A sub-inhibitory concentration of amphotericin B enhances candidastatic activity of interferon-gamma- and interleukin-13-treated murine peritoneal macrophages

We studied the effects of interferon-gamma (IFN-gamma), a Th1 cytokine, and interleukin-13 (IL-13) or interleukin-4 (IL-4), Th2 cytokines, on the antifungal activity of resident murine peritoneal macrophages against Candida albicans 'in vitro'. IFN-gamma, IL-13 and IL-4 treatment enhanced the candidastatic functions of the macrophages. Reactive oxygen intermediates (ROIs) seem to be directly involved in the increase of anti-Candida activity in macrophages treated with Th1 or Th2 cytokines. Study of unopsonized C. albicans phagocytosis showed that IFN-gamma reduces the uptake process whereas the Th2 cytokines increase it. This difference is correlated to mannose receptor expression, which is decreased by IFN-gamma but increased by the Th2 cytokines. So, the effects on phagocytosis and candidastatic activity of IFN-gamma-treated macrophages are dissociated. In contrast, the phagocytic ability of macrophages pretreated 'in vitro' with IL-4 or IL-13 played a complementary role to the ROIs, in reduction of yeast proliferation by macrophages. In consequence, the macrophages treated with IL-13 and IL-4 develop a higher fungistatic activity than macrophages activated by IFN-gamma. Amphotericin B associated with IL-13 or IFN-gamma, but not with IL-4, enhanced the yeast growth inhibition activity of macrophages. The ROIs were involved in the additive effect of IFN-gamma with amphotericin B, whereas another mechanism was implicated in the increase of candidastatic activity of macrophages treated with IL-13 in association with amphotericin B.     

Recombinant interleukin 12 cures mice infected with Leishmania major

Resistant C57BL/6 mice infected with Leishmania major are self-healing, whereas susceptible BALB/c mice fail to contain cutaneous infection and subsequently undergo fatal visceral dissemination. These disparate outcomes are mediated by dissimilar expansions of T helper type 1 (Th1) and Th2 CD4+ T lymphocyte subsets in vivo during cure and progression of disease. Because interleukin 12 (IL-12) has potent T cell growth and interferon gamma (IFN-gamma) stimulatory effects, we studied its effect on CD4+ T cell differentiation during murine leishmaniasis. Treatment with recombinant murine (rMu)IL-12 during the first week of infection cured 89% of normally susceptible BALB/c mice, as defined by decreased size of infected footpads and 1,000-10,000-fold reduced parasite burdens, and provided durable resistance against reinfection. Cure was associated with markedly depressed production of IL-4 by lymph node cells cultured with antigen or mitogen, but preserved or increased production of IFN-gamma relative to untreated mice. IL-4 and IFN-gamma mRNA associated with CD4+ T lymphocytes isolated from infected lymph nodes showed similar reciprocal changes in response to rMuIL-12 therapy. A single injection of anti-IFN-gamma monoclonal antibody abrogated the protective effect of rMuIL-12 therapy and restored Th2 cytokine responses. We conclude that rMuIL-12 prevents deleterious Th2 T cell responses and promotes curative Th1 responses in an IFN-gamma- dependent fashion during murine leishmaniasis. Since BALB/c leishmaniasis cannot be cured with rMuIFN-gamma alone, additional direct effects of IL-12 during T cell subset selection are suggested. Because rMuIL-12 is uniquely protective in this well-characterized model of chronic parasitism, differences in IL-12 production may underlie heterogenous host responses to L. major and other intracellular pathogens.     

A trial of immunotherapy against Leishmania amazonensis infection in vitro and in vivo with Z-100, a polysaccharide obtained from Mycobacterium tuberculosis, alone or combined with meglumine antimoniate

