Altered circadian rhythms of the stress hormone and melatonin response in lupus-prone MRL/MP-fas(Ipr) mice

The immune system interacts with the hypothalamo-pituitary-adrenal axis via so-called glucocorticoid increasing factors, which are produced by the immune system during immune reactions, causing an elevation of systemic glucocorticoid levels that contribute to preservation of the immune reactions specificities. Previous results from our laboratory had already shown an altered immuno-neuroendocrine dialogue via the hypothalamo-pituitary-adrenal axis in autoimmune disease-prone chicken and mouse strains. In the present study, we further investigated the altered glucocorticoid response via the hypothalamo-pituitary-adrenal axis in murine lupus. We established the circadian rhythms of corticosterone, dehydroepiandrosterone-sulfate, adrenocorticotropic hormone and melatonin, as well as the time response curves after injection of interleukin-1 of the first three parameters in normal SWISS and lupus-prone MRL/MP-fas(Ipr) mice. The results show that lupus-prone MRL/ MP-fas(Ipr) mice do not react appropriately to changes of the light/dark cycle, circadian melatonin rhythms seem to uncouple from the light/dark cycle, and plasma corticosterone levels are elevated during the resting phase. Diurnal changes of dehydroepiandrosterone-sulfate and adrenocorticotropic hormone were normal compared to healthy controls. These data indicate that MRL/ MP-fas(Ipr) mice not only show an altered glucocorticoid response mediated via the hypothalamo pituitary adrenal axis to IL-1, but are also affected by disturbances of corticosterone and melatonin circadian rhythms. Our findings may have implications for intrathymic T cell development and the emergence of autoimmune disease.     

Lupus-prone MRL/faslpr/lpr mice display increased AID expression and extensive DNA lesions,comprising deletions and insertions,in the immunoglobulin locus: Concurrent upregulation of somatic hypermutation and class switch DNA recombination

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies. These autoantibodies are mutated and class-switched, mainly to IgG, indicating that immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) are important in their generation. Lupus-prone MRL/fas^lpr/lpr mice develop a systemic autoimmune syndrome that shares many features with human SLE. We found that Ig genes were heavily mutated in MRL/fas^lpr/lpr mice and contained long stretches of DNA deletions and insertions. The spectrum of mutations in MRL/fas^lpr/lpr B cells was significantly altered, including increased dG/dC transitions, increased targeting of the RGYW/WRCY mutational hotspot and the WGCW AID-targeting hotspot. We also showed that MRL/fas^lpr/lpr greatly upregulated CSR, particularly to IgG2a and IgA in B cells of the spleen, lymph nodes and Peyer's patches. In MRL/fas^lpr/lpr mice, the significant upregulation of SHM and CSR was associated with increased expression of activation-induced cytidine deaminase (AID), which mediates DNA lesion, the first step in SHM and CSR, and translesion DNA synthesis (TLS) polymerase (pol) θ, pol η and pol ζ, which are involved in DNA synthesis/repair process associated with SHM and, possibly, CSR. Thus, in lupus-prone MRL/fas^lpr/lpr mice, SHM and CSR are upregulated, as a result of enhanced AID expression and, therefore, DNA lesions, and dysregulated DNA repair factors, including TLS polymerases, which are involved in the repair process of AID-mediated DNA lesions.     

IL-17 producing CD4 T cells mediate accelerated ischemia/reperfusion-induced injury in autoimmunity-prone mice

Elements of the innate and adaptive immune response have been implicated in the development of tissue damage after ischemic reperfusion (I/R). Here we demonstrate that T cells infiltrate the intestine of C57BL/6 mice subjected to intestinal I/R during the first hour of reperfusion. The intensity of the T cell infiltration was higher in B6.MRL/lpr mice subjected to intestinal I/R and reflected more severe tissue damage than that observed in control mice. Depletion of T cells limited I/R damage in B6.MRL/lpr mice, whereas repletion of B6.MRL/lpr lymph node-derived T cells into the I/R-resistant Rag-1^−/− mouse reconstituted tissue injury. The tissue-infiltrating T cells were found to produce IL-17. Finally, IL-23 deficient mice, which are known not to produce IL-17, displayed significantly less intestinal damage when subjected to I/R. Our data assign T cells a major role in intestinal I/R damage by virtue of producing the pro-inflammatory cytokine IL-17.     

