Dose dependency of the responses in draining lymph nodes and skin to repeated applications of oxazolone. A quantitative and histological study in mice.

The skin reactivity and lymph node responses to a large scale of different doses of oxazolone, were followed under continuous stimulation for a period of 12 days. It was found that the lack of a continuous high blast cell activity in the paracortex was a physiological response that could be observed with high dosage as well as with very low dosage. Low doses gave distinct stimulation of the paracortex without any detectable reaction in the cortex and in the medulla. Manifest stimulation of these compartments required considerably higher doses. A marked paracortical response was the only lymph node change required for development of a typical delayed type skin response. When germinal centres and plasma cells had developed, the skin reactivity was also found to change characteristically.     

Some requirements for the response of separated T-cell sub-populations to the mitogens phytohaemagglutinin and concanavalin A.

The proliferative response of various separated populations of mouse spleen and thymus lymphocytes to the mitogen phytohaemagglutinin (PHA) was not a direct function of the level of responsive T cells, but was governed by other regulatory effects. These included a stimulation by adherent macrophages, an inhibition by a separate population of adherent cells and an adherent cell independent restriction of proliferation at high cell concentration. In contrast, the proliferative response to Concanavalin A (Con A) was more closely related to the level of responsive T cells. All density and electrophoretically isolated sub-sets of splenic T cells appeared capable of a proliferative response to PHA and Con A, although under some conditions the PHA responsiveness of certain fractions was suppressed. In the thymus, the minor low theta sub-population appeared capable of response to both mitogens, and accounted for all the activity of the unfractioned thymus cells. No response to either mitogen could be obtained from the major, high theta thymocyte population.     

Dynamic and morphological responses of draining lymph nodes to single and repeated applications of oxazolone to guinea-pig skin

Auricular lymph nodes in guinea-pigs were studied for 3 weeks under continuous stimulation with oxazolone applied to the ear skin. Quantitation of 3H-Tdr labelled paracortical lymphocytes following pulse labeling, demonstrated a marked, but only transitory rise in the proportion of cells in DNA synthesis. In spite of this, the total number of cells in S-phase continued to rise during the remaining part of the observation time, as a result of a steady increase of the paracortical cell mass. The variation in the proportion of large, pyroninophilic blast cells, revealed a pattern similar to that of the labelling index. A high proportion of blast cells was found only in the initial phase of the reaction, while the chronic response was characterized by a low proportion, no different from the starting level. Paracortical enlargement appeared to be the most reliable morphological criterion on which a chronically stimulated paracortex could be distinguished from an "unstimulated". The initial paracortical response, similar to a primary reaction, was followed by an equally pronounced development of germinal centres and plasma cells. These were also persistent features under continued stimulation. The reported changes are most likely specific responses to oxazolone stimulation.     

Changes in the mouse thymus observed during puberty

The changes in the female mouse thymuses during puberty were investigated. During this period the lymphatic cell number in the thymus and its proliferative activity were found to decrease while the mitogen reactivity to PHA to increase. These changes cannot be observed in females ovariectomized before puberty. The role of ovarian hormones in the observed phenomenon is discussed.     

Uterine response of golden hamsters to estrogenic stimulation in postnatal ontogeny

The proliferative activity of the cells of uterine epithelium and stroma of the developing golden hamsters and the activity of acid phosphatase isozymes in these tissues was studied. The dynamics of changes of these indices during the normal development and upon estrogenic stimulation was followed. The uterine cells were shown to react to estrogenic stimulation was followed. The uterine cells were shown to react to estrogenic stimulation by the increase of proliferation from the first days of postnatal development. Characteristic changes in the ratio of the acid phosphatase isozymes in the epithelial cells in response to estrogenic stimulation appeared in the 2nd week of postnatal development only. The definite level of differentiation of cells-targets appears to be indispensable for the realization of certain specific effects of the hormone.     

Stimulation of energy plastic metabolism by phytohemagglutinin in lymphoid organs and adrenal glands

It has been established that the concentration of nucleic acids, protein, glycogen, ATP, copper, manganese, zinc as well as the activity of cytochromoxidase and succinate dehydrogenase in tissues of the thymus gland and spleen of albino rats a day after stimulation by phytohemagglutinin increase considerably reaching the maximal values three days later. Taking a prolonged term (up to 7 days) after phytohemagglutinin administration it is found that the content and activity of the studied indices in the thymus and spleen tissues lower regularly, but fail reaching normal values even 14 days after stimulation. In this case changes in the test indices in the spleen are less pronounced than in the thymus in all the studied periods. In the tissues of adrenals a tendency of changes in the manganese and copper content is like that in lymphoid organs in all periods after stimulation by phytohemagglutinin; other test indices have an opposite tendency.     

