4ß-Hydroxywithanolide E isolated from Physalis pruinosa calyx decreases inflammatory responses by inhibiting the NF-κB signaling in diabetic mouse adipose tissue.

BACKGROUND: Chronic inflammation in adipose tissue together with obesity induces insulin resistance. Inhibitors of chronic inflammation in adipose tissue can be a potent candidate for the treatment of diabetes; however, only a few compounds have been discovered so far. The objective of this study was to find a novel inhibitor that can suppress the inflammatory response in adipose tissue and to elucidate the intracellular signaling mechanisms of the compound. METHODS: To find the active compounds, we established an assay system to evaluate the inhibition of induced MCP-1 production in adipocyte/macrophage coculture in a plant extract library. The active compound was isolated by performing high-performance liquid chromatography (HPLC) and was determined as 4ß-hydroxywithanolide E (4ßHWE) by nuclear magnetic resonance (NMR) and mass spectroscopy (MS) spectral analyses. The effect of 4ßHWE on inflammation in adipose tissue was assessed with adipocyte culture and db/db mice. RESULTS: During the screening process, Physalis pruinosa calyx extract was found to inhibit production of MCP-1 in coculture strongly. 4ßHWE belongs to the withanolide family of compounds, and it has the strongest MCP-1 production inhibitory effect and lowest toxicity than any other withanolides in coculture. Its anti-inflammatory effect was partially dependent on the attenuation of NF-κB signaling in adipocyte. Moreover, in vivo experiments showed that the oral administration of 4ßHWE to db/db mice resulted in the inhibition of macrophage invasion and cytokine expression in adipose tissue after 2 weeks of treatment; improved the plasma adiponectin, non-esterified fatty acids and MCP-1 concentrations; and increased glucose tolerance after 3 to 4 weeks of treatment. CONCLUSIONS: These results suggest that 4ßHWE has anti-inflammatory effect via inhibition of NF-κB activation in adipocyte. Moreover, the attenuation of inflammation in adipocyte has an effect on the inhibition of macrophage accumulation in obese adipose tissue. Consequently, 4ßHWE improves impaired glucose tolerance. Thus, 4ßHWE is a useful natural anti-inflammatory compound to attenuate progression of diabetes and obesity.

Pharmacologic profiling reveals lapatinib as a novel antiviral against SARS-CoV-2 in vitro.

The emergence of SARS-CoV-2 virus has resulted in a worldwide pandemic, but effective antiviral therapies are not widely available. To improve treatment options, we conducted a high-throughput screen to uncover compounds that block SARS-CoV-2 infection. A minimally pathogenic human betacoronavirus (OC43) was used to infect physiologically-relevant human pulmonary fibroblasts (MRC5) to facilitate rapid antiviral discovery in a preclinical model. Comprehensive profiling was conducted on more than 600 compounds, with each compound arrayed across 10 dose points. Our screening revealed several FDA-approved agents that can attenuate both OC43 and SARS-CoV-2 viral replication, including lapatinib, doramapimod, and 17-AAG. Importantly, lapatinib inhibited SARS-CoV-2 RNA replication by over 50,000-fold. Further, both lapatinib and doramapimod could be combined with remdesivir to improve antiviral activity in cells. These findings reveal novel therapeutic avenues that could limit SARS-CoV-2 infection.

A novel HLA-DRB4 allele, DRB4*01:08, identified by sequence-based typing.

New allele DRB4*01:08 showed one nucleotide difference with DRB4*01:01:01:01 at codon 87 (GAG>AAG).

Identification of bacterial pathogens in pediatric community-acquired lower respiratory tract infection using a simplified procedure of sputum sampling and examination: comparison between hospitalized children with and without underlying diseases.

