Intra-allantoic injection of calcium promotes hatching of chick embryos grown in shell-less culture.

The hatch rate of chick embryos cultured outside of the eggshell with 350 mg calcium l-lactate hydrate (CaL) and 3.5 mL water is fourfold greater in cultures in which the chorioallantoic membrane (CAM) surrounds the egg contents by incubation day 17.5 (E17.5) an event which occurs in ovo by E13. It was first investigated whether decreasing the volume of water added with 350 mg CaL would promote CAM expansion due to the smaller volume to enclose. When 350 mg CaL was present, the CAM did not surround the egg contents by E13. By E17.5, the CAM surrounded the egg contents in 53%-74% of cultures; however, CAM expansion was not significantly different when 0, 1, 2, or 3.5 mL water was present. The hatch rate with 2 or 3.5 mL water was greater than 50% but was not improved with less water. Second, it was investigated whether CaL or water inhibits CAM expansion. In the absence of CaL, the CAM surrounded the egg contents in up to two-thirds of cultures by E13, whether 2 mL water was present or not. Thus CaL, but not water, inhibits expansion of the CAM by E13, even though CaL promotes hatching. Finally, it was investigated whether injection of aqueous CaL into the allantoic fluid, in conjunction with not adding CaL to culture hammocks, would promote CAM expansion. Allantoic injection of CaL starting at E13 did not promote CAM expansion at E17.5 but resulted in hatch rates of approximately 30%. Allantoic injection is a novel route for supplementation of calcium in cultured chick embryos.

Shell-less culture system for chick embryos from the blastoderm stage to hatching.

Since 2014, methods have been described to hatch chick embryos from shell-less culture after egg contents are first incubated within shells for 55-70 h. The present report describes for the first time a shell-less culture system for chick embryos from the blastoderm stage to hatching. For the first 69-70 h, egg contents suspended in polymethylpentene kitchen wrap (F.O.R. Wrap, Riken Fabro, Tokyo, Japan) supported in 6.35 or 6.67 cm inside diameter tripods and covered with a disc of immobilized Milli-Wrap, were rotated back and forth through 90° at 16 or 22 cycles per minute (CPM). Subsequently, the Milli-Wrap disc was removed and culture tripods were transferred to environmental chambers, which were rocked ±20° through incubation day 8.5 (E8.5). From E9, environmental chambers were maintained in the horizontal position through to hatching with controlled O2 and CO2 . To provide supplemental calcium, an aqueous solution containing 100 mg/mL of calcium l-lactate hydrate was injected through the plastic wrap into the albumen at E9 (2.5 mL) and at E13 (1.0 mL) or E15 (1.0 mL). After incubation for 69-70 h at 16 or 22 CPM, 80%-83% of previously unincubated egg contents yielded apparently normal embryos. Hatch rate of normal embryos resulting from turntable incubation at 16 or 22 CPM was approximately 43%. Of note, egg contents remained in the same culture tripod from blastoderm stage to hatching. This technique may find use as an educational tool and in basic investigations of early embryogenesis, teratogenesis, and gene transfer experiments.

Supplemental calcium increases hatch rate but not hatchling mass of chick embryos in shell-less culture.

A method is described for culturing 64-70 h-old chicken embryos and egg contents outside of the eggshell through to hatching. Cultured egg contents were suspended in polymethylpentene kitchen wrap (F.O.R. wrap; Riken Fabro) supported in polyvinyl chloride tripods. Tripods were incubated in Plexiglas environmental chambers which were rocked automatically through an angle of ±20°. The concentration of CO2 was maintained at 2% throughout incubation, while that of O2 was increased from ambient to 50%, and relative humidity was decreased from 90%-92% to 83%-84% at incubation Day 9. Cultured embryos not supplemented with calcium did not hatch. The Hatch rate increased when supplemental calcium L-lactate hydrate was increased between 250 and 350 mg. A maximal hatch rate of 54.8% was achieved when cultures were supplemented with 350 mg of calcium L-lactate hydrate and 3.5 ml of sterile water. Adding 400 or 450 mg of calcium L-lactate hydrate did not increase the hatch rate further. The mass of cultured hatchlings (including the retracted yolk) and yolk-free carcass wet and dry mass and length of the right third toe were significantly less than the corresponding parameters observed in hatchlings in ovo. No statistically significant differences in hatchling mass, yolk-free carcass wet or dry mass, or length of the right third toe were noted among cultured hatchlings supplemented with 250-450 mg of calcium L-lactate hydrate. Failure to completely absorb albumen was the most common abnormality observed in cultures which failed to hatch. The present technique allows a unique approach to study the physiology of the developing chicken embryo.

Mycobacterium grossiae sp. nov., a rapidly growing, scotochromogenic species isolated from human clinical respiratory and blood culture specimens.