Methoxychlor-Induced Alterations in the Histological Expression of Angiogenic Factors in Pituitary and Uterus

Within the reproductive system, oestrogenic stimulation of uterine and pituitary tissue typically causes a proliferative response accompanied by an angiogenic induction of new blood vessels from existing ones, thereby providing nutrients and oxygen to the growing tissue. The pro-oestrogenic pesticide methoxychlor (MXC), however, has shown a differential effect on proliferative activity. An increase in uterine growth is present, while the pituitary undergoes a decrease in size, even though the effect is accompanied by a characteristic oestrogen-induced elevation in pituitary prolactin concentration. The focus of the current study was whether the observed differences in tissue growth between uterus and pituitary in response to MXC administration were paralleled by a corresponding disparity in the expression of those growth factors (members of the vascular endothelial growth factor (VEGF) and angiopoietin families and their receptors) that are involved in the angiogenic cascade. Ovariectomized adult Sprague-Dawley female rats were administered MXC (0-200 mg/kg, oral) for 1 or 3 weeks. Immunohistochemical staining of uteri and pituitaries was performed under strictly controlled conditions for VEGF and its receptor VEGFR2, Angiopoietin-1 (Ang1) and angiopoietin-2 and their tyrosine kinase receptor Tie2, and platelet endothelial adhesion factor (as an index of vascularity). Image acquisition and densitometric assessments of staining intensity were conducted under blind conditions. The results showed uterine MXC-induced increases in the expression of VEGFR2 and Ang1, changes consistent with a normal proliferative response to oestrogenic stimulation. For VEGF, staining tended to be most pronounced in the stromal region, although there did not appear to be a progressive increase with dose. VEGFR2 expression showed significant dose-related trends in luminal and glandular epithelia by 1 week. Similar effects at 1 week were evident for Ang1 in glandular epithelium. In the anterior pituitary, a dose-related increase in VEGF was present for the 1 and 3 week treatments, and the number of pituitary vessels per unit area was also increased after 3 weeks. The effects indicate that even though the insecticide has not been found to cause an augmentation in pituitary growth, a dose-related rise in the expression of at least one principal angiogenic factor is present that may be associated with an increase in vascular density.     

Synergy among rat T cells in the proliferative response to alloantigen.

A synergistic interaction in the proliferative response to alloantigen is described for mixtures of rat thymus and lymph node cells. The optimal conditions for synergy are quantitatively defined. Regression analysis of the slope of the dose-response curve has been utilized to estimate the degree of interaction in thymus-lymph node cell mixtures. The slope of the response of cell mixtures was noted to be significantly greater than the slope for the response of lymph node cells alone. Irradiation was shown to have a differential effect on the response of thymus and lymph node cells in mixtures. Irradiated thymus cells retained the capacity for synergy in mixtures, whereas irradiated lymph node cells did not. Additional studies have demonstrated that both de novo protein synthesis and specific antigen recognition by both responding cell populations in mixtures was required for maximal synergy. These studies demonstrate that synergy cannot be explained as an artifact of altered cell density in vitro. They establish that thymus cells and lymph node cells represent distinct subsets which manifest qualitatively different functions in the proliferative response to alloantigen. Thymus cells can respond directly to alloantigen by proliferation but also have the capacity to amplify the proliferative response of lymph node cells—a capacity which is resistant to X irradiation but requires recognition of alloantigen and de novo protein synthesis. Lymph node cells may similarly respond by proliferation to alloantigen but lack the amplifier activity of thymus cells. Synergy for rat lymphoid cells, like mouse lymphoid cells, has been shown to involve an interaction of thymus-derived lymphocytes.     

Role of bone marrow glycosaminoglycans in the reaction of the hematopoietic tissue to extreme conditions

The authors studied medullary glucosaminoglycans of rats and mice in extreme effects (hypoxia, hyperbaric oxygenation, inflammation, radiation, cooling, hemorrhage). Four types of reaction were determined which cause different changes of proliferation of hematopoietic cells. Hematopoiesis stimulation is accompanied by regular change of types of reaction of hematopoietic microenvironment. Its disturbance affects the changes of hematopoiesis and blood regeneration rate. It was shown that the increase of neutral glucosaminoglycans concentrations in bone marrow depended on the play ground and proliferative activity of erythroid sprout and that of acid glucosaminoglycans--primarily of granulocytic sprout.     