Loss of circadian rhythm and light‐induced suppression of pineal melatonin levels in Cry1 and Cry2 double‐deficient mice

Cryptochrome 1 and 2 (Cry1 and Cry2) are considered essential for generating circadian rhythms in mammals. The role of Cry1 and Cry2 in circadian rhythm expression and acute light‐induced suppression of pineal melatonin was assessed using Cry1 and Cry2 double‐deficient mice (Cry1^?/?/Cry2^?/?) developed from the C3H strain that synthesizes melatonin. We examined the circadian variation of pineal melatonin under a 12:12‐h light–dark (LD) cycle and constant darkness (DD). Light suppression of pineal melatonin concentration was analyzed by subjecting a 30‐min light pulse at the peak phase of melatonin concentration. Wild‐type mice showed significant rhythmicity in pineal melatonin concentration with the highest level at Zeitgeber time 22 (ZT22, where time of light on was defined as ZT0) under LD or ZT18 on the first day of DD. In contrast, Cry1^?/?/Cry2^?/? mice did not show significant circadian rhythmicity, with only a small peak observed at ZT22 in LD. Nevertheless, a significant daily variation could be observed under DD, with a small increase at ZT6 and ZT18 h. Melatonin concentration was significantly suppressed by acute light pulse at ZT22 in wild‐type mice but not in Cry1^?/?/Cry2^?/? mice. The present results suggest that Cry genes are required for regulating pineal melatonin synthesis via circadian and photic signals from the suprachiasmatic nucleus of the hypothalamus (SCN).     

Courtship Bout Duration in per Circadian Period Mutants In Drosophila melanogaster

Mutations on the period locus of Drosophila melanogaster influence circadian periods as well as the rhythm in inter-pulse intervals in male courtship song; per ^L mutations produce long circadian periods and courtship song rhythms and per ^s mutations produce short circadian periods and courtship song rhythms. Thus, these mutations influence timing mechanisms over both long and short behavioral time horizons. We examined if the mean courtship duration of male Drosophila melanogaster cycles rhythmically, and if mutations at the period locus influence courtship bout duration. We measured the courtship bout durations of the following: (1) wild type Canton-S (per ⁺) males; (2) per ^L males; and (3)per ^s males. Rhythmicity of courtship bout duration could not be mathematically determined. Mean courtship bout duration did not differ among the three groups; thus, mutations at the period locus did not influence mean courtship duration. There was a nonsignificant trend for per ⁺ males that were successful at mating to have longer mean courtship duration than unsuccessful males.     

Spontaneous occurrence of chronic non-suppurative destructive cholangitis and antimitochondrial autoantibodies in MRL/lpr mice: Possible animal model for primary biliary cirrhosis

MRL/MP mice bearing the lymphoproliferative gene lpr (known as MRL/MP‐lpr/lpr or MRL/Ipr mice) are known to spontaneously develop severe autoimmune diseases such as glomerulonephritis, arteritis and arthritis at an early stage of their life. They have also been reported to develop severe sialadenitis, suggesting that this mouse could be a model for Sjögren’s syndrome. Primary biliary cirrhosis, an autoimmune disease characterized by chronic non‐suppurative destructive cholangitis and the occurrence of antimitochondrial antibodies, is frequently associated with Sjögren’s syndrome. In this study, we examined whether cholangitis and/or antimitochondrial antibodies occur in this mouse model, using more than 100 young and old MRL/Ipr mice. We frequently found portal inflammation associated with cholangitis of small intrahepatic bile ducts, especially in older mice. There was also infiltration of inflammatory cells (monocytes) as well as CD4‐positive T cells. Epithelioid granuloma and bile‐duct loss were also occasionally found. These histological features resemble primary biliary cirrhosis. In addition, antimitochondrial antibodies were shown by immunocytochemistry to be present in the sera of MRL/Ipr mice. There is currently no established animal model for primary biliary cirrhosis. Therefore, further studies on MRL/Ipr mice, with respect to pathogenesis of primary biliary cirrhosis, are warranted.     