A previously undescribed, rapidly growing, scotochromogenic species of the genus Mycobacterium (represented by strains PB739T and GK) was isolated from two clinical sources - the sputum of a 76-year-old patient with severe chronic obstructive pulmonary disease, history of tuberculosis exposure and Mycobacterium avium complex isolated years prior; and the blood of a 15-year-old male with B-cell acute lymphoblastic leukaemia status post bone marrow transplant. The isolates grew as dark orange colonies at 25-37 °C after 5 days, sharing features in common with other closely related species. Analysis of the complete 16S rRNA gene sequence (1492 bp) of strain PB739T demonstrated that the isolate shared 98.8 % relatedness with Mycobacterium wolinskyi. Partial 429 bp hsp65 and 744 bp rpoB region V sequence analyses revealed that the sequences of the novel isolate shared 94.8 and 92.1 % similarity with those of Mycobacterium neoaurum and Mycobacterium aurum, respectively. Biochemical profiling, antimicrobial susceptibility testing, HPLC/gas-liquid chromatography analyses and multilocus sequence typing support the taxonomic status of these isolates (PB739T and GK) as representatives of a novel species. Both isolates were susceptible to the Clinical and Laboratory Standards Institute recommended antimicrobials for susceptibility testing of rapidly growing mycobacteria including amikacin, ciprofloxacin, moxifloxacin, doxycycline/minocycline, imipenem, linezolid, clarithromycin and trimethropin/sulfamethoxazole. Both isolates PB739T and GK showed intermediate susceptibility to cefoxitin. We propose the name Mycobacterium grossiae sp. nov. for this novel species and have deposited the type strain in the DSMZ and CIP culture collections. The type strain is PB739T (=DSM 104744T=CIP 111318T).

The acute phase reactant orosomucoid-1 is a bimodal regulator of angiogenesis with time- and context-dependent inhibitory and stimulatory properties.

BACKGROUND: Tissues respond to injury by releasing acute phase reaction (APR) proteins which regulate inflammation and angiogenesis. Among the genes upregulated in wounded tissues are tumor necrosis factor-alpha (TNFα) and the acute phase reactant orosomucoid-1 (ORM1). ORM1 has been shown to modulate the response of immune cells to TNFα, but its role on injury- and TNFα-induced angiogenesis has not been investigated. This study was designed to characterize the role of ORM1 in the angiogenic response to injury and TNFα. METHODS AND RESULTS: Angiogenesis was studied with in vitro, ex vivo, and in vivo angiogenesis assays. Injured rat aortic rings cultured in collagen gels produced an angiogenic response driven by macrophage-derived TNFα. Microarray analysis and qRT-PCR showed that TNFα and ORM1 were upregulated prior to angiogenic sprouting. Exogenous ORM1 delayed the angiogenic response to injury and inhibited the proangiogenic effect of TNFα in cultures of aortic rings or isolated endothelial cells, but stimulated aortic angiogenesis over time while promoting VEGF production and activity. ORM1 inhibited injury- and TNFα-induced phosphorylation of MEK1/2 and p38 MAPK in aortic rings, but not of NFκB. This effect was injury/TNFα-specific since ORM1 did not inhibit VEGF-induced signaling, and cell-specific since ORM1 inhibited TNFα-induced phosphorylation of MEK1/2 and p38 MAPK in macrophages and endothelial cells, but not mural cells. Experiments with specific inhibitors demonstrated that the MEK/ERK pathway was required for angiogenesis. ORM1 inhibited angiogenesis in a subcutaneous in vivo assay of aortic ring-induced angiogenesis, but stimulated developmental angiogenesis in the chorioallantoic membrane (CAM) assay. CONCLUSION: ORM1 regulates injury-induced angiogenesis in a time- and context-dependent manner by sequentially dampening the initial TNFα-induced angiogenic response and promoting the downstream stimulation of the angiogenic process by VEGF. The context-dependent nature of ORM1 angioregulatory function is further demonstrated in the CAM assay where ORM1 stimulates developmental angiogenesis without exerting any inhibitory activity.

Robotic surgical telepathology between the Iron Mountain and Milwaukee Department of Veterans Affairs Medical Centers: a 12-year experience.

