Increase of mouse resistance to Candida albicans infection by thymosin alpha 1.

Studies were carried out to assess the ability of thymosin alpha 1 to prolong the survival of mice challenged with Candida albicans. Two- to four-month-old mice were treated with graded doses of thymosin alpha 1 before, after, or before and after intravenous challenge with C. albicans. Significant resistance ot lethal infection was afforded by 100 micrograms of thymosin alpha 1 per kg given before or before and after challenge, whereas no protection was found in mice treated with thymosin alpha 1 administered at any dose level after inoculation. Pretreatment with thymosin alpha 1 also prevented the increased susceptibility to C. albicans infection of mice pretreated with cyclophosphamide on day -6. The results showed that thymosin alpha 1 was capable of protecting untreated or cyclophosphamide-pretreated mice from C. albicans infection at an optimal dose and schedule of administration.     

Increased susceptibility to systemic candidiasis in interleukin-6 deficient mice.

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates multiple aspects of the innate immune response. It has been recently shown that endogenous IL-6 is crucial for an efficient defence against severe infections with Gram-negative and Gram-positive bacteria. The aim of the present study was to investigate the role of endogenous IL-6 in the defence against infection with the yeast Candida albicans. During experimental candidemia, IL-6 deficient mice (IL-6-/-) had a decreased survival and an increased fungal load in their organs when compared with IL-6+/+ controls, despite increased plasma concentrations of tumour necrosis factor-alpha (TNF), interleukin-1 alpha (IL-1 alpha) and IL-1 beta, IL-6-/- mice were not able to mount an efficient neutrophil response during the infection. When mice were rendered neutropenic by cyclophosphamide, neutropenic IL-6-/- mice were equally susceptible to C. albicans when compared to neutropenic IL-6+/+ mice, implying that neutrophils mediate the beneficial effect of endogenous IL-6. In conclusion, IL-6-/- mice are more susceptible to disseminated candidiasis, and the effect of IL-6 is most likely mediated by neutrophils.     

Role of granulocytes in increased host resistance to Candida albicans induced by recombinant interleukin-1.

The effect of human recombinant interleukin-1 alpha (IL-1 alpha) on a systemic candidal infection in mice under various conditions of immunosuppression was investigated. In normal mice and in mice pretreated with cyclophosphamide, hydrocortisone acetate, or sublethal total body irradiation, the outgrowth of Candida albicans in the kidney was significantly reduced by the administration of a single intraperitoneal dose of 80 ng of IL-1 (P less than 0.05). In mice treated with either cyclophosphamide or irradiation, IL-1 also significantly reduced the outgrowth of C. albicans in the spleen. The protective effect of IL-1 was present when given 24 h before injection of C. albicans but also when IL-1 was given simultaneously with or 6 h after injection of C. albicans in cyclophosphamide-treated mice. The effect of IL-1 was independent of the presence or recruitment of granulocytes, since IL-1 inhibited the outgrowth of C. albicans in the kidneys and spleens of mice that were rendered severely granulocytopenic (less than 50 granulocytes per mm3) throughout the duration of the infection by either repeated injections of cyclophosphamide or sublethal total body irradiation. These results indicate that the enhancement of host resistance by IL-1 is not due solely to increased granulocytopoiesis or chemotaxis of granulocytes but strongly suggest that other mechanisms play a role in the protective effect of IL-1 against systemic infections.     

Efficacy of interleukin-1 beta against systemic Candida albicans infections in normal and immunosuppressed mice.

Prophylactic treatments with either recombinant human interleukin-1 beta (rHuIL-1 beta) or a muramyl dipeptide analog ([Abu1]MDP) enhanced the resistance of mice to systemic infection with Candida albicans. The optimum treatment regimen in both normal and cyclophosphamide-treated mice was intraperitoneal administration of 100 ng of rHuIL-1 beta or 1.6 mg of [Abu1]MDP per mouse once daily for 3 consecutive days before infection. Neither rHuIL-1 beta nor [Abu1]MDP was efficacious when started after the infection or when given before cyclophosphamide to mice infected subsequently. Continuing to treat after the infection with either drug neither enhanced nor antagonized the efficacy of prophylactic treatments.     

