Changes of host cell infiltration into Meth A fibrosarcoma tumor during the course of regression induced by injections of a BCG nucleic acid fraction.

MY-1, which consists of DNA and RNA extracted and purified from Mycobacterium bovis strain BCG, causes the regression of various experimental syngeneic tumors when injected intratumorally. In order to identify the host cells involved in the antitumor mechanism(s) of MY-1, we examined Meth A tumors inoculated intradermally to BALB/c mice, which were given multiple injections of MY-1 following tumor inoculation. Histological and immunohistochemical examinations were performed at several time points. On day 4 after inoculation, the MY-1-treated tumors were heavily infiltrated with a heterogeneous population of mononuclear cells with low density nuclei. The MY-1-injected tumors contained asialo-GM1-positive cells and Mac-1-positive cells, which indicated that the infiltrating mononuclear cells were natural killer cells and macrophages. On day 14 after inoculation, the tumors were infiltrated with a large number of L3T4-positive cells and fewer Lyt-2-positive cells, both of which were more abundant in the MY-1-treated tumors than in the control tumors. The observed sequence of host cell infiltration corresponded well with our previous studies which have indicated that the antitumor mechanism of MY-1 is divided into two phases, i.e. the early phase when natural killer cells and macrophages inhibit tumor growth, and the late phase when L3T4-positive cells act to induce tumor regression via a delayed-type hypersensitivity against tumor cells.     

Structure-activity relationship of mycoloyl glycolipids derived from Rhodococcus sp. 4306

Novel mycoloyl glycolipids with short carbon chains were isolated and purified from Rhodococcus sp. 4306, a soil origin of Actinomycetales. Their chemical structures were identified as trehalose 6,6'-dimycolate (TDM), trehalose 6-monomycolate, glucose 6-monomycolate, mannose 6-monomycolate and fructose 6-monomycolate. The length of carbon chains and number of double bonds of mycolic acids were C(34), C(36)and C(38)saturated, monoenoic and dienoic molecular species, which were much shorter than those of Mycobacterium tuberculosis (C(78-88)monoenoic and dienoic). Among them, only TDM could induce prominent granulomatous inflammation of the lung and spleen in mice. By contrast, other mycoloyl glycolipids induced mild lesions. The small-sized TDM of Rhodococcus possessed granulomatogenic activity, however, the toxicity was much lower than that of M. tuberculosis. Rhodococcal TDM was composed of mycolic acid with the shortest carbon chains, when compared to granulomatogenic TDM of Mycobacterium, Nocardia and Rhodococcus reported previously. Our results imply that rhodococcal TDM is a pathogenetic factor similar to that of M. tuberculosis, although rhodococcal TDM exhibits low toxicity.     

Determination of molecular species composition of C80 or longer-chain alpha-mycolic acids in Mycobacterium spp. by gas chromatography-mass spectrometry and mass chromatography

The molecular species composition of alpha-mycolic acids ranging from C68 to C86 in 13 rapidly growing and 12 slowly growing mycobacterial species was determined by gas chromatography, gas chromatography-mass spectrometry, and mass chromatography. In gas chromatographic analysis, the molecular species of alpha-mycolic acids were well separated as trimethylsilyl ether derivatives of the methyl esters, according to their total carbon numbers. The total carbon and double-bond numbers of mycolic acids at each peak on gas chromatograms were determined from the [M]+, [M - 15]+, and [M - 90]+ ions on the mass spectrum, and straight and branched chain structures were identified by the mass fragment ions [A]+, due to C2--C3 cleavage [R-CH-O-Si(CH3)3]+, and [B]+, due to C3--C4 cleavage [(CH3)3-Si-O-CH-CH(R')-COOCH3]+. The concentration of odd- and even-carbon-numbered mycolic acids, which often overlap each other on gas chromatograms, and the composition of three homologous mycolic acids with different alpha units (C22:0, C24:0, and C26:0) were clearly determined by mass chromatography monitoring [M - 15]+ ions and [B - 29]+ ions, respectively. The molecular species composition of alpha-mycolic acids and their average carbon numbers (av. cn.) as a simple expression of the composition were calculated from the mass chromatograms. Each mycobacterial species examined was demonstrated to possess a characteristic profile of alpha-mycolic acid composition, and based on this the species were classified approximately into eight groups: C68 to C76 (av. cn. 72), dienoic, possessing a C20 alkyl branch at the 2 position (C22 alpha-unit) for Mycobacterium diernhoferi and Mycobacterium sp. strain 3707, a chromogenic rapid grower; C72 to C78 (av. cn. 75), dienoic with both C22 and C24 alpha units, containing a small or a large amount of odd-carbon-numbered molecules, for M. vaccae, M. rhodesiae, and M. phlei (chromogenic rapid growers); C72 to C80 (av. cn. 75 to 77), dienoic with C24 alpha-unit, containing a moderate or a large amount of odd-carbon-numbered molecules, for M. smegmatis, M. chitae, M. chelonae (M. chelonei), and M. fortuitum (nonchromogenic rapid growers); C78 to C82 (av. cn. 80), even-carbon-numbered dienoic with C24 alpha unit for M. agri and M. thermoresistible (rapid growers); C75 to C81 (av. cn. 77 to 79), odd-carbon-numbered dienoic with C24 alpha unit for M. nonchromogenicum complex (M. nonchromogenicum, M. terrae, and "M. novum") (slow growers); (vi) C76 to C84 (av. cn. 79 to 81), even-carbon-numbered dienoic with C24 alpha unit for MAIS complex including M. scrofulaceum, M. avium, and M. intracellulare (slow growers); (vii) C72 to C80 (av. cn. 77 to 79), even-carbon-numbered dienoic with C24 alpha unit for M. szulgai, M. gordonae, and M. kansasii (chromogenic slow growers); and (viii) C76 to C86 (av. cn. 79 to 81), even-carbon-numbered dienoic with C26 alpha unit M. bovis Ravenol and BCG and M. tuberculosis H37Rv. This study demonstrated that gas chromatography-mass spectrometric analysis of the molecular species composition of alpha-mycolic acid can give rapid, important, and very precise information for the identification of pathogenic and nonpathogenic mycobacterial species.     

