Phenotypic mapping of the chicken embryonic thymic microenvironment developing within an organ culture system

The chicken thymic microenvironment, as it developed in an embryonic thymus organ culture system, was phenotypically mapped using a panel of mAb defining both epithelial and nonepithelial stromal cell antigens. We have previously reported that thymocyte proliferation and differentiation with proceed for up to 6-8 days in thymus organ culture, hence demonstrating the functional integrity of the thymic microenvironment in vitro. During this time, the stromal component reflected that of the normal embryo with cortical and medullary epithelial areas readily identifiable by both morphology and surface-antigen expression. An abundance of subcapsular and cortical epithelial antigens was detected in the cultured thymus, particularly those normally expressed by the epithelium lining the capsule, trabeculae, and vascular regions (type I epithelium) in the adult and embryonic thymus. Medullary epithelial antigens developed in organ culture, although were present in lower frequency than observed in the age-matched embryonic thymus. MHC class II expression by both epithelial and nonepithelial cells was maintained at high levels throughout the culture period. With increasing time in culture, the ratio of epithelial to nonepithelial cells decreased, concurrent with a decrease in thymocyte frequency and suggestive of a bidirectional interaction between these two cell types. Thus, a functionally intact thymic microenvironment appears to be maintained in embryonic thymus organ culture, a model that is currently being exploited to assess the role of stromal antigens, as defined by our mAb, in the process of thymopoiesis.     

Multiple system organ failure: a study of outcome

One hundred patients with Multiple System Organ Failure (MSOF) were studied. The precipitating factors were infections, poisoning, metabolic disorders, surgical disorders and cardiac arrest resulting in an overall mortality of 65%. Mean inpatient stay was 3.86 days, being significantly longer in patients who survived (6.25 days). Age, sex, addictions and premorbid health did not affect outcome. GIT (89%), CNS (81%) and Liver failure (62%) were seen most commonly. Highest mortalities were observed with RS (81.2%), CVS (80.37%) and CNS (76.5%). The mortality with 2,3,4,5,6 and 7 OSF was 8.3%, 18.7%, 70%, 92%, 100% and 100% respectively. The mortality was highest (50.76%) on the first day of MSOF and during the initial 48 hours of the total duration of disease. The method proposes an easily reproducible way to evaluate severity of illness and predicting outcome in acute MSOF.     

Gene expression of steroidogenic enzymes in chicken embryonic gonads

The monoclonal antibody Ki-S2 binds to a recently characterized proliferation-specific protein, p100. To assess its distribution pattern under physiologic and pathologic conditions, we performed immunohistochemical analyses on an exhaustive spectrum of normal tissues, 624 miscellaneous solid cancers, and 95 hematologic malignancies, and compared the results with Ki-67 immunostaining on consecutive sections. In addition, Ki-S2 expression was related to the DNA content by dual parameter flow cytometric analysis in parallel with Ki-67 labeling using a human cancer cell line. Immunoreactivity was enhanced at the G1/S transition and persisted through G2 and M phase. After adequate antigen retrieval, the antibody was found to yield identical results on fresh and formalin-fixed, paraffin-embedded material. The antibody specifically labeled actively proliferating cells, which constitute a subset of the population recognized by Ki-67. In normal human tissues, Ki-S2 immunolabeling hardly ever exceeded 40% of the Ki-67+ cell fraction. Immunoreactive scores of the two antibodies exhibited a linear correlation, but statistically significant differences in the ratio of Ki-S2-positive to Ki-67-positive cells were nevertheless observed between different tissue types. In contrast, the ratio of Ki-S2 and Ki-67 immunoreactive scores varied widely in neoplastic cells and tissues, occasionally attaining a ratio of almost 1:1. This suggests that loss of growth regulatory mechanisms in malignant cells might result in an extreme reduction of the G1 phase fraction and thus in a significantly shorter doubling time. Therefore, antibody Ki-S2 is likely to allow a more precise evaluation of the cell fraction that will complete a division cycle and a more confident appraisal of the malignancy potential of a neoplastic process.     