OBJECTIVES: To determine the efficacy and the immunomodulatory function of Z-100 alone or combined with meglumine antimoniate on Leishmania amazonensis infection. METHODS: The effect of the compounds was evaluated by microscopic counting of intracellular amastigotes in macrophages stained with Giemsa, or axenic promastigotes, and IC(50) was determined by linear regression. The antileishmanial effect of the compounds was assessed in infected BALB/c mice by a limiting dilution analysis and the production of gamma interferon (IFN-gamma), interleukin 10 (IL-10), IL-4, IgG1 and IgG2a was measured by ELISA. RESULTS: In vitro, Z-100 showed antileishmanial activity against intracellular amastigotes of L. amazonensis with an IC(50) of 13 mg/L. Moreover, infected macrophages treated with Z-100 (12 mg/L) showed smaller parasitophorous vacuoles with fewer parasites than the control. In addition, the efficacy of Z-100 plus meglumine antimoniate [14 mg/L pentavalent antimony (Sb(v))] was higher (46% inhibition) than either Z-100 or meglumine antimoniate alone. Nevertheless, no effect of Z-100 on axenic promastigotes was observed. Infected BALB/c mice treated with Z-100 (100 microg/kg) alone did not show any antileishmanial effects in comparison with the control group, and IFN-gamma, as well as IL-10 and IL-4, was up-regulated by the treatment. In addition, both IgG1 and IgG2a were also increased by the Z-100 treatment. Although Z-100 plus meglumine antimoniate (14 or 28 mg/kg Sb(v)) controlled both the parasite load and the footpad swelling in comparison with control mice, no significant differences were found with meglumine antimoniate alone. CONCLUSIONS: In vitro, Z-100 alone or combined with meglumine antimoniate showed an antileishmanial effect on L. amazonensis. However, no effect was observed in infected BALB/c mice treated with Z-100, suggesting that the up-regulation of IL-10 and IL-4 production by the treatment could be interfering with the development of a protective Th1-type response. For further understanding of the effects of Z-100 in vivo, another strain of mice such as C57BL/6 should be tested in future.     

Modulation of the cellular immune response after oral or subcutaneous immunization with microparticles containing Brucella ovis antigens.

An antigenic extract (HS) from Brucella ovis was encapsulated in either poly-epsilon-caprolactone (PEC) or poly-lactic-co-glycolic acid 75:25 (PLGA) microparticles containing beta-cyclodextrin and Pluronic F-68 as stabilising agents. The resulting microparticles displayed sub-5 microm sizes. Antigen loading was 5.2 and 3.8 microg/mg for HS-PEC and HS-PLGA microparticles, respectively. Specific HS cytokine profiles were determined after subcutaneous and oral immunisation of BALB/c mice. Gut distribution studies of the formulations after oral administration showed that HS-PEC microparticles interacted more strongly with mucosa and Peyer's patches than HS-PLGA. Accordingly, oral immunisation with HS-PLGA induced a negligible immune response, whereas HS-PEC elicited a Th1 response although of low intensity. Subcutaneous immunisation with HS-PEC induced high IFN-gamma and IL-2 release; in contrast, HS-PLGA particles induced a Th2 profile characterized by significant levels of IL-4. Splenic cells from free-HS immunised mice released IFN-gamma and IL-2 but not IL-4. A less intense Th1 pattern was also found from HS stimulated nai;ve splenic cells. These results suggest that the HS itself possesses Th1 immunopotentiating properties, required to control brucellosis, that can be specifically increased by encapsulation in PEC microparticles. In contrast, PLGA microparticles modulate the response toward a Th2 pathway.     

Innate immune response in Th1- and Th2-dominant mouse strains

C57BL/6 and BALB/c mice are prototypical Th1- and Th2-type mouse strains, respectively. In the present study, we attempted to characterize the innate immune response of macrophages from these mouse strains. Macrophages from C57BL/6 mice produced higher levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12 than those from BALB/c mice after stimulation with macrophage-activating lipopeptide-2 (MALP-2, a synthetic TLR-2 ligand) or lipopolysaccharide (LPS, a TLR-4 ligand). The augmented IL-12 production by C57BL/6 macrophages increased interferon-gamma and, in contrast, decreased IL-13 production by CD4+ T cells. On stimulation with MALP-2 or LPS, C57BL/6 macrophages produced lysosomal enzyme and nitric oxide, effector molecules for bacterial killing, whereas BALB/c macrophages did not. Bactericidal activity of BALB/c macrophages was impaired relative to C57BL/6 macrophages when cells were infected with live bacteria in vitro. In a murine model of septic peritonitis induced by cecal ligation and puncture (CLP), BALB/c mice failed to facilitate bacterial clearance relative to C57BL/6 mice despite an augmented peritoneal leukocyte infiltration that was associated with increased peritoneal levels of cytokines/chemokines. BALB/c mice exhibited increased plasma and hepatic levels of cytokines/chemokines, resulting in an exaggerated systemic inflammation as determined by acute-phase proteins. Finally, BALB/c mice were vulnerable to CLP-induced lethality relative to C57BL/6 mice. Altogether, innate immune response of macrophages is different between these mouse strains, which may affect the development of Th1 and Th2 adaptive immunity in these strains. Reduced systemic inflammatory response in C57BL/6 mice that may result from an eminent local response appears to be beneficial during sepsis.     