Control of feeding rhythm in the terrestrial slug Incilaria bilineata

A modulatory neuron of feeding rhythm was newly identified in the buccal ganglia of the isolated central nervous system (CNS) of the terrestrial slug Incilaria bilineata. This neuron was termed the “feeding rhythm modulator” (FRM). Its morphological and electrical properties were compared with those of the MGC (metacerebral giant cell, a cerebral modulatory neuron of feeding rhythm). There was no direct connection between FRM and MGC. In order to investigate the control mechanism of the buccal central pattern generator, feeding rhythm was observed by varying the activities of MGC and FRM simultaneously. At a lower level of activity of MGC, feeding rhythm was not only sensitive to the activity of MGC but also to that of FRM. As the level of activity of MGC increased, feeding rhythm was exclusively controlled by the activity of MGC, and became unaffected by the activity of FRM. This indicates that cerebral neurons such as MGC primarily control feeding rhythm and modulate the contribution of FRM in a hierarchical manner.     

Developing network adequacy standards for VA Community Care

ObjectivesTo inform how the VA should develop and implement network adequacy standards, we convened an expert panel to discuss Community Care Network (CCN) adequacy and how VA might implement network adequacy standards for community care.Data Sources/Study settingData were generated from expert panel ratings and from an audio‐recorded expert panel meeting conducted in Arlington, Virginia, in October 2017.Study DesignWe used a modified Delphi panel process involving one round of expert panel ratings provided by nine experts in network adequacy standards. Expert panel members received a list of network adequacy standard measures used in commercial and government market and were provided a rating form listing a total of 11 measures and characteristics to rate.Data Collection MethodsItems on the rating form were individually discussed during an expert panel meeting between the nine expert panel members and VA Office of Community Care leaders. Attendees addressed discordant views and generated revised or new standards accordingly. Recorded audio data were transcribed to facilitate thematic analysis regarding opportunities and challenges with implementing network adequacy standards in VA Community Care.Principal FindingsThe five highest ranked standards were network directories for Veterans, regular reporting of network adequacy data to VA, maximum wait time/distance standards, minimum ratio of providers to enrolled population, and qualitative assessments of network adequacy. During the expert panel discussion with VA Community Care leaders, opportunities and challenges implementing network adequacy standards were highlighted.ConclusionsOur expert panel shed light on priorities for network adequacy to be implemented under CCN contracts, such as developing comprehensive provider directories for Veterans to use when selecting community providers. Remaining questions focus on whether the VA could reasonably develop and implement network adequacy standards given current Congressional restraints on VA reimbursement to community providers.     

Confluent BALB/c 3T3 cells monolayer provides selective and efficient growth of neoplastic cells

In an attempt to further characterize non-irradiated contact-inhibited confluent monolayer of BALB/c 3T3 cells (= Contact-Sensitive Plates; CSP) as substrata for in vitro drug sensitivity testing, we compared the efficiency of colony formation with panels of cell lines on CSP with that on plastic dishes or in agar. Tumorigenicity in athymic nude mice was also examined. We found that: (1) HeLa cells, 2 esophageal cancer lines, rat 3Y1 fibroblasts transformed by either adenovirus type 12, mouse polyoma virus, Rous avian sarcoma virus, or plasmid DNA carrying v-Ha-ras oncogene all formed colonies on CSP and in agar and at the same time was tumorigenic. The efficiency of colony formation on CSP proved always to be higher than that in agar. (2) None of the 4 "normal" fibroplastic cell lines formed colonies on CSP or in agar and were tumorigenic. (3) Simian virus 40 and adenovirus E1A gene transformed rat 3Y1 fibroblasts formed colonies on CSP but not in agar, and were not tumorigenic. Therefore, CSP was found to provide selective and efficient growth of neoplastic cells when compared to other substrata and is also helpful in detecting incompletely transformed cells.     

