Biological relevance of colony morphology and phenotypic switching by Burkholderia pseudomallei

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42 degrees C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.     

Histological alterations of intestinal villi in chickens fed dried Bacillus subtilis var. natto

Two experiments were conducted. In experiment 1, chickens were fed dried Bacillus subtilis var. natto for 3 or 28 days. Growth performance and internal organs were not different from controls, but feed efficiency tended to be improved in the 28-day feeding. In these birds, blood ammonia concentration was decreased (P<0.05). Blood glucose concentration, and amylase and lipase activity in the intestinal content were not significantly different among dietary groups. These results suggest that the B. subtilis natto depressed ammonia concentration. In experiment 2, chickens were fed dietary B. subtilis natto for 28 days. These birds had a tendency to display greater growth performance and intestinal histologies, such as villus height, cell area and cell mitosis, than the controls. Flat cell outline on the duodenal villus surface in controls developed large, protruded cell clusters and cell protuberances after feeding of dietary B. subtilis natto. These results indicate that intestinal function was activated by the depressed blood ammonia concentration in the body of the chicken. The present results may suggest that the B. subtilis natto has the potential to be a beneficial microorganism in chickens.     

Repeated oocyte pick up in prepubertal swamp buffalo (Bubalus bubalis) calves after FSH superstimulation

The objective in this study was to investigate the effect of repeated oocyte collection by transvaginal, ultrasound-guidance, oocyte pick-up (OPU) in nine, prepubertal (8-12 months), swamp buffaloes. Animals were treated with FSH for 3 days and received GnRH on the third day, 24 h before OPU. This session was repeated on five occasions at 2 weekly intervals. Over the five sessions of hormone treatment followed by OPU, 39/42 (92.9%) animals responded and had 6.6+/-3.6 follicles with a follicular diameter of 5.0+/-2.0 mm. The oocyte recovery rate was 5.4+/-3.7 and averaged 82.8% oocytes, except for session 4, when oocyte recovery was around 75.0%. Most oocytes were denuded (39.5%), whilst 28.8% had a substantial cumulus mass. There were no differences in the ovarian responses and the recovery rates between the collections. It was concluded that five repeat cycles of FSH and OPU did not influence the follicular response to superstimulation or the number of oocytes recovered from prepubertal, buffalo calves.     

Long-term result of autologous cultivated oral mucosal epithelial transplantation for severe ocular surface disease

The present study aimed to investigate the clinical outcomes of autologous cultivated oral mucosal epithelial transplantation (COMET) on human amniotic membrane (AM) for corneal limbal stem cell deficiency (LSCD). In this prospective, noncomparative case series, 20 eyes (18 patients) with bilateral severe ocular surface disease were chosen to undergo COMET on human AM. The primary outcome was clinical success, and the secondary outcomes were the best-corrected visual acuity difference, corneal opacification, symblepharon formation, and complications. The mean patient age was 48.2 ± 15.5 years. The mean follow-up time was 31.9 ± 12.1 months (range 8–50 months). All except one eye exhibited complete epithelialization within the first postoperative week. A successful clinical outcome, defined as a stable ocular surface without epithelial defects, a clear cornea without fibrovascular tissue invasion at the pupillary area, and no or mild ocular surface inflammation, was obtained in 15 of 20 eyes (75 %). The clinical success rate at 1 year was 79.3 %, and that at 4 years (end of follow-up) was 70.5 %. Fourteen of 20 (70 %) eyes exhibited improvement in visual acuity after COMET, and some required subsequent cataract surgery (2 eyes), penetrating keratoplasty (3 eyes), or keratoprosthesis implantation (1 eye). Preoperative symblepharon was eliminated in most eyes (8 of 13, 61.5 %) after COMET combined with eyelid reconstruction when needed. The only complication was corneal perforation (1 eye) induced by a severe eyelid abnormality; treatment with a tectonic corneal graft was successful. COMET can successfully restore ocular surface damage in most eyes with corneal LSCD.     

GD2-specific chimeric antigen receptor-modified T cells targeting retinoblastoma – assessing tumor and T cell interaction

A novel disialoganglioside 2 (GD2)-specific chimeric antigen receptor (CAR)-modified T cell therapy against retinoblastoma (RB) were generated. GD2-CAR consists of a single-chain variable fragment (scFv) derived from a monoclonal antibody, hu3F8, that is linked with the cytoplasmic signaling domains of CD28, 41BB, a CD3ζ, and an inducible caspase 9 death fusion partner. GD2 antigen is highly expressed in Y79RB cell line and in several surgical RB tumor specimens. In vitro co-culture experiments revealed the effective killing of Y79RB cells by GD2-CAR T cells, but not by control CD19-CAR T cells. The killing activities of GD2-CAR T cells were diminished when repeatedly exposed to the tumor, due to an attenuated expression of GD2 antigen on tumor cells and upregulation of inhibitory molecules of the PD1 and PD-L1 axis in the CAR T cells and RB tumor cells respectively. This is the first report to describe the potential of GD2-CAR T cells as a promising therapeutic strategy for RB with the indication of potential benefit of combination therapy with immune checkpoint inhibitors.     

Determination of diazolidinyl urea in a topical cream by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of diazolidinyl urea (DU) in a cream formulation is described. The aqueous phase of the emulsion was separated by centrifugation, removed, filtered, diluted and applied onto the HPLC system. DU was detected by ultraviolet absorption at a wavelength of 214 nm. The calibration curve was linear over the range of 79–553 μg/ml, and identical when determined on consecutive days. The relative standard deviation for repeat determinations was less than 0.5%. Recoveries were 97.74–101.72%. This analytical method is useful for quantitation of DU in cream formulations.