Modulation of the phosphoinositide 3-kinase pathway alters innate resistance to polymicrobial sepsis

We examined the effect of modulating phosphoinositide 3-kinase (PI3K) activity in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Inhibition of PI3K activity with wortmannin increased serum cytokine levels and decreased survival time in septic mice. We have reported that an immunomodulator, glucan phosphate, induces protection in murine polymicrobial sepsis. We observed that glucan stimulated tissue PI3K activity, which positively correlated with increased survival in septic mice. We investigated the effect of PI3K inhibition on survival in septic mice treated with glucan. Treatment of mice with the PI3K inhibitors, wortmannin and LY294002, completely eliminated the protective effect of glucan, indicating that protection against septic mortality was mediated through PI3K. Inhibition of PI3K resulted in increased serum levels of IL1-beta, IL-2, IL-6, IL-10, IL-12, and TNF-alpha in septic mice. Apoptosis is thought to play a central role in the response to septic injury. We observed that inhibition of PI3K activity in septic mice resulted in increased splenocyte apoptosis and a change in the anatomic distribution of splenocyte apoptosis. We conclude that PI3K is a compensatory mechanism that suppresses proinflammatory and apoptotic processes in response to sepsis and/or inflammatory injury. Thus, PI3K may play a pivotal role in the maintenance of homeostasis and the integrity of the immune response during sepsis. We also observed that glucan phosphate decreased septic morbidity and mortality through a PI3K-dependent mechanism. This suggests that stimulation of the PI3K pathway may be an effective approach for preventing or treating sepsis and/or septic shock.     

Postirradiation glucan administration enhances the radioprotective effects of WR-2721

Based on murine survival studies, endogenous hemopoietic spleen colony formation (E-CFU), and recovery of bone marrow and splenic granulocyte-macrophage colony-forming cells (GM-CFC), it was demonstrated that the postirradiation administration of glucan, an immunomodulator and hemopoietic stimulant, enhances the radioprotective effects of WR-2721. LD50/30 dose reduction factors for mice treated with WR-2721 (200 mg/kg approximately 30 min before irradiation), glucan (250 mg/kg approximately 1 h after irradiation), or both agents were 1.37, 1.08, and 1.52, respectively. Enhanced survival in mice treated with both agents appeared to be due in part to glucan's ability to accelerate hemopoietic regeneration from stem cells initially protected from radiation-induced lethality by WR-2721. Following a 10-Gy radiation exposure, E-CFU numbers in mice treated with saline, WR-2721, glucan, or both WR-2721 and glucan were 0.05 +/- 0.03, 6.70 +/- 1.05, 0.95 +/- 0.24, and 33.90 +/- 2.96, respectively. Similarly, bone marrow and splenic GM-CFC numbers were greater in mice treated with both WR-2721 and glucan than in mice treated with either agent alone. These results demonstrated at least additive radioprotective effects when mice were given WR-2721 prior to irradiation and glucan following irradiation. These effects appeared to depend on the sequential cell protection mediated by WR-2721 and hemopoietic repopulation mediated by glucan.     

Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice

Hyperoxia generates an oxidative stress in the mouse lung, which activates the major stress-inducible kinase pathways, including c-Jun NH2-terminal kinase (JNK). We examined the effect of Jnk1 gene deletion on in vivo responses to hyperoxia in mice. The survival of Jnk1-/- mice was reduced relative to wild-type mice after exposure to continuous hyperoxia. Jnk1-/- mice displayed higher protein concentration in bronchoalveolar lavage (BAL) fluid and increased expression of heme oxygenase-1, a stress-inducible gene, after 65 h of hyperoxia. Contrary to other markers of injury, the leukocyte count in BAL fluid of Jnk1-/- mice was markedly diminished relative to that of wild-type mice. The decrease in BAL leukocyte count was not associated with any decrease in lung myeloperoxidase activity at baseline or after hyperoxia treatment. Pretreatment with inhaled lipopolysaccharide increased BAL neutrophil content and extended hyperoxia survival time to a similar extent in Jnk1-/- and wild-type mice. Associated with increased mortality, Jnk1-/- mice had increased pulmonary epithelial cell apoptosis after exposure to hyperoxia compared with wild-type mice. These results indicate that JNK pathways participate in adaptive responses to hyperoxia in mice.     