Ontogeny of proliferative and cytotoxic responses to interleukin 2 and concanavalin A in murine fetal thymus

The ontogeny of proliferative and cytotoxic responses to concanavalin A (Con A) and interleukin 2 (IL 2) in C57BL/6J (B6) fetal thymus (FT) was investigated. Embryonic thymocytes were either taken from embryos at different times of gestation or from 14 day B6 FT that were maintained as organ cultures for various times. It was found that the B6 FT could proliferate to Con A and EL4 SN (an IL 2 containing culture supernatant) in a synergistic fashion. This synergy between Con A and EL4 SN was first observed at the 16th to 17th day of gestation. A similar differentiation process took place in 14-day FT that had been maintained as organ cultures; the synergy between Con A and EL4 SN was first observed after 3 days in organ culture. This synergy increased with increasing time of organ culture, and was most evident after 10 days. The synergy between Con A and EL4 SN was also observed when the EL4 SN was replaced with IL 2 which had been purified from crude EL4 SN to apparent homogeneity. B6 FT could also form cytotoxic T lymphocytes (CTL) on stimulation with Con A and EL4 SN. Con A-activated CTL (polyspecific) were detected by including phytohemagglutinin in the assay medium. CTL response was first detected in the 17-day fetal thymus by using this assay. In organ cultures, CTL responses were first detected after 4 days in organ culture, and reached peak levels after 12 to 14 days. The CTL precursor (CTL-P) frequencies in the B6 FT after 2, 5, 10, and 14 days in organ culture were less than 1/10,000, 1/2232, 1/297, and 1/70, respectively; the corresponding CTL-P frequency in adult thymus was 1/60. After 6 days in organ culture, B6 FT could also form CTL in response to Con A and pure IL 2. This finding suggests that the ability to synthesize other differentiation factors that are required for CTL responses is acquired at an early time of thymic differentiation.     

Participation of peptide hydra morphogen in the regulation of proliferation processes in different tissues of white rats]

The PMH influence on proliferation processes in some tissues of white rats was studied. PMH injections in doses 10.0 mkg/kg and 100.0 mkg/kg stimulated the processes of DNA--synthesis after 24 hours in corneal and tongue epithelium. The direct dose-dependent effect was revealed. PMH in dose 10.0 mkg/kg activated the proliferative processes in the stomach epithelium and thymus cortex. after 24 hours too. The given dose of PMH caused trustworthy decreasing of proliferative activity in thymus after 4 hours. The stimulation of DNA-synthesis and acceleration of mitosis were found after 5-times application on white rat's corneal with 100 nM PMH solution.     

Immunofluorescence of histone H1 in stimulated lymphocytes measured by flow cytophotometry.

The modification of histones or their redistribution during the transition from actively transcribing chromatin to the heterochromatic chromosomes seems to play a major in regulation of gene expression. The purpose of this study was to monitor the change in immunofluorescence of histone HI during phytohemagglutinin stimulation in peripheral lymphocytes. The histone antigens were prepared from pig thymus, proven to be pure by gel electrophoresis and repeatedly injected as RNA-complexes into rabbits. The antihistone HI antiserum titer was 1:4000, and there was no cross-reactivity with other histone fractions as shown by microcomplement fixation tests. Affinity chromatography purified antibody after being labeled with fluorescein isothiocyanate was able to differentially stain HeLa cells as controls and those, where histone HI had been extracted by perchloric acid treatment. The measurements were done on a Los Alamos Scientific Laboratories-flow cytophotometer cell sorter. Staining peripheral lymphocytes resulted in a bimodal distribution. The increase in number of cells with high fluorescence intensity had its maximum about 20 hr before the maximum proliferative activity of the lymphocytes as measured by number of cells in S phase with the DNA-stain mithramycin.     

Amplification of the proliferative response to alloantigen by a factor present in an extract of syngeneic thymic lymphoid cells: demonstration of optimal conditions and target cell for factor activity.

