Burn healing in organ cultures of embryonic chicken skin: a model system

Burn wounds cause complex damage to the skin. Unlike simple cuts, burns cause a graded damage at the margin of the wound and leave biochemically complex debris in the wound, two factors that complicate the process of wound healing. To develop an in vitro system for studying cellular responses to burns in whole tissue, we have applied a standardized burn wound to organ cultures of embryonic chicken skin and evaluated cellular changes in response to the burn damage. This simplified system is not subject to uncontrolled infection and does not involve angiogenesis and granulation. Thus, these cultures provide a simplified model of how cells of the dermis and epidermis in and around a burn wound respond to heat damage early in the process of healing.     

Voltage associated with spontaneous embryonic motility in the developing chicken: an automated characterization during mid-late embryogenesis

Movement of developing chicken embryos and their associated membranes generates voltage detectable with electrodes inserted just beneath the eggshell. Use of such voltages as a motility indicator offers an embryonic behavioral assessment method less subjective and invasive than observational methods using windows that disrupt substantial portions of the eggshell. We used a computerized signal recording and processing procedure to compare voltages from embryonic Day 12 (E12), E15, and E18 chicken eggs with embryos, assessed on the same day. Larger voltages were recorded from E18 subjects than from E12 or E15 subjects. Because this could have been due to embryonic size (mass) and/or proximity to the electrodes, making age comparisons uninterpretable, we used standard deviation-normalized and Z-score-based data transformations, comparing groups for relative deviations from basal voltages. E18 subjects still appeared more active than E12 subjects, with E15 a transitional age, in contrast to results from earlier window-based studies. The automated assessment method we used could enhance behavioral teratology studies of avian species.     

The effect of hyperoxia on embryonic and organ mass in the developing chick embryo

OBJECTIVES: We investigated the sensitivity and specificity of exercise-induced T wave normalization (TWN) in infarct-related electrocardiographic leads (IRLs) for detection of residual viability in the infarct area. BACKGROUND: The meaning of exercise-induced TWN on IRLs is not yet well understood. Recent reports suggest that TWN during dobutamine echocardiography could indicate the presence of viable myocardium. METHODS: We evaluated 40 consecutive patients with a recent acute myocardial infarction and negative T waves in at least two IRLs. All patients underwent exercise testing; positron emission tomography (PET) with nitrogen-13 ammonia and fluorine-18 fluorodeoxyglucose; and coronary angiography. RESULTS: Twenty-four patients showed exercise-induced TWN: 18 at a work load < or =50 W (group la) and 6 at a work load > or =75 W (group 1b); 16 patients did not show TWN (group 2). On the PET study, viability in the infarct area was present in 17 patients (94%) from group la, in only 1 (16%) from group 1b and in 4 (25%) from group 2 (p < 0.0001). The sensitivity, specificity and diagnostic accuracy of exercise-induced TWN, in comparison with residual viability, were, respectively, 82%, 67%, 75% for TWN at every work load and 77%, 94%, 85% for TWN at a work load < or =50 W. Moreover, the sensitivity and diagnostic accuracy of TWN at the low work load were higher for anterior infarctions (87% and 88%, respectively). CONCLUSIONS: Exercise-induced TWN on IRLs at low work loads is a sensitive and specific index for the presence of residual viability in the infarct area. Sensitivity and diagnostic accuracy of this sign are higher for anterior infarctions.     

Control of the thymic microenvironment by growth hormone/insulin-like growth factor-I-mediated circuits