Bile acid conjugation in fetal hepatic organ cultures

A new technique has been developed in which mammalian fetal liver can be maintained in organ culture for prolonged periods with intact structure and function. Near-term rat fetal liver explants were incubated in vitro for periods of up to 3 wk with preservation of normal cellular morphology and intercellular (organ) relationships. [14C]cholate was incorporated into tissue and medium conjugates at a constant rate during 21 days in vitro. During a 24-h incubation with radioactively labeled cholic acid, bile acid conjugates accumulated in tissues to a maximum value by 6 h and maintained this value through 24 h. During the same 24-h incubation with [14C]cholate, conjugates were secreted into the medium at a constant rate. Addition of 8 X 10(-4) M taurine to the medium during a 4-day incubation produced a threefold enhancement in the rate of conjugate formation in tissues and medium. Enhanced conjugation in the presence of additional taurine was due almost entirely to increased taurocholate formation and no significant difference was observed in the amount of glycocholate formed. Exposure of explants to 3.6 X 10(-4) M cycloheximide for prolonged periods resulted in inhibition of conjugate formation, but when this concentration of cycloheximide was maintained for only 24 h a significantly (P less than 0.001) increased rate of conjugate formation was observed. The results indicate that metabolic processes in the organ-culture system are in a state of dynamic equilibrium and that morphologic integrity and specific hepatocytic function are maintained after 21 days in vitro. Preferential taurocholate formation was demonstrated in rat fetal liver, and the data suggest that glycine and taurine interact with separate enzymatic systems in bile acid conjugation. The possible mechanisms that mediate the effect of cycloheximide are discussed.     

Peritoneal healing after fibrin glue application: a comparative study in a rat model

The influence of fibrin glue on adhesion formation and peritoneal healing is evaluated in a prospective, randomized, controlled study. In all, 20 Wistar rats underwent microsurgical suturing of two silicone sheets, one covered with a fibrin glue barrier, to the anterior peritoneum. Each animal thus served as its own control. After 10 days, adhesions and peritoneal healing were evaluated by a blinded observer through a second-look laparotomy. Adhesions were scored using a modification of the classification of Diamond. Tissue around the silicone sheet was examined histologically and by scanning electron microscopy to evaluate the inflammatory reaction and peritoneal healing (ingrowth of blood vessels and quality of peritoneal cells). Adhesion scores for treated and control sides were (mean +/- SD) 2.89 +/- 4.68 and 6.79 +/- 9.09 (P = 0.181) respectively, and the percentage of the sheet covered by peritoneum was 26.25 +/- 31.50 and 29.21 +/- 40.21 (P = 0.226) respectively. Using the paired Wilcoxon rank test, the P values for the ingrowth of blood vessels and peritoneal healing evaluated by histology and scanning electron microscopy were 0.842, 0.692 and 0.695 respectively. We conclude that although the mean adhesion score was reduced by > 50% by fibrin glue, there is no statistically significant difference concerning adhesion formation or peritoneal healing with the use of fibrin glue.     

Early embryonic formation of the human vomeronasal organ

We studied the origin and development of the vomeronasal system in early human embryos of 10-18 mm crown-rump length under normal and pathological conditions. The formation of the vomeronasal organ from the vomeronasal groove and placode in a 10-mm embryo is described. We propose that the vomeronasal cavity originates in the schizocoel way. We studied the development of the vomeronasal and olfactory nerves. Attention is paid to the development of the nasolacrimal duct and the epithelial plug. The possible use of the vomeronasal system by embryos during the first seven weeks of development is discussed.     

