Small interfering RNAs targeted to interleukin-4 and respiratory syncytial virus reduce airway inflammation in a mouse model of virus-induced asthma exacerbation.

Asthma exacerbations are caused primarily by viral infections. Antisense and small interfering RNA (siRNA) technologies have gained attention as potential antiasthma and antiviral approaches. In this study we analyzed whether gene silencing of interleukin (IL)-4 expression and respiratory syncytial virus (RSV) replication by RNA interference is able to suppress allergen- and virus-induced responses in a mouse model of virus-induced asthma exacerbation. Knockdown efficacy of IL-4 siRNA molecules was analyzed in the human HEK293T cell line by cotransfection of six different siRNAs with a plasmid carrying mouse IL-4. The most potent siRNA was then used in a mouse model of RSV-induced asthma exacerbation. BALB/c mice were sensitized intraperitoneally with ovalbumin (OVA) and then infected 12 days later intranasally with RSV Long strain (1×10(6) TCID50/mouse), followed 1 day later by intranasal challenge with OVA for 3 days. Mice were pretreated intranasally three times with either siRNA to IL-4 or GFP control, 2 days before, and on the first two OVA challenge days. siRNAs to RSV or rhinovirus control were inoculated intranasally once, 3 hr before RSV infection. Combined anti-IL-4 and anti-RSV siRNAs were able to significantly reduce total cell counts and eosinophilia in bronchoalveolar lavage fluid, development of airway hyperresponsiveness, and airway inflammation and to downregulate IL-4 mRNA expression and RSV viral RNA, but to upregulate IFN-γ levels in lung tissues. We conclude that anti-helper T cells type 2 and antiviral siRNAs may constitute a new therapeutic approach for treatment of virus induced asthma exacerbations.

Cytokine gene expression in the skin and peripheral blood of atopic dermatitis patients and healthy individuals.

OBJECTIVES: Atopic dermatitis (AD) is an increasingly common, chronic, relapsing, inflammatory skin disease characterized by impaired epidermal barrier function and cutaneous inflammation. The prevalence of AD has steadily increased during the past few decades. The aim of this study was to comparatively investigate cytokine gene expression in the skin and peripheral blood of atopic dermatitis patients and healthy individuals. RESULTS: In the skin of patients with AD, a significant increase of the level of gene expression was observed for interleukin (IL)-2r (p < 0.0023), IL-5 (p = 0.002), IL-6 (p < 0.0023), IL-8 (p = 0.01), IL-12B (p < 0.0023), IL-10 (p < 0.0023), IL-23 (p = 0.002), IL-29 (p < 0.0023), and transforming growth factor beta (tGFbeta) (p < 0.0023) as compared to healthy individuals. In contrast, no difference between AD patients and healthy donors was detected with respect to cytokine gene expression in the peripheral blood. METHODS: Samples of skin and peripheral blood from 48 severe AD patients (SCORAD = 78.5 [57;89], IGA = 4.2 [3,9;4,7]) at the age of 17 to 45 years and 20 healthy donors aged from 19 to 32 years were analyzed for gene expression of cytokines using real-time reverse transcription polymerase chain reaction (RT-PCR). CONCLUSIONS: Activity of markers of chronic inflammation and Th1 immune response in severe AD, namely IL-2r, IL-8, IL-12B, IL-23, IL-29 and TGFbeta, as well as activity of anti-inflammatory IL-5 were predominant in the skin but not in the blood of AD patients.

Muropeptides trigger distinct activation profiles in macrophages and dendritic cells.