Histopathologic effects of soluble glucan and WR-2721, independently and combined in C3H/HeN mice.

Soluble glucan, an immunomodulator, and Walter Reed (WR)-2721, a radioprotectant, increase postirradiation survival when administered before and after exposure, respectively. Combined, these agents act synergistically through WR-2721's ability to spare hematopoietic stem/progenitor cells from radiation injury and glucan's ability to subsequently stimulate spared cells to proliferate. In this study, the histopathologic effects of WR-2721 (200 mg/kg, ip) and glucan (250 mg/kg, iv), at doses capable of increasing survival in lethally irradiated mice, were evaluated in unirradiated and irradiated female C3H/HeN mice. After treatment, whole body weights and wet organ weights of liver, spleen, and kidney, as well as gross and histologic changes in these and other tissues, were monitored on Days 1, 4, 7, 11, 15, 21, and 28. Morphometric studies of splenic white and red pulps were also performed. Soluble glucan, with or without WR-2721, in unirradiated groups, was associated with splenomegaly, transient morphometrically determined perturbations of white and red pulp areas, and histologic alterations of white pulp. In irradiated mice, splenic weight loss was initially dampened in glucan groups and accompanied by morphologic and histologic changes similar to those seen in unirradiated counterparts. The subsequent rebound of splenic parameters in irradiated mice was limited to WR-2721-treated mice and was associated with hematopoietic reconstitution. Glucan, with or without WR-2721, in unirradiated groups was associated with transient hepatomegaly and associated histologic changes. Similar changes in irradiated animals were seen only in the combined treatment group.     

Glucan-induced modification of the increased susceptibility of cyclophosphamide-treated mice to Staphylococcus aureus infection

Summary Glucan, a 1–3-polyglucosidic component of the cell wall of Saccharomyces cerevisiae, was evaluated for its ability to modify experimentally induced S. aureus septicemia in an immunosuppressed mouse model. AKR/J mice were injected with glucan (0.45 mg), cyclophosphamide (0.6 mg), isovolumetric saline (0.5 ml), or glucan (0.45 mg) and cyclophosphamide (0.6 mg) on days –10, –7, –4 and –1 prior to intravenous challenge with 1.0×10⁹ S. aureus on day 0.In contrast to the significant (P<0.05) decrease in leukocytes observed in the cyclophosphamide-treated mice, the administration of glucan to cyclophosphamide-treated mice resulted in maintenance of the peripheral leukocyte counts. Furthermore, glucan, as a single pretreatment regimen, resulted in a median survival time of 12.5 days, as against only 7.5 days in the saline control group. A 1.4-day median survival was observed in mice pretreated with cyclophosphamide and subsequently challenged with S. aureus. However, when glucan and cyclophosphamide were administered together, a median survival time of 9.0 days was observed.Histopathologic examination revealed that glucan administration inhibited the renal necrosis observed in both normal and cyclophosphamide-treated mice following staphylococcal challenge. Glucan also produced a marked reduction of hepatic pathology in cyclophosphamide-treated mice following S. aureus challenge. These data denote that glucan administration is effective in altering morbidity and mortality due to systemic S. aureus disease in cyclophosphamide-treated mice.     

The effect of total gnotobiological isolation and antimicrobial preparations on the survival of mice with acute radiation sickness

In experiments on 406 CBA mice the effect of antimicrobial preparations, used in combination with total gnotobiotic isolation, on the survival rate of totally irradiated animals was studied. The use of antimicrobial preparations was shown to considerably enhance the survival rate of these animals. Cyprofloxacin, a new preparation of the kinolone row, exhibited a good effect ensuring selective decontamination of the intestine. The use of cyprofloxacin prior to irradiation with the subsequent isolation of the animals in gnotobiotic chambers increased the survival rate of these animals by 2.7 times. But the maximum survival rate of irradiated mice was observed in those cases when antimicrobial preparations were used both before and after irradiation in combination with the total gnotobiotic isolation of the animals.     

