Translocation of Enterococcus faecalis strains across a monolayer of polarized human enterocyte-like T84 cells

We used a two-chamber system to study transcytosis of Enterococcus faecalis across monolayers of human colon carcinoma-derived T84 cells, which show structural resemblance to the native intestine. Among 16 E. faecalis isolates from different sources, the well-characterized strain OG1RF and 8 other isolates (2 endocarditis isolates, 1 urine isolate, and all 5 fecal isolates) showed translocation in this assay, while 6 clinical isolates (3 endocarditis and 3 urine isolates), the recipient strain JH2-2, and the control, Escherichia coli DH5alpha, had no detectable translocation. Of two OG1RF mutants involving the previously studied epa (enterococcal polysaccharide antigen) gene cluster, known to be needed for virulence and resistance to killing by polymorphonuclear leukocytes, one epa mutant (TX5179) was unable to translocate, while TX5180, with an epa disruption farther downstream, showed a moderate decrease in translocation relative to that of the wild-type strain OG1RF (P < 0.01), indicating that the epa gene cluster is important for translocation across a T84 monolayer. This observation was confirmed by complementation of the epa mutant (TX5179) with epa genes and restoration of its translocation ability. In conclusion, we have demonstrated translocation of at least some strains of E. faecalis across T84 monolayers, although strains differ considerably in this ability, and we have demonstrated that epa mutations can cause marked changes in successful translocation. These results suggest that this model may be a useful in vitro system for studying the process of translocation from the intestinal tract.     

Intratracheal administration of liposomal clodronate accelerates alveolar macrophage reconstitution following fetal liver transplantation

To facilitate study of alveolar macrophages in vivo, we developed a method to rapidly and efficiently replace resident alveolar macrophages with macrophages of a different (donor) genotype. Chimeric mice were generated by lethal irradiation followed by fetal liver transplantation (FLT) using green fluorescent protein (GFP) transgenic reporter mice as donors. Kinetics of peripheral blood monocyte (PBM) and alveolar macrophage reconstitution was determined 4 and 10 weeks post-FLT by quantifying the percentage of GFP+ cells. To enhance the recruitment of donor monocytes into the lung after FLT, mice were treated with intratracheal administration of liposomal clodronate to deplete host alveolar macrophages at 6 weeks post-FLT. PBM reconstitution occurred by 4 weeks after FLT (85.7+/-1.6% of CD11b+/Gr-1+ monocytes were GFP+), and minimal alveolar macrophage repopulation was observed (9.5% GFP+). By 10 weeks following FLT, 48% of alveolar macrophages were GFP+ by immunostaining of macrophages on lung tissue sections, and 55.1 +/- 1.6% of lung lavage macrophages were GFP+ by fluorescein-activated cell sorter analysis. Clodronate treatment resulted in a significant increase in GFP+ alveolar macrophages 10 weeks after FLT. By immunostaining, 90% of macrophages were GFP+ on lung tissue sections and 87.5 +/- 1.1% GFP+ in lung lavage (compared with GFP-transgenic controls). The ability of newly recruited alveolar macrophages to clear Pseudomonas aeruginosa and activate nuclear factor-kappaB in response to Eschericia coli lipopolysaccharide demonstrated normal macrophage function. Optimizing this methodology provides an important tool for the study of specific genes and their contribution to alveolar macrophage function in vivo.     

肾上腺器官内淋巴管的解剖学研究

采用墨汁硝酸银水溶液局部动脉灌注方法,研究了胎儿、犬和家兔肾上腺(总计80例)器官内的淋巴管.所见结果表明:胎儿和犬肾上腺被膜内及腺体内的较粗大静脉周围存在淋巴管和毛细淋巴管;家兔肾上腺的淋巴管仅分布于被膜内;在肾上腺皮质和髓质的实质内,无论胎儿、犬和兔,仅有血管并无淋巴管或毛细淋巴管.     

Rosiglitazone, an agonist of peroxisome proliferator-activated receptor γ, reduces pulmonary inflammatory response in a rat model of endotoxemia

Objective: The effect of rosiglitazone, a potent peroxisome proliferator-activated receptor γ (PPAR-γ) agonist, on pulmonary inflammation in endotoxemia was investigated. Materials and methods: Male Wistar rats were given either lipopolysaccharide (LPS, 6 mg/kg i.v.) or saline, pretreated with rosiglitazone (0.3 mg/kg i.v.) or its vehicle (dimethyl sulphoxide) 30 min before LPS. The selective PPAR-γ antagonist GW9662 (0.3 mg/kg i.v.) was given 20 min before rosiglitazone. Wet/dry weight (W/D) ratio, myeloperoxidase (MPO) activity, malondialdehyde (MDA) as well as TNF-α and CINC-1 concentrations were measured in lung tissues 4 h after LPS injection. Expression of ICAM-1, NF-κB p65 and PPAR-γ were also determined by immunohistochemistry or Western blot analysis. Results: Rosiglitazone pretreatment significantly attenuated the increases in W/D ratio, MPO activity and MDA levels, and reduced pulmonary overproduction of TNF-α and CINC-1 as well as expression of ICAM-1 following endotoxemia. Rosiglitazone also inhibited the nuclear localization of NF-κB and up-regulated the expression of PPAR-γ protein. The specific PPAR-γ antagonist GW9662 abolished the effect of rosiglitazone. Conclusion: These findings suggest that PPAR-γ agonists might be used as therapeutic agents in the therapy of inflammatory lung injury related to endotoxemia. Received 8 January 2005; returned for revision 6 July 2005; returned for final revision 20 July 2005; accepted by M. Katori 31 July 2005     

Secondary sulphonylurea failure: what pathogenesis is responsible?