Synergistic effect on mortality in mice with murine cytomegalovirus and Pseudomonas aeruginosa, Staphylococcus aureus, or Candida albicans infections.

A synergistic effect on mortality was demonstrated in a combined infection of mice with murine cytomegalovirus (MCMV) and Pseudomonas aeruginosa, Staphylococcus aureus, or Candida albicans. Mice infected intraperitoneally with a 0 to 20% lethal dose inoculum of MCMV 3 days prior to the intravenous injection of a 0 to 20% lethal dose inoculum of either the bacteria or fungus demonstrated a striking enhancement of mortality. MCMV-infected mice given Pseudomonas or Staphylococcus exhibited a 90 to 100% mortality within 24 to 48 h, whereas 80% of viral-infected animals injected with Candida died in 5 days. Injection of the bacteria or fungus at various times during the MCMV infection resulted in enhanced mortality on days 0,1,2, and 3 of the viral infection. Greatest synergism was observed on day 3, with a progressive decline in death rates thereafter. Immunization with MCMV abrogated the synergistic effect on mortality in all three combined infections. Immunization with Pseudomonas reduced mortality in the combined MCMV-Pseudomonas infection. These results indicate that mice exhibit a markedly enhanced susceptibility to bacterial and fungal infections during the course of the MCMV infection and suggest that the enhancement may be related to viral-induced alterations in host resistance.     

Trehalose dimycolate enhances resistance to infection in neutropenic animals.

Bacterial infections are lethal complications of neutropenia, and antibiotics alone are inadequate therapy for these infections. Irradiated mice become severely neutropenic and remain susceptible to infection for 2 to 3 weeks, depending on the dose and quality of radiation. Some bacterial cell wall derivatives stimulate nonspecific host defense mechanisms against a variety of microbes which might cause postirradiation infection. In this study we determined if the cell wall glycolipid trehalose dimycolate (TDM), derived from Mycobacterium phlei, or a synthetic preparation of TDM was able to (i) enhance survival in mice when given before or after lethal doses of 60Co radiation and (ii) increase nonspecific resistance to postirradiation infection. Treatment with TDM oil-in-water emulsions and with synthetic TDM significantly enhanced survival before and after lethal doses of 60Co irradiation. This result correlated with the ability of TDM to reduce the translocation of intestinal bacteria and to stimulate hematopoiesis. With respect to nonspecific resistance to infection, TDM injected 1 h after sublethal irradiation increased resistance to a lethal Klebsiella pneumoniae challenge (10 50% lethal doses of K. pneumoniae in 30 days [LD50/30]) 4 or 14 days later. Increasing the dose of K. pneumoniae to 5,000 LD50/30 on day 4 overwhelmed the ability of TDM-treated mice to overcome infection. However, TDM treatment 1 h postirradiation combined with ceftriaxone antibiotic therapy (days 5 through 14) enhanced survival, even when the higher dose of bacteria (5,000 LD50/30) was used. These results indicate that in irradiated mice, TDM can be used to enhance survival and, as a potent stimulant of nonspecific resistance to infection in neutropenic mice, can act synergistically with antibiotic therapy to reduce sepsis and mortality.     

Roles of tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, platelet-activating factor, and arachidonic acid metabolites in interleukin-1-induced resistance to infection in neutropenic mice.