Circadian disruption,melatonin rhythm perturbations and their contributions to chaotic physiology

The aim of this report is to summarize the data documenting the vital nature of well-regulated cellular and organismal circadian rhythms, which are also reflected in a stable melatonin cycle, in supporting optimal health. Cellular fluctuations in physiology exist in most cells of multicellular organisms with their stability relying on the prevailing light:dark cycle, since it regulates, via specialized intrinsically-photoreceptive retinal ganglion cells (ipRGC) and the retinohypothalamic tract, the master circadian oscillator, i.e., the suprachiasmatic nuclei (SCN). The output message of the SCN, as determined by the light:dark cycle, is transferred to peripheral oscillators, so-called slave cellular oscillators, directly via the autonomic nervous system with its limited distribution. and indirectly via the pineal-derived circulating melatonin rhythm, which contacts every cell. Via its regulatory effects on the neuroendocrine system, particularly the hypothalamo-pituitary-adrenal axis, the SCN also has a major influence on the adrenal glucocorticoid rhythm which impacts neurological diseases and psychological behaviors. Moreover, the SCN regulates the circadian production and secretion of melatonin. When the central circadian oscillator is disturbed, such as by light at night, it passes misinformation to all organs in the body. When this occurs the physiology of cells becomes altered and normal cellular functions are compromised. This physiological upheaval is a precursor to pathologies. The deterioration of the SCN/pineal network is often a normal consequence of aging and its related diseases, but in today's societies where manufactured light is becoming progressively more common worldwide, the associated pathologies may also be occurring at an earlier age.     

A functional subdivision of the circadian clock is revealed by differential effects of melatonin administration

The biological clock of the suprachiasmatic nuclei drives numerous physiological and behavioural circadian rhythms. In this study, we addressed the question as to whether different components of the clock may control separately various circadian functions. Using the rat transpineal microdialysis tool, we analysed the effect of clock perturbation by exogenous melatonin injection on two hormonal clock outputs: pineal melatonin and adrenal corticosterone secretions. As already reported, a single melatonin injection at the light/dark transition induces a marked increase in the endogenous pineal melatonin peak for the two following days. In the same animals, by contrast, the amplitude of the corticosterone rhythm was not altered following melatonin injection. These data show that the melatonin injection does not display an overall effect on the circadian clock, but rather influences a subpopulation of melatonin-sensitive neurons involved, among other functions, in the circadian control of the pineal pathway.     

Aberrant Wound Healing and TGF-β Production in the Autoimmune-Prone MRL/+ Mouse

Wound healing is a complex process that involves inflammation, apoptosis, growth, and tissue remodeling. The autoimmune-prone inbred mouse strain MRL/+ manifests accelerated and extensive healing to ear punch wounds, suggesting a link between immune defects and wound healing. Prior studies with lupus-prone mice have shown that hematopoietic cells of lupus-prone strains can transfer disease to otherwise non-autoimmune-prone recipients. In this study we performed reciprocal bone marrow transfers between MRL and the control strain B10.BR and found that radioresistant MRL/+ host cells, rather than hematopoietic cells, are required for the healing response. We have also made the novel observations that, compared to normal controls, MRL/+ hematopoietic cells overproduce TGF-β1 and manifest impaired inflammatory responses to lipopolysaccharide challenge. These features suggest that the aberrant wound healing phenotype of MRL mice is independent of their propensity to develop autoimmunity.     

Circadian and ultradian rhythms inperiod mutants ofDrosophila melanogaster

Using digital techniques for signal analysis—the correlogram and a high-resolution analysis of time series, maximum-entropy spectral analysis (MESA)—we have detected both circadian and ultradian rhythms in the locomotor activity of free-runningper ⁰ males and in females lacking theper locus (per ^–; heterozygous for two deficiencies, each of which deletes the gene). Over half theper ⁰ individuals and half theper ^– individuals tested were rhythmically active, with dominant periods ranging from 4 to 22 h; most of the significantly rhythmicper ⁰ andper ^– flies clearly exhibited multiple periodicities. This novel pattern of rhythmic behavior is thoroughly distinct from the wild-type pattern. One hypothesis suggested by our observations is that theper ⁺ gene products mediate the coupling of multiple ultradian oscillators to produce wild-type circadian rhythms.This work was supported in part by NIH Grant GM-33205 to J.C.H.     

Pattern of individual biorhythms of the rectal temperature of rats in health and during starvation

Individual cosinor-analysis with the use of special software demonstrates that the rectal temperature of intact rats exhibits two clear-cut rhythms: the circadian rhythm and a 96-h infradian rhythm. Under conditions of total starvation, including a lethal outcome, the spectrum of the biorhythms of the rectal temperature alters markedly: the circadian rhythm is completely eliminated in the majority of starving animals, the amplitude of the 4-day rhythm declines or this rhythm is also eliminated, and ultradian and infradian components appear in the biorhythmic pattern of the rectal temperature. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 6, pp. 656–657, June, 1994 Presented by Yu. A. Romanov, Member of the Russian Academy of Medical Sciences     

Role of the pineal gland in immunity: II. Melatonin enhances the antibody response via an opiatergic mechanism.