Bacterial peptidoglycan and its muropeptide derivatives potently activate mammalian innate immune system and are promising immunomodulators and vaccine adjuvants. However, their effects on human antigen-presenting cells, such as dendritic cells (DCs) and Mphi, are not fully understood. Lysozyme treatment of PG from Salmonella typhi yielded three muropeptides, GlcNAc-MurNAc-L-Ala-D-isoGlu-meso-DAP (GM-3P), GlcNAc-MurNAc-L-Ala-D-isoGlu-meso-DAP-D-Ala (GM-4P), and a dimer (GM-4P)(2), in which two GM-4P monomers are linked through their peptidic moieties. All three muropeptides induced TNF-alpha and IL-6 production by Mphi (GM-3P>GM-4P>>(GM-4P)(2)), but failed to trigger TNF-alpha, IL-6 and IL-12p70 production by immature DCs. At the same time, muropeptide-stimulated DCs abundantly produced inflammatory chemokines IL-8, MIP-1 alpha and MIP-1 beta, as well as displayed signs of phenotypic and functional maturation. Thus, muropeptide-dependent pro-inflammatory cytokine production is repressed in DCs. While this defect may be partly compensated in vivo by muropeptide-activated Mphi, neither Mphi nor DCs produce Th1- or Th17-polarizing cytokines upon muropeptide stimulation, which may contribute to the preferential induction of Th2 responses by muropeptides and should be taken into account when designing muropeptide-based immunomodulators and adjuvants.

Functional changes of macrophages induced by dimeric glycosaminylmuramyl pentapeptide.

Under the influence of dimeric glucosaminylmuramyl pentapeptide (diGMPP), a component of bacterial cell wall, macrophages undergo certain changes similar to those associated with dendritic cell (DC) maturation. The effect of diGMPP on DCs resulted in maturation and expression of CD83. Macrophages treated with diGMPP displayed reduced phagocytic activity and elevated ability to kill ingested bacteria. Reduced phagocytosis may be due to phenotypic changes that occur in macrophages during the maturation process, such as reduced expression of receptors that mediate ingesting of microorganisms (CD16, CD64, and CD11b). Down-regulated expression of pattern-recognizing receptors (TLR2, TLR4, and CD206) was accompanied by elevated expression of antigen-presenting (HLA-DR) and costimulating molecules (CD86 and CD40), similar to alterations observed in maturating DCs. In addition, diGMPP treatment of macrophages resulted in enhanced synthesis of IL-12, TNF-alpha, and IL-1beta.

Study of interaction between the polyoxidonium immunomodulator and the human immune system cells.

Polyoxidonium (PO) is a high-molecular weight physiologically active compound with pronounced immunomodulating activity, an N-oxidized polyethylene-piperazine derivative. The aim of our work was to study cellular and molecular mechanisms of the action of PO on the human peripheral blood leukocytes. By means of flow cytometry it was established that the binding of fluorescein-isothiocyanate-labeled PO (FITC-labeled PO) occurs more rapidly with monocytes and neutrophils than with lymphocytes (7- to 8-fold weaker as compared with monocytes). Using colloidal gold-labeled PO and electron microscopy it was shown with that the preparation penetrates into leukocytes by endocytosis. PO is localized in endoplasmic vesicles of cellular cytosol. Analysis of one of the crucial signal transducer, the intracellular Ca(2+), performed with the Fluo-3 fluorescent dye, showed that PO does not induce Ca(2+) mobilization from the intracellular calcium stores and influx of extracellular Ca(2+). The study of the intracellular hydrogen peroxide (H(2)O(2)) production with the 2',7'-dichlorfluorescein indicator demonstrated that PO significantly increases the level of intracellular H(2)O(2) in monocytes and neutrophils, however, this increase is much less as compared with phorbol myristate acetate stimulation. The analysis of immunomodulating effect produced by PO proved its stimulating activity on some cytokines production in vitro, e.g. interleukin 1beta (IL-1beta), tumor necrosis factor (TNF)-alpha and IL-6. A dose-dependent increase in the intracellular killing by blood phagocytes was established under the action of PO.

Effect of polyoxidonium on the phagocytic activity of human peripheral blood leukocytes.

