Inhibition of the CXCR3-mediated pathway suppresses ultraviolet B-induced pigmentation and erythema in skin.

BACKGROUND: Skin pigmentation by ultraviolet (UV) B radiation is caused in part by inflammation mediated by chemokines and cytokines secreted by keratinocytes in the irradiated area. However, such inflammatory processes have not been well documented. OBJECTIVES: To elucidate the inflammation processes caused by UVB irradiation using skin-lightening agents that suppress melanin synthesis after UVB irradiation. METHODS: Utilizing a three-dimensional (3D) skin model, agents that suppressed formation of sunburn cells (SBC) after UVB irradiation were screened. Molecules whose expression was upregulated by UVB irradiation and attenuated by pretreatment with the agent were then screened by gene microarray to explore the mechanism of UVB irradiation. Messenger RNA expression of the molecules identified to be responsible for melanin biosynthesis was knocked down with a Tet-off shRNA lentivirus construct to confirm the involvement of the molecule in the pigmentation pathway following UVB irradiation. RESULTS: Paeonia suffruticosa Andrews (PSA) pretreatment suppressed SBC formation in the 3D skin model, and erythema formation and pigmentation in volunteers exposed to UVB irradiation. Comprehensive gene analysis after UVB irradiation showed upregulation of CXCR3 and its ligands, CXCL9/monokine induced by interferon (IFN)-γ (MIG), CXCL10/10-kDa IFN-γ-induced protein (IP-10) and CXCL11/inducible T-cell α-chemoattractant (I-TAC). Upregulation of these genes was partially suppressed by PSA pretreatment. Melanin biosynthesis increased upon stimulation of CXCR3 ligands (MIG, IP-10 or I-TAC) and decreased following CXCR3 downregulation by shRNA knockdown. CONCLUSIONS: UVB irradiation activates CXCR3-mediated signalling that leads to melanin synthesis. PSA pretreatment shows a lightening effect partly by attenuating CXCR3-mediated signalling at the transcriptional level.

Vitamin E ointment at high dose levels suppresses contact dermatitis in rats by stabilizing keratinocytes.

OBJECTIVE: The pharmacological effect of vitamin E ointment at high dose levels was investigated in rats and mice during the development of contact dermatitis. MATERIALS AND METHODS: Allergic or irritant contact dermatitis was induced in sensitized or unsensitized animals by topical application of chemical agent(s). Cultured keratinocytes were prepared from dorsal skin of rats. RESULTS: The vitamin E ointment at 20-40% suppressed allergic and irritant contact dermatitis, exerting a comparable effect to that of 0.5% prednisolone ointment. Microscopic findings revealed that 20% vitamin E ointment reduced the keratinocyte damage, whereas 0.5% prednisolone was ineffective. The protective action of vitamin E on keratinocyte damage was also confirmed in a cell culture experiment. Furthermore, 20% vitamin E ointment blocked down-regulation of skin barrier function induced by contact dermatitis, although 0.5% prednisolone ointment was inactive. CONCLUSIONS: These results indicate that 20% vitamin E ointment suppresses contact dermatitis by stabilizing keratinocytes, concomitantly with novel, interesting properties.

Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies.

Mutations of receptor tyrosine kinases are implicated in the constitutive activation and development of human malignancy. An internal tandem duplication (ITD) of the juxtamembrane (JM) domain-coding sequence of the FLT3 gene (FLT3/ITD) is found in 20% of patients with acute myeloid leukemia (AML) and is strongly associated with leukocytosis and a poor prognosis. On the other hand, mutations of the c-KIT gene, which have been found in mast cell leukemia and AML, are clustered in 2 distinct regions, the JM domain and D816 within the activation loop. This study was designed to analyze the mutation of D835 of FLT3, which corresponds to D816 of c-KIT, in a large series of human hematologic malignancies. Several kinds of missense mutations were found in 30 of the 429 (7.0%) AML cases, 1 of the 29 (3.4%) myelodysplastic syndrome (MDS) cases, and 1 of the 36 (2.8%) acute lymphocytic leukemia patients. The D835Y mutation was most frequently found (22 of the 32 D835 mutations), followed by the D835V (5), and D835H (1), D835E (1), and D835N (1) mutations. Of note is that D835 mutations occurred independently of FLT3/ITD. An analysis in the 201 patients newly diagnosed with AML (excluding M3) revealed that, in contrast to the FLT3/ITD mutation (n = 46), D835 mutations (n = 8) were not significantly related to the leukocytosis, but tended to worsen disease-free survival. All D835-mutant FLT3 were constitutively tyrosine-phosphorylated and transformed 32D cells, suggesting these mutations were constitutively active. These results demonstrate that the FLT3 gene is the target most frequently mutated to become constitutively active in AML.