Sulphonylurea (SU) stimulates insulin secretion by pancreatic beta-cells and is generally used as a first-line treatment for type 2 diabetes. However, after long-term SU treatment (six months or over), some patients begin to show an increase in blood glucose once again (secondary SU failure). Two theories have been put forward to explain this failure--dysfunction of the proinsulin conversion machinery or insulin resistance. However, the primary pathogenesis behind secondary SU failure still needs to be investigated. Using a reliable technique that specifically identifies intact proinsulin (IPI), total proinsulin (TPI) and specific insulin (SI), this study aims to discover if a defect in the proinsulin converting mechanism plays a role in SU failure. Three groups were recruited for this study: healthy controls (n=8), SU responders (n=38) and secondary SU failures (n= 46). Serum concentrations of insulin-related molecules released in response to a standard glucose challenge test were compared between the groups. It was found that total SI was lower in the patient groups (P<0.05 compared to the control group), while TPI and IPI showed no distinct difference between the three groups (P>0.05). TPI:SI ratio and IPI:SI ratio showed marked increases in the patient groups (P<0.05 compared to control group), with no obvious quantitative difference between SU responders and secondary SU failures (P>0.05). Similar results for the Homa Insulin Resistant Index were found between the two patient groups. Interestingly, blood glucose at 180 mins after glucose challenge was significantly higher in the secondary SU failure group (P<0.05), with no correlation to SI, while the SU responder group showed good correlation between the parameters (P<0.05). We conclude that type 2 diabetes is associated with obvious dysfunction in the proinsulin-converting process and shows severe SI deficiency in responding to glucose challenge. Dysfunction of the proinsulin conversion mechanism was not an extra cause responsible for SU failure.     

Prediction of apatite lattice constants from their constituent elemental radii and artificial intelligence methods

Lattice constants (LCs) of all possible 96 apatite compounds, A(5)(BO(4))(3)C, constituted by A[double bond]Ba(2+), Ca(2+), Cd(2+), Pb(2+), Sr(2+), Mn(2+); B[double bond]As(5+), Cr(5+), P(5+), V(5+); and C[double bond]F(1-), Cl(1-), Br(1-), OH(1-), are predicted from their elemental ionic radii, using pattern recognition (PR) and artificial neural networks (ANN) techniques. In particular, by a PR study it is demonstrated that ionic radii predominantly govern the LCs of apatites. Furthermore, by using ANN techniques, prediction models of LCs a and c are developed, which reproduce well the measured LCs (R(2)=0.98). All the literature reported on 30 pure and 22 mixed apatite compounds are collected and used in the present work. LCs of all possible 66 new apatites (assuming they exist) are estimated by the developed ANN models. These proposed new apatites may be of interest to biomedical research especially in the design of new apatite biomaterials for bone remodeling. Similarly these techniques may also be applied in the study of interface growth behaviors involving other biomaterials.     

Progressive decrease of proinsulin secretion in sulphonylurea-treated type 2 diabetes

Progressive deterioration of beta-cell function is proposed as a disease-related factor of sulphonylurea (SU) failure in type 2 diabetes. If it gradually worsens over time then disease duration may mirror the progressive beta-cell deterioration. The aim of the present study is to assess whether or not disease duration is influential in remodelling the secretion pattern of insulin-like molecules and in glucose control of SU-treated type 2 diabetes. A research model is used to investigate proinsulin secreting capacity over time, using two groups of patients: i) disease duration <5 years (n=62), comprising SU responders (SUr; n=48) and SU failures (SUf; n=14); and ii) disease duration > or = 5 years (n= 37), comprising an SUr group (n=17) and an SUf group (n=20). Blood samples are taken at 0 h, 0.5 h 1 h, 2 h and 3 h during a standard oral glucose tolerance test and measured for glucose, total proinsulin (TPI), intact proinsulin (IPI) and specific insulin (SI) concentrations. Pairwise comparison of estimated marginal means of blood glucose, SI, IPI and TPI levels at each time point are carried out between groups and subgroups. (SUr vs. SUf). Homa insulin resistance index (IR index) is applied to analyse IR between the groups. It was found that patients with shorter disease duration had higher proinsulin (TPI and IPI) levels at all time points (P<0.05), together with a lower glucose level at 2 h and 3 h (P<0.05). Homa insulin index analysis showed no difference between the two groups (P=0.26). Results also showed that the SUr group had a significantly lower glucose level at Oh and 3h (P<0.05), although no significant difference in insulin and proinsulin levels was found between the SUr and SUf groups. In conclusion, proinsulin may play an important role in glucose control in SU-treated type 2 diabetes, but the effect is reduced in SUf patients.