Since mid-1996, we have operated a diagnostic robotic telepathology (TP) system at the Iron Mountain, MI, Department of Veterans Affairs Medical Center (VAMC) from the Milwaukee, WI, VAMC, located some 220 miles away. No on-site pathologist is present in Iron Mountain. Instead, an experienced, well-trained pathologist assistant, under direction of pathologists located in Milwaukee, is responsible for tissue grossing and sectioning. The pathologist assistant places slides onto the stage of the robotic microscope, which is then controlled by pathologists in Milwaukee. Each case read by TP is subsequently read by light microscopy (LM) by the same pathologist. Three distinct phases of TP have been recognized. Our experience during phase I (mid-1996 to early 1999) has been published previously. During phase II (early 1999 to mid-2004), 1 of the 2 senior telepathologists in phase I retired, and 3 junior pathologists were hired. During phase III (mid-2004 to June 2008), 2 new junior pathologists were hired, and ASAP Imaging (Apollo Telemedicine, Inc., Falls Church, VA) was implemented. The number of TP case opportunities in phases I, II, and III was 2200, 5841, and 3512, respectively, resulting in a total of 11 553. A total of 1834 cases were deferred to LM for a variety of reasons. The number of TP diagnoses rendered in phases I, II, and III was 2144, 4636, and 2939, respectively, resulting in a total of 9719. The major discordance rates in phases I, II, and III were 0.33%, 0.45%, and 0.20%, respectively, with an overall rate of 0.35%. Pathologist-specific discordance rates were not significantly different and ranged from a low of 0.12% to a high of 0.77%, whereas case deferral rates were significantly different (P < .0001) and ranged from 2.5% to 28.7%. In general, no relationship between deferral rate and discordance rate was noted. Iron Mountain clinicians have expressed great satisfaction with the services provided by their off-site pathologist colleagues.

Robotic surgical telepathology between the Iron Mountain and Milwaukee Department of Veterans Affairs Medical Centers: a twelve year experience.

Since mid-1996 we have operated a diagnostic robotic telepathology (TP) system at the Iron Mountain, Michigan, Department of Veterans Affairs Medical Center (VAMC) from the Milwaukee, Wisconsin VAMC, located some 220 miles away. No on-site pathologist is present in Iron Mountain. Instead, an experienced, well-trained pathologist assistant, under direction of pathologists located in Milwaukee, is responsible for tissue grossing and sectioning. The pathologist assistant places slides onto the stage of the robotic microscope, which is then controlled by pathologists in Milwaukee. Each case read by TP is subsequently read by light microscopy (LM) by the same pathologist. Three distinct phases of TP have been recognized. Our experience during Phase I (mid-1996 through early 1999) has been published previously. During Phase II (early 1999 through mid-2004), one of the two senior telepathologists in Phase I retired and three junior pathologists were hired. During Phase III (mid-2004 though June 2008), two new junior pathologists were hired and ASAP Imaging (Apollo Telemedicine, Inc., Falls Church, VA) was implemented. The number of TP case opportunities in Phases I, II and III was 2,200; 5,841 and 3,512; respectively resulting in a total of 11,553. A total of 1,834 cases were deferred to LM for a variety of reasons. The number of TP diagnoses rendered in Phases I, II and III was 2,144; 4,636 and 2,939; respectively, for a total of 9,719. The major discordance rates in Phases I, II and III were 0.33%, 0.45% and 0.20%, respectively with an overall rate of 0.35%. Pathologist-specific discordance rates were not significantly different and ranged from a low of 0.12% to a high of 0.77%, while case deferral rates were significantly different (P < 0.0001) and ranged from 2.5% to 28.7%. In general, no relationship between deferral rate and discordance rate was noted. Iron Mountain clinicians have expressed great satisfaction with the services provided by their off-site pathologist colleagues.

Chick chorioallantoic membrane as an in vivo model to study vasoreactivity: characterization of development-dependent hyperemia induced by epoxyeicosatrienoic acids (EETs).

Shell-less culture of chick chorioallantoic membrane (CAM) of developing chicken embryos is a useful model to evaluate the effects of vascular agents. We assessed the response of CAM vessels to epoxyeicosatrienoic acids (EETs), derivatives of the essential fatty acid arachidonic acid, that have a number of important biological functions, including dilation of microvessels in the coronary, cerebral, renal, and mesenteric circulations. Three of four regioisomers of EETs, 14,15-, 11,12-, and 8,9-EET, induced a characteristic dose-dependent acute hyperemia within 4 min after application on 10-day-old CAMs. This response was marked in early stages of development (between days 8 and 10), but the frequency and intensity of the response were reduced after 11 days of development. Histological examination demonstrated that the hyperemia was not due to extravasation of erythrocytes. However, many capillaries were distended and contained densely packed erythrocytes as compared to uniformly arranged vessels and erythrocytes in untreated CAMs. Transmission electron microscopy showed the basal laminae surrounding capillaries remained intact, similar to those in vehicle-treated or untreated CAM tissue. The hyperemia was specific to EETs since we did not observe it to be induced by other vasodilators such as nitric oxide or prostacyclin. In conclusion, we report a novel vascular response to EETs using the CAM as an in vivo model. These lipids specifically distend a subset of capillaries in a dose- and development-dependent manner.