The aim of this study is to confirm the usefulness of sputum sampling from the hypopharynx through the nose to identify causative bacteria of pediatric community-acquired lower respiratory tract infection (CA-LRTI) and compare its features between the patients with and without underlying diseases. A retrospective study was performed on 244 pediatric patients hospitalized for CA-LRTI of suspected bacterial etiology. Sputum sample was obtained from these patients by aspirating airway secretion through the nose or the tracheostomy orifice, or coughing up by themselves. Sputum samples were assessed as suitable in 119 (74.4%) of 160 patients with CA-LRTI of suspected pure bacterial etiology. Ninety-six (70.1%) of 137 samples suctioned from the hypopharynx through the nose were suitable for bacterial examination. Seventy-eight (73.6%) of 106 patients identified with causative bacteria had some underlying diseases, and the other 28 patients did not have any underlying diseases. Proportions and antibiotics susceptibility profiles of the identified causative bacteria were almost similar in the patients with and without underlying diseases. Sputum sampling from the hypopharynx through the nose might be significant in pediatric CA-LRTI of suspected bacterial etiology. The initial treatment for patients without underlying diseases would be applicable to those with underlying diseases in the CA-LRTI of children.

Characteristics of extracted ion beam from a cesium-free negative ion source using sheet plasma.

We report the characteristics of extracted beam current in the test plasma produced by direct current for sheet plasma upgrade producing negative ions by volume production without cesium (Cs) seeding. The negative hydrogen ion beam is extracted by a two-grid extraction system, which is located at the periphery of the sheet plasma. Experimental observations show that (i) negative hydrogen ions are successfully extracted from the sheet plasma by single/multi-aperture grids and (ii) the ratio of the extracted electron current IEG(e) and the hydrogen negative ion current IEG(H-), IEG(e)/Ic(H-), decreases from 8.0 to 2.0 with an increase in the height of the electron fence (HEF), which is a filter that prevents electron diffusion from the extraction region.

Spin fluctuations probed by NMR in paramagnetic spinel LiV(2)O(4): a self-consistent renormalization theory.

Low-frequency spin fluctuation dynamics in paramagnetic spinel LiV(2)O(4), a rare 3d-electron heavy-fermion system, is investigated. A parametrized self-consistent renormalization (SCR) theory of the dominant AFM spin fluctuations is developed and applied to describe temperature and pressure dependences of the low-T nuclear spin-lattice relaxation rate 1/T(1) in this material. The experimental data for 1/T(1) available down to ∼1 K are well reproduced by the SCR theory, showing the development of AFM spin fluctuations as the paramagnetic metal approaches a magnetic instability under the applied pressure. The low-T upturn of 1/T(1)T detected below 0.6 K under the highest applied pressure of 4.74 GPa is explained as the nuclear spin relaxation effect due to the spin freezing of magnetic defects unavoidably present in the measured sample of LiV(2)O(4).

Effect of Bacillus subtilis C-3102 on loose stools in healthy volunteers.

Ingestion of Bacillus subtilis C-3102 spores (C-3102) has relieved the symptoms of diarrhoea in piglets and changed the composition of gut microbiota in humans. Recently, it was suggested that the composition of the human gut microbiota affects stool consistency. In this study, a double-blind, randomised, placebo-controlled trial was conducted to assess the preventive effects of chronic diarrhoea in healthy volunteers with loose stools by ingestion of C-3102. The results showed that oral doses of C-3102 tablets significantly decreased the Bristol Stool Scale score and stool frequency, and also significantly improved abdominal sounds. With regard to gut microbiota, the relative abundance of Lachnospira, Actinomyces and SMB53 were significantly changed. This study shows that C-3102 could be effective for treating loose stools (Trial registration: UMIN000022583, http://tinyurl.com/ya4refqn ).

Isolation of transforming and early antigen-inducing Epstein-Barr virus from nasopharyngeal carcinoma hybrid cells (NPC-KT).

The biologic activities of Epstein-Barr (EB) virus from an epithelioid-nasopharyngeal carcinoma hybrid cell line (NPC-KT) and three subclones from the NPC-KT cells were examined. Much infectious virus was released by treatment with 5-iodo-2'-deoxyuridine. All virus preparations from NPC-KT cells and the subclones were found to possess both transforming and EB virus-induced early antigen (EA)-inducing activities. The lymphoblastoid cell lines, which were established by infection of human cord-blood lymphocytes with the virus, were diploid with normal karyotypes. The cell lines were positive for EB virus-associated nuclear antigen but negative for EA and EB viral capsid antigen.

Tumorigenicity of nasopharyngeal carcinoma hybrid cell line.