Age-related development of gamma-aminobutyric acid (GABA)B receptor functions in various brain regions of spontaneously hypertensive rats.

The age-related development of GABAB receptors and their coupling to adenylate cyclase were studied in the brains of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Compared with WKY rats, the specific [3H]GABA binding to GABAB receptors showed a significant decrease not only in the posterior hypothalamus, midbrain, hippocampus and striatum of eleven-week-old SHR, which maintain a hypertensive state, but also in the posterior hypothalamus of four-week-old normotensive SHR. Similarly, the GABAB receptor agonists (baclofen and DN-2327)-induced suppression of adenylate cyclase activity showed a decrease in the posterior hypothalamus of four-week-old SHR as well as in the posterior hypothalamus and striatum of eleven-week-old SHR. These results suggest that the functions of the GABAB receptor in the brain of SHR may be decreased independently from the occurrence of blood pressure elevation and that such changes may even be involved in the pathogenesis of SHR.

Age-related decrease of gamma-aminobutyric acid (GABA) release in brain of spontaneously hypertensive rats.

We studied the developmental alteration of GABA release in spontaneous ly hypertensive(SHR) and normotensive Wistar-Kyoto rat(WKY) brain. The release of [3H]GABA observed under high potassium(30 mM) in eleven-week-old(hypertensive) SHR hippocampus and the spontaneous release of [3H]GABA in the same aged SHR medulla oblongata were lower than those of age-matched WKY. We conclude that the GABAergic mechanisms may be different in SHR and WKY brain and may be associated with the development of hypertension.

Cloning of the ALL-1 fusion partner, the AF-6 gene, involved in acute myeloid leukemias with the t(6;11) chromosome translocation.

Reciprocal chromosome translocations involving 11q23 are frequently associated with acute leukemias, with the t(4;11) translocation predominating among acute lymphoblastic leukemias, and the t(9;11), t(11;19) and t(6;11) translocations most common among acute myeloid leukemias. In each of these translocations the ALL-1 gene, located at 11q23 and constituting the human homologue of Drosophila trithorax, fuses to a specific gene on the partner chromosome to produce a chimeric protein. Here we report the cloning and the characterization of the partner gene from chromosome 6 (AF-6). AF-6 is expressed in a variety of cell types and encodes a protein of 1612 amino acids. The protein contains short stretches rich in prolines, charged amino acids, serines, or glutamines. In addition, the AF-6 protein contains the GLGF motif shared with several proteins of vertebrates and invertebrates thought to be involved in signal transduction at special cell-cell junctions.

Distribution of taurine-like immunoreactivity in the mouse liver during ontogeny and after carbon tetrachloride or phenobarbital intoxication.

The ontogenic pattern of development of taurine-like immunoreactivity (TLI) was studied in the mouse liver. The effect on adult mice of carbon tetrachloride or phenobarbital treatment was also examined. Light-microscopically, granules of TLI were first found in the liver from 17-day-old embryos, diffusely distributed throughout the lobules. These positive granules increased with age, were most numerous in the two-week-old mouse, and were notably decreased in the central region of some lobules in the three-week-old mouse. In mature mice, hepatocytes containing TLI-positive granules were distributed unevenly in each liver lobule, and were located predominantly in the peripheral region. Electron-microscopically, TLI was observed in small vesicles in the cytoplasm of hepatocytes and was found mainly in the cisternal lumen of smooth-surfaced endoplasmic reticulum. Some taurine-positive vesicles surrounding the reticulum seemed to associate with the protoplasm. Similar positive vesicles were often located near the bile canaliculi. In carbon tetrachloride-intoxicated mature mice, TLI was no longer limited to the peripheral region of lobules; hepatocytes situated in the central region of lobules also contained intense TLI. In mice injected with a small and repeated dose of phenobarbital, the distribution pattern of TLI was similar to that in the untreated group. However, in mice injected with a large dose of phenobarbital, TLI was markedly increased, especially in the central region of lobules. The results demonstrate that the distribution pattern of TLI in mouse liver changes during development, and that the pattern in mature mice is affected by intoxication with carbon tetrachloride or a toxic dose of phenobarbital.

