frameworks only the "abbreviated" CDRs, the stretches of CDR residues that contain the specificity-determining residues that are essential for the surface complementarity of the Ab and its ligand. In competition RIAs, the recombinant variant completely inhibited the binding of radiolabeled murine and humanized COL-1 to CEA. The HuCOL-1 and its variant showed no difference in their binding ability to the CEA expressed on the surface of a CEA-transduced tumor cell line. Compared with HuCOL-1, the HuCOL-1 variant showed lower reactivity to patients' sera carrying anti-V region Abs to COL-1. The final variant of the HuCOL-1, which retains its Ag-binding reactivity and shows significantly lower serum reactivity than that of the parental Ab, can serve as a prototype for the development of a potentially useful clinical reagent.

Perspectives on biotechnological applications of archaea.

Many archaea colonize extreme environments. They include hyperthermophiles, sulfur-metabolizing thermophiles, extreme halophiles and methanogens. Because extremophilic microorganisms have unusual properties, they are a potentially valuable resource in the development of novel biotechnological processes. Despite extensive research, however, there are few existing industrial applications of either archaeal biomass or archaeal enzymes. This review summarizes current knowledge about the biotechnological uses of archaea and archaeal enzymes with special attention to potential applications that are the subject of current experimental evaluation. Topics covered include cultivation methods, recent achievements in genomics, which are of key importance for the development of new biotechnological tools, and the application of wild-type biomasses, engineered microorganisms, enzymes and specific metabolites in particular bioprocesses of industrial interest.