The thymus gland is a central lymphoid organ in which bone marrow-derived T cell precursors undergo maturation, eventually leading to the migration of positively selected thymocytes to the T-dependent areas of peripheral lymphoid organs. This process occurs under the influence of the thymic microenvironment, by means of secretory polypeptides and cell-cell contacts. The thymic microenvironment is a tridimensional cellular network composed of epithelial cells (its major component), macrophages, dendritic cells, fibroblasts and extracellular matrix elements. The epithelial reticulum is a heterogeneous tissue, in which a particular lymphoepithelial structure has been isolated in vitro: the thymic nurse cell complex, which possibly creates particular microenvironmental conditions for thymocyte differentiation. Additionally, thymic nurse cells are useful tools to study mechanisms involved in intrathymic T cell migration, including neuroendocrine influences. Previous data showed that thymic hormonal function can be modulated by hormones and neuropeptides, including growth hormone. Interestingly, GH acts pleiotropically on the thymic epithelium increasing cell growth and expression of extracellular matrix ligands and receptors, the latter resulting in an enhancement of thymocyte adhesion to the epithelial cells and thymocyte release from thymic nurse cells. The role of GH on thymus development is further stressed by the findings obtained with GH-deficient dwarf mice. Besides the precocious decline in serum thymulin found in these animals, a progressive thymic hypoplasia occurs, with decreased numbers of CD4+CD8+thymocytes, both defects being largely restored by long-term GH treatment. The effects of GH in the thymus are apparently mediated by IGF-1. Enhancement of thymulin secretion induced by GH, as well as the stimulation of thymocyte adhesion to thymic epithelial cells can be prevented in vitro by treatment with antibodies for IGF-I or IGF-I receptor. Moreover, in both systems IGF-I alone can yield similar effects. Also, the enhanced concanavalin-A mitogenic response and IL-6 production by thymocytes observed in GH-treated mice can be detected in animals treated with IGF-I. Lastly, mouse substrains selected for high or low IGF-I circulating levels exhibited differential thymus developmental patterns correlating with IGF-I levels. A further conceptual aspect concerning the GH-IGF-I-mediated control of thymus physiology is the recent demonstration of an intrathymic production of these molecules, leading to the hypothesis that, in addition to the classical endocrine pathway, GH-IGF-I-mediated paracrine and autocrine pathways may also be implicated in the control of thymus physiology. In any case, such control is exerted pleiotropically, with modulation in the expression of several genes in different cell types of the organ. In this respect, it is exciting to imagine a role of GH-IGF-I loops in shaping the intrathymically generated T cell repertoire.     

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.     

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.     

Human immunodeficiency virus infection of the human thymus and disruption of the thymic microenvironment in the SCID-hu mouse

Infection with the human immunodeficiency virus (HIV) results in immunosuppression and depletion of circulating CD4+ T cells. Since the thymus is the primary organ in which T cells mature it is of interest to examine the effects of HIV infection in this tissue. HIV infection has been demonstrated in the thymuses of infected individuals and thymocytes have been previously demonstrated to be susceptible to HIV infection both in vivo, using the SCID-hu mouse, and in vitro. The present study sought to determine which subsets of thymocytes were infected in the SCID-hu mouse model and to evaluate HIV-related alterations in the thymic microenvironment. Using two different primary HIV isolates, infection was found in CD4+/CD8+ double positive thymocytes as well as in both the CD4+ and CD8+ single positive subsets of thymocytes. The kinetics of infection and resulting viral burden differed among the three thymocyte subsets and depended on which HIV isolate was used for infection. Thymic epithelial (TE) cells were also shown to endocytose virus and to often contain copious amounts of viral RNA in the cytoplasm by in situ hybridization, although productive infection of these cells could not be definitively shown. Furthermore, degenerating TE cells were observed even without detection of HIV in the degenerating cells. Two striking morphologic patterns of infection were seen, involving either predominantly thymocyte infection and depletion, or TE cell involvement with detectable cytoplasmic viral RNA and/or TE cell toxicity. Thus, a variety of cells in the human thymus is susceptible to HIV infection, and infection with HIV results in a marked disruption of the thymic microenvironment leading to depletion of thymocytes and degeneration of TE cells.     

An alternate pathway for T cell development supported by the bone marrow microenvironment: recapitulation of thymic maturation

In the principal pathway of alpha/beta T cell maturation, T cell precursors from the bone marrow migrate to the thymus and proceed through several well-characterized developmental stages into mature CD4+ and CD8+ T cells. This study demonstrates an alternative pathway in which the bone marrow microenvironment also supports the differentiation of T cell precursors into CD4+ and CD8+ T cells. The marrow pathway recapitulates developmental stages of thymic maturation including a CD4+CD8+ intermediary cell and positive and negative selection, and is strongly inhibited by the presence of mature T cells. The contribution of the marrow pathway in vivo requires further study in mice with normal and deficient thymic or immune function.     