The effect of polyoxidonium on the functional activity of human peripheral blood phagocytes was studied in vitro. Polyoxidonium is an N-oxidized polyethylene-piperazine derivative, a water-soluble high-molecular synthetic immunomodulator. It was established that a one-hour incubation of leukocytes with polyoxidonium increases the ability of leukocytes to kill the ingested Staphylococcus aureus in a dose-dependent manner. This increase was observed in leukocytes obtained from healthy individuals and from patients with chronic granulomatous disease. The study of phagocyte spontaneous and stimulated chemiluminescence showed a significant decrease in the quantity of chemiluminescent impulses in the extracellular space in the presence of polyoxidonium both in luminol- and lucigenin-dependent chemiluminescence assays. Polyoxidonium proved to have an antioxidant activity at all doses tested (100, 250, and 500 &mgr;g/ml). Evaluation of the intracellular hydrogen peroxide (H(2)O(2)) level with a fluorescent indicator dichlorofluorescein showed that incubation with polyoxidonium leads to a higher luminescence intensity of dichlorofluorescein, thus indicating an increase in the intracellular H(2)O(2) level. This increase was not as substantial as in the case of stimulation with phorbol myristate acetate. When polyoxidonium was used at a dose of 500 &mgr;g/ml, the difference with the control was significant for neutrophils and monocytes. Polyoxidonium can be used as adjuvant in combined treatment of acute and chronic infections of any etiology, and in the treatment of chronic granulomatous disease and secondary immunodeficiences along with etiotropic drugs.

Some Peculiarities of Humoral Antibacterial Immunity in HIV-infected and AIDS Patients.

The investigation was outlined to study antibodies against some antigens of extracellular microbes associated with inflammation in broncho-pulmonary system and accessory nasal sinus - Staphylococcus aureus, Streptococcus pneumoniae, Branhamella catarrhalis - in individuals (18 patients) with different stages of HIV-infection. The level of antibodies was measured by ELISA and their Ab affinity was assessed by sodium thiocyanate-induced alteration of antibody-antigen interaction. To determine interrelations between antibody production and CD4(+) T lymphocyte number flow cytometry was employed. At the early stages of HIV-infection the levels of antibodies against Streptococcus pneumoniae and GMGM decreased, in comparison with HIV-negative donors. During HIV-infection course levels of antibodies against Staphylococcus aureus peptidoglycan, its antigen determinants and Streptococcus pneumoniae somatic antigen increased. Time affinity of antibodies against these antigens decreased. At all stages of HIV-infection and at all forms of its complications, we observed an increase of titer of antibodies to GMDP, antigenic determinant of peptidoglycan, which carried immunostimulating and adjuvant activities. HIV patients with CD4(+) T lymphocyte number <200 cells/&mgr;l displayed higher level of antibodies to bacterial antigens than that in patients with CD4(+) T lymphocyte number 200-400 per ml. The development of humoral immune response against some of extracellular bacteria is characterized, on the one hand, by their increased levels, and on the other hand, decreased affinity.

Competitive Specificity Analysis of Natural Antibodies against Epitope of Bacterial Cell Wall Peptidoglycoan: Glucosaminylmuramyl Dipeptide Carrying Adjuvant Activity.

The natural antibodies against glucosaminylmuramyl dipeptide (GMDP), the epitope of peptidoglycan of bacterial cell wall, isolated from human serum by thermal extraction possess a capability to cross-react with determinant of glycan chain - tetrasaccharide consisting of N-acetylglucosamine and N-acetylmuramic acid. The intensity of interaction of natural anti-GMDP-antibodies with specific ligand is significantly higher than with tetrasaccharide. The natural antibodies against tetrasaccharide carry properties of heteroclitic antibodies, i.e. the intensity of their interaction with heterologous ligand, GMDP, is significantly higher than with homologous one, tetrasaccharide GMGM. GMDP is supposed to be specific antigenic peptidoglycan determinant against which the antibodies reacting with various intensity to homologous (GMDP) and relative (tetrasaccharide) hapten are formed in the process of natural immunization.

Affinity of Serum Natural Antibodies against Peptidoglycan Bacterial Cell Component with Adjuvant Activity.

We studied affinity of natural antibodies of human serum against glucosaminylmuramyl dipeptide (GMDP), the epitope peptidoglycan bacterial cell wall component which carries adjuvant activity. Antibodies against GMDP were isolated from the blood sera of healthy donors using thermal extraction of antibodies from specific ligand on plastic. Determination of the dissociation constant (K(d)) showed equal K(d) in the serum and affinity-purified anti-GMDP-antibodies, i.e. extraction by this method led to the isolation of all subpopulations of antibodies in spite of their affinity. K(d) of serum, affinity-purified and monoclonal anti-GMDP-antibodies proved of low value - 10(-6) M, and according to this index anti-GMDP-antibodies may be classified between anti-protein and anti-carbohydrate antibodies.