Short-term inhalation of sargramostim with concomitant high-dose steroids does not hasten recovery in moderate COVID-19 pneumonia: a double-blind, randomised, placebo-controlled trial.

BACKGROUND: Granulocyte-macrophage colony stimulating factor (GM-CSF) inhalation may alleviate pulmonary inflammation caused by viral pneumonia. To investigate this, we evaluated its efficacy on COVID-19 pneumonia. METHODS: This double-blind, randomised, placebo-controlled study (ClinicalTrials.gov: NCT04642950) evaluated patients in the first half of 2021 at seven Japanese hospitals. Hospitalised patients with COVID-19 pneumonia with moderate hypoxaemia inhaled sargramostim or placebo for 5 days. The primary endpoint was days to achieve a ≥ 2-category improvement from baseline on a modified 7-category ordinal scale. Secondary endpoints included degree of oxygenation, defined by amount of oxygen supply, and serum CCL17 level. RESULTS: Seventy-five patients were randomly assigned in a 2:1 ratio to receive sargramostim or placebo, of which 47 and 23 were analysed, respectively. No difference was observed between groups regarding the primary endpoint (8.0 and 7.0 days for sargramostim and placebo, respectively) or in the secondary endpoints, except for CCL17. A post hoc sub-analysis indicated that endpoint assessments were influenced by concomitant corticosteroid therapy. When the cumulative corticosteroid dose was ≤500 mg during Days 1-5, recovery and oxygenation were faster in the sargramostim group than for placebo. Bolus dose corticosteroids were associated with temporarily impaired oxygenation and delayed clinical recovery. The increase in serum CCL17, a candidate prognostic factor, reflected improvement with sargramostim inhalation. The number of adverse events was similar between groups. Two serious adverse events were observed in the sargramostim group without causal relation. CONCLUSIONS: Inhaled sargramostim was likely to be effective for COVID-19 pneumonia unless the concomitant corticosteroid dose was high.

Chromosome-specific segmentation revealed by structural analysis of individually isolated chromosomes.

Analysis of structural rearrangements at the individual chromosomal level is still technologically challenging. Here we optimized a chromosome isolation method using fluorescent marker-assisted laser-capture and laser-beam microdissection and applied it to structural analysis of two aberrant chromosomes found in a lung cancer cell line. A high-density array-comparative genomic hybridization (array-CGH) analysis of DNA samples prepared from each of the chromosomes revealed that these two chromosomes contained 296 and 263 segments, respectively, ranging from 1.5 kb to 784.3 kb in size, derived from different portions of chromosome 8. Among these segments, 242 were common in both aberrant chromosomes, but 75 were found to be chromosome-specific. Sequences of 263 junction sites connecting the ends of segments were determined using a PCR/Sanger-sequencing procedure. Overlapping microhomologies were found at 169 junction sites. Junction partners came from various portions of chromosome 8 and no biased pattern in the positional distribution of junction partners was detected. These structural characteristics suggested the occurrence of random fragmentation of the entire chromosome 8 followed by random rejoining of these fragments. Based on that, we proposed a model to explain how these aberrant chromosomes are formed. Through these structural analyses, it was demonstrated that the optimized chromosome isolation method described here can provide high-quality chromosomal DNA for high resolution array-CGH analysis and probably for massively parallel sequencing analysis.

Establishment of an in vitro cell line experimental system for the study of inhalational anesthetic mechanisms.

General anesthesia affects the expression of clock genes in various organs. Expression of Per2, a core component of the circadian clock, is markedly and reversibly suppressed by sevoflurane in the suprachiasmatic nucleus (SCN), and is considered to be a biochemical marker of anesthetic effect in the brain. The SCN contains various types of neurons, and this complexity makes it difficult to investigate the molecular mechanisms of anesthesia. Here, we established an in vitro experimental system using a cell line to investigate the mechanisms underlying anesthetic action. Development of the system comprised two steps: first, we developed a system for application of inhalational anesthetics and incubation; next, we established cultures of anesthetic-responsive cells expressing mPer2 promoter-dLuc. GT1-7 cells, derived from the mouse hypothalamus, responded to sevoflurane by reversibly decreasing mPer2-promoter-driven bioluminescence. Interestingly, the suppression of bioluminescence was found only in the serum-starved GT1-7 cells, which showed neuron-like morphology, but not in growing cells, suggesting that neuron-like characteristics are required for anesthetic effects in GT1-7 cells.

Characterization of sevoflurane effects on Per2 expression using ex vivo bioluminescence imaging of the suprachiasmatic nucleus in transgenic rats.

