Improved Regeneration of Autologous Transplanted Lymph Node Fragments by VEGF‐C Treatment

Secondary lymphedema is a common complication after removal of lymph nodes in combination with radiation therapy in the treatment of breast cancer, cervical cancer, and melanomas. Only symptomatic therapies are available at the moment, and lymphedema is for most patients a lifelong condition involving psychological and physical disabilities. Animal models exist to study the pathophysiology of lymphedema but not to study surgical treatments. The aim of this study was to show that regeneration of autologous transplanted lymph node fragments is possible in rats that were irradiated previously locally in the groin and to examine the effects of vascular endothelial growth factor (VEGF)‐C injections on the rate of regeneration of transplanted lymph nodes. In all of the animals, inguinal and popliteal lymph nodes and adjacent lymphatic vessels were unilaterally removed and the inguinal region irradiated by a single dose of 15 Gy. Afterward, lymph node fragments were transplanted subcutaneously in the irradiated region. Half of the animals were treated by local VEGF‐C injections after transplantation. Four weeks after transplantation, drainage of the leg was tested by injection of blue dye, and the transplanted fragments were removed and examined immunohistologically. We could show that regeneration of autologous transplanted lymph node fragments is possible in areas treated with radiotherapy in the rat. We also documented that transplants can achieve a connection to the lymphatic collectors of the leg. The results suggest that the outcome of regeneration can be improved by injection of VEGF‐C in the transplantation area. Thus, lymph node fragment regeneration may be relevant for lymphedema prevention and therapy. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Differential regulation of TRPV4 mRNA levels by acclimation salinity and extracellular osmolality in euryhaline tilapia

Prolactin (PRL) cells of the euryhaline Mozambique tilapia, Oreochromis mossambicus, are osmoreceptors. Hyposmotically-induced PRL release is mediated by the inward movement of extracellular Ca(2+) through a stretch-activated Ca(2+) channel, which has been recently identified as the transient receptor potential vanilloid 4 (TRPV4). In the present study, changes in plasma PRL, as well as PRL and TRPV4 mRNA expression from the rostral pars distalis (RPD), were measured in fish transferred from seawater (SW) to fresh water (FW) and in fish transferred from FW to SW. The in vitro effects of osmolality on PRL release and on PRL and TRPV4 mRNA expression in dispersed PRL cells were compared between fish adapted to SW and FW. Both the release and expression of PRL fell when fish were transferred to SW and rose when fish were transferred to FW. By contrast, TRPV4 expression increased by 48h after fish were transferred from FW to SW and declined as early as 6h after transfer from SW to FW. A similar pattern was observed in vitro where TRPV4 expression responded positively to an increase in medium osmolality while PRL expression declined. Incubation with the Ca(2+) ionophore, A23187, and the phosphodiesterase inhibitor, IBMX, stimulated PRL release. While both IBMX and A23187 inhibited TRPV4 expression, only A23187 reduced PRL expression. Together, these findings indicate that the expression of TRPV4 mRNA is osmosensitive, increasing as extracellular osmolality rises. Furthermore, these data suggest that TRPV4 expression may be regulated through the same second messenger pathways involved in hyposmotically-induced PRL release.     

Colonic fermentation as affected by antibiotics and acidic pH: Application of an in vitro model.

Antimicrobial substances such as vancomycin or metronidazole suppress normal gut flora, thereby preventing physiological fermentation of colonic substrates that may promote mucosal inflammation. This study was designed to establish an in vitro model of microbial metabolism in the colon under control and disturbed conditions (acidic pH) to investigate specific effects of vancomycin and metronidazole on the production of short chain fatty acids (SCFA), which play a pivotal role in maintaining homeostasis in the colon. The experiments were carried out with the colon simulation technique (Cositec) representing an in vitro model for the semi-continuous incubation of defined colon contents. Inocula and fermentable substrates were sampled from cecal contents of fistulated pigs. Disturbed microbial metabolism was generated by reduction of pH in the fermentation vessels from 6.7 to 5.8 and 5.1. In general, application of either vancomycin or metronidazole resulted in a significant decrease of SCFA production rates indicating substantial disturbance of the homeostasis of microbial metabolism. With low doses of vancomycin acetate and butyrate production rates were reduced and with high doses of the antibiotic propionate production was inhibited to a greater extent. Treatment with metronidazole inhibited butyrate production almost completely. Similarly, low pH caused a reduction in total SCFA production, which was mainly due to respective decrease of acetate synthesis. Metronidazole effects were not consistently changed at low pH. The Cositec system provides an excellent facility to test the effects of different antibiotics under defined conditions. In this study, both vancomycin and metronidazole affected microbial metabolism to a considerable extent. Both substances may thus be responsible for disturbances of colon function in vivo.     