Immunoreactivity of neural crest-derived cells in thymic tissue developing under the rat kidney capsule

In order to study the functional development of a thymus in an experimental model, small pieces of adult rat thymic tissue were cultured for 9 days and implanted under the kidney capsule of littermates. The tissues were examined with a panel of antibodies raised against thymic and neural factors and neural crest cells at intervals from 5 to 13 days. At 5 days post-implantation, there were groups of L1+ cells within the implants that reacted with antibodies raised against neural and neural crest cell markers. L1+ cells were highly mitotic, rounded cells measuring 8.7 +/- 0.6 micrometer in diameter. Double immunostaining with different combinations of antibodies showed that 94% of the L1+ cells were also TH+, and many were HNK-1/NCAM+, PGP 9.5+, NGF+, chromogranin A+, VIP+, S100+, CGRP+, GAD+, and A2B5+. A few were also pan-cytokeratin+. These results indicate that these cells are derived from neural crest derived cells and belong to the neuroepithelial line of development. The L1+ cells were most numerous before nerves appeared (about Day 9) and reduced in number and extent as the thymus differentiated. The neural crest cells occasionally had long cytoplasmic extensions, but it was not possible to decide if they formed the nerves that appeared in the implants. Adult thymuses also contained a population of L1+ and HNK-1/NCAM+ cells, mainly in the subcapsular cortex, the septa, and the medulla. These cells could be a source of neural crest cells able to repopulate the implant. The adult thymus may always contain a reservoir of cells potentially capable of producing neuropeptides and transmitter factors required for thymic growth and regeneration.     

The influence of fluoride exposure on dentin mineralization using an in vitro organ culture model

This study aimed to characterize fluoride-induced alterations in dentin mineralization within a dentin-pulp organ culture system. Tooth sections derived from male Wistar rat incisors were cultured in Trowel-type culture for 14 days, in the presence of 0 mM, 1 mM, 3 mM and 6 mM sodium fluoride. Tooth sections were processed and analyzed for uptake of fluoride, its subsequent effect on dentin mineralization by tetracycline hydrochloride incorporation and mineral composition, expressed as calcium/phosphorous (Ca/P) ratios. Tetracycline hydrochloride incorporation was demonstrated to decrease with increased fluoride exposure, accompanied by significant increases in both Ca/P ratios and fluoride incorporation. These findings provide further evidence that the established alterations in dentin formation during fluorosis are a consequence of disruption to the mineralization process, and provide a model system with which to investigate further the potential role the extracellular matrix plays in inducing the apparent changes in mineral composition.     

Biosynthesis of proteoglycogen: modulation of glycogenin expression in the developing chicken

Glycogenin, the autoglucosyltransferase that primes the biosynthesis of proteoglycogen, is found in the polysaccharide linked proteoglycogen form in mammals and chicken. Glycogenin was released from proteoglycogen and its activity was measured, together with that of glycogen synthase as well as glycogen content, in muscle, liver, and brain during chicken development. The specific activity of glycogenin, expressed per protein, increased with development only in muscle and was higher than the specific activities measured in liver and brain at any time. Concomitant with the rise in activity, an enhanced expression of the protein was observed with Western blot. The specific activity of glycogen synthase increased with development in muscle and liver, while glycogen accumulation was noticeable only in liver. The results indicate that the molar concentration of proteoglycogen is higher in muscle than in liver. The high glycogen content of liver may indicate that the size of the polysaccharide moiety of proteoglycogen is larger in liver than in muscle. This is the first report of developmental modulation of de novo biosynthesis of glycogen at the level of the primer that initiates glucose polymerization.     

Microbial parasites versus developing T cells: an evolutionary 'arms race' with implications for the timing of thymic involution and HIV pathogenesis

The thymus attempts to ensure that T cells which emerge from it are able to discriminate self from nonself. As such, it is a potential 'backdoor' through which microbial parasites can enter, manipulate the host into perceiving them as 'self', and thereby avoid immune surveillance. It is proposed that the host has evolved to overcome this parasitic strategy by rapidly producing large numbers of long-lived T cells very early in life (closing the backdoor), before the developing individual has significant contact with infectious organisms, and while still under the protection of its mother's intact immune system. Hence the capacity of the thymus to function efficiently early in the lifespan would have been strongly favored by natural selection. It is well established in evolutionary biology that strong selection favoring enhanced early function easily accommodates, through pleiotropy, the accumulation of later occurring negative effects, and it is through this process that thymic involution and subsequent immune system senescence may have evolved. Once a large pool of competent T cells has been produced, even those microbes capable of contaminating the thymus usually can be eliminated, or at least contained. However, microbes that both destroy peripheral T cells (particularly peripheral T cells that are activated against them), and contaminate the thymus (leading to deletion of potential replacements of the destroyed peripheral cells), may be able to eventually overcome the immune system, thus producing disease after a long period of apparent latency. Human immunodeficiency virus, which is initially well controlled by the immune system, may become unleashed via this process.     