The pineal gland constitutes a major neuroendocrine organ in the brain. It transduces exogenous signals such as circadian and seasonal variations of light and temperature into proper hormonal changes which adjust and adapt internal endocrine functions. These pineal activities seem to be exerted via circadian synthesis and release of the indoleamine melatonin, a neurohormone secreted by the pineal itself. Alteration of circadian rhythms have been associated with affective disorders, psychosomatic diseases, cancer and many other pathologies. We have reported that functional and pharmacologic inhibition of melatonin synthesis results in depressed immune functions in vivo and that exogenous, evening administration of melatonin enhances antibody formation via an antigen-activated process and also antagonizes the immunosuppressive effects of corticosterone. We communicate here findings demonstrating that (a) three different inbred strains of mice possess a clear-cut cycle of melatonin levels in serum, (b) that melatonin administered in the evening enhances primary antibody response (IgM and IgG immunoglobulins) in vivo according to a dose-response behaviour and that (c) the opioid receptors blocker naltrexone antagonizes the immunostimulatory effect of melatonin. These findings point to a fundamental immunoregulatory role of circadian melatonin and to an activity of the neurohormone via opioid peptides.     

Extrapineal melatonin and its role in the neuroendocrine regulation of homeostasis

The hormone of melatonin is the main regulator of biological rhythms. It was first found in the pineal gland in 1958. Melatonin is involved in the regulation of many vital physiological processes: maturation and development of genitalias, metabolism of pigments and free radicals, immune response, mood and sleep, and cell proliferation and differentiation. The pineal gland is not the only organ synthesizing melatonin. Extrapineal melatonin is widely dis-tributed in humans and animals. Melatonin-producing cells are found in the gastrointestinal and respiratory tracts, pancreas, adrenal and thyroid glands, thymus, cerebellum, urogenital system, placenta, and other organs. Melatonin is intensely synthesized in non-endocrine cells: mast cells, natural killers, eosinophilic leukocytes, platelets, and endotheliocytes. Such a wide distribution of melatonin reflects its key role as an intercellular neuroendocrine regulator and coordinator of many complex and interrelated biological processes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 4, pp. 364–730, April, 1999     

Promoter Methylations of p16 and p14 Genes in Early and Advanced Gastric Cancer. Correlations of the Modes of Their Occurrence with Histologic Type

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16^INK4a and p14^ARF. These share an exon using different reading frames, and act through Rb and p53 pathways. Recently, it has been found that silencing of p16^INK4a and p14^ARF expressions by aberrant methylation of the CpG islands in the promoter regions is an alternative mechanism that inactivates possible tumor suppressor functions in various tumors. To clarify the features of gastric cancers with promoter methylation of p16^INK4a and p14^ARF, we investigated the methylation status in gastric cancer cell lines and primary gastric cancers using methylation-specific PCR (MSP), and correlated the methylation status with microsatellite instability (MSI), DNA ploidy pattern, p53 immunohistochemistry, and various clinicopathologic factors, paying attention to the correlations with the histologic types. Of 10 cell lines studied, silencing of the expression of p16^INK4a and p14^ARF due to promoter methylation was detected by MSP and RT-PCR in six (60%) and two (20%) cell lines, respectively. p14^ARF silencing was detected only in cell lines derived from gastric cancer of the diffuse type, while p16^INK4a silencing was found in cell lines derived from both diffuse and intestinal types. In 59 primary gastric cancers, promoter methylation of p16^INK4a and p14^ARF was found in 10 (17%) and 14 (24%) of the tumors independently, there being an association with DNA diploidy, but not with p53 immunohistochemistry. p16^INK4a methylation was found irrespective of tumor stages and histology. Whereas p14^ARF methylation was found more frequently in intestinal type cancers in an early stage and in diffuse type cancers in an advanced stage, MSI tended to be related especially to p14^ARF methylation in cancers of the intestinal type. Thus, the significance of p14^ARF methylation differed between intestinal and diffuse types, while such a difference was not observed in p16^INK4a methylation.     

