Antibacterial and antibiofouling clay nanotube-silicone composite.

INTRODUCTION: Invasive medical devices are used in treating millions of patients each day. Bacterial adherence to their surface is an early step in biofilm formation that may lead to infection, health complications, longer hospital stays, and death. Prevention of bacterial adherence and biofilm development continues to be a major healthcare challenge. Accordingly, there is a pressing need to improve the anti-microbial properties of medical devices. MATERIALS AND METHODS: Polydimethylsiloxane (PDMS) was doped with halloysite nanotubes (HNTs), and the PDMS-HNT composite surfaces were coated with PDMS-b-polyethylene oxide (PEO) and antibacterials. The composite material properties were examined using SEM, energy dispersive spectroscopy, water contact angle measurements, tensile testing, UV-Vis spectroscopy, and thermal gravimetric analysis. The antibacterial potential of the PDMS-HNT composites was compared to commercial urinary catheters using cultures of E. coli and S. aureus. Fibrinogen adsorption studies were also performed on the PDMS-HNT-PEO composites. RESULTS: HNT addition increased drug load during solvent swelling without reducing material strength. The hydrophilic properties provided by PEO were maintained after HNT addition, and the composites displayed protein-repelling properties. Additionally, composites showed superiority over commercial catheters at inhibiting bacterial growth. CONCLUSION: PDMS-HNT composites showed superiority regarding their efficacy at inhibiting bacterial growth, in comparison to commercial antibacterial catheters. Our data suggest that PDMS-HNT composites have potential as a coating material for anti-bacterial invasive devices and in the prevention of institutional-acquired infections.

Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis.

Recent studies have suggested that T cells play a critical role in the pathogenesis of psoriasis. Guttate psoriasis is a well-defined form of psoriasis frequently associated with streptococcal throat infection. This study tested the hypothesis that T cells in acute guttate psoriasis skin lesions may be activated by streptococcal superantigens. Peripheral blood as well as lesional and perilesional skin biopsies were analyzed for T cell receptor V beta repertoire using monoclonal antibodies against 10 different V beta families. Skin biopsies from all patients with acute guttate psoriasis, but not skin biopsies from patients with acute atopic dermatitis or inflammatory skin lesions induced in normal subjects with sodium lauryl sulfate, demonstrated selective accumulation of V beta 2+ T cells (P < 0.05). The expansion of V beta 2+ T cells occurred in both the CD4+ and the CD8+ T cell subsets. Sequence analysis of T cell receptor beta chain genes of V beta 2-expressing T cells from skin biopsies of patients with guttate psoriasis showed extensive junctional region diversity that is more compatible with a superantigen rather than a conventional (nominal) antigen-driven T cell response. All streptococcal isolates from patients with guttate psoriasis secreted streptococcal pyrogenic exotoxin C, a superantigen known to stimulate marked V beta 2+ T cell expansion. These data support the concept that acute guttate psoriasis is associated with superantigenic stimulation of T cells triggered by streptococcal superantigen(s).

Epidermal HLA-DR and the enhancement of cutaneous reactivity to superantigenic toxins in psoriasis.

Streptococcal and staphylococcal superantigens (SAg's) have been implicated in the pathogenesis of inflammatory skin diseases, but the mechanisms by which these toxins act are unknown. The present study assessed the ability of nanogram quantities of topically applied purified toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxin type B, and streptococcal pyrogenic enterotoxin types A and C to induce inflammatory reactions in clinically uninvolved skin of normal controls and subjects with psoriasis, atopic dermatitis, and lichen planus. These SAg's triggered a significantly greater inflammatory skin response in psoriatics than in normal control subjects or in subjects with atopic dermatitis or lichen planus. Surprisingly, skin biopsies did not exhibit the T-cell receptor Vbeta stimulatory properties predicted for SAg-induced skin reactions. By 6 hours after patch testing with SAg's, TNF-alpha mRNA had increased in the epidermis (but not the dermis) in biopsies from psoriatics, compared with controls. Immunohistochemical studies revealed significantly higher HLA-DR expression in keratinocytes from psoriatics than from controls. However, a mutant TSST-1 protein that fails to bind HLA-DR did not elicit an inflammatory skin reaction. These results indicate that keratinocyte expression of HLA-DR enhances inflammatory skin responses to SAg's. They may also account for previous studies failing to demonstrate selective expansion of T-cell receptor Vbetas in psoriatics colonized with SAg-producing Staphylococcus aureus, and they identify a novel T cell-independent mechanism by which SAg's contribute to the pathogenesis of inflammatory skin diseases.