Subgroup F adenovirus growth in foetal intestinal organ cultures

An in vitro foetal intestinal organ culture system was employed to determine the permissiveness of human intestinal cells for subgroup F adenovirus infection. Ad40 and Ad41 growth, monitored through group-specific hexon antigen production, was poor in comparison to that of Ad2 in these cultures, further demonstrating their fastidious nature in most human cells. The low growth capability of these viruses in culture, in relation to their association with gastrointestinal disease is discussed.     

Hydrosalpingeal fluid inhibits in-vitro embryonic development in a murine model

Recent evidence describing a suboptimal clinical outcome in women with hydrosalpinges who undergo in-vitro fertilization (IVF) and embryo transfer suggests a potential deleterious effect of this fluid on in-utero embryo development. Consequently, we evaluated in-vitro mouse embryo development in the presence of hydrosalpingeal fluid (HF) collected from 10 infertile women of reproductive age. Chemical analyses showed both similarities and differences of these fluids to reported values for fluids collected from non-diseased Fallopian tubes. The HF had a significant deleterious effect upon mouse embryo cleavage and development to the expanded and hatched blastocyst stage, although the effect was variable among patients. Dilution of HF to 30% concentration with culture medium failed to negate this effect. This argues against the effect resulting from a relative lack of critical, supportive component(s) in the HF. Additionally, further experiments performed with cultures under an oil overlay significantly reduced the embryotoxicity of the HF. This evidence suggests there may be a lipophilic factor that can impair embryo development. The relatively poor IVF-embryo transfer success in women with proximally patent hydrosalpinges may be explained, at least in part, by reflux of a lipophilic embryotoxic factor(s) into the uterine cavity.     

Identification of the blood-borne somatotroph-differentiating factor during chicken embryonic development

Somatotrophs become a significant population by day 16 of chicken embryonic development. We have previously demonstrated that an earlier induction of GH cell differentiation is possible with the addition of day 16 embryonic serum to cultures of day 12 pituitary cells, an age when somatotrophs are rare. The present study was designed to identify the blood-borne signal(s) responsible for the serum activity, using reverse hemolytic plaque assays to identify individual GH-secreting cells. The activity was found to be a heat-stable, ether-soluble compound(s) that is bound or inhibited by a trypsin-sensitive protein. The extent of GH cell differentiation was greater (P < 0.05; n = 3) in response to the ether phases of heated day 16 (14.1 +/- 0.4% of all cells) and day 12 sera (9.3 +/- 0.4%) than with untreated serum from days 16 and 12 (6.1 +/- 0.4% and 0.82 +/- 0.4%, respectively). Furthermore, ether-extracted day 16 serum was more effective than ether-extracted day 12 serum, which was also different from basal (0.85 +/- 0.4%; P < 0.05). Based on this biochemical profile, the abilities of various steroids to stimulate differentiation were tested. Three steroids were found to stimulate somatotroph differentiation in vitro: 17beta-estradiol, corticosterone, and progesterone. However, the estradiol receptor antagonist, tamoxifen, while abolishing the effect of estradiol, had no effect on the induction of differentiation by day 16 serum. In contrast, RU486, a specific glucocorticoid receptor antagonist in chickens, blocked the stimulatory effects of corticosterone, progesterone, and day 16 serum on somatotroph differentiation. We next tested whether the active compound in day 16 embryonic serum was corticosterone, the predominant glucocorticoid in chickens. Incubation of day 16 serum with corticosterone antiserum, but not control antiserum, suppressed day 16 serum-induced GH cell differentiation. Therefore, we conclude that corticosterone is the blood-borne signal capable of stimulating somatotroph differentiation in vitro. The present findings together with previous reports indicate that somatotroph differentiation during embryonic development may result from an increase in circulating glucocorticoid concentrations.     