The effect of heat-killed streptococci on the survival of heart grafts in inbred strains of mice.

Studies with inbred strains of mice revealed that exposure to type 12, Group A, beta-hemolytic streptococci affects the host response to heart grafts implanted in the ear. Intraperitoneal injection of streptococci 10 days before grafting led to curtailed survival of syngrafts without altering the normal rejection time of allografts. Similar sensitization, combined with local injection of streptococci into the graft site at the time of grafting, was followed by rapid rejection of both syngrafts and allografts. The time interval between exposure and grafting was critical. Injection with streptococci 5 days before grafting led to a prolonged survival of allografts and no demonstrable effect on syngrafts. In contrast, injection of streptococci 15 days before grafting did not alter survival of either type of graft. The data indicate heart grafts implanted in the ear may serve as a useful model for the study of the host responses to streptococcal antigens.     

Role of radiation-induced granulocyte colony-stimulating factor in recovery from whole body gamma-irradiation

The purpose of this study was to further elucidate the radioprotective role of granulocyte colony-stimulating factor (G-CSF) induced in response to irradiation. The induction of G-CSF and interleukin-6 (IL-6) in response to radiation exposure was evaluated in mice. The level of cytokine in serum was determined by multiplex Luminex. The role of G-CSF on survival and tissue injury after total body gamma-irradiation was evaluated by administration of neutralizing antibody to G-CSF before radiation exposure. An isotype control was used for comparison and survival was monitored for 30 d after irradiation. Jejunum samples were used for immunohistochemistry. Ionizing radiation exposure induced significant levels of the hematopoietic cytokines G-CSF and IL-6, in mice receiving 9.2 Gy radiation. Maximal levels of G-CSF were observed in peripheral blood of mice 8h after irradiation. IL-6 levels were maximum at 12h after irradiation. Administration of G-CSF antibody significantly enhanced mortality in irradiated mice. G-CSF antibody-treated mice had higher numbers of CD68(+) cells and apoptotic cells in intestinal villi. Our results confirm that radiation exposure induces elevations of circulating G-CSF and IL-6. Neutralizing antibody to G-CSF exacerbates the deleterious effects of radiation, indicating that G-CSF induced in response to irradiation plays an important role in recovery.     

Chemoprotective activity of an extract of Phyllanthus amarus against cyclophosphamide induced toxicity in mice.

The effect of 75% methanolic extract of the plant Phyllanthus amarus (P. amarus) was studied against cyclophosphamide (CTX) induced toxicity in mice. Administration of CTX (25 mg/kg b.wt, i.p.) for 14 days produced significant myelosuppression as seen from the decreased WBC count and bone marrow cellularity. Administration of P. amarus extract at doses 250 and 750 mg/kg b.wt significantly reduced the myelosuppression and improved the WBC count, bone marrow cellularity as well as the number of maturing monocytes. CTX treatment also reduced the activity of glutathione system and increased the activity of phase I enzyme that metabolize CTX to its toxic side products. P. amarus administration was found to decrease the activity of phase I enzyme. Administration of P. amarus also increased the cellular glutathione (GSH) and glutathione-S-transferase (GST), thereby decreasing the effect of toxic metabolites of CTX on the cells. Administration of P. amarus did not reduce the tumor reducing activity of CTX. In fact, there was a synergistic action of CTX and P. amarus in reducing the solid tumors in mice. Results indicated that administration of P. amarus can significantly reduce the toxic side effects of CTX and is not interfering with the antitumor efficiency of CTX.     