Penicillin G-induced cutaneous anaphylaxis in the guinea pig.

Allergic cutaneous responses were induced by intradermal injection of penicillin G (PCG) and PCG-bovine serum albumin (BSA) conjugates onto the back of guinea pig actively immunized with PCG potassium (25 mg/animal) incorporated in Freund's complete adjuvant. The PCG-induced response was characterized macroscopically by erythema and edema with a maximum level at 24 hrs after elicitation and microscopically by the infiltration with basophils, macrophages and lymphocytes following with neutrophils. In addition, intensity of macrophage-infiltration shared a similar time course change with those of erythema and edema. These suggest that this response is associated to a delayed type hypersensitivity of Jones-Mote type. On the other hand, in the PCG-BSA-induced response the edema with erythema at the early phase was a noticeable observation and this response disappeared within 12 hrs, although the erythema continued by 24 hrs. Microscopically, the degranulation of mast cells and severe infiltration with neutrophils in the early phase and the infiltration of eosinophils in the late phase accompanying the infiltration with monocytes, basophils and lymphocytes were characteristic findings, which suggest that PCG-BSA response is a similar hypersensitivity to an atopic dermatitis. As mentioned above, we confirmed two types of allergic cutaneous responses in the guinea pig immunized with PCG.

Development of postoperative intrathoracic chemo-thermotherapy for lung cancer with objective of improving local cure.

From April 1985 through March 1988, 24 lung cancer patients with carcinomatous pleuritis were treated with a new combined treatment modality consisting of pulmonary resection and postoperative intrathoracic chemo-thermotherapy (PICT). They consisted of 15 male patients and nine female patients. A majority of the patients (79%%) had adenocarcinoma. The PICT was started 10 to 14 days after the operation. Immediately after a bolus intrathoracic injection of cisplatin (CDDP) 50-100 mg, thermotherapy was carried out using 13.56-MHz or 8.00-MHz radiofrequency waves for 60 minutes. The peripleural temperature was precisely monitored in 20 patients. The temperature was successfully maintained above 42 degrees C for 40 minutes in each of two or three treatment courses in 13 patients, with no complications. However, in the other seven patients the therapy resulted in incomplete treatment because of development of a side effect. Cytologic examination of the pleural effusion, which was performed after the completion of PICT, gave a negative result in 16 of 20 patients examined. The median follow-up period was 16 months. Local relapse was recognized in only three cases who received incomplete treatment or in whom no temperature measurement was performed. The overall survival of the treated patients (n = 24) was significantly prolonged in comparison with a historical control group treated by surgery only (n = 17) or exploratory thoracotomy (n = 11). Of those 17 patients treated by surgery only, ten patients (59%) died of local relapse. These results suggest that this new treatment modality consisting of pulmonary resection and PICT is useful for the treatment of lung cancer patients with carcinomatous pleuritis, especially in the light of improved local control.

[Antigenicity of penicillin G in the guinea pig].

Antigenicity of penicillin G (PCG) was studied in guinea pigs. PCG 5 mg, 10 mg or 25 mg with Freund's complete adjuvant each on days 0, 7 and 21 was injected to a guinea pig: intramuscularly into both thighs and intracutaneously into four locations on the back. A remarkable antigenicity was induced in animals immunized with 25 mg although only low antigenicity in 5 mg and 10 mg. A maximum serum level of the antibody was observed about 2 weeks after last immunization and all of animals immunized with 25 mg died in active systemic anaphylaxis test. As mentioned above, it has been firstly demonstrated that a remarkable antigenicity of PCG can be produced by immunizing with a high dose of 25 mg in the guinea pig model in which PCG itself is used as immunogen.

Naloxone blocks the interferon-alpha induced changes in hypothalamic neuronal activity.

The effects of recombinant human interferon-alpha (IFN-alpha) and naloxone (NLX) were studied on the single neuron activities of the preoptic and anterior hypothalamus (PO/AH) and the ventromedial hypothalamus (VMH) in tissue slices. Perfusion of IFN-alpha in physiological doses (10-5000 units/ml) decreased and increased the firing rate of the majority of PO/AH and VMH neurons respectively. The IFN-alpha-induced changes in firing rate of PO/AH and VMH neurons were reversibly blocked or attenuated by simultaneous application of NLX. The results suggest that IFN-alpha may exert its action through opiate receptors in the hypothalamus.