Microrespirometer chamber for determinations of viability in cell and organ cultures

The effects of chemical, physical, and infectious cytotoxic agents on primary and cultured cells were evaluated by measurements of oxygen uptake for various time periods. A newly developed respirometer used a Clark oxygen electrode in a 1.0-ml chamber, with provisions for constant mixing and for temperature control of both the sample and electrode chambers. The device was unique because the electrode and instrumentation were provided by a clinical blood-gas analyzer. Oxygen uptake by blank controls was negligible, whereas cells and tissue consumed oxygen at rates of approximately 1 to 5 mul/h in a dose- and temperature-dependent fashion. Cyanide, heat, and freeze-thaw lysis reduced the oxygen uptake to less than 0.6 mul/mg per h. Infection of trachea organ cultures with Mycoplasma pneumoniae significantly reduced relative ciliary activity, tetrazolium reduction capacity, and oxygen consumption in a coordinated fashion.     

Chicken keratin-19: cloning of cDNA and analysis of expression in the chicken embryonic gut

From many recent studies, it has been argued that keratins (cytokeratins) play important roles in the morphogenesis and differentiation of organ development. To learn the role of keratin in digestive tract development, a cDNA of the chicken homolog of keratin-19 (GK-19) was cloned and its expression pattern was analyzed in the digestive tract of chicken embryos. The GK-19 full-length sequence was approximately 1.6 kb and showed more than 80% similarity to human and mouse keratin-19. The result of in situ hybridization with the proventriculus (glandular stomach) of different developmental stages showed that GK-19 expression disappeared specifically in the glandular epithelium from day 6 to day 9 of incubation. Furthermore, GK-19 was localized in the notochord, floor plate, anterior lobe of the pituitary gland and mesonephros. These results suggest the possibility that GK-19 may have multiple roles in organogenesis during embryogenesis.     

Safety standards for stab-resistant body armour: a computer tomographic assessment of organ to skin distances

Biliary drainage has long been called the Achilles' heel of liver transplantation, and biliary complications compromise the success of liver transplantation by increasing graft loss and the rates of a required second operation, morbidity, and mortality. One cause of complications is unrecognized anomalous biliary anatomy. We examined 73 intraoperative donor duct cholangiograms (IODDCs) to assess our ability to identify biliary anomalies intraoperatively. Normal anatomy was seen in 42% (31/73); some part of the right-sided biliary system drained into the left bile duct in 22% (16/73); trifurcated systems with a single branch point for the right posterior, right anterior, and left ducts appeared in 16% (12/73); low insertion of a right segmental duct to the hepatic duct was seen in 11% (8/73); and drainage of a right segmental duct into the cystic duct or into the hepatic duct at the cystic duct origin was noted in 8% (6/73). It was believed that the last group represented a condition that dictated extra caution in biliary reconstruction. The incidence of radiographic recognition of these anomalies was more than twice the clinical recognition in our patient population, implying that many such "problem" ducts usually go unrecognized. IODDCs facilitate training of transplant fellows. Costs are low, and morbidity is nil.     

Preservation of human skin structure and function in organ culture

Human keratinocytes can be maintained in monolayer culture under serum-free conditions for an extended period of time. Under low Ca2+ conditions (e.g., 0.05-0.15 mM), an undifferentiated state is maintained and the cells proliferate optimally. When the Ca2+ concentration is raised to approximately 1.0 mM, differentiation occurs and growth shows. Human dermal fibroblasts can also be maintained in monolayer culture under serum-free conditions, but in contrast to keratinocytes, a physiological level of extracellular Ca2+ (above approximately 1.0 mM) is required. A variety of growth factors stimulate proliferation of both cell types but do not replace the Ca2+ requirement of the fibroblast population. All-trans retinoic acid also promotes proliferation of both cell types and, most interestingly, replaces the requirement-for a physiological level of Ca2+ in the fibroblast cultures. Human skin can be maintained in organ culture for an extended period of time under serum-free conditions. Conditions optimized for fibroblast proliferation (either physiological Ca2+ or all-trans retinoic acid) are required. In the presence of culture conditions optimized for the epithelial cell component, both the epidermis and dermis rapidly lyse. These data suggest that the fibroblast is the critical component in maintaining homeostasis of skin, and that maintenance of the epidermis as well as the dermis depends on the viability and functioning of these cells.     