Endotheliopathy of liver sinusoidal endothelial cells in liver disease

Liver is the largest solid organ in the abdominal cavity, with sinusoid occupying about half of its volume. Under liver disease, hemodynamics in the liver tissue dynamically change, resulting in injury to liver sinusoidal endothelial cells (LSECs). We discuss the injury of LSECs in liver diseases in this article. Generally, in noninflamed tissues, vascular endothelial cells maintain quiescence of circulating leukocytes, and unnecessary blood clotting is inhibited by multiple antithrombotic factors produced by the endothelial cells. In the setting of inflammation, injured endothelial cells lose these functions, defined as inflammatory endotheliopathy. In chronic hepatitis C, inflammatory endotheliopathy in LSECs contributes to platelet accumulation in the liver tissue, and the improvement of thrombocytopenia by splenectomy is attenuated in cases with severe hepatic inflammation. In COVID-19, LSEC endotheliopathy induced by interleukin (IL)-6 trans-signaling promotes neutrophil accumulation and platelet microthrombosis in the liver sinusoids, resulting in liver injury. IL-6 trans-signaling promotes the expression of intercellular adhesion molecule-1, chemokine (C-X-C motif) ligand (CXCL1), and CXCL2, which are the neutrophil chemotactic mediators, and P-selectin, E-selectin, and von Willebrand factor, which are involved in platelet adhesion to endothelial cells, in LSECs. Restoring LSECs function is important for ameliorating liver injury. Prevention of endotheliopathy is a potential therapeutic strategy in liver disease.     

Nutrient-induced thermogenesis (NIT) following amino acid infusion

Nutrient-induced thermogenesis (NIT) induced by parenteral infusion of amino acid (AA) mixtures of different composition and of the same AA mixtures given via different routes (parenteral or intraportal infusion) were investigated in rats using a small animal indirect calorimeter. When 8 different AA solutions of differing composition but with the same total concentration were infused parenterally, both standard NIT (each AA is assumed to generate 3.28 kcal/g) and specific NIT (heat energy of each AA is calculated assuming that it is oxidized to carbon dioxide and water, and metabolised to urea and sulphuric acid) values of the leucine (Leu)-rich and the glycine (Gly)-rich solutions were significantly greater than those of the control solution. Removal of Leu or Gly from the respective AA solutions reversed the increase of both NIT values down to control levels. When the parenteral and portal infusion routes were used in one rat, both NIT values for parenteral infusion of the Leu-rich solution were again significantly greater than those of the control. Likewise, both NIT values for intrportal infusion of the Leu-rich solution were also significantly greater than those of the control. However, no difference in NIT values was found between parenteral and portal infusion of either solution. The result of this study indicated that Leu and Gly may be thermogenic AAs, and the thermogenic effect of Leu is not dependent upon the route of infusion.     

Use of a pericardial fat pad flap for preventing bronchopleural fistula: An experimental study focusing on the angiogenesis and cytokine production of the fat pad

An experimental study was conducted to determine whether pericardial fat tissue could induce neovascularization and produce cytokines related to tissue repair. Neovascularization was examined using chick chorioallantoic membranes. Pieces of pericardial fat tissue, omentum, and intercostal muscle were individually placed on a number of chorioallantoic membranes and neovascularization induced by each material was assayed 6 days after the implantation. The intensity of neovascularization was in the order of pericardial fat omentum > muscle. Cytokines, such as interleukin 1 (IL-1) and , tumor necrosis factor- (TNF), interferon- (IFN-), and interleukin 6 (IL-6) were assayed in a culture supernatant of pericardial fat tissue. The latter was obtained 24h after the addition of lipopolysaccharide (LPS) following various incubation times. All cytokines other than IFN are known to play a part in tissue repair, whereas IFN is negatively related to tissue repair because it inhibits fibroblast growth. The pericardial fat tissue incubated with LPS produced a certain amount of IL-1 on day 1, and TNF on days 1 and 8, whereafter these values decreased to an undetectable level. Irrespective of the addition of LPS, a large amount of IL-6 was observed in the supernatant of pericardial fat tissue and it was detectable until day 29. On the contrary, INF was not detected at any assay time. These observations suggest that a pericardial fat pad flap could possibly be beneficial in the prevention of bronchopleural fistula after pulmonary resection.