Allogenic stem cell transplant-associated acute graft versus host disease: a computational drug discovery text mining approach using oral and gut microbiome signatures.

PURPOSE: Acute graft versus host disease (aGVHD) is a major cause of non-relapse morbidity and mortality post-allogenic hematopoietic stem cell transplant (HSCT). Using conventional literature search and computational approaches, our objective was to identify oral and gut bacterial species associated with aGVHD, potentially affecting drug treatment via lipopolysaccharide (LPS) pathways. METHODS: Medline, PubMed, PubMed Central, and Google Scholar were searched using MeSH terms. The top 100 hits per database were curated, and 25 research articles were selected to examine oral and gut microbiomes associated with health, HSCT, and aGVHD. Literature search validation, aGVHD drug targets, and microbial metabolic pathway identification were completed using BioReader, MACADAM, KEGG, and STRING programs. RESULTS: Our review determined that (1) oral genera Rothia, Solobacterium, and Veillonella were identified in HSCT patients' stool and associated with aGVHD; (2) shifts in gut enterococci profiles were determined in HSCT-associated aGVHD; (3) gut microbiome dysbiosis prior or during HSCT and lower Shannon diversity index at time of HSCT were also associated with increased risk of aGVHD and transplant related death; and (4) Coriobacteriaceae family was negatively correlated with gut aGVHD, whereas Eubacterium limosum was associated with decreased risk of chronic GVHD relapse. Additionally, we identified molecular pathways related to TLR4/ LPS, including candidate aGVHD drug targets, impacted by oral and gut bacterial taxa. CONCLUSION: Reduced microbial diversity reflects higher severity and mortality rate in HSCT patients with aGVHD. Multi-omics approaches to decipher oral and gut microbiome associations will be critical for developing aGVHD preventive therapies.

Efficacy and safety of orally/sublingually, intranasally, and intraperitoneally administered recombinant murine interferon in the treatment of murine encephalomyocarditis virus.

Interferons (IFN) have been shown to be effective in protecting animals against lethal viral infections when administered systemically in relatively high doses. Intraperitoneal (i.p.) injection of mice with encephalomyocarditis virus (EMCV) gives rise to a rapidly progressive fatal disease characterized by central nervous system involvement and encephalitis. IFN-alpha has been shown to be effective in protecting mice against lethal EMCV infection when given via parenteral and oral/sublingual routes. The current study was designed to explore the ability of orally/sublingually and intranasally (i.n.) administered IFN-alpha to treat mice infected with EMCV in support of a planned clinical trial to evaluate efficacy of oral IFN-alpha in human viral infections. The primary objective of the study was to determine the efficacy of recombinant murine IFN-alpha (rMuIFN-alpha) in the treatment of mice infected with 100 LD(50) EMCV following oral, i.n., and i.p. administration at doses of 20,000 and 100,000 IU. The results of the current experiment did not indicate protection from infection with EMCV in mice that received IFN by the i.n. or oral/sublingual routes. The negative controls, infection of mice with 100 LD(50) of EMCV followed by treatment with excipient via all three routes, resulted in death of nearly all mice, as expected. The positive control, treatment of EMCV-infected (100 LD(50)) mice with rMuIFN-alpha via the i.p. route, was successful in protecting a significant number of mice from death compared with matched controls. This study points out the need to determine the optimum conditions for administration of oral/sublingual or i.n. IFN to insure maximum efficacy against viral infections.

Immune alterations in three mouse strains following 2-deoxy-D-glucose administration.

Using 2-deoxy-D-glucose (2-DG)-induced stress, our laboratory has developed studies to define stress effects on immune responses. Here, we report effects of increasing doses of 2-DG on the immune response of BALB/c, C57BL/6 and BDF(1) mice 2 h after three injections of 0 to 2000 mg/kg of 2-DG. Female 4- to 5-week-old mice were euthanized and blood and spleens were collected. A suspension of partially purified mature T splenocytes was obtained by negative selection using J11.d2 antibodies. Glucose and corticosterone levels were measured in the plasma of each mouse. Splenocyte and mature T splenocyte suspensions were tested in in vitro proliferation assays with or without concanavalin A. Splenocytes were analyzed for the following cell-surface markers: CD3, TCR alpha/beta, CD4, CD8 and major histocompatibility complex (MHC) Class II. Significant increases in blood glucose levels were observed in C57BL/6 and BALB/c strains with the highest 2-DG dose (p<0.05). Corticosterone levels were higher in BDF(1) mice and C57BL/6 mice following the administration of 1000 and 2000 mg/kg of 2-DG, respectively (p<0.01). In vitro proliferation of mature T splenocytes in the presence of concanavalin A was decreased in BDF(1) (p<0.05) but not in BALB/c and C57BL/6 mice. In addition, in BDF(1) mice the decrease was highly correlated with an increase of CD3+ and TCR alpha/beta+ cells in the spleen. These results demonstrated high variability in the response of different mouse strains to 2-DG-induced stress.