Myelination in cerebellar slice cultures: development of a system amenable to biochemical analysis

Myelin deposition and maintenance are critical to proper function of the mammalian nervous system. Previous investigations of myelination in the central nervous system (CNS) were hampered by the lack of an in vitro system that can faithfully reproduce in vivo events yet is amenable to biochemical investigation. We have developed a procedure, based on organotypic cultures, which permits efficient preparation of large numbers of cerebellar slice cultures that can be easily manipulated. Cultures have been examined to document myelination biochemically (by incorporation of [35S]sulfate into sulfolipids), immunohistochemically (by labeling the myelin components myelin basic protein and galactocerebroside), and morphologically (by both light and electron microscopy). We tested the effects of biologically active peptides and antibodies on myelination in the thin slices. The results indicate that the cultures provide an in vitro system that can be used to examine specific cellular events that occur during CNS myelination.     

An ultrastructural analysis of chick embryonic skin development in vitro

Behaviour of 6-day chick embryo thigh skin in vitro in two different nutrients was studied electron microscopically. In explants supported with chicken serum containing medium epithelium keratinized faster than and in a similar way to that in ovo, except for the absence of corpuscola cribriformia in pericytes. It did not in explants supported with chick embryo extract containing medium, but underwent a noticeable ultrastructural evolution, mainly of granular reticulum and Golgi complexes. In the two series of cultures mesenchyme presented the same ultrastructural characteristics, especially as far as collagen fibers were concerned. The above data rule out the suggested regulatory role of collagen fibers in epidermal differentiation and support a possible epidermal two-step differentiative process. They are discussed in relation to the general mechanisms implicated in skin evolution.     

Effect of testosterone on long-term organ cultures of canine prostate

Organ cultures of rodent and human prostate glands have shown marked differences in their morphological response to testosterone. In this study, explants from 19 canine prostate glands were cultivated for a minimum of 9 days in Trowell's T-8 medium. Groups of explants were exposed to media containing from 0.05 to 100 mum testosterone. While the higher testosterone levels (50 and 100 mum) markedly decreased explant viability, explants cultivated at lower levels (0.05 to 5 mum) appeared similar to control explants in testosterone-free Trowell's T-8 medium. Atmospheric mixtures containing either 95% or 50% oxygen were equally effective. Shortly after the cultures were initiated, large amounts of secretory product were liberated into the lumen. After 9 or more days in vitro, glandular epithelium appeared cuboidal and never revealed the acid phosphatase-rich secretory granules seen in the preculture control. However, the epithelium exhibited an increase in alkaline phosphatase and lipid content following cultivation.     

Expression of heat shock proteins in mouse skin during wound healing

Wound healing conditions generate a stressful environment for the cells involved in the regeneration process and are therefore postulated to influence the expression of heat shock proteins (Hsps). We have examined the expression of four Hsps (Hsp27, Hsp60, Hsp70 and Hsp90) and a keratin (keratin 6) by immunohistochemistry during cutaneous wound repair from Day 1 to Day 21 after wounding in the mouse. Hsps were constitutively expressed in normal mouse epidermis and their patterns of expression were modified during the healing process. The changes were not directly linked to the time course of the healing process but rather were dependent on the location of cells in the regenerating epidermis. In the thickened epidermis, Hsp60 was induced in basal and low suprabasal cells, Hsp70 showed a reduced expression, and Hsp90 and Hsp27 preserved a suprabasal pattern with an induction in basal and low suprabasal cells. All Hsps had a uniform pattern of expression in the migrating epithelial tongue. These observations suggest that the expression of Hsps in the neoepidermis is related to the proliferation, the migration, and the differentiation states of keratinocytes within the wound.