Albumin nanoparticles improved the stability, nuclear accumulation and anticytomegaloviral activity of a phosphodiester oligonucleotide.

The goal of this study was to evaluate the potential of albumin nanoparticles as a delivery system for antisense oligonucleotides. Nanoparticles were prepared by a coacervation process and cross-linkage with glutaraldehyde. Phosphodiester (PO) and phosphorotioate (PS) oligonucleotides were either adsorbed on the surface of nanoparticles (PO-NPA and PS-NPA) or incorporated in the nanoparticle matrix (PO-NPB and PS-NPB). When PO-loaded nanoparticles were incubated with phosphodiesterase, only NPB was able to keep the oligonucleotide hybridization capability for at least 60 min. The antiviral activity was evaluated in MRC-5 fibroblasts infected with human cytomegalovirus at a MOI of 0.0035. Both PO nanoparticle formulations significantly increased the antiviral activity of free PO (P<0.001) and NPB showed slightly higher efficacies than NPA (P<0.05). On the other hand, PS exhibited significant higher activity than free PO (P<0.001), however, no significant differences were found between PS-nanoparticle and PO-nanoparticle formulations. These findings were well correlated with the intracellular distribution observed for fluorescent oligonucleotide-loaded albumin nanoparticles. Even these carriers delayed and decreased the uptake of PO by MRC-5 cells, they finally induced a diffused cytoplasmic distribution and major nuclear accumulation. In summary, albumin nanoparticles partially protected a PO against enzymatic degradation and improved their presence in the nucleus and thus, increased its efficiency.     

Interleukin 12 is effective treatment for an established systemic intracellular infection: experimental visceral leishmaniasis

When administered at or near the initiation of experimental intracellular infection caused by Leishmania major, Toxoplasma gondii, or Cryptococcus neoformans, treatment with the immuno-regulatory cytokine interleukin 12 (IL-12), induces protective antimicrobial activity. In contrast, once infections are established, IL-12 exerts considerably less or no effect in the face of a suppressive Th2 cell- associated response (L. major) or rapidly progressive fatal infection (T. gondii). To test the efficacy of IL-12 in an established intracellular protozoal infection but under quite different immunologic conditions (Th1 cell response, acquired resistance), L. donovani- infected BALB/c mice were treated starting 2 wk after challenge coincident with the onset of the Th1 cell response. In this environment, 7 d of IL-12 treatment reduced liver parasite burdens by 47%, an effect comparable to that induced by exogenous interferon (IFN) gamma. The in vivo mechanism responsive to IL-12 was complex, and required both CD4+ and CD8+ T cells as well as natural killer cells and the action of multiple endogenous antileishmanial cytokines (IFN-gamma, IL-2, tumor necrosis factor alpha). Early treatment with IL-12 before the expression of the Th1 cell response was also effective and induced an accelerated, near-cure response via an IFN-gamma-dependent mechanism. These results extend the antimicrobial-inducing capacity of IL-12 beyond prophylaxis by indicating that IL-12 can exert clear-cut therapeutic activity in an established intracellular infection.     

Enhancement of T helper type 1 immune responses against hepatitis B virus core antigen by PLGA nanoparticle vaccine delivery.