A factor extracted from syngeneic thymic lymphoid cells (thymocytes) is shown to amplify the proliferative (MLC) response of syngeneic lymphoid cells to alloantigen in vitro. The optimal conditions for an effect of the thymus factor are quantitatively defined by kinetic and dose-response studies. Other variables that could potentially influence the activity of the thymus factor, such as the presence of 2-mercaptoethanol and the source of alloantigen, are identified. Factor activity can be recovered from semi-allogeneic thymocytes, as well as syngeneic thymocytes. The factor appears to predominantly effect the proliferative response of T cells localized in peripheral lymphoid organs. As such, this factor appears to be distinct from the variety of previously described factors derived from thymic reticuloepithelial elements that are thought to primarily induce the differentiation of T cell precursors found predominantly in bone marrow. Several possible mechanisms of action of this thymocyte-derived factor are considered.     

The annual involution and regeneration of the thymus in hibernating animals and perspectives of its studies in gerontology and stem cell proliferation

Data on a unique phenomenon of annual involution and neogenesis of thymus gland in hibernating animals are reviewed. In accordance with morphological findings, the annual thymus involution in hibernating animals is close to the age-dependent thymus involution occurring in all mammals once in a lifetime. In opposite, thymus involution in hibernating animals is totally different from the accidental involution. During hibernation, the thymus tissue is substituted by the brown fat tissue. In the spring, thymus gland neogenesis stats with intensive growth of epithelial tissue followed by lymphocyte infiltration and exhaustion of brown tissue. Morphological changes in the thymus gland within the annual cycle were compared with seasonal dynamics of structural and functional changes in peripheral lymphoid organs (spleen, lymphoglandular, peritoneal fluid). A general regularity was observed involving a decreased functional activity of immune cells in autumn, its sharp depression during winter hibernation, and obvious increase in summer with the onset of a season of animal activity. It is supposed that a sharp increase in the tumor necrosis factor (TNF) production observed during short-term awakenings in winter may serve an important link in this unique immune adaptation mechanism. The season changes in cellular TNF secretion suggest a mobilization of protective resources in hibernating animals in autumn and winter, i.e. in seasons when the thymus gland activity is depressed. The annual involution of thymus gland cannot be related to droppings in the environmental or body temperatures, as it comes long before their fall. Additionally, it is not related to ageing, as it occurs already in young hibernating animals. The role of hormones, including melatonine and corticosteroids, in mechanisms regulating thymus gland involution in hibernating animals is discussed.     

Apparent stimulation of calf thymus DNA polymerase alpha by ATP.

The mechanism by which millimolar concentrations of ATP stimulate the activity and increase the processivity of calf thymus DNA polymerase alpha has been investigated with poly(dA)/oligo(dT) as template/primer to eliminate possible effects due to primer synthesis. The effect of ATP on the rate of DNA synthesis with this template/primer was found to be dependent upon whether or not the ATP was neutralized and the species of buffer used in the reaction. The present studies suggest that ATP stimulation of calf thymus DNA polymerase can be attributed to changes in the pH of the reaction mixture, a shift in the magnesium ion optimum, or both. Furthermore, effects of ATP on the processivity of DNA polymerase alpha could be mimicked by lowering the pH of the reaction mixture.     

Thymus involution and inhibition of spleen growth accompanies streptozotocin-induced diabetes in rats; possible relationship of these changes to the elevated hydrolase levels in diabetic plasma

Diabetes in man and rats is accompanied by increased levels of acid hydrolases in plasma and urine, the increase being reversed by insulin. In investigating possible tissue sources of the additional enzymes, the unexpected observation was made that streptozotocin-induced diabetes in rats is accompanied by substantial involution of the thymus and the failure of the spleen to grow at its normal rate. One week after streptozotocin treatment, the average thymus weight of diabetic rats was 30% less than that of age-matched control rats. Eight weeks after the induction of diabetes, only 10 to 20% of the thymus remained. At that time, the spleen weight was only 50% that of age-matched controls. The organ weight changes were paralleled by changes in the organ levels of acid hydrolases. The changes were all reversed by insulin treatment. Since it appeared possible that corticosteroids might be the mediators of the streptozotocin-induced changes, the effects of streptozotocin were determined in adrenalectomized rats. The decreases in thymic and splenic weights again were observed, as well as decreases in most organ hydrolases and increases in most plasma hydrolases. Clearly, the principal streptozotocin effects are not adrenal dependent. Although analyses of hexosaminidase A and B levels in various tissues and plasma suggested that the thymus and spleen may contribute to the additional hydrolases found in diabetic plasma, other factors must also be involved.     