Treatment with a single low dose (80 to 800 ng) of interleukin-1 (IL-1) 24 h before a lethal bacterial challenge in granulocytopenic and in normal mice enhances nonspecific resistance. The mechanism behind this protection has only partially been elucidated. Since IL-1 induces production of tumor necrosis factor alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-activating factor (PAF), and arachidonic acid metabolites, we investigated the potential role of these substances in IL-1-induced protection. Low doses of murine TNF-alpha but not of human TNF-alpha enhanced survival, suggesting an effect via the type II TNF receptor rather than the type I TNF receptor, which has little species specificity. In line with this TNF-alpha-induced protection from infection, pretreatment with a low dose of a rat anti-murine TNF-alpha monoclonal antibody tended to inhibit IL-1-induced protection, suggesting a role of TNF-alpha as a mediator of IL-1-induced enhanced resistance to infection. Pretreatment with higher doses of anti-TNF-alpha, however, showed a dose-related protective effect per se, which could be further enhanced by a suboptimal dose of IL-1. A combination of optimal doses of anti-TNF-alpha and IL-1 produced an increase in survival similar to that produced by separate pretreatments. This lack of further enhancement of survival by combined optimal pretreatments suggests a similar mechanism of protection, most likely attenuation of deleterious effects of overproduced proinflammatory cytokines like TNF-alpha during lethal infection. Pretreatment with different doses of GM-CSF before a lethal Pseudomonas aeruginosa challenge in neutropenic mice did not enhance survival. Different doses of WEB 2170, a selective PAF receptor antagonist, of MK-886, a selective inhibitor of leukotriene biosynthesis, or of several cyclooxygenase inhibitors did not reduce the protective effect of IL-1 pretreatment. We conclude that IL-1-induced nonspecific resistance is partially mediated by induction of TNF-alpha and not by GM-CSF, PAF, and arachidonic acid metabolites. The mechanism of action of IL-1 seems to be similar to that of anti-TNF-alpha.     

Prophylactic efficacy of a basidiomycetes preparation AHCC against lethal Candida albicans infection in experimental granulocytopenic mice]

The prophylactic effects of a Basidiomycetes preparation, AHCC, against lethal Candida albicans infection were investigated in non treated or immunosuppressed mice. In the cyclophosphamide-or 5-fluorouracil (5-FU)-treated leukopenic mice and nontreated mice, the intraperitoneal administration of AHCC prior to C. albicans infection clearly prolonged the survival periods of the infected mice. In doxorubicin-treated mice, AHCC was less but significantly effective. On the other hand, in prednisolone-treated mice, AHCC was not effective. Oral administration of AHCC also protected the 5-FU-treated mice from lethal Candida infection, as indicated by prolongation of the survival periods and inhibition of Candida growth in the kidneys of these mice and by the increase in a number of neutrophils in their peripheral blood. These results suggested that AHCC may display a protective role against opportunistic fungal infection in leukopenic hosts.     

Comparison of anidulafungin's and fluconazole's in vivo activity in neutropenic and non-neutropenic models of invasive candidiasis

We compared the rate and extent of anidulafungin's and fluconazole's activity in neutropenic and non-neutropenic mice with Candida albicans invasive candidiasis. In immunocompetent mice, anidulafungin significantly improved survival vs. controls and fluconazole, and significant reductions in (1→3)-β-D-glucan and fungal burden were observed. In neutropenic animals, the highest doses of anidulafungin (5 mg/kg) and fluconazole (10 mg/kg) also improved survival and reduced fungal burden. However, there were no differences in survival between these antifungals as anidulafungin's activity was attenuated in this model. These results demonstrate that the extent of anidulafungin in vivo efficacy may be dependent on host immune status.     

Tumor necrosis factor alpha has a protective role in a murine model of systemic candidiasis.