Currently, there is a need for therapeutic vaccines that are effective in inducing robust T helper type 1 (Th1) immune responses capable of mediating viral clearance in chronic hepatitis B infection. Hepatitis B therapeutic vaccines were designed and formulated by loading the hepatitis B core antigen (HBcAg) into poly(D,L-lactic-acid-co-glycolic acid) (PLGA) nanoparticles with or without monophospholipid A (MPLA), a Th1-favoring immunomodulator. These particles were around 300 nm in diameter, spherical in shape and had approximately 50% HBcAg encapsulation efficiency. A single immunization with a vaccine formulation containing (MPLA+HBcAg) coformulated in PLGA nanoparticles induced a stronger Th1 cellular immune response with a predominant interferon-gamma (IFN-gamma) profile than those induced by HBcAg alone, free (HBcAg+MPLA) simple mixture or HBcAg-loaded nanoparticles in a murine model. More importantly, the level of HBcAg-specific IFN-gamma production could be increased further significantly by a booster immunization with the (HBcAg+MPLA)-loaded nanoparticles. In summary, these results demonstrated that codelivery of HBcAg and MPLA in PLGA nanoparticles promoted HBcAg-specific Th1 immune responses with IFN-gamma production. These findings suggest that appropriate design of the vaccine formulation and careful planning of the immunization schedule are important in the successful development of effective HBV therapeutic vaccines.     

Cutaneous leishmaniasis. The defect in T cell influx in BALB/c mice

Local cellular responses to cutaneous infection with Leishmania mexicana amazonensis were examined in susceptible (BALB/c) and resistant (C57BL/6) mouse strains by immunocytochemical and electron microscopic studies. Infection during the first 8 wk in both animal strains was characterized by progressively enlarging lesions, epidermal thickening and ulceration, and accumulation of eosinophils and Ia+ infected macrophages. Healing of C57BL/6 mouse lesions began after 12 wk of infection and was associated with local influx of both Th (L3T4+) and T cytotoxic/suppressor (Lyt-2+) cells into the dermis, and Ia antigen expression on epidermal keratinocytes. T lymphocyte infiltration was marked and intracellular parasites were scarce by 21 wk of C57BL/6 infection. Similarly, granulomas in C57BL/6 livers contained L3T4+ and Lyt-2+ T lymphocytes and no visible intracellular parasites by 21 wk of infection. In contrast, BALB/c mouse lesions continued to enlarge and never healed. Throughout the entire course of infection, T lymphocyte influx into the heavily infected dermis was minimal. Keratinocyte Ia expression was absent in BALB/c lesions. BALB/c livers were heavily infected by 18 wk of cutaneous infection, with few demonstrable T lymphocytes. A systemic absence of T cells could not be demonstrated in BALB/c mice. Both L3T4+ and Lyt-2+ T cells were found in the peripheral blood in normal numbers in both mouse strains. Our results support the role of T cells as important local effector cells in the healing response of murine cutaneous leishmaniasis. We suggest that local T lymphocyte infiltration may provide lymphokines, particularly IFN-gamma, that can activate infected macrophages to destroy the intracellular parasites. Alternatively, T cells may play a cytotoxic role, killing infected macrophages and allowing local humoral factors to destroy released extracellular parasites.     

T cell and non-T cell compartments can independently determine resistance to Leishmania major