Investigations into the loss of glutathione from lenses in organ culture

PURPOSE: To investigate possible causes and implications of the decrease in glutathione concentration in rat lenses during organ culture. METHODS: Freshly excised lenses were incubated in modified TC-199 medium. Ellman's Reagent or the GSH-400 assay were used to assay glutathione levels in lenses cultured for different times and under a variety of altered culture conditions. RESULTS: In lenses from young rats the glutathione decrease was not ameliorated by reduction of oxygen tension in the incubator, nor by supplementation of the culture medium with various antioxidants or sulfhydryl compounds, nor with the amino acid precursors of glutathione. Addition of 2-mercaptoethanol stimulated cysteine transport into the lens but had only a modest effect in maintaining the level of glutathione. The decrease in glutathione concentration was less in cultured lenses from older rats. Lenses from rhesus monkeys exhibited no decrease in glutathione levels when maintained in organ culture for up to 48 h. CONCLUSIONS: The basis for the decreased glutathione in cultured young rat lenses is still uncertain. The data from the present study indicate a definite relationship between glutathione loss and age for cultured rat lenses, with young lenses being much more susceptible. The resistance of cultured monkey lenses to loss of glutathione demonstrates species differences in this property which may be relevant to previously reported differences in susceptibility to oxidative damage.     

A novel promoting action of fullerene C60 on the chondrogenesis in rat embryonic limb bud cell culture system

Fullerene C60 was solubilized with polyvinylpyrrolidone (PVP) in water, and the aqueous solution was applied to the rat limb bud cell differentiation system. By the incubation with various concentrations of C60 with poly(vinylpyrrolidone), cell differentiation was extremely promoted, up to the levels of a 3.2-fold increase of the controls. Although poly(vinylpyrrolidone) alone inhibited the cell differentiation in proportion to the concentration, the observed promoting action by C60 surpassed the action of poly(vinylpyrrolidone). This specific promoting action on the chondrogenesis is the novel significant activity of C60.     

Modification of an in vitro technique in order to grow adult rat anterior pituitary in organ culture

The study of adult rat anterior pituitary maintained in vitro was made possible by a modification of the suspension system used in the watch-glass method of organ culture. The integrity and health of the tissue was observed with the electron microscope. Somatotrophs and gonadotrophs were seen routinely. A cell with large (400-750 nm) granules and thought to be a luteotroph (mammotroph) was also seen. These results support the use of this method for the growth of adult rat anterior pituitary, or other large explants, for short periods of time in organ culture.     

Blocking N-cadherin function disrupts the epithelial structure of differentiating neural tissue in the embryonic chicken brain

The cell adhesion molecule N-cadherin is ubiquitously expressed in the early neuroepithelium, with strongest expression in the ependymal lining. We blocked the function of N-cadherin during early chicken brain development by injecting antibodies against N-cadherin into the tectal ventricle of embryos at 4-5 d of incubation [embryonic day 4 (E4)-E5]. N-cadherin blockage results in massive morphological changes in restricted brain regions. At approximately E6, these changes consist of invaginations of pieces of the ependymal lining and the formation of neuroepithelial rosettes. The rosettes are composed of central fragments of ependymal lining, surrounded by an inner ventricular layer and an outer mantle layer. Radial glia processes are radially arranged around the ependymal centers of the rosettes. The normal layering of the neural tissue is thus preserved, but its coherent epithelial structure is disrupted. The observed morphological changes are restricted to specific brain regions such as the tectum and the dorsal thalamus, whereas the ventral thalamus and the pretectum are almost undisturbed. At E10-E11, analysis of late effects of N-cadherin blockage reveals that in the dorsal thalamus, gray matter is fragmented and disorganized; in the tectum, additional layers have formed at the ventricular surface. Together, these results indicate that N-cadherin function is required for the maintenance of a coherent sheet of neuroepithelium in specific brain regions. Disruption of this sheet results in an abnormal morphogenesis of brain gray matter.     

Development of the diffuse endocrine system in the chicken proventriculus

The study was aimed at comparison of chemiluminescence of granulocytes during their incubation with suspensions of Pseudomonas strains isolated from various sources. The study was performed on 136 strains isolated from clinical material. The results were correlated with resistance of these bacteria to bactericidal activity of serum and their ability to produce proteolytic enzymes. It was found that strains isolated from blood and pus weakly activate granulocytes in contrary to bacteria isolated from urine and feces. Ability to pronounced activation of granulocytes was without relation to susceptibility of a given strain to action of serum. But it correlated negatively with ability to production of proteolytic enzymes. These observations indicate existence of relation between characteristics of strain and type of clinical material from which it was isolated.