The inhalation anesthetic sevoflurane suppresses Per2 expression in the suprachiasmatic nucleus (SCN) in rodents. Here, we investigated the intra-SCN regional specificity, time-dependency, and pharmacological basis of sevoflurane-effects. Bioluminescence image was taken from the SCN explants of mPer2 promoter-destabilized luciferase transgenic rats, and each small regions of interest (ROI) of the image was analyzed. Sevoflurane suppressed bioluminescence in all ROIs, suggesting that all regions in the SCN are sensitive to sevoflurane. Clear time-dependency in sevoflurane effects were also observed; application during the trough phase of the bioluminescence cycle suppressed the subsequent increase in bioluminescence and resulted in a phase delay of the cycle; sevoflurane applied during the middle of the ascending phase induced a phase advance; sevoflurane on the descending phase showed no effect. These results indicate that the sevoflurane effect may depend on the intrinsic state of circadian machinery. Finally, we examined the involvement of GABAergic signal transduction in the sevoflurane effect. Co-application of both GABAA and GABAB receptor antagonists completely blocked the effect of sevoflurane on the bioluminescence rhythm, suggesting that sevoflurane inhibits Per2 expression via GABAergic signal transduction. Current study elucidated the anesthetic effects on the molecular mechanisms of circadian rhythm.

Rapid modulation of hypothalamic Kiss1 levels by the suckling stimulus in the lactating rat.

In mammals, lactation suppresses GnRH/LH secretion resulting in transient infertility. In rats, GnRH/LH secretion is rescued within 18-48 h after pup separation (PS) and rapidly re-suppressed by subsequent re-exposure of pups. To elucidate the mechanisms underlying these rapid modulations, changes in the expression of kisspeptin, a stimulator of GnRH secretion, in several lactating conditions (normal-lactating; 4-h PS; 18-h PS; 4-h PS +1-h re-exposure of pups; non-lactating) were examined using in situ hybridization. PS for 4 h or 18 h increased Kiss1 expressing neurons in both the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC), and subsequent exposure of pups re-suppressed Kiss1 in the AVPV. A change in Kiss1 expression was observed prior to the reported time of the change in GnRH/LH, indicating that the change in GnRH/LH results from changes in kisspeptin. We further examined the mechanisms underlying the rapid modulation of Kiss1. We first investigated the possible involvement of ascending sensory input during the suckling stimulus. Injection of the anterograde tracer to the subparafascicular parvocellular nucleus (SPFpc) in the midbrain, which relays the suckling stimulus, revealed direct neuronal connections between the SPFpc and kisspeptin neurons in both the AVPV and ARC. We also examined the possible involvement of prolactin (PRL). Administration of PRL for 1 h suppressed Kiss1 expression in the AVPV but not in the ARC. These results indicate that suckling stimulus rapidly modulates Kiss1 expression directly via neuronal connections and indirectly through serum PRL, resulting in modulation in GnRH/LH secretion.

Epigenetic suppression of mouse Per2 expression in the suprachiasmatic nucleus by the inhalational anesthetic, sevoflurane.

BACKGROUND: We previously reported that sevoflurane anesthesia reversibly suppresses the expression of the clock gene, Period2 (Per2), in the mouse suprachiasmatic nucleus (SCN). However, the molecular mechanisms underlying this suppression remain unclear. In this study, we examined the possibility that sevoflurane suppresses Per2 expression via epigenetic modification of the Per2 promoter. METHODS: Mice were anesthetized with a gas mixture of 2.5% sevoflurane/40% oxygen at a 6 L/min flow for 1 or 4 h. After termination, brains were removed and samples of SCN tissue were derived from frozen brain sections. Chromatin immunoprecipitation (ChIP) assays using anti-acetylated-histone antibodies were performed to investigate the effects of sevoflurane on histone acetylation of the Per2 promoter. Interaction between the E'-box (a cis-element in the Per2 promoter) and CLOCK (the Clock gene product) was also assessed by a ChIP assay using an anti-CLOCK antibody. The SCN concentration of nicotinamide adenine dinucleotide (NAD(+)), a CLOCK regulator, was assessed by liquid chromatography-mass spectrometry. RESULTS: Acetylation of histone H4 in the proximal region of the Per2 promoter was significantly reduced by sevoflurane. This change in the epigenetic profile of the Per2 gene was observed prior to suppression of Per2 expression. Simultaneously, a reduction in the CLOCK-E'-box interaction in the Per2 promoter was observed. Sevoflurane treatment did not affect the concentration of NAD(+) in the SCN. CONCLUSIONS: Independent of NAD(+) concentration in the SCN, sevoflurane decreases CLOCK binding to the Per2 promoter E'-box motif, reducing histone acetylation and leading to suppression of Per2 expression.