In experimental murine cutaneous leishmaniasis caused by Leishmania major (Lm), the cellular determinants governing development of protective or exacerbative T cells are not well understood. We, therefore, attempted to determine the influence of T cell and non-T cell compartments on disease outcome. To this end, T cell chimeric mice were constructed using adult thymectomized lethally irradiated, bone marrow-reconstituted (ATXBM) animals of genetically resistant, C57BL/6, or susceptible, BALB/c, backgrounds. These hosts were engrafted with naive T cell populations from H-2-congenic susceptible, BALB.B6-H-2b, or resistant, C57BL/6.C-H-2d, animals, respectively. Chimeric mice were then infected with Lm, and disease outcome was monitored. BALB/c T cell chimeric mice, BALB/c ATXBM hosts given naive C57BL/6.C-H-2d T cells, resolved their infections as indicated by reductions in both lesion size and parasite numbers. Furthermore, the mice developed typical Th1 (interferon[IFN]-gamma hiinterleukin[IL]-4lo) cytokine patterns. In contrast, both sham chimeric, BALB/c ATXBM hosts given naive BALB/c T cells, and control irradiated euthymic mice succumbed to infection, producing Th2 profiles (IFN-gamma loIL-4hiIL-10hi). C57BL/6 T cell chimeras, C57BL/6 ATXBM hosts given naive BALB.B6-H-2b T cells, resolved their infections as did C57BL/6 sham chimeras and euthymic controls. Interestingly, whereas C57BL/6 control animals produced Th1 cytokines, chimeric animals progressed from Th0 (IFN-gamma hiIL-4hiIL- 10hi) to Th2 (IFN-gamma loIL-4hiIL-10hi) cytokine profiles as cure ensued. Both reconstitution and chimeric status of all mice were confirmed by flow cytometry. In addition, T cell receptor V beta usage of Lm-specific blasts was determined. In all cases, V beta use was multiclonal, involving primarily V beta 2, 4, 6, 8.1, 8.2, 8.3, 10, and 14, with relative V beta frequencies differing between H-2b and H-2d animals. Most importantly, however, these differences did not segregate between cure and noncure outcomes. These findings indicate that: (a) genetic traits determining cure in Lm infection can direct disease outcome from both T cell and non-T cell compartments; (b) the presence of the curing genotype in only one compartment is sufficient to confer cure; (c) curing genotype T cells autonomously assume a Th1 cytokine profile-mediating cure; (d) noncuring genotype T cells can mediate cure in a curing environment, despite the onset of Th2 cytokine production; and lastly, (e) antigen specificity of responding T cells, as assessed by V beta T cell receptor diversity, is not a critical determinant of disease outcome.     

Improving peptide-based assays to differentiate between vaccination and Mycobacterium bovis infection in cattle using nanoparticle carriers for adsorbed antigens.

The development of diagnostic tests to differentiate between vaccinated animals and those infected with Mycobacterium bovis is required so that test and slaughter control strategies can continue alongside vaccination. In this work, the peptide antigen, ESAT-6, p45, derived from the N-terminal sequence of the ESAT-6 protein, was adsorbed onto a range of microparticulate and nanoparticulate substrates to enhance the in vitro immune response of blood lymphocytes previously sensitised to M. bovis. Two types of hydroxyapatite (HA) nanoparticles (both approximately 300 nm in linear dimension), carbonate hydroxyapatite nanospheres (CHA, approximately 50 nm), two sizes of polystyrene nanospheres ( approximately 500 and 40 nm), calcium carbonate microparticles (0.3-1.0 microm) and glass microspheres (1.0-3.0 microm) were incubated in a solution of the peptide in PBS. Peptide adsorption increased on the nanoparticle carriers in the order HA (2.5+/-0.12%w/w), CHA (4.9+/-0.12) polystyrene (500 nm, 6.8+/-0.15%, 40 nm, 9.2+/-0.07) and these systems exhibited fairly low levels of desorption (approximately 10-15% peptide release) over a 24-h incubation period in PBS at 37 degrees C. HA, CHA and polystyrene carriers with adsorbed peptide were subsequently tested in the BOVIGAM assay to investigate the efficiency of the immune response of blood lymphocytes in terms of interferon-gamma (IFN-gamma) production. A general elevation of IFN-gamma production resulted for particle-bound peptide relative to free peptide at high peptide concentrations (>10 microg/ml). Only HA-adsorbed peptide resulted in consistently higher immune responses at low peptide concentration (<0.1 microg/ml) compared with the free peptide, indicating that peptide antigens adsorbed to hydroxyapatite nanoparticles may be useful, in diagnostic assays, for differentiating between tuberculosis (TB)-infected and vaccinated animals.     

Macrophages are a significant source of type 1 cytokines during mycobacterial infection