The effects of recombinant interleukin-1 and recombinant tumor necrosis factor on non-specific resistance to infection

Natural and synthetic immunomodulators that increase non-specific resistance to infection induce the production of interleukin-1 (IL-1) and tumor necrosis factor (TNF). Therefore, we investigated the effect of IL-1 and of TNF on the survival of lethally-infected mice. Mice were injected with 1 x 10(6) Klebsiella pneumoniae in the thigh muscle. When recombinant human IL-1 beta was given as a single i.p. injection 24 h before the infection, survival was increased. Using 80 ng IL-1 beta per mouse, survival compared to control animals was 80% versus 20% 48 h after the infection (p less than 0.001). No effect of IL-1 was observed when it was given 1/2 h before or 6 h after the infection. IL-1 alpha proved to be at least as potent as IL-1 beta. Numbers of bacteria cultured from the blood, thigh muscle, liver, spleen, and kidney were similar in IL-1-treated and control animals. Protection against death by IL-1 was also investigated in granulocytopenic mice with a Pseudomonas aeruginosa infection. Administration of the cyclooxygenase-inhibitor, ibuprofen, did not affect the beneficial effect of IL-1. In this model human recombinant TNF was at least tenfold less active than IL-1 beta. Pretreatment with IL-1 also had a significant effect on survival of mice that received a high dose of bacterial lipopolysaccharide.     

Plasmodium falciparum and Plasmodium berghei: effects of ornithine decarboxylase inhibitors on erythrocytic schizogony

Five ornithine decarboxylase inhibitors: alpha-difluoromethylornithine (DFMO) (eflornithine); alpha-monofluoromethyl-3,4-dehydroornithine; alpha-monofluoromethyl-3,4-dehydroornithine methyl ester; alpha-monofluoromethyl-3,4-dehydroornithine ethyl ester; and (2R,5R)-delta-methyl-alpha-acetylenic putrescine were shown to inhibit erythrocytic schizogony of Plasmodium falciparum in vitro and reduced spermidine levels in infected erthrocytes. Only DFMO was effective at limiting erythrocytic schizogony of P. berghei in vivo. Administration of DFMO as a 2% solution in the drinking water for 4 days reduced parasitemia in mice by 50% in a 4-day suppression test but did not increase survival time of infected mice. This is the first demonstration of an effect of DFMO on plasmodial erythrocytic schizogony in vivo and suggests that interference with polyamine biosynthesis may, in fact, be a viable chemotherapeutic target in erythrocytic malaria.     

山苍子油对小鼠系统性白念珠菌感染的影响

目的研究山苍子油对小鼠系统性白念珠菌感染的治疗作用。方法建立小鼠系统性白念珠菌感染模型,观察给药后小鼠中位生存时间,并检测给药后小鼠肾脏菌落形成单位计数。结果山苍子油能明显延长小鼠的中位生存时间,降低肾脏菌落形成单位计数。结论山苍子油对小鼠系统性白念珠菌感染具有治疗作用,值得进一步研究和开发。     

Nanoscopic cell-wall architecture of an immunogenic ligand in Candida albicans during antifungal drug treatment

The cell wall of Candida albicans is composed largely of polysaccharides. Here we focus on β-glucan, an immunogenic cell-wall polysaccharide whose surface exposure is often restricted, or “masked,” from immune recognition by Dectin-1 on dendritic cells (DCs) and other innate immune cells. Previous research suggested that the physical presentation geometry of β-glucan might determine whether it can be recognized by Dectin-1. We used direct stochastic optical reconstruction microscopy to explore the fine structure of β-glucan exposed on C. albicans cell walls before and after treatment with the antimycotic drug caspofungin, which alters glucan exposure. Most surface-accessible glucan on C. albicans yeast and hyphae is limited to isolated Dectin-1–binding sites. Caspofungin-induced unmasking caused approximately fourfold to sevenfold increase in total glucan exposure, accompanied by increased phagocytosis efficiency of DCs for unmasked yeasts. Nanoscopic imaging of caspofungin-unmasked C. albicans cell walls revealed that the increase in glucan exposure is due to increased density of glucan exposures and increased multiglucan exposure sizes. These findings reveal that glucan exhibits significant nanostructure, which is a previously unknown physical component of the host–Candida interaction that might change during antifungal chemotherapy and affect innate immune activation.     