Amplification of the ABCB1 region accompanied by a short sequence of 200bp from chromosome 2 in lung cancer cells.

Lung cancer sublines No15-80-1 and No15-80-6 were selected by treatment of cell line NCI-H460 with paclitaxel at stepwise increasing concentrations from 50 nmol/L to 800 nmol/L. The two sublines exhibited amplifications of the ABCB1 region (previously MDR1) with different copy number profiles, but shared a common amplification pattern, which has been observed in amplification mediated by the breakage-fusion-bridge (BFB) cycle. Sequence analysis of the distal ends of the amplified regions, which were probably generated in a break-and-fusion of the initial round of the BFB cycle, revealed a head-to-head fused sequence of chromosome 7. The sequence was identical in the two sublines. A short sequence of 200bp derived from chromosome 2 was incorporated, suggesting translocation between chromosomes 2 and 7. The copy number of the short sequence was comparable to that of the neighboring sequence, suggesting coamplification. The timing of the occurrence of the putative translocation and the initiation of BFB-cycle-driven amplification during the stepwise selection were determined by using the unique junction sequences specific to these events as indicators. The results demonstrated that the translocation occurred at the step of 100 nmol/L treatment and the BFB cycle initiated in the step of 400 nmol/L-treatment. It is likely that the translocation, preceding amplification by several selection steps, activated ABCB1 gene expression. The diversity in amplification profiles between the two sublines was generated by the separately operating BFB cycles, after an initial break-and-fusion that probably occurred in a single cell.

Expression of porcine FSHbeta subunit promoter-driven herpes simplex virus thymidine kinase gene in transgenic rats.

A transgenic rat was established using the construct of porcine FSHbeta subunit promoter, the -852/+10 bp region, fused to a Herpes simplex virus thymidine kinase (HSV-TK) gene. Integration of the transgene was confirmed by PCR of tail DNA. RT-PCR of total RNAs of the pituitary, gonad, cerebellum, liver, kidney, adrenal gland, prostate, and uterus revealed that FSHbeta was only expressed in the pituitary. Analysis of the expression of reporter gene, HSV-TK, using two specific primer sets revealed that different transcripts were present in the pituitary and testis. The transcript initiated at the transcription initiation site of the porcine FSHbeta gene was detected in the pituitary, and another within the TK gene was found in the testis, indicating ectopic testis-specific expression. Immunohistochemistry of the pituitary glands of the transgenic rats for FSH and HSV-TK demonstrated that the FSH-producing cells also produced HSV-TK. The results indicated that the -852/+10 bp region of the FSHbeta promoter contains an element(s) that determines the tissue-specific expression. We succeeded in producing FSHbeta promoter-driven HSV-TK transgenic rats and were the first time to do so using an animal other than the mouse. The transgenic rats show male infertility that involves abnormal spermatogenesis. We also observed a decrease in the weight of the testis and epididymis, and both motile and living spermatozoa were absent in the epididymis. Consequently, the FSHbeta-HSV-TK transgenic rat will provide a useful model for studies on FSH function and male infertility.

High level expression of Prop-1 gene in gonadotropic cell lines.

Prop-1 acts as an upstream regulator for the Pit-1 gene to induce development of Pit-1 lineage pituitary cell lines, GH-, PRL-, and TSH-producing cells, in the early stage of pituitary organogenesis. Furthermore, Prop-1 is presumed to be involved in the function of FSH/LH-producing cells, gonadotropes, since the defective Prop-1 gene shows hypogonadism. Recently, we reported evidence that Prop-1 directly regulates expression of the porcine FSHbeta gene, thus providing a novel advance in understanding the function of Prop-1 in FSH/LH production and hypogonadism. This study was intended to demonstrate the expressions of Prop-1 gene in pituitary tumor-derived cell lines. RT-PCR analyses were conducted of Pit-1, glycoprotein alpha subunit (alphaGSU), GnRH receptor, and cyclophilin A (a ubiquitously expressing gene). We observed expression of the Pit-1 gene in alphaT1-1, TalphaT1, MtT/S, GH3, and TtT/GF cells, expression of the alphaGSU gene in alphaT1-1, alphaT3-1, LbetaT2, LbetaT4, TalphaT1, and GH3 cells, and expression of GnRH receptor gene in alphaT3-1, LbetaT2, LbetaT4, and GH3 cells, respectively. These expression profiles were in accord with their cell lineages, with only a few exceptions. To accurately measure the expression level of the Prop-1 gene, a quantitative analysis was performed using the real-time PCR method. This analysis demonstrated that the LbetaT2 and LbetaT4 gonadotrope cell lines, which express the FSHbeta gene, express the Prop-1 gene. Taken together with our previous observation that Prop-1 is present in the adult porcine pituitary gonadotropes, Prop-1 might also be involved in development of gonadotropes and hormone production.