T-helper 1 (Th1) cells are believed to be the major producer of the type 1 cytokine interferon-gamma (IFN-gamma) in cell-mediated immunity against intracellular infection. We have investigated the ability of macrophages to release type 1 cytokines and their regulatory mechanisms using both in vivo and in vitro models of pulmonary mycobacterial infection. During pulmonary infection by live Mycobacterium bovis bacilli Calmette-Guérin (BCG) in wild-type mice, lung macrophages released interleukin-12 (IL-12), IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha), and expressed surface activation markers. However, macrophages in infected IL-12(-/-) mice released TNF-alpha but not IFN-gamma and lacked surface activation makers. In freshly isolated lung macrophages from naive IL-2(-/-) mice, mycobacteria alone released TNF-alpha but not IFN-gamma, whereas exogenously added IL-12 alone released a minimum of IFN-gamma. However, these macrophages released large quantities of IFN-gamma upon stimulation with both mycobacteria and IL-12. In contrast, mycobacteria and exogenous IFN-gamma released only a minimum of endogenous IFN-gamma. Endogenous IL-18 (IFN-gamma-inducing factor) played little role in IFN-gamma responses by macrophages stimulated by mycobacteria and IL-12. Our data reveal that macrophages are a significant source of type 1 cytokines during mycobacterial infection and that both IL-12 and intracellular pathogens are required for the release of IFN-gamma but not TNF-alpha. These findings suggest that macrophages regulate cell-mediated immunity by releasing not only IL-12 and TNF-alpha but also IFN-gamma and that full activation of IFN-gamma response in macrophages is tightly regulated.     

Reciprocal expression of interferon gamma or interleukin 4 during the resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets

We purified poly(A)+ mRNA from the spleen and lymph nodes at designated times after infection with Leishmania major in genetically susceptible BALB/c and resistant C57BL/6 mice. The steady-state levels of IL-2, IFN-gamma, IL-4, and IL-1 beta mRNA were determined using Northern hybridizations. IL-2 mRNA levels in the infected organs of BALB/c and C57BL/6 mice were comparable after infection, but IFN-gamma and IL-4 mRNA levels were reciprocally expressed. Levels of IFN-gamma mRNA in C57BL/6 draining nodes and spleen were significantly greater than in BALB/c mice except at 4 and 6 wk of infection, when splenic IFN-gamma mRNA levels were transiently comparable. In contrast, IL-4 mRNA was apparent only in BALB/c and not in C57BL/6 nodes and spleen. Tissue levels of IL-1 beta mRNA were 10-20-fold greater in BALB/c mice. BALB/c mice were pretreated with GK1.5 mAb, a manipulation that promotes healing of subsequent infection by transiently depleting L3T4+ cells. At 8 wk of infection, by which time lymphoid organs were repopulated with L3T4+ cells, GK1.5-pretreated BALB/c mice produced IFN-gamma, but not IL-4 message. Serum levels of IgE were markedly elevated in infected BALB/c, but not in infected C57BL/6 or GK1.5-pretreated BALB/c mice, consistent with in vivo biologic activity of IL-4 in nonhealing mice. Treatment of infected BALB/c mice with neutralizing anti-IL-4 antibody abolished the elevation of serum IgE and significantly attenuated the progression of disease as assessed by size and ulceration of the lesion, and by reduction in the number of tissue parasites. Both protective and deleterious responses to Leishmania infection have previously been shown to be L3T4+ cell dependent. Our findings are consistent with the differential expansion of protective, IFN-gamma-producing Th1 cells in healing mice, and the expansion of deleterious, IL-4-producing Th2 cells in nonhealing mice. The inverse relationship of IFN-gamma and IL-4 gene expression during leishmaniasis may underlie the divergence of cellular and humoral immunity that occurs during chronic infection with Leishmania and possibly other intracellular parasites.     

Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation.

Expression of the macrophage mannose receptor is inhibited by interferon gamma (IFN-gamma), a T helper type 1 (Th-1)-derived lymphokine. Interleukin 4 (IL-4), a Th-2 lymphocyte product, upregulates major histocompatibility class II antigen expression but inhibits inflammatory cytokine production by macrophages. We have studied the effect of IL-4 on expression of the macrophage mannose receptor (MMR) by elicited peritoneal macrophages. We found that recombinant murine IL-4 enhances MMR surface expression (10-fold) and activity (15-fold), as measured by the respective binding and degradation of 125I-mannose-bovine serum albumin. Polymerase chain reaction analysis of cDNAs from purified primary macrophage populations revealed that MMR, but not lysozyme or tumor necrosis factor alpha, mRNA levels were markedly increased by IL-4. The above effects were associated with morphologic changes. These data establish IL-4 as a potent and selective enhancer of murine MMR activity in vitro. IL-4 induces inflammatory macrophages to adopt an alternative activation phenotype, distinct from that induced by IFN-gamma, characterized by a high capacity for endocytic clearance of mannosylated ligands, enhanced (albeit restricted) MHC class II antigen expression, and reduced proinflammatory cytokine secretion.     