Hypothalamic control of pituitary and adrenal hormones during hypothermia.

In order to investigate neuroendocrinological mechanisms of hypothermia, we determined the changes in plasma concentrations of corticosterone (CS), prolactin (PRL), and thyrotropin (TSH), and their correlations with alterations in hypothalamic dopamine (DA) and thyrotropin releasing hormone (TRH), in rats restrained and immersed in a water bath at various temperatures. A graded decrease of body temperature induced a progressive increase in the plasma level of CS, whereas that of PRL showed a drastic decrease. The plasma level of TSH also showed an increase during mild hypothermia (about 35 degrees C), but this increase was not evident during profound hypothermia (below 24 degrees C). The changes in these hormones were readily reversed by rewarming animals. Although DA content in the hypothalamus was not affected, its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), showed an increase following the decrease of body temperature. Pretreatment of the animals with sulpiride, a D2-antagonist, prevented the hypothermia-induced inhibition of PRL release. Hypothalamic TRH was significantly decreased during mild hypothermia, and it returned to control levels after rewarming. These results suggest that the decrease in plasma PRL induced by hypothermia may be associated with the activation of hypothalamic DA neurons, whereas the increase in plasma TSH during mild hypothermia seems to be caused by the increased release of TRH in the hypothalamus.

Morphine induced alterations of gamma-aminobutyric acid and taurine contents and L-glutamate decarboxylase activity in rat spinal cord and thalamus: possible correlates with analgesic action of morphine.

Acute administration of morphine induced significant increases of gamma-amino-butyric acid (GABA) content and L-glutamate decarboxylase (GAD) activity at the dorsal parts of the dorsal horn and surroundings of the central canal in the rat spinal cord, in which GABA inhibitory interneurons may play significant roles. In the thalamus, morphine also induced significant increases of GABA content and GAD activity in the vicinity of the ventrolateral part of the ventral nucleus (VM), entopeduncular nucleus (EP), nucleus reuniens thalami (RE), nucleus parafascicularis thalami (PF) and interpeduncular nucleus (IP), respectively. The most significant increase of GABA was observed in the VM and PF, which are known to receive neuronal inputs from secondary neurons involved in the perception of pain. In spite of well-known involvement of periaqueductal gray matter (PVG) in the occurrence of morphine analgesia, GABA content in this area did not change following acute administration of morphine. The above mentioned increases of GABA in the spinal cord and thalamus were antagonized by the pretreatment with levallorphan, a narcotic antagonist, and were not observed when an analgesic dose of sodium salicylate or pentazocine was administered. On the other hand, acute administration of morphine failed to alter the microdistribution of taurine (2-aminoethanesulfonic acid) in the rat spinal cord and thalamus, in which significant increases of GABA content were observed. Contrary to the results obtained in acutely morphine-treated rats, animals rendered dependent by the implantation of a morphine pellet showed significant increases of taurine content in the spinal cord, whereas no change in GABA contents was detected in both spinal cord and thalamus. The present results suggest that morphine analgesia may involve mechanisms intensifying the inputs of GABA inhibitory neurons at the levels of the spinal cord and thalamus, where the primary and secondary neurons involved in the perception of pain are terminated respectively. Possible involvement of alterations in spinal taurine contents in the occurrence of morphine dependence are also suggested.

Ethanol oxidation: effect on the redox state of brain in mouse.

Administration of a single large dose of ethanol to mice results in increases, for concentrations in the brain, of ratios of lactate to pyruvate, of aglycerophosphate to dihydroxyacetone phosphate, of malate to oxaloacetate, and of glutamate to the product of alpha-ketoglutarate and ammonium ion. These changes are noticed as early as 5 minutes after the single dose is given. Ethanol administration for 30 days also produces these changes in metabolite concentrations in the brain. However, in contrast to the single alcohol dose, long-term alcohol administration results in a marked decrease in the concentration of adenosine triphosphate in brain and increases in those of adenosine diphosphate and adenosine monophosphate. Pyrazole, an inhibitor of alcohol dehydrogenase, prevents the effects of ethanol on the concentration of brain metabolites. These results may provide new insight into the biochemical and pharmacological effects of alcohol on brain metabolism and the importance of alcohol dehydrogenase activity in the brain.