Effects of TGF-beta and glucocorticoids on map kinase phosphorylation, IL-6/IL-11 secretion and cell proliferation in primary cultures of human lung fibroblasts

Transforming growth factor-beta1 (TGF-beta1) is crucially involved in the fibrotic events characterizing interstitial lung diseases (ILDs), as well as in the airway remodeling process typical of asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal and fibrotic human lung fibroblasts (HLFs), the effects of TGF-beta1 on mitogen-activated protein kinase (MAPK) phosphorylation, cell proliferation, and production of interleukins 6 (IL-6) and 11 (IL-11), in the presence or absence of a pretreatment with budesonide (BUD). MAPK phosphorylation was detected by Western blotting, cell viability and proliferation were evaluated using Trypan blue staining and [(3)H]-thymidine incorporation assay, respectively, and the release of IL-6 and IL-11 into cell culture supernatants was assessed by ELISA. TGF-beta1 (10 ng/ml) significantly stimulated MAPK phosphorylation (P < 0.01), and also enhanced cell proliferation as well as the secretion of both IL-6 and IL-11, which reached the highest increases at the 72nd h of cell exposure to this growth factor. All such effects were prevented by BUD (10(-8) M) and, with the exception of IL-6 release, also by a mixture of MAPK inhibitors. Therefore, our findings suggest that the fibrotic action exerted by TGF-beta1 in the lung is mediated at least in part by MAPK activation and by an increased synthesis of the profibrogenic cytokines IL-6 and IL-11; all these effects appear to be prevented by corticosteroids via inhibition of MAPK phosphorylation.     

Block of mitochondrial apoptotic pathways in lizard ovarian follicle cells as an adaptation to their nurse function

Pyriforms are ovarian follicle nurse cells that undergo apoptosis at the end of previtellogenesis and are completely eliminated by the epithelium. This event is accompanied by the active transfer of organelles and macromolecules to the oocyte via an intercellular bridge. Since it would be a nonsense for damaged mitochondria to reach the oocyte, we have postulated that pyriform cells have adapted their apoptotic machinery to prevent mitochondrial degradation. To verify this hypothesis, we have studied mitochondrial morphology and functionality during follicle cell regression. Cytological and biochemical evidence indicates that mitochondria in pyriforms maintain their size, organization and membrane potential. This clearly indicates that they are not involved in apoptosis signalling/progression. This block would favour both the oocyte, by increasing the pool of organelles available from follicle cells, and also the regressing pyriforms, by maintaining the energy resources required for completion of their nurse function. The block is probably attributable to an over-expression of Bcl-2 and might be carried out by sequestering cytochrome c inside the organelles. As demonstrated by in vitro experiments, the mitochondrial apoptosis pathway can be activated by stress induction, such as serum deprivation, but not following physiological pro-apoptotic signalling, such as treatment with gonadotrophin-releasing hormone. These studies were supported by a grant from the MIUR (PRIN project: Molecular responses of embryonic, differentiated and tumoral cells exposed to cadmium intoxication).     

Organisation of cervical cytology screening in Croatia: past, present and future

This presentation highlights strengths and weaknesses of cervical cytology screening in Croatia, with particular reference to the opportunistic screening, the use of conventional Papanicolaou (Pap) test and the analysis of some organizational, educational and performance issues that are associated with it. Its aim is to propose measures to improve the efficacy of cervical cytology screening in order to reduce cervical cancer mortality. Currently, in excess of 450,000 Pap tests/ year are examined at 35 laboratories scattered throughout the country. All of these laboratories use standard operating procedures including internal and external quality control. They employ a total of 68 cytologists and 91 cytotechnologists. The sensitivity of cervical screening in Croatia is 90.0%, specificity 98.6%, positive predictive value 92.3%, negative predictive value 98.1% and overall diagnostic accuracy 97.2%. The high diagnostic accuracy of cervical cytology is attributed to the long-standing tradition of education and training of cytologists (postgraduate MSc course since 1967, independent residency since 1974) and cytotechnologists (since 1968). This tradition spanning more than half a century means that today in Croatia there is a developed network of cytology laboratories staffed by highly competent cytologists and trained cytotechnologists. The high accuracy of cancer detection through Pap tests provides strong evidence in support of cervical cytology screening remaining the basic method of prevention for cervical carcinoma. However, some modifications to the current situation are needed. These relate primarily to opportunistic screening. The current screening coverage rate is 68%, although there is capacity, which would allow for all women at risk, i.e. those aged 25-64, to be screened once in three years. The screening coverage relates mainly to those women visiting gynecological out patient clinics for unrelated conditions. A properly organized and controlled national screening programme should replace this. This should be accompanied by the introduction of alternative, highly sensitive methods of sample collection and preparation, such as are available through the introduction of new technologies, e.g. liquid based cytology.     