Genetic variability in the immune-inflammatory response after major burn injury

Thermal injury induces immune dysfunction and alters numerous physiological parameters. Studies have proposed that genetics influence the outcome after traumatic injury and/or sepsis, however, the contribution of genetics to the immune-inflammatory response postburn has not been investigated. In this study, mice of three distinct genetic backgrounds (C57BL/6NCrlBR, BALB/cAnNCrlBR, and 129S6/SvEvTac) were subjected to thermal injury or a sham procedure, and 3 days later, blood and splenic immune cells (splenocytes and macrophages) were isolated for analysis. Splenocytes from the C57BL/6NCrlBR strain displayed suppressed splenic T cell proliferation postinjury, whereas the other strains were unaffected. Burn injury also induced a shift toward a Th2-type T-cell response (suppressed IFN-gamma production) in the C57BL/6NCrlBR strain, but not in the other strains. Macrophages from C57BL/6NCrlBR and 129S6/SvEvTac mice were highly proinflammatory with elevated productive capacity for TNF-alpha and nitric oxide, whereas no such changes were observed in macrophages for BALB/cNCrlBR mice. C57BL/6NCRLBR macrophages produced increased IL-10 levels postburn, and BALB/cNCrlBR macrophages had suppressed IL-10 production postinjury. No differences in fasting blood glucose and insulin were observed after thermal injury. However, significant postburn weight loss was observed in the BALB/cNCrlBR and 129S6/SvEvTac strains, but not in the C57BL/6NCrlBR strain. In summary, these findings support the concept that the immune-inflammatory response postburn is influenced by genetic make-up. Further elucidation of the influence of genetics under such conditions is likely to contribute to the improvement in existing, and development of new, therapeutic regimes for burn patients.     

Intracellular killing of Brucella melitensis in human macrophages with microsphere-encapsulated gentamicin

OBJECTIVES: Treatment of human brucellosis demands antibiotic targeting into the mononuclear-phagocytic system. The aim of this work was to prepare and characterize particulate carriers containing gentamicin and to study their interactions with phagocytic cells and bactericidal activity against intracellular Brucella melitensis. METHODS: Different poly(lactide-co-glycolide) (PLGA) polymers with free carboxylic end-group were used to formulate micro- and nanoparticles containing gentamicin, by a water-oil-water solvent-evaporation technique. PLGA 502H and 75:25H microparticles were selected because they showed the highest gentamicin loadings as well as good physico-chemical properties and sustained release in vitro. RESULTS: Gentamicin-containing microspheres of both polymers were successfully phagocytosed by infected THP-1 human monocytes, and immunocytochemistry studies revealed that the antibiotic reached Brucella-specific compartments. A dose of 30 microg of encapsulated gentamicin was able to reduce intracellular Brucella infection by 2.2 log. CONCLUSIONS: Altogether, these results suggest that 502H and 75:25H microspheres are suitable carriers for gentamicin targeting inside human macrophages and thus for brucellosis treatment.     

Stimulation with cytokines enhances penetration of azithromycin into human macrophages.

An effective intracellular concentration of an antimicrobial agent is essential for therapy of infections caused by organisms of the Mycobacterium avium complex. We previously reported on the effect of the combination of azithromycin and tumor necrosis factor (TNF) against M. avium infection in macrophages. We now report that stimulation of macrophages either with recombinant human gamma interferon (IFN-gamma, 10(2) U/ml) or with recombinant human TNF-alpha (10(2) U/ml) resulted in an increase in the intracellular concentration of azithromycin by approximately 200% within 3 h, compared with the concentration in unstimulated macrophages. Infection of macrophages with M. avium complex led to a decrease in the uptake of [14C]azithromycin by infected cells, compared with that by uninfected controls. Stimulation of infected macrophages with recombinant IFN-gamma or TNF-alpha overcame the inhibitory effect associated with infection. These results suggest that the increased bactericidal activity of the TNF-alpha-azithromycin or IFN-gamma-azithromycin combination against M. avium is related to enhanced uptake of the antibiotic by the stimulated phagocyte.