Influence of farnesol on the morphogenesis of Aspergillus niger

Farnesol was the first quorum-sensing regulator to be found in eukaryotic cells. In Candida albicans, a dimorphic fungal human pathogen, farnesol blocks the yeast-to-filamentous growth transition. Here we show that in Aspergillus niger farnesol acts as an inhibitor of conidiation: Colonies grown on media containing farnesol were unable to develop conidia. Although farnesol treated A. niger cultures exhibited a colony morphology resembling the "fluffy" phenotype of A. nidulans, which is caused by a hyperactive G-protein/cAMP pathway, the intracellular level of cAMP in A. niger mycelia grown in presence of farnesol is greatly diminished. Furthermore, whereas inhibiting adenylyl cyclase led to a farnesol-like effect, the addition of external cAMP inhibited conidiation without causing a "fluffy" phenotype. This suggests that the mechanisms regulating conidiation in A. niger and A. nidulans are different.     

Reactions of N-quaternary 1-phenyl-3,4-dihydroisoquinolinium compounds with nucleophiles - products and their isomerism - part 1

Variously substituted 1-phenyl-3,4-dihydroisoquinolinium compounds were prepared, in order to study their reactions with hydroxide ions concerning ring-chain isomerism of their products. Thereby was observed a dependence on the electrophilic properties of C-1 for forming the pseudo-bases and a ring opening tendency to the amino ketones by electron-withdrawing substituents at the nitrogen atom. Moreover steric effects caused by voluminous substituents in 1- and 2-positions also importantly affected the isomeric distribution. So the N-ethyl derivatives showed an enhanced dissociation of the hemiaminal to the quaternary iminium base. From a solution of the dihydroisoquinolinium salt 35 in [D6]DMSO after addition of NaOD until pH 10 resulted an equilibrium of the quaternary iminium base 36q, the pseudo-base 36a and the amino ketone 36b. These species were detected and assigned by NMR measurements.     

Preventive Effects of the Probiotic Escherichia coli Strain Nissle 1917 on Acute Secretory Diarrhea in a Pig Model of Intestinal Infection

Pretreatment with the probiotic Escherichia colistrain Nissle 1917 (EcN) was assessed in a pig model of intestinal infection to prevent acute secretory diarrhea. In the model 10¹⁰ colony forming units of the porcine enterotoxigenic Escherichia coli Abbotstown (EcA) was given via orogastric tube to weaned piglets at day 21 postpartum (−EcN/+EcA group, n = 7). Forty-eight hours after challenge electrophysiological parameters of isolated intact jejunal epithelia were characterized in Ussing chambers. In agreement with clinical signs of diarrhea, tissues of challenged animals showed an overshoot of secretory response after stimulation of the cAMP-mediated second messenger pathway by forskolin, indicating higher excitability of chloride secretory systems under infected conditions. The data were compared with respective measurements from animals that got a daily dose of 10¹⁰ cfu of the probiotic EcN over 10 days before EcA challenge (+EcN/+EcA group; n = 4), from a group that received only EcN (+EcN/–EcA; n = 4), or from a group that remained totally untreated (−EcN/−EcA; n = 6). EcN pretreatment completely abolished clinical signs of secretory diarrhea in +EcN/+EcA animals. Furthermore, jejunum epithelia of these animals did not exhibit an overshoot of secretory response upon stimulation with forskolin. Our studies demonstrate for the first time the efficacy of prophylactic EcN in pig small intestine for preventing an effect of toxigenic EcA. This infection model with freshly weaned piglets may be predestinated to further characterize EcN effects on the cellular level, i.e., involved second messenger pathways, or it may also be useful to examine the efficacy of other substrates or microbe strains against secretory stimuli.     

The colon carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is actively secreted in the distal colon of the rat: an integrated view on the role of PhIP transport and metabolism in PhIP-induced colon carcinogenesis

Epidemiological studies show that a positive correlation exists between the consumption of strongly heated meat and fish and the development of colorectal tumours. In this context, it has been postulated that the uptake of toxic substances formed during meat and fish processing such as heterocyclic aromatic amines (HCAs) may be causally related to colon carcinogenesis. In a previous study, we have shown that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundantly formed HCA in the above-mentioned food items, is mainly absorbed in the small intestine (i.e. proximal jejunum) of the rat. In the present study, we analysed whether PhIP can actively be secreted by enterocytes in the rat proximal jejunum and distal colon. Unidirectional PhIP flux rates from the mucosal-to-the serosal compartment (J _ms ) and in the opposite direction (J _sm ) were examined in Ussing chambers with ¹⁴C-PhIP as radiotracer and in the absence of electrochemical gradients. Under these experimental conditions, significant negative net flux rates (J _net  = J _ms  ? J _sm ) can only be explained by an active secretion of PhIP into the luminal compartment, and such an effect was observed in the rat distal colon, but not in the proximal jejunum. Moreover, the data obtained suggest that the breast cancer resistance protein, the multidrug resistance protein 4 and P-glycoprotein are not involved in the active secretion of PhIP in the rat distal colon. The potential role of PhIP transport in colon carcinogenesis is discussed.