Luplatex and its radioprotective properties

In experimental conditions the radioprotective properties of the placental complex Luplatex created in Scientific production complex "Biotechindustry" was studied. In experiments on mice F1(CBA x C57Bl) it was shown that Luplatex injected intraperitoneally in dose 0.5 ml 5-10 min before or after whole body gamma-irradiation with 8 Gy (LD80/30) increased the survival up to 40% as compared to the control group. In white mice protected by oral administration of Luplatex 30 min before exposure to 7.5 Gy, which is absolute minimal lethal dose for this type of mice (LD100/30), the effect was 48.3%.     

Effects of IL-1 on hematopoietic progenitors after myelosuppressive chemoradiotherapy

Recently it has been recognized that IL-1 plays an important role in hematopoietic regulation. Administration of 5-fluorouracil (5-FU) to mice causes prolonged neutropenia. rHIL-1 injected to mice after 5-FU, accelerated the recovery of hematopoietic progenitors and blood neutrophils. The combination of rhIL-1 and rhG-CSF reduced the neutropenic period significantly. Sublethal irradiation of mice induced profound neutropenia for 3 weeks which was associated with 80% mortality. Administration of rhIL-1 20 hours prior to or 2 hours post irradiation resulted in a significantly improved survival and rapid recovery of the neutrophil count. IL-1 administered alone or in combination with other colony stimulating factors to spontaneous breast tumor bearing mice following 5-FU therapy resulted in a rapid recovery of neutrophils, improved survival, and markedly reduced the tumor mass. Experiments in primates demonstrated that rhIL-1 administered to 5-FU treated animals shortened the neutropenic period from 30 to 17 days and increased the number of marrow progenitors responsive to other CSFs. Prolonged administration of IL-1 (14 days) to these animals resulted in a delayed neutrophil recovery as compared to animals receiving short courses of IL-1. rhIL-1 administered to primates receiving marrow grafts after lethal irradiation, did not result in rapid hematopoietic recovery. In humans, studies with CD-34 positive marrow cells showed that IL-1 had a radioprotective effect on a committed and early marrow progenitors. These data show the therapeutic potential of IL-1 in the treatment of chemoradiotherapy induced myelosuppression.     

Protective effect of RH-3 with special reference to radiation induced micronuclei in mouse bone marrow

Effect of pre-irradiation administration of different doses of RH-3, the herbal preparation of an Indian medicinal plant Hippophae rhamnoides, 30 min before 10 Gy whole body gamma irradiation was studied. Doses between 25 to 35 mg/kg body wt. were found to render > 80 % survival in mice. In order to investigate whether RH-3 protected against radiation induced genotoxicity, mice were administered different doses of RH-3, 30 min before 2 Gy dose and compared with untreated, RH-3 treated and irradiated controls. The bone marrow cells were collected at different time intervals following various treatments and processed for scoring micronuclei (MN). Administration of RH-3 alone did not enhance the MN frequency as compared to the control, and radiation dose of 2 Gy significantly enhanced the MN frequency (3.1 %, P < 0.01). Pre-irradiation treatment with RH-3, however, reduced the radiation induced MN frequency in a drug dose dependent manner suggesting its radioprotective efficacy. The protective effect of RH-3 on radiation induced perturbations in cell cycle progression was studied flowcytometrically in mouse bone marrow cells. RH-3 treatment (30 mg/kg body wt.) enhanced DNA synthesis (S-phase) in unirradiated controls and also countered radiation induced depression of S-phase to facilitate replenishment of cells lost due to radiation injury.     

Role of TNF-alpha-induced reactive oxygen species in endothelial dysfunction during reperfusion injury