Size analysis of heterogeneous nuclear RNA in an aqueous gel system.

We have analyzed the size distribution of heterogeneous nuclear RNA (hnRNA) from cultured murine cells in an aqueous gel system consisting of 1.8% polyacrylamide and 0.5% agarose. hnRNA, labeled in vivo with 32P at a low specific activity, was labeled in vitro at its 3'-end with NaB3H4, and fractionated by gel electrophoresis. The ratio of log 32P/3H cpm was determined for each gel fraction and shown to be linearly related to distance migrated. The ratio of 32P/3H cpm was used to predict the molecular weight of 28-S and 18-S RNAs and shown to be within approx. 10% of the known molecular weight. Denaturation of hnRNA with heat or dimethyl sulfoxide gave similar number and weight average molecular weight when analyzed by gel electrophoresis. The same hnRNA analyzed by sucrose gradient velocity sedimentation had a similar number average molecular weight but much higher weight average molecular weight.

A potential role for superantigens in the pathogenesis of psoriasis.

Psoriasis is a complex inflammatory skin disease in which local vascular changes, T-cell activation, abnormal keratinocyte proliferation and differentiation, and neutrophil activation all contribute to the ongoing disease process. Because of recent interest in T-cell activation as a trigger for psoriatic lesions, we hypothesized that psoriasis may be triggered by superantigens, e.g., toxins of microbial origin that stimulate T cells expressing particular T-cell receptor (TCR) beta chain variable (V beta) gene segments. Lesional skin biopsies and peripheral blood from two patients with acute exacerbations of their psoriasis that appeared to be triggered by infection were analyzed for TCR V beta gene expression using monoclonal antibodies directed against V beta 5.1, 5.2, 6.7, 8.1, and 12. Skin biopsies from both patients demonstrated a different pattern of V beta expansion that correspond to the V beta pattern expected to be induced by the type of superantigen expressed during the infection. In contrast, using immunofluorescence and flow cytometry, peripheral blood T cells from these patients did not demonstrate any expansion of the 5 V beta subsets studied. These observations support the hypothesis that local activation of cutaneous T cells in psoriasis may be caused by a superantigen and provides a new direction for investigating the pathogenesis of this complex and fascinating skin disorder.

Cutaneous exposure to the superantigen staphylococcal enterotoxin B elicits a T-cell-dependent inflammatory response.

We analyzed the impact of superantigens secreted by skin-colonizing Staphylococci on the skin and the associated lymphoid tissue following epicutaneous application and intracutaneous injection of small amounts of staphylococcal enterotoxin B (SEB). A single intracutaneous injection of 50 ng of SEB elicited a strong inflammatory response in the skin of BALB/c mice. Three to 6 h later, we observed langerhans cell activation, mast cell degranulation, vasodilation, upregulation of ICAM-1, and induction of VCAM-1 on dermal blood vessels, with vascular adhesion of granulocytes. by 12 to 24 h, cell infiltration of the dermis increased, reaching the epidermis. Among the infiltrating leukocytes, a substantial number of eosinophils was found. After 48 h, the infiltrate was dominated by mononuclear cells. The response to SEB was dose-dependent, and signs of inflammation slowly disappeared over 5 to 7 days. Although the induction of VCAM-1 on dermal blood vessels suggested a role for interleukin-1/tumor necrosis factor-alpha in this reaction, the activation of monocytes/macrophages was not able to substitute for lymphocytes, as severe combined immunodeficiency (SCID) mice (which are lymphocyte-deficient) did not mount an inflammatory skin response to intradermal injection of SEB. The fact that