Norepinephrine as a growth stimulating factor in bacteria--mechanistic studies.

Catecholamines (norepinephrine, epinephrine, dopamine) enhance the growth of several species of gram-negative bacteria. Since catechol rings are known siderophores in bacteria, the administration of catecholamines may enhance growth by improving iron uptake in growth-limiting media, serving as auxiliary siderophores. We have tested the iron content in bacterial growth media which are known to support rapid growth and "slow growth" media. Additionally, we have examined the uptake of 3H-norepinephrine, to determine whether the catecholamine is actually taken into the bacteria or is merely adsorbed to the outside of the bacteria. Finally, we have been examining the supernatants produced by culturing bacteria with norepinephrine. These supernatants have been shown to have the capacity to enhance growth of naive cultures of bacteria, and are suggested to contain an "autoinducer of growth". We have found that both fast-growth and slow-growth media contain similar concentrations of iron, and that these levels do not change in most supernatants from NE-supplemented bacterial cultures. Examination of culture supernatants from NE-supplemented bacteria under different temperature conditions reveals some interesting differences. First, culture supernatant from NE-treated Escherichia coli, cultured at 37 degrees C, when examined by HPLC, exhibits a change in the norepinephrine content over time which is not seen in supernatant from 21 degrees C cultures or other media treatments. Second, the 37 degrees C culture NE-supplemented E. coli supernatant was significantly more effective in enhancing growth of three bacterial species than any other culture method other than NE-supplementation itself (this includes supernatant from NE-supplemented cultures of the other two species as well as supernatants from unsupplemented cultures of all three species).

Inhibition of interferon, cytokine, and lymphocyte proliferative responses in elite swimmers with altitude exposure.

To determine the immunologic consequences of athletic training at altitude, blood samples were taken at rest from 10 swimmers and 8 control nontraining but altitude-exposed members of the 1996 Australian Olympic Swimming Team, near the start and completion of a 21-day training camp at 2102 m. Blood leukocyte numbers dropped in both groups (p < 0.05), with the decrease greater in the swimmers (-38% swimmers, -3% controls). Concanavalin A (ConA)-induced blastogenesis decreased in both groups (p < 0.01), but the drop was greater in the control group (-32% swimmers, -56% controls, p < 0.05). Lipopolysaccharide (LPS)-induced blastogenesis more than doubled in both groups (281% swimmers, 249% controls, p < 0.01). Increases in mitogen-induced interleukin-1beta (IL-1beta), IL-4, and interferon-gamma (IFN-gamma) production and a decrease in IL-2 levels were observed in both groups after altitude exposure (all p < 0.05). The percentage of cells expressing HLA-DR fell (-33% swimmers, -20% controls, p < 0.01), whereas those expressing CD-4 expression increased (16% swimmers only, p < 0.01). Although training at medium-level altitude alters some immunologic parameters, the training-induced changes may be secondary to those induced by altitude alone.

Effects of 2-deoxy-D-glucose administration on cytokine production in BDF1 mice.