We hypothesized that neutralization of TNF-alpha at the time of reperfusion exerts a salubrious role on endothelial function and reduces the production of reactive oxygen species. We employed a mouse model of myocardial ischemia-reperfusion (I/R, 30 min/90 min) and administered TNF-alpha neutralizing antibodies at the time of reperfusion. I/R elevated TNF-alpha expression (mRNA and protein), whereas administration of anti-TNF-alpha before reperfusion attenuated TNF-alpha expression. We detected TNF-alpha expression in vascular smooth muscle cells, mast cells, and macrophages, but not in the endothelial cells. I/R induced endothelial dysfunction and superoxide production. Administration of anti-TNF-alpha at the onset of reperfusion partially restored nitric oxide-mediated coronary arteriolar dilation and reduced superoxide production. I/R increased the activity of NAD(P)H oxidase and of xanthine oxidase and enhanced the formation of nitrotyrosine residues in untreated mice compared with shams. Administration of anti-TNF-alpha before reperfusion blocked the increase in activity of these enzymes. Inhibition of xanthine oxidase (allopurinol) or NAD(P)H oxidase (apocynin) improved endothelium-dependent dilation and reduced superoxide production in isolated coronary arterioles following I/R. Interestingly, I/R enhanced superoxide generation and reduced endothelial function in neutropenic animals and in mice treated with a neutrophil NAD(P)H oxidase inhibitor, indicating that the effects of TNF-alpha are not through neutrophil activation. We conclude that myocardial ischemia initiates TNF-alpha expression, which induces vascular oxidative stress, independent of neutrophil activation, and leads to coronary endothelial dysfunction.     

Effects of dietary β – 1,3 – glucan on the growth,survival, physiological and immune response of marron,Cherax tenuimanus (smith, 1912)

Six isonitrogenous and isocalorific diets supplemented with five different levels of beta –1,3 – glucan (0.08%, 0.1%, 0.2%, 0.4% and 0.8%) were formulated and tested for marron (Cherax tenuimanus) growth, survival, organosomatic indices, osmoregulatory capacity and immunological parameters (total and differential haemocyte counts, haemolymph clotting time and bacteraemia). The sixth diet without any beta – 1,3 – glucan was used as a control. Each diet was provided to 18 marron (0.47 ± 0.02 g initial weight) replicated 3 times in individual 250 L fiberglass cylindrical tanks. Each tank was provided with a biological filtration recirculating water system. After 84 days of culture, the survival and yield were higher in the marron fed 0.1% beta glucan supplemented diet. The different levels of beta glucan did not alter any of the physiological parameters of marron. However, dietary supplementation with beta glucan resulted in significantly higher (P < 0.05) total haemocyte count (THC) and granular cells. The bacteraemia rank was lower in all diets having beta glucan supplemented with more than or equal to 0.1% compared to the control and 0.08% beta glucan supplemented diets. Results suggest that dietary beta – 1,3 – glucan at a minimum concentration of 0.1–0.2% can improve the immune system of marron.     

Effects of endocrine glands and hormone replacement on the mast cell count of the Harderian gland of mice

There are marked sex differences in the Harderian gland of the C3H/He mouse strain, the females having a larger number of mast cells than the males as one of the major differences. Mast cell counts of the Harderian gland were made on male mice subjected to combinations of adrenalectomy, gonadectomy and administration of sex steroid hormones. Castration alone caused a significant increase in the count resulting in about three times the number found in intact males. Castration plus adrenalectomy increased the count over 6-fold, to levels close to those found in female mice. Administration of testosterone or estrone to the mice which had been castrated and adrenalectomized prevented the increase, while progesterone treatment had no effect on the count. Although the number of mast cells in the male Harderian gland was necessarily small compared to either the female gland or that of castrated and adrenalectomized males, no obvious dimorphism could be found microscopically.     

The immunoadjuvant effect of soluble glucan derivatives in mice

We examined the effect of soluble derivatives of yeast glucan on the humoral immune response of various strains of inbred mice after administration of different doses according to various schedules. Glucan was injected i.v. or s.c. in a single dose or repeatedly. The immune response was examined by determining the titres of serum hemagglutinins against sheep erythrocytes (SRBC-Ab). The immunoadjuvant effect of glucan derivatives depends on the inbred strain used, on the dose of glucan, mode and time of administration with respect to antigen injection. The results have shown that the stimulatory effect of glucan derivatives occurred already after a single injection, the optimum dose being 10–20 mg/kg. Intravenous injection was more efficient than the subcutaneous one. In some cases, a slight increase of the spleen mass was observed. Translated by I. Miler