Stable enzyme biosensors based on chemically synthesized Au-polypyrrole nanocomposites

This work describes development and optimization of a generic method for the immobilization of enzymes in chemically synthesized gold polypyrrole (Au-PPy) nanocomposite and their application in amperometric biosensors. Three enzyme systems have been used as model examples: cytochrome c, glucose oxidase and polyphenol oxidase. The synthesis and deposition of the nanocomposite was first optimized onto a glassy carbon electrode (GCE) and then, the optimum procedure was used for enzyme immobilization and subsequent fabrication of glucose and phenol biosensors. The resulting nanostructured polymer strongly adheres to the surface of the GCE electrode, has uniform distribution and is very stable. The method has proved to be an effective way for stable enzyme attachment while the presence of gold nanoparticles provides enhanced electrochemical activity; it needs very small amounts of pyrrole and enzyme and the Au-PPy matrix avoids enzyme leaking. The preparation conditions, Michaelis-Menten kinetics and analytical performance characteristics of the two biosensors are discussed. Optimization of the experimental parameters was performed with regard to pyrrole concentration, enzyme amount, pH and operating potential. These biosensors resulted in rapid, simple, and accurate measurement of glucose and phenol with high sensitivities (1.089 mA/M glucose and 497.1 mA/M phenol), low detection limits (2 x 10(-6)M glucose and 3 x 10(-8)M phenol) and fast response times (less than 10s). The biosensors showed an excellent operational stability (at least 100 assays) and reproducibility (R.S.D. of 1.36%).     

Antibiotic resistance in long-term care facilities

Long-term-care facilities (LTCFs) comprise a heterogeneous group of institutions that provide a wide variety of services to diverse groups of patients, most of whom are elderly. Infections are common in LTCFs and these are complicated by antimicrobial-resistant pathogens. The residents in LTCFs have a high frequency of colonization with antimicrobial-resistant organisms, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, penicillin-resistant pneumococci, extended spectrum P-lactamase-producing gram-negative organisms, and fluoroquinolone-resistant gram-negative organisms. Although several control programs have been published, up to now there still is a long way to go in this area of health care. This review will briefly touch upon the clinical relevance of antimicrobial resistance in LTCFs.     

An anti-infective peptide that selectively modulates the innate immune response

We show that an innate defense-regulator peptide (IDR-1) was protective in mouse models of infection with important Gram-positive and Gram-negative pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Salmonella enterica serovar Typhimurium. When given from 48 h before to 6 h after infection, the peptide was effective by both local and systemic administration. Because protection by IDR-1 was prevented by in vivo depletion of monocytes and macrophages, but not neutrophils or B- and T-lymphocytes, we conclude that monocytes and macrophages are key effector cells. IDR-1 was not directly antimicrobial: gene and protein expression analysis in human and mouse monocytes and macrophages indicated that IDR-1, acting through mitogen-activated protein kinase and other signaling pathways, enhanced the levels of monocyte chemokines while reducing pro-inflammatory cytokine responses. To our knowledge, an innate defense regulator that counters infection by selective modulation of innate immunity without obvious toxicities has not been reported previously.     

The ingestion of a Nectandra membranacea extract changes the bioavailability of technetium-99m radiobiocomplex in rat organs

The radiobiocomplexes labeled with technetium-99m (Tc-99m) have been widely used in nuclear medicine in single photon emission computed tomography and in basic research. The aim of this study was to assess the influence of a Nectandra membranacea extract on the bioavailability of the sodium pertechnetate (Na(99m)TcO(4)) radiobiocomplex in rat organs. The animals were treated with a N. membranacea extract (30 mg/ ml), for 6 days. Na(99m)TcO(4) was injected, the organs were isolated and weighed, and the radioactivity was determined in each organ (%ATI/organ). The %ATI/organ was divided by the mass of each organ to calculate the %ATI/g. A significant increase of the %ATI/organ of Na(99m)TcO(4) was observed in muscle and thyroid as well as in the %ATI/g in the heart, kidney and thyroid. These findings could result from the interaction between components of the plant extract and the radiobiocomplex which may influence the uptake Na(99m)TcO(4) in rat organs. Therefore, precaution is suggested in the interpretation of nuclear medicine results in patients using this herb.