Physical exercise and diet changes have been shown to affect immune parameters, and similar effects are also induced by the administration of a nonmetabolizable glucose analog, 2-deoxy-D-glucose (2-DG). The present study was designed to characterize the effects of glucoprivation induced by 2-DG administration on concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in the blood and interferon-gamma (IFN-gamma), IL-2, and IL-4 in vitro production by partially purified T splenocytes in BDF1 mice. Mice (n = 8 per group) were injected intraperitoneally one or three times with 0, 500, 750, or 1000 mg/kg of 2-DG, and blood and spleens were collected 2 h after the last injection. Partially purified T splenocytes were cultured 24 h in the presence of concanavalin A (ConA). A significant increase in the corticosterone levels with the amount of 2-DG injected was observed after one or three injections (p<0.05). The amount of 2-DG injected was associated with an increase in TNF-alpha, IL-1beta, and IL-6 concentrations in the blood of mice after one or three injections of 2-DG (p<0.05). A significant decrease in in vitro proliferation of partially purified splenocytes in the presence of ConA was associated with a decrease in IFN-gamma production in the culture supernatants and an increase in IL-1 receptor expression on the cell surface (p<0.05).

Effects of social conflict on immune responses and E. coli growth within closed chambers in mice.

Social conflict has been shown to affect the neuroendocrine stress response in rodents. The current study was designed to characterize the effects of social conflict on leukocyte subset distribution and function as well as in vivo bacterial growth. Male DBA/2 mice implanted or not implanted with a closed chamber containing Escherichia coli were repeatedly challenged by temporary placement in the territory of a dominant CF-1 mouse five times a day for 2 consecutive days. Nonstressed animals were similarly handled, but were not exposed to social conflict. Effects on immune responses and E. coli growth were analyzed 13 h after the last social conflict session. Social conflict alone was associated with an increase in plasma corticosterone concentration and decreases in thymocyte numbers and splenocyte ability to proliferate in vitro in the presence of lipopolysaccharide (p < 0.05). After social conflict, immature CD4+CD8+ thymocytes decreased, whereas mature T cells increased (p < 0.05). In the presence of E. coli, social conflict induced a significant increase in plasma concentration of interleukin-1beta, and a decrease in the number of thymocytes and the percentage of CD4+CD8+ T cells in the thymus (p < 0.05). In addition to the lymphocyte subpopulation changes observed with social conflict alone, the proportion of CD3+ and major histocompatibility complex (MHC) class II IAd+ cells were significantly higher in stressed mice implanted with a closed chamber containing E. coli (p < 0.05). Social conflict tended to favor E. coli growth in the closed chamber, indicating possible direct bacterial-neuroendocrine hormone interactions. Taken together, these results suggest that stress may modulate the host immune response by altering both bacterial growth and resistance to infection.

Influence of exercise mode and carbohydrate on the immune response to prolonged exercise.

The influence of exercise mode and 6% carbohydrate (C) versus placebo (P) beverage ingestion on lymphocyte proliferation, natural killer cell cytotoxicity (NKCA), Interleukin (IL)-1beta production, and hormonal responses to 2.5 hr of intense running and cycling (approximately 75% VO2max) was measured in 10 triathletes serving as their own controls. The C versus P condition (but not exercise mode) resulted in higher plasma glucose concentrations, lower plasma cortisol concentrations, reduced postexercise lymphocytosis and NKCA, and a lessened T-cell reduction during recovery, No condition or mode effects were observed for concanavalin A and phytohemagglutinin-induced lymphocyte proliferation. Significant mode (but not condition) effects were observed for lipopolysaccharide-induced IL-1beta production over time. However, when expressed per monocyte, the mode effect was abolished and a sustained suppression in IL-1beta/monocyte was observed in all sessions throughout recovery. These data indicate that carbohydrate ingestion significantly affects plasma glucose and cortisol concentrations, blood lymphocyte counts, and NKCA, whereas exercise mode has no effect on these parameters.

Catecholamine enhancement of Aeromonas hydrophila growth.

Several species of bacteria have been shown to respond to the administration of norepinephrine and other catecholamines with increased growth (in culture) and virulence. In this study, we examined the effects of catecholamines on the growth of cultures of Aeromonas hydrophila, a Gram-negative bacillus found in brackish water. Bacterial cultures were maintained in tryptic soy both, then washed free of medium and transferred to a bovine serum-supplemented minimal salts medium. Treatment of A. hydrophila cultures with 10(-3)to 10(-5)M norepinephrine resulted in dramatic increases in growth at 24 h and longer, as assessed by spot plate analysis on tryptic soy agar plates. Norepinephrine-treated cultures had 4.5 log greater bacterial numbers than control cultures. Epinephrine, dopamine and isoproterenol were shown to be similarly effective in enhancing growth of A. hydrophila, over narrower concentration ranges. Acetylcholine supplementation of cultures did not alter the growth of A. hydrophila. Serotonin slightly enhanced Aeromonas growth when administered at very high concentrations (10(-3)M). The increased growth observed after catecholamine administration may alter the capacity to infect an animal under stressful conditions, and is another potential mechanism by which a stress response can affect susceptibility to disease.