Patterns of interaction between the immune and endocrine systems in the manifestation of hereditary autoimmune disorders in NZB mice

Specific features of immune-endocrine interrelationships and of pituitary-adrenal system functioning are studied in NZB mice with hereditary autoimmune pathology. It is established that the development of disease is accompanied by a weakened response to stress, reduced blood corticosteroid level, and decreased adrenal reactivity to exogenous adrenocorticotropic hormone. Moreover, a loss of sensitivity to interleukin-2 is observed. These facts provide evidence of disturbed interaction between the immune and endocrine systems as well as of inhibition of pituitary-adrenal system activity as the autoimmune disease develops. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 4, pp. 394–397, April, 1995     

Identification of a Mutation in the Clock1 Gene Affecting Zebrafish Circadian Rhythms

As part of an ongoing program to identify genes involved in maintaining circadian rhythms of zebrafish, 6,500 mutagenized genomes were screened for dominant mutants affecting circadian locomotor activity. Molecular analysis of one of these mutant lines, Clk1^dg3, revealed an I254N mutation in the PAS domain of the Clock1 protein. This isoleucine is tightly conserved in the Clock genes of several different species, and the I254N was not seen in any of the wild-type zebrafish population tested. Analysis of circadian activity rhythms as well as melatonin rhythms in homozygotes revealed the biological clock runs with a shortened period. The effect of this Clock1 mutation was characterized in vitro using a cell culture system where it appears to enhance the transactivation ability of the I254N Clock1 protein compared with that of the normal gene product.     

Effect of an inverted light regime on the biological rhythms of the mitotic index of mouse esophageal epithelium in different periods

Changes of the mitotic index in the esophageal epithelium were studied every 20 min for 24 hours in control mice and in mice kept under conditions of an inverted light regime for 10 days. A monophasic circadian rhythm was revealed, which was virtually completely resynchronized to the new light regime after 10 days of photoinversion. Circahoralian fluctuations of the mitotic index with 1–2-hour periods were noted. In controls the period of these fluctuations was shorter in the active phase of the circadian rhythm than during the passive phase. After photoinversion this regularity was no longer observed. Hence, in contrast to the circadian rhythm, the hierarchical structure of the temporal organization of the mitotic index rhythm of the esophageal epithelium in different periods was not restructured during the 10 days of the new light/dark regime. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 1, pp. 94–97, January, 1996     

Diurnal time course of hexenal narcosis in experimental hepatosis

A statistically significant circadian rhythm of hypnogenic effect of hexenal is revealed in intact Wistar rats, with the maximum recorded in the daytime and the minimum at night, and an amplitude of at least 30% of the mesor. Circadian rhythms of the analgetic action of hexenal and of α-tocopherol concentrations in the blood serum are found to be in reciprocal relationship. Experimental hepatosis induced by intragastric administration of CCl₄ is attended by alteration of the time organization of the antitoxic function of the liver and of the concentrations of iron, α-tocopherol, and lipid peroxidation products in the blood collected from the hepatic and portal veins. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 5, pp. 540–543, May, 1995 Presented by E. D. Gol'dberg, Member of the Russian Academy of Medical Sciences     

High-resolution analysis of locomotor activity rhythms indisconnected,a visual-system mutant ofDrosophila melanogaster

Free-running locomotor activity and eclosion rhythms ofDrosophila melanogaster, mutant at thedisconnected (disco) locus, are substantially different from the wild-type phenotype. Initial periodogram analysis revealed little or no rhythmicity (Dushayet al., 1989). We have reanalyzed the locomotor activity data using high-resolution signal analysis (maximum-entropy spectral analysis, or MESA). These analyses, corroborated by autocorrelograms, uncovered significant residual circadian rhythmicity and strong ultradian rhythms in most of the animals tested. In this regard thedisco mutants are much like flies expressing mutant alleles of theperiod gene, as well as wild-type flies reared throughout life in constant darkness. We hypothesize that light normally triggers the coupling of multiple ultradian oscillators into a functional circadian clock and that this process is disrupted indisco flies as a result of the neural lesion.This work was supported in part by NIH Grant FM-33205.     

Effect of insulin and adrenocorticotropic hormone on rat brain cortex in the early postnatal period

Administration of insulin and adrenocorticotropic hormone to 1-, 2-, and 3-day-old rats leads to an increase in the RNA and total protein contents of cortical neurons, while administration of adrenocorticotropic hormone alone increases the number of dying neurons in layer V of the cortex. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 9, pp. 305–307, September, 1995 Presented by V. A. Trufakin, Member of the Russian Academy of Medical Sciences