The role of tumor necrosis factor alpha (TNF-alpha) in host defense against systemic Candida albicans infection was evaluated in a murine model of systemic candidiasis in which uniform death occurred between 5 and 6 days after infection. TNF-alpha was first detected at 16 h postinfection and progressively increased thereafter. Peak levels (700 to 900 pg/ml) were measured in mice near death. Administration of 0.5 to 1.0 mg of polyclonal immunoglobulin G (IgG) TNF-alpha antibody (TNF-alpha Ab) to mice 2 h preinfection neutralized serum TNF-alpha for up to 30 h. However, this regimen shortened survival from a mean of 5.5 days for IgG controls to 3.4 days (P = 1.9 x 10(-12)). Semiquantitative cultures of spleen, lung, liver, and kidney conducted at 1, 2, and 3 days postinfection found colony counts of spleen and kidney to be significantly higher for TNF-alpha Ab recipients but only for the first 48 h. Administration of 1.5 and 1.0 mg of TNF-alpha Ab at 2 h before and 48 h after fungal injection, respectively, shortened the mean survival from 4.9 to 2.3 days (P = 5.2 x 10(-8)). This regimen neutralized serum TNF-alpha throughout infection. With this regimen, colony counts of all organs were significantly higher in TNF-alpha Ab recipients at 1, 2, and 3 days postinfection. Histopathologic studies showed an increase in the number and size of C. albicans foci in tissues. Peripheral leukocyte counts and inflammatory response in tissue were similar for TNF-alpha Ab and IgG sham recipients. In vitro, incubation of C. albicans with four to eight times the peak serum levels of TNF-alpha for up to 24 h did not inhibit the rate of germ tube or pseudohypha formation. Thus, TNF-alpha that was produced during infection with C. albicans augmented host resistance against this organism and prolonged survival. The protective effect of TNF-alpha was not mediated by increased leukocytes in blood or tissues nor by a direct anticandidal effect of TNF-alpha. This study suggests that the administration of exogenous TNF-alpha may enhance host resistance against systemic C. albicans infection and may improve host survival.     

A protective role of platelet-activating factor in murine candidiasis.

Platelet-activating factor (PAF) is a potent phospholipid-derived modulator of immunological and inflammatory processes. In this study, the role of exogenous and endogenous PAF in resistance to infection with Candida albicans was investigated. Administration of PAF following a lethal challenge of C. albicans significantly protected mice from death and reduced the number of organisms in the kidneys. Neutralization of endogenous PAF with the PAF antagonist BN50739 shortened the mean survival time and increased the number of C. albicans cells per kidney. Shortly after infection of mice (30 min), significant levels of PAF were detected in the serum. PAF-induced protection appears to be mediated through the actions of tumor necrosis factor alpha (TNF-alpha), since pretreatment with anti-TNF-alpha before each injection of PAF abrogated the majority of PAF-induced enhanced resistance. Administration of PAF in vivo elevated serum TNF-alpha levels and TNF-alpha mRNA expression in the kidney. Production of TNF-alpha was markedly diminished by pretreatment with the PAF antagonist BN50739 prior to infection with C. albicans. We conclude that PAF, which is produced during infection with C. albicans, plays an important role in determining the level of resistance to this infectious microorganism. This effect of PAF appears to be mediated, at least in part, through the induction of TNF-alpha.     

Combined effect of fluconazole and thymosin alpha 1 on systemic candidiasis in mice immunosuppressed by morphine treatments.

Treatment of systemic infection with Candida albicans with a combination of an antifungal agent (i.e. fluconazole) and a thymus-derived immunostimulant (i.e. thymosin alpha 1 (T alpha 1)) in mice immunosuppressed by morphine treatments was investigated. In normal mice, fluconazole given after infection with 10(6) C. albicans cells was more effective than in mice treated with morphine. Combination treatment with fluconazole and T alpha 1 prolonged survival and reduced the fungal burden in the kidneys of immunosuppressed mice. We also investigated the influence of this combined treatment on killing properties of polymorphonuclear leucocytes (PMN) and natural killer (NK) cell activity, inhibited by morphine administrations. Treatment with T alpha 1 or fluconazole as single agents promoted a recovery of normal NK cell activity and intracellular killing of C. albicans by PMN, while the combination significantly increased both of these responses, probably through the modulation of lymphokine production. Our data suggest that the additive effect of T alpha 1 and fluconazole is due to a direct antifungal action and activation of the immunocompetence.     