Antigen-specific regulation of T lymphocyte proliferative responses to contact-sensitizing chemicals in the guinea pig

Skin painting of guinea pigs with either 4-ethoxymethylene-2-phenyloxazol-5-one or 2,4-dinitrofluorobenzene induced not only a primary proliferative response in the draining lymph node but also the systemic suppression of subsequent proliferative responses to topically applied hapten. The inhibition of lymphocyte proliferation, as assessed by the incorporation of [3H]-thymidine and the presence of large pyroninophilic cells in the paracortex, was hapten-specific and long-lasting. This study demonstrates that, in common with the mouse, the sensitization of guinea pigs results in the induction of a hapten-specific suppressor mechanism, which serves to control the proliferative response following reexposure to hapten. However, the antigen-nonspecific suppression of proliferation observed in the mouse following exposure to some potent contact sensitizers was not, under the conditions employed, detectable in the guinea pig.     

The influence of various cell kinetic conditions on functional differentiation in the small intestine of the rat. A study of enzymes bound to subcellular organelles

The process of cell maturation and cell ageing of absorptive epithelial cells was investigated in normal rat duodenum. The development of a number of enzymes bound to subcellular organelles was studied by using microchemical analyses on various cell compartments dissected from crypts and villi from freeze-dried cryostat sections. The development of the ultrastructural features of the absorptive epithelium was investigated by electron microscopy of various cell positions along the whole length of the crypt and the base of the villus. The data obtained were related to cell position along the crypt and villus and to cell age during migration from the bottom of the crypt to the tip of the villus.The influence of changes in the life-span of the cells and of increasing proliferative activity was studied by comparing normal rat duodenum with that from germfree rats and rats recovering from low radiation doses (72 hr after 400 R).Our data show that the specific activity of nonspecific esterases mainly localized in the endoplasmic reticulum increases when the cells migrate along the upper half of the crypt and the basal part of the villus. Activity of alkaline phosphatase, measured as a marker for the microvilli, is absent in the crypt, but increases linearly from the base of the villus to the tip. The longer life-span of villus cells in germfree animals does not result in a higher activity of these enzymes than in normal animals. An increased proliferative activity in the crypt, as present 72 hr after X-irradiation, is accompanied by a decreased activity of both enzymes but the pattern of activity during cell migration remains the same. The specific activity of enzymes bound to mitochondria or lysosomes (monoamineoxidase and β-N-acetylglucosaminidase) are not affected by changing crypt cell kinetics.Electrophoretic analyses of isolated cell compartments showed that the increase during normal differentiation or the decrease after X-irradiation of esterase activity is due to changes in overall activity, not to the appearance or disappearance of specific isoenzymes. Electron microscopy showed that in the normal intestine there is a gradual development of ultrastructural features during migration of the cell along the crypt while the most drastic changes in cell structure occur at the moment the cell enters the villus. Contrary to our expectation, the ultrastructural development was not influenced by increased proliferative activity in the crypt 72 hr after irradiation, and hence the decrease in enzyme activity found cannot be related to changes in ultrastructure.     

The role of periodic changes in cyclase and protein kinase activity in the mechanism of the proliferative effect of ACTH

A prolonged effect of ACTH on the state of adenylate and guanylate cyclase systems in the adrenal glands of experimental animals was investigated. It was found that in guinea pigs injected with ACTH (4 units daily for 1-50 days) the weight of adrenal glands and the DNA content in these organs increased 2.0-2.5-fold by the end of experiment; the increase in both values was stepwise. The corticosteroid level in the blood varied throughout the experiment: the changes in the DNA content in adrenals and in the corticosteroid content in the blood were oppositely directed. This was accompanied by cyclic changes in the basal and stimulated activities of adenylate and guanylate cyclases and proteinases in the adrenal glands occurring with a periodicity of 6-15 days. The activity peaks for cyclases and protein kinases preceded the rise in the DNA content in the adrenals. A clearcut correlation between the changes in the enzyme activity and the hormone dose was observed. The changes in the basal and stimulated activities of guanylate cyclase seem to be due to the control of cAMP level in the cell (stimulation of cGMP-dependent cAMP phosphodiesterase). Apparently, the periodic changes in the activity of cAMP-dependent protein kinases in the cytoplasmic and nuclear fractions and a relatively high activation of nuclear protein kinases (by 30-60%) in comparison of cytoplasmic ones (8-10%) are related to stimulation of DNA synthesis. It is concluded that the changes in the activity of cyclases and protein kinases play a role in the mechanism of proliferative effect of ACTH.