Effects of 2-deoxy-D-glucose administration on immune parameters in mice.

Physical exercise and diet alterations have been shown to affect immune parameters. Similar effects are also induced by the administration of the non-metabolizable glucose analog, 2-deoxy-D-glucose (2-DG). The current study was designed to characterize the effects of glucoprivation induced by 2-DG administration on leukocyte subset distribution and function. BDF1 mice (n = 8 per group) were injected intraperitoneally one or three times with 0, 500, 750, 1000 or 1500 mg/kg of 2-DG. Two hours after the last injection of 2-DG, immunological parameters were analyzed. A dose-dependent increase in plasma glucose concentrations of mice injected once with up to 1500 mg/kg of 2-DG was observed (p < 0.001). After either one or three injections of up to 1500 mg/kg of 2-DG, corticosterone levels, leukocyte counts in the spleen, and CD3+ cells in the thymus increased. In vitro proliferation of partially purified lymphocytes from the spleen in the presence of both concanavalin-A and lipopolysaccharide decreased in a dose dependent manner (p < 0.05). In addition, after three injections, the proportion of both thymocytes and splenocytes bearing alphabeta-TCR increased as the concentration of 2-DG increased (p < 0.01). These results demonstrate that 2-DG administration induced dose-dependent changes in both thymus and spleen cell distribution and function.

Immune alterations in male and female mice after 2-deoxy-D-glucose administration.

Administration of 2-deoxy-D-glucose (2-DG) induces acute cellular glucoprivation. In the current study, we examined differences in immune parameters after 2-DG administration in both sexes. Male and female BDF1 mice were injected three times, 48 h apart, either with a saline solution (control group) or with 2-DG in saline (500 mg/kg). Two hours after the last injection, blood and spleens were collected. Plasma levels of interleukin-1beta, and interferon-gamma levels were measured. Additionally, the levels of the specific leukocyte antigens CD3, CD4, CD8, T cell receptor (TCR) alpha/beta, I-Ad, and H-2Ld/H-2Db were evaluated by flow cytometry on both blood and spleen cells. The blastogenic response of leukocytes from both tissues to mitogens was assessed. Levels of glucose, corticosterone, testosterone, progesterone, 17beta-estradiol, follicle-stimulating hormone, and luteinizing hormone were also determined. Increases in the percentage of cells bearing TCR alpha/beta and I-Ad in the blood and H-2Ld/H-2Db in the spleen were observed in the 2-DG-treated group for both sexes. In contrast, higher corticosterone and IL-1beta plasma concentrations, as well as higher percentages of splenocytes bearing TCR alpha/beta and I-Ad, and lower mitogen-induced proliferation of mature T splenocytes (79%) were observed in female but not in male mice injected with 2-DG compared with those injected with saline (p < 0.05). Taken together, these results suggest that female mice are more sensitive than male mice to immune alterations induced by 2-DG administration.

Induction of Carbon Starvation-Induced Proteins in Vibrio vulnificus.

By using two-dimensional polyacrylamide gel electrophoresis of pulse-labelled proteins, carbon starvation-induced (Sti) proteins produced by Vibrio vulnificus were identified. At least 34 proteins were induced over a 26-h period of carbon starvation. Although the total rate of protein synthesis over the 26-h starvation period suggests that there is a dramatic decrease in total protein synthesis within the first hour of starvation, at least 23 of the Sti proteins were induced within the first 20 min of carbon depletion. Six temporal classes of proteins could be identified, with class A(ii) encompassing the largest (21 proteins) group. All of the proteins in this group could be characterized by one of two patterns of protein synthesis. The addition of chloramphenicol at sequential times throughout starvation revealed that proteins required for starvation survival are made within the first 4 h of starvation.

A comprehensive community-based youth diversion program.

The authors describe a community-based program to divert youths from the juvenile justice system. The program's special features and (1) data collection that permits evaluation and accountability, and (2) liaison with justice personnel, service agencies, and the community.