Nondetectable levels of interferon gamma is a critical host defense during the first day of herpes simplex virus infection

Previous studies have demonstrated the importance of IFN alpha/beta in resistance to primary viral infections. However, the role of IFN gamma in primary infections is unclear. The present studies were undertaken to determine whether IFN gamma induction was an important early host defense against primary HSV infection. The approach was to block the IFN gamma response with antibodies to IFN gamma prior to infection and at various times post-infection (p.i.). The data indicates that treatment of mice with anti-IFN gamma prior to infection enhanced mortality (89% vs 37%). Anti-IFNs given at various times post HSV challenge proved most effective within the first 24 h of infection. The above results suggest for the first time that IFN gamma mediates important host defense(s) early during primary HSV infection. Similar results were obtained using antibody to IFN alpha/beta.     

Experimental Candida albicans infection in conventional mice and germfree rats.

Swiss-Webster white mice were intravenously infected with various doses of Candida albicans, and the viable units in their spleens, livers, lungs, and kidneys were determined at various intervals after challenge. The results showed that C. albicans multiplied to a greater extent in the kidneys of mice than in their spleens, lungs, or livers. The infection in mice was chronic; increasing numbers of C. albicans were observed in their kidneys until about 17 to 24 days postchallenge. Clearance of C. albicans from infected kidneys was not symmetrical, since the number of viable C. albicans in one kidney did not coincide with the viable counts observed in the opposite kidney of that same animal. Male and female mice did not differ in their overall susceptibility (50% lethal dose test) or in the number of viable C. albicans in the kidneys at various time intervals after infection. C. albicans also multiplied in the kidneys of germfree rats; however, the peak of the C. albicans infection in their kidneys occurred earlier than in those of conventional mice.     

Protection against lethal bacterial infection in mice by monocyte-chemotactic and -activating factor.

Chemotactic factors regulate the recruitment of neutrophils, lymphocytes, or monocytes-macrophages to infectious and inflammatory sites. The purpose of this study was to determine whether monocyte-chemotactic and -activating factor (MCAF [MCP-1], a JE gene product) also influences the host defense mechanism against microbial infection. We evaluated the effect of recombinant human MCAF on the survival rate of mice systemically infected with Pseudomonas aeruginosa or Salmonella typhimurium. The administration of 2.5 micrograms of MCAF 6 h before infection completely protected the mice from lethal infection. Mice with cyclophosphamide-induced leukopenia exhibiting increased susceptibility to P. aeruginosa were also endowed with resistance by the same dose of MCAF. Administration of MCAF at -6 h was critical, since MCAF given either earlier or later than -6 h failed to rescue mice from lethal infection. The in vivo effect on the survival of mice paralleled the reduced recovery of viable P. aeruginosa or S. typhimurium from the peritoneal cavity, i.e., the number of recovered bacteria from the MCAF (2.5 micrograms per mouse)-treated mice was reduced to less than 2% of control mice for P. aeruginosa and 4% of control mice for S. typhimurium at 24 h. Since MCAF exhibited chemotaxis on murine macrophages as well as enhanced phagocytosis and killing of bacteria in vitro, the activation of macrophages, followed by the recruitment into the peritoneal cavity, is responsible for eliminating bacteria and thus enhancing the survival rate.     

Comparison of the effects of recombinant interleukin 6 and recombinant interleukin 1 on nonspecific resistance to infection

Interleukin 1 (IL 1) is a potent enhancer of nonspecific resistance to infection in mice. Since IL 1 also induces interleukin 6 (IL 6), we tested the hypothesis that IL 6 mediates the effect of IL 1 on nonspecific resistance. In a lethal Pseudomonas aeruginosa infection in granulocytopenic mice, in which 80 ng of recombinant human IL 1 alpha protects against death, IL 6 appeared to be much less effective. Dosages of 8 ng, 80 ng and 320 ng IL 6 did not differ from the control, whereas 800 ng had a marginal protective effect (0.05 less than p less than 0.1). IL 1 and IL 6 did not potentiate each other in animals treated with suboptimal dosages of both cytokines. Numbers of bacteria cultured from the blood, thigh muscle, liver, spleen, and kidney were similar in animals treated with 800 ng IL 6 and in control animals, arguing against activation of microbicidal mechanisms. The serum concentration profile of IL 6 after an i.p. injection of 80 ng IL 1 was similar to that after 80 ng IL 6 i.p. Only minute amounts of IL 1 were detected in serum after an i.p. injection of IL 6. Taken these data together, it appears that increased resistance to infection induced by IL 1 is not mediated by IL 6.     