Transcriptional activity of the 5' upstream region of the porcine glycoprotein hormone alpha subunit gene.

Gene expression of the porcine glycoprotein hormone alpha subunit (p-alphaGSU) was examined in LbetaT2 cells, which were established from the anterior pituitary lobe of the immortalized transgenic mouse and produce alphaGSU, and in CHO cells cloned from Chinese hamster ovaries. Expression of the reporter gene fused with p-alphaGSU gene upstream in LbetaT2 cells showed that the distal regions -540/-240 and -798/-541 are important for the activation of gene expression. In contrast, the transcriptional activity of the distal region of p-alphaGSU gene was repressed in CHO cells. The region -540/-240 contains an adequate enhancer, called pituitary glycoprotein hormone basal element, whereas the region -798/-541 has no distinguished element. Transfection of the expression vector containing cDNA of a pan-pituitary activator, Ptx1, whose putative binding sites are present scatted in the distal region of the p-alphaGSU gene, revealed unexpectedly that this factor significantly suppressed the expression of p-alphaGSU gene in LbetaT2 cells, indicating that Ptx1 is unrelated to the upregulation in the region -798/-541. Thus, this study demonstrated for the first time that the distal region -798/-541of the p-alphaGSU gene is indispensable for prominent expression of this gene in which an as yet unidentified factor may participate.

Pituitary transcription factor Prop-1 stimulates porcine follicle-stimulating hormone beta subunit gene expression.

Molecular cloning of the transcription factor that modulates the expression of porcine follicle-stimulating hormone beta subunit (FSHbeta) gene was performed by the yeast one-hybrid cloning system using the -852/-746 upstream region (Fd2) as a bait sequence. We eventually cloned a pituitary transcription factor, Prop-1, which has been identified as an upstream transcription factor of Pit-1 gene. Binding ability of Prop-1 to the bait sequence was confirmed using recombinant Prop-1, and the binding property was investigated by DNase I footprinting, revealing that Prop-1 certainly bound to the large AT-rich region throughout the Fd2. Co-transfection of Prop-1 expression vector together with a reporter gene fused with Fd2 in CHO cells demonstrated an attractive stimulation of reporter gene expression. Immunohistochemistry of adult porcine pituitary confirmed the colocalization of the Prop-1 and FSHbeta subunit. This study is the first to report that Prop-1 participates in the regulation of FSHbeta gene. The present finding will provide new insights into the development of pituitary cell lineage and combined pituitary hormone deficiency (CPHD), since why the defect of Prop-1 causes CPHD including gonadotropins (FSH and LH) has yet to be clarified.

Prominent expression of spinocerebellar ataxia type-1 (SCA1) gene encoding ataxin-1 in LH-producing cells, LbetaT2.

The LH-producing cell line, LbetaT2, and non LH-producing cell line, alphaT3-1 cells, established from a pituitary tumor, were employed for cDNA subtraction cloning to identify genes with expression unique to LH producing cells. Several cDNAs that code for known as well as for many unidentified clones were discovered. Most clones were the spinocerebellar ataxia type-1 (SCA1) gene encoding ataxin-1, the abnormality of which causes neurodegeneration and loss of cerebellar Purkinje cells. We examined whether the expression of SCA1 gene in LbetaT2 cells is related to hormone production. We also compared the expression of SCA1 with that in various other pituitary tumor derived cell lines, and confirmed the prominent expression of SCA1 in LbetaT2 cells. The effect of gonadal factor(s) for SCA1 gene expression was examined. The expression level in female rats was low and did not change during the estrus cycle, but increased significantly after ovariectomy and did not return to the normal level under low and high doses of estrogen. In the male pituitary SCA1 gene expression increased markedly after castration and was not decreased by estrogen or testosterone. The Ontogeny of SCA1 gene expression was investigated in porcine fetal and postnatal pituitaries and revealed biphasic and sexually dimorphic expression. Transient expression of SCA1 gene was observed at fetal day 50 and 65 in males and day 40 in females, followed by a decline and increased expression before birth in both genders. Thus the expression of SCA1 gene is prominent in LH-producing cells and is not under direct control of gonadal factor(s) in both genders. In addition to the variable expression of SCA1 gene during the fetus period, the present results provide a novel aspect to the understanding of Boucher-Neuhauser syndrome (Ataxia Hypogonadism Choroidal Dystrophy).