The spontaneous production of Epstein-Barr virus (EBV) and tumorigenicity in the BALB/c mutant nude mouse were studied with the use of the following 4 cell lines derived from the human nasopharynx: a) Ad-AH, an 8-azahypoxanthine-resistant epithelioid cell line; b) A2L, an EBV-carrying lymphoblastoid cell line; c) A2L/AH, an EBV-carrying epithelioid hybrid cell line established by fusion of Ad-AH cells with A2L cells; and d) NPC-KT, an EBV-carrying epithelioid hybrid cell line established by fusion of Ad-AH cells with nasopharyngeal carcinoma (NPC) primary culture cells. The NPC-KT hybrid cell line was the only cell line to produce EBV antigens of the lytic cycle and virus particles. Cloning efficiency in agarose was clearly related to tumorigenicity in nude mice. Especially high, frequent tumor formation was observed in the heterotransplantation of NPC-KT hybrid cells that presented poorly differentiated carcinoma, and the tumor cells were positive for EBV-associated nuclear antigen. These NPC-KT hybrid cells maintained the characteristics of a histologic type of NPC tumor cells. Thus experimental systems of NPC were established in vitro and in vivo by the application of NPC-KT hybrid cells to NPC model cells.

Heterogeneity of Epstein-Barr virus derived from a nasopharyngeal carcinoma that has transforming and lytic properties.

The biological activities of Epstein-Barr virus (EBV) from a human epithelial hybrid cell line derived from a nasopharyngeal carcinoma (NPC-KT) were studied. Low concentrations of virus from the NPC-KT cell line stimulated DNA synthesis of human cord blood lymphocytes (CBL) and induced CBL to form EBV nuclear antigen-positive continuous cell lines. The CBL exposed to higher doses of virus showed stimulated DNA synthesis 2 days after infection, followed by cell lysis. Virus from the NPC-KT cell line induced EBV-specific antigens in nonproducer Raji cells and reduced the colony-forming ability of the super-infected cells. The ratio of transforming activity to early antigen-inducing ability was not constant during cell passage. The data obtained suggest that NPC-KT clone 1 cells are producing virus with cytotoxic and transforming properties.

Rescue of a biologically active Epstein-Barr virus from nonproducer cells.

We recently established an epithelial/hybrid cell line, A2L/AH, by fusion of 8-azaguanine-resistant epithelial cells, Ad-AH, with a nonproducer lymphoblastoid cell line, A2L, using lymphocytes derived from the human nasopharynx transformed with the B95-8 strain of Epstein-Barr virus. The treatment of the parental A2L lymphoid cells with iododeoxyuridine led to the expression of Epstein-Barr virus early antigen, but neither virus capsid antigen or induction of Epstein-Barr virus DNA synthesis was observed. However, the treatment of the A2L/AH hybrid cells with iododeoxyuridine led to early antigen, virus capsid antigen and virus DNA synthesis, and the formation of virus particles. The virus rescued from the A2L/AH hybrid cells transforms human cord blood lymphocytes but is not able to induce early antigen in superinfected Raji cells.

Superinfection of epithelial hybrid cells (D98/HR-1, NPC-KT, and A2L/AH) with Epstein-Barr virus and the relationship to the C3d receptor.

Three Epstein-Barr virus (EBV) genome-positive epithelial/hybrid cell lines (D98/HR-1, NPC-KT, and A2L/AH) were superinfected with EBV derived from P3HR-1 (HR-1), NPC-KT, and B95-8 cells. The HR-1 virus is lytic and induces early antigen in superinfected Raji cells; the virus is not capable of transforming B-lymphocytes. Virus preparations from NPC-KT cells have both transforming and early antigen-inducing properties, while B95-8 virus can only transform B-lymphocytes. It was possible to demonstrate EBV antigens after superinfection of D98/HR-1 cells with both HR-1 virus and NPC-EBV. The NPC-KT hybrid cells, which were originally prepared by fusing human adenoid epithelial cells (Ad-AH) and EBV genome-positive NPC explanted epithelial cells, were susceptible to superinfection with HR-1 virus but not to NPC-EBV. The A2L/AH hybrid cells, which were prepared by fusion between Ad-AH cells and lymphocytes transformed by B95-8 virus, could not be superinfected with any of the virus preparations. In order to further investigate the nature of the EBV receptor as it relates to other cell membrane components, we examined cell surface markers on Ad-AH, NPC-KT, A2L/AH, and D98/HR-1 cells using monoclonal antibodies and by rosette formation. We found that the NPC-KT, A2L/AH, and Ad-AH cell lines express the OKB2 antigen and that the common acute lymphoblastic leukemia antigen is expressed on the A2L/AH cells. We also found that NPC-KT parental cells and a clone of NPC-KT cells express erythrocyte antibody complement b and erythrocyte antibody complement d, as determined by rosette formation, but were negative for C3b and C3d when monoclonal antibodies against these two markers were used. The D98/HR-1 cells were also confirmed to be negative for C3b and C3d. The data suggest that the C3d receptor may be part of the EBV receptor but that the C3d receptor, by itself, is not the only receptor to which EBV can bind.