Toll-like receptor 2- and 6-mediated stimulation by macrophage-activating lipopeptide 2 induces lipopolysaccharide (LPS) cross tolerance in mice,which results in protection from tumor necrosis factor alpha but in only partial protection from lethal LPS doses

Patients or experimental animals previously exposed to lipopolysaccharide (LPS) become tolerant to further LPS challenge. We investigated the potential of the macrophage-activating lipopeptide 2 (MALP-2) to induce in vivo cross tolerance to tumor necrosis factor alpha (TNF-alpha) and LPS. MALP-2-induced tolerance could be of practical interest, as MALP-2 proved much less pyrogenic in rabbits than LPS. Whereas LPS signals via Toll-like receptor 4 (TLR4), MALP-2 uses TLR2 and TLR6. LPS-mediated cytokine release was studied in mice pretreated with intraperitoneal injections of MALP-2. No biologically active TNF-alpha could be detected in the serum of MALP-2-treated animals when challenged with LPS 24 or 72 h later, whereas suppression of LPS-dependent interleukin (IL)-6 lasted for only 24 h. Protection from lethal TNF-alpha shock was studied in galactosamine-treated mice. Dose dependently, MALP-2 prevented death from lethal TNF-alpha doses in TLR4(-/-) but not in TLR2(-/-) mice, with protection lasting from 5 to 24 h. To assay protection from LPS, mice were pretreated with MALP-2 doses of up to 10 micro g. Five and 24 h later, the animals were simultaneously sensitized and challenged by intravenous coinjection of galactosamine and a lethal dose of 50 ng of LPS. There was only limited protection (four of seven mice survived) when mice were challenged 5 h after MALP-2 pretreatment, and no protection when mice were challenged at later times. The high effectiveness of MALP-2 in suppressing TNF-alpha, the known ways of biological inactivation, and low pyrogenicity make MALP-2 a potential candidate for clinical use.     

Tumor necrosis factor and interleukin-6 in Candida albicans infection in normal and granulocytopenic mice.

We administered a neutralizing monoclonal antibody to tumor necrosis factor (TNF) during infection with Candida albicans in normal and granulocytopenic mice. Mice were rendered granulocytopenic (less than 0.1 x 10(9) granulocytes per liter) with cyclophosphamide. Growth of C. albicans from the kidneys was significantly increased in normal mice treated with the antibody to TNF, compared with that in control mice, after 36 h (3.6 x 10(4) +/- 1.2 x 10(4) CFU per kidney versus 9.1 x 10(3) +/- 6.2 x 10(3) CFU per kidney; P less than 0.05) and after 72 h (3.7 x 10(6) +/- 2.7 x 10(6) CFU per kidney versus 2.3 x 10(4) +/- 1.3 x 10(4) CFU per kidney; P less than 0.01). In granulocytopenic mice, the antibody to TNF had no effect on the growth of C. albicans from the kidneys. Furthermore, our study showed that the cytokines TNF and interleukin-6 (IL-6) were produced in a dose-dependent manner during C. albicans infection. TNF was detectable between 6 and 60 h, with peak levels at 24 h. Both TNF and IL-6 levels were significantly higher in cyclophosphamide-treated mice than in normal mice. Heat-inactivated C. albicans induced a TNF response different from that induced by viable C. albicans, with an early peak occurring at 3 to 4 h and declining to non-detectable levels after 15 to 24 h. Peak levels of TNF obtained with heat-inactivated C. albicans were lower than those obtained with viable C. albicans. Our study demonstrates that TNF and IL-6 are produced systemically during C. albicans infection and suggests that TNF is essential for granulocyte antifungal activity in vivo.