Differentiation of keratinocytesin vitro: a new culture vessel mimicking thein vivo situation

A culture vessel consisting of two independent chambers separated only by the growth substrate is described. Cells may be cultured on both sides of the growth substrate. Culture medium and gas exposure can independently be controlled in both compartments. Human hair follicles have been used as source of keratinocytes and the bovine eye lens capsule has been explored as growth substrate. The presence of 5% CO₂ in air in the lower compartment appears to have a significant effect on the morphology of the cultures. When the cultures are being exposed to air with 5% CO₂, the culture medium being applied in the lower compartment, formation of corneocytes characteristic for adult stratum corneum is induced, as evidenced by light and electron microscopy. To the knowledge of the authors, this stage of differentiationin vitro has not been obtained with previously described systems. Differentiation of the lower cell layers has been characterised with specific antibodies. The possible use of the system for applied and pure scientific research is discussed.     

Vascular Multiplicity Should Not Be a Contra-Indication for Live Kidney Donation and Transplantation

BackgroundWhether vascular multiplicity should be considered as contraindication and therefore ‘extended donor criterion’ is still under debate.MethodsData from all live kidney donors from 2006–2013 (n = 951) was retrospectively reviewed. Vascular anatomy as imaged by MRA, CTA or other modalities was compared with intraoperative findings. Furthermore, the influence of vascular multiplicity on outcome of donors and recipients was studied.ResultsIn 237 out of 951 donors (25%), vascular multiplicity was present. CTA had the highest accuracy levels regarding vascular anatomy assessment. Regarding outcome of donors with vascular multiplicity, warm ischemia time (WIT) and skin-to-skin time were significantly longer if arterial multiplicity (AM) was present (5.1 vs. 4.0 mins and 202 vs. 178 mins). Skin-to-skin time was significantly longer, and complication rates were higher in donors with venous multiplicity (203 vs. 180 mins and 17.2% vs. 8.4%). Outcome of renal transplant recipients showed a significantly increased WIT (30 vs. 26.7 minutes), higher rate of DGF (13.9% vs. 6.9%) and lower rate of BPAR (6.9% vs. 13.9%) in patients receiving a kidney with AM compared to kidneys with singular anatomy.ConclusionsWe conclude that vascular multiplicity should not be a contra-indication, since it has little impact on clinical outcome in the donor as well as in renal transplant recipients.     

Employment of a noninvasive magnetic method for evaluation of gastrointestinal transit in rats

ABSTRACT: AC Biosusceptometry (ACB) was previously employed towards recording gastrointestinal motility. Our data show a reliable and successful evaluation of gastrointestinal transit of liquid and solid meals in rats, considering the methods scarcity and number of experiments needed to endorsement of drugs and medicinal plants. ACB permits real time and simultaneous experiments using the same animal, preserving the physiological conditions employing both meals with simplicity and accuracy.     

Rotavirus enterotoxin NSP4 binds to the extracellular matrix proteins laminin-beta3 and fibronectin

Rotavirus is the most important cause of viral gastroenteritis and dehydrating diarrhea in young children. Rotavirus nonstructural protein 4 (NSP4) is an enterotoxin that was identified as an important agent in symptomatic rotavirus infection. To identify cellular proteins that interact with NSP4, a two-hybrid technique with Saccharomyces cerevisiae was used. NSP4 cDNA, derived from the human rotavirus strain Wa, was cloned into the yeast shuttle vector pGBKT7. An intestinal cDNA library derived from Caco-2 cells cloned into the yeast shuttle vector pGAD10 was screened for proteins that interact with NSP4. Protein interactions were confirmed in vivo by coimmunoprecipitation and immunohistochemical colocalization. After two-hybrid library screening, we repeatedly isolated cDNAs encoding the extracellular matrix (ECM) protein laminin-beta3 (amino acids [aa] 274 to 878) and a cDNA encoding the ECM protein fibronectin (aa 1755 to 1884). Using deletion mutants of NSP4, we mapped the region of interaction with the ECM proteins between aa 87 and 145. Deletion analysis of laminin-beta3 indicated that the region comprising aa 726 to 875 of laminin-beta3 interacts with NSP4. Interaction of NSP4 with either laminin-beta3 or fibronectin was confirmed by coimmunoprecipitation. NSP4 was present in infected enterocytes and in the basement membrane (BM) of infected neonatal mice and colocalized with laminin-beta3, indicating a physiological interaction. In conclusion, two-hybrid screening with NSP4 yielded two potential target proteins, laminin-beta3 and fibronectin, interacting with the enterotoxin NSP4. The release of NSP4 from the basal side of infected epithelial cells and the subsequent binding to ECM proteins localized at the BM may signify a new mechanism by which rotavirus disease is established.     

Chronic exposure of diesel exhaust particles induces alveolar enlargement in mice

Background

Diesel exhaust particles (DEPs) are deposited into the respiratory tract and are thought to be a risk factor for the development of diseases of the respiratory system. In healthy individuals, the timing and mechanisms of respiratory tract injuries caused by chronic exposure to air pollution remain to be clarified.

Methods

We evaluated the effects of chronic exposure to DEP at doses below those found in a typical bus corridor in Sao Paulo (150 μg/m³). Male BALB/c mice were divided into mice receiving a nasal instillation: saline (saline; n = 30) and 30 μg/10 μL of DEP (DEP; n = 30). Nasal instillations were performed five days a week, over a period of 90 days. Bronchoalveolar lavage (BAL) was performed, and the concentrations of interleukin (IL)-4, IL-10, IL-13 and interferon-gamma (INF-γ) were determined by ELISA-immunoassay. Assessment of respiratory mechanics was performed. The gene expression of Muc5ac in lung was evaluated by RT-PCR. The presence of IL-13, MAC2+ macrophages, CD3+, CD4+, CD8+ T cells and CD20+ B cells in tissues was analysed by immunohistochemistry. Bronchial thickness and the collagen/elastic fibers density were evaluated by morphometry. We measured the mean linear intercept (Lm), a measure of alveolar distension, and the mean airspace diameter (D0) and statistical distribution (D2).

Results

DEP decreased IFN-γ levels in BAL (p = 0.03), but did not significantly alter IL-4, IL-10 and IL-13 levels. MAC2+ macrophage, CD4+ T cell and CD20+ B cell numbers were not altered; however, numbers of CD3+ T cells (p ≤ 0.001) and CD8+ T cells (p ≤ 0.001) increased in the parenchyma. Although IL-13 (p = 0.008) expression decreased in the bronchiolar epithelium, Muc5ac gene expression was not altered in the lung of DEP-exposed animals. Although respiratory mechanics, elastic and collagen density were not modified, the mean linear intercept (Lm) was increased in the DEP-exposed animals (p ≤ 0.001), and the index D2 was statistically different (p = 0.038) from the control animals.

Conclusion

Our data suggest that nasal instillation of low doses of DEP over a period of 90 days results in alveolar enlargement in the pulmonary parenchyma of healthy mice.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0172-z) contains supplementary material, which is available to authorized users.     

A new species of Mesocoelium (Digenea: Mesocoeliidae) found in Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia

Mesocoelium lanfrediaesp. nov. (Digenea: Mesocoeliidae) inhabits the small intestine of Rhinella marina (Amphibia: Bufonidae) and is described here, with illustrations provided by light, scanning electron microscopy and molecular approachs. M. lanfrediae sp. nov. presents the typical characteristics of the genus, but is morphometrically and morphologically different from the species described previously. The main diagnostic characteristics of M. lanfrediae sp. nov. are (i) seven pairs of regularly-distributed spherical papillae on the oral sucker, (ii) ventral sucker outlined by four pairs of papillae distributed in a uniform pattern and interspersed with numerous spines, which are larger at the posterior margin and (iii) small, rounded tegumentary papillae around the opening of the oral sucker, which are morphologically different from those of the oral sucker itself, some of which are randomly disposed in the ventrolateral tegumentary region of the anterior third of the body. Addionally, based on SSU rDNA, a phylogenetic analysis including Brachycoeliidae and Mesocoeliidae taxa available on GenBank established the close relationship between M. lanfrediae sp. nov. and Mesocoelium sp.     

Psoriatic hair follicle cells I. Biochemistry and behaviour in culture

It is generally accepted that in psoriasis there is an alteration of epidermal cell proliferation. It has been reported that an increased rate of thymidine incorporation into keratinocytes is found in the upper part of the hair follicle in involved skin, but this is not the case in the lower part. Here we show that cells from psoriatic hair follicles could be brought in culture under the same conditions as those of normal hair follicles. Cells, whether originating from the upper or lower part of the hair follicle sheath either from involved or uninvolved psoriatic skin, show a faster rate of outgrowth in the first days of culture. Moreover, a large number of psoriatic cells have an increased motility in the early stages of culture, as compared to control cells. These properties can no longer be observed after several days in culture. The activity of glucose-6-phosphate dehydrogenase known to be increased in psoriatic plaques is normal in hair follicles isolated from these plaques. Protein gel electrophoretic investigations showed that there is no difference in gel patterns between normal and psoriatic hair follicles.In conclusion, the isolation of human hair follicles represents a simple method that allows psoriatic keratinocytes to be brought in culture and permits the study of certain aspects of the disease.     

Biosynthesis of chlorophyll P-680 of photosystem II in greening leaves of plants adapted to heat shock

In etiolated pea and maize leaves illuminated after incubation at 38 degreesC, a new dark reaction was shown manifested in the bathochromic shift of spectral bands and accompanied by esterification of the product of protochlorophyllide photochemical reduction--Chld 684/676: Chld 684/676 --> Chl 688/680. After completion of the reaction a rapid (20-30 sec) quenching of the fluorescence of the reaction product (Chl 688/680) was observed. The reaction Chld 684/676 --> Chl 688/680 is inhibited under anaerobic conditions and in the presence of cyanide; the reaction accompanied by Chl 688/680 fluorescence quenching is not observed in pea mutants with impaired function of photosystem II reaction centers. The spectral properties of the formed Chl form with the absorption maximum at 680 nm, fluorescence quenching, and simultaneous synthesis of pheophytin suggest that the reaction is connected with the chlorophyll of photosystem II reaction center--P-680.