Biochemical characterization of U937 cells resistant to L-asparaginase: the role of asparagine synthetase.

A human histiocytic lymphoma cell line, U937, is highly sensitive to L-asparaginase with an ID50 of about 0.0001 U/ml after 72 hr of culture. When U937 cells were made resistant to either L-asparaginase (1 U/ml) or asparagine deprivation, the activity of asparagine synthetase increased to 80- or 7-fold of the wild type, respectively. The phenotype of the resistance to L-asparaginase turned out to be stable under nonselective conditions for over several months. The hybrids between L-asparaginase sensitive (Molt4) and resistant (HL-60) cell lines revealed the latter phenotype in terms of L-asparaginase sensitivity and the activity of asparagine synthetase. Furthermore, U937 cells resistant to L-asparaginase could survive in glutamine-free media with 1.5-fold elevation of glutamine synthetase activity. These results altogether clarify the role of asparagine synthetase in L-asparaginase toxicity and have a good implication for the clinical use of L-asparaginase.

Changes in sensitivity to anticancer drugs during TPA-induced cellular differentiation in a human T-lymphoblastoid cell line (MOLT-4).

After four days of treatment with 10(-8) M TPA, differentiation of the human T-lymphoblastoid cell line MOLT-4 was induced along the T cell lineage, confirmed by a fall in adenosine deaminase and 5'-ectonucleotidase and a rise in purine nucleoside phosphorylase activity. TPA-treated cells became resistant to the cytotoxic effects of 1-beta-D-arabinofuranosylcytosine (Ara-C), 9-beta-D-arabinofuranosyladenine (Ara-A), and 2-chlorodeoxyadenosine. This was, in part, due to the altered cell cycle distribution (accumulation of cells in the G1 phase), since the toxicity of Ara-C and Ara-A is S phase specific. The diminished rate of Ara-C transport concomitant with Ara-CTP formation after TPA treatment is considered to be the biochemical basis for this acquired resistance.

Cell cycle related change of Ara-C transport in HL-60 cells after differentiation induction.

Using a promyelocytic leukemia cell line, HL-60, we studied the membrane transport of Ara-C before and after differentiation induced by retinoic acid (RA). In RA-treated cells, Ara-C transport was reduced and there was a concomitant increase of the ID50 values of Ara-C in comparison with the controls. By three different procedures to synchronize untreated cells, i.e. density arrest G1 phase enrichment, aphidicolin-induced S phase accumulation and the double isoleucine block method, we found that Ara-C transport was 30-50% higher in the S phase than in the G1 phase. Therefore, the observed decrease in Ara-C transport is, in part, due to the retarded growth accompanied by an accumulation of cells in the G1 phase after differentiation induction.

Distribution of cerebellothalamic neurons projecting to the ventral nuclei of the thalamus: an HRP study in the cat.

Distribution of cerebellothalamic neurons projecting to the ventral nuclei of the thalamus was examined in the cat, using the horseradish peroxidase (HRP) method. After injections of HRP within the lateral or ventrolateral portions of the ventro-anterior and ventrolateral nuclear complex of the thalamus (VA-VL), neurons labeled retrogradely with HRP were seen contralaterally in the cerebellar nuclei; many of them were situated in the nucleus interpositus anterior and nucleus interpositus posterior, and a moderate number of them were located in the nucleus lateralis. Labeled neurons in the nucleus interpositus posterior were observed mainly in the medial and ventral portions of the nucleus. On the side ipsilateral to the injections, a few labeled neurons were seen in the nucleus interpositus anterior, nucleus interpositus posterior, and nucleus lateralis. Virtually no labeled neurons were found in the nucleus medialis of the cerebellum. After HRP injections into the medial or dorsomedial portions of the VA-VL, many labeled neurons were found contralaterally in the ventral and ventrolateral portions of the nucleus interpositus posterior, as well as in the nucleus lateralis, especially in its ventral and lateral portions. On the side ipsilateral to the injections, labeled neurons in the nucleus lateralis and nucleus interpositus posterior were small in number. In the nucleus medialis only a few labeled neurons were found bilaterally in the caudal levels of the nucleus. After HRP injections centered on the ventromedial nucleus of the thalamus, many labeled neurons occurred bilaterally in the caudal portions of the nucleus madialis, with a slight contralateral preponderance, and contralaterally in the lateral and ventral portions of the nucleus lateralis. A few labeled neurons were also seen contralaterally in the ventrolateral and lateral portions of the nucleus interpositus posterior, and ipsilaterally in the nucleus lateralis.

Pathology review for paediatric non-Hodgkin's lymphoma patients in Japan; a report from the Japan association of childhood leukaemia study (JACLS).

A central pathology review system with an immunophenotyping laboratory was established in Japan to support the clinical trial, the Japan Association of Childhood Leukaemia Study (JACLS) NHL-98, for patients with paediatric non-Hodgkin's lymphoma (NHL). Pathology samples from 155 clinically-suspected NHL cases were evaluated centrally initially using the Revised European-American Lymphoma (REAL) classification in a rapid review (within 2 weeks after surgery/biopsy) and then later at the consensus review (once a year). The samples were subsequently re-classified according to the new World Health Organisation (WHO) classification. After the pathology review, 96 (62%) patients were eligible for the study, and 58 of them (60%) had extra-nodal primaries. These NHL cases included B-cell lymphomas (precursor B-cell, 11; Burkitt, 18; diffuse large B-cell, 18; not otherwise specified, 3) and T/Natural Killer (NK)-cell lymphomas (precursor T-cell, 23; anaplastic large cell, 20; others, 3). There was excellent concordance in making the diagnoses (95/96, 99%) and typing (93/96, 97%) of NHL between the rapid and consensus reviews. Five cases, initially diagnosed as diffuse large B-cell lymphoma by the review, were re-classified as Burkitt lymphoma according to the immunocytochemical criteria by the WHO classification. A total of 59 (38%) cases were excluded from the study: they were Hodgkin lymphoma (7), leukaemias (11), reactive lymphoid hyperplasia (20), necrotizing lymphadenitis (7), no consensus diagnosis (1), insufficient materials (2), and others (11). This is the first report of the central pathology review from the paediatric NHL group study in Japan. Because various diseases, either neoplastic or reactive, mimicked NHL, clinically and histopathologically, the central pathology review system was critical and essential for patient enrollment and protocol assignment in our clinical trial. Through the two-step review system, highly reliable data were generated to support this study.

The human parainfluenza virus type-1 prototypic strain contains a heat-labile hemagglutinin-neuraminidase protein.

The virus yield of human parainfluenza virus type-1 (hPIV-1) in cultured cells at 38 degrees C is reduced more than 100-fold compared to 34 degrees C, while the virus yield of Sendai virus (SV, Enders strain), a murine parainfluenza virus type-1 with high homology to hPIV-1 was almost equal at both temperatures. To understand the basis for the differences in the temperature growth characteristics of the two viruses, we examined the heat-stability of hPIV-1 and SV glycoproteins expressed from cDNAs by pulse-chase experiments. The hemagglutinin-neuraminidase (HN) protein of hPIV-1 was stable after a 6-h chase at 34 degrees C, while at 38 degrees C prominent protein degradation was observed starting at 3 h chase and by 6 h HN was reduced by 65%. In contrast, SV HN protein was stable at both 34 and 38 degrees C. The other hPIV-1 glycoprotein, the fusion (F) protein was stable at both temperatures. To identify the amino acids which are responsible for the heat-lability of hPIV-1 HN, mutant HN proteins were constructed by site-directed mutagenesis. Mutant hPIV-1 HN which had substitutions at positions 461 and 462 became heat-stable at 38 degrees C. These data indicate amino acids around 461 are responsible for the heat-lability of the wild type hPIV-1 HN protein and the reduced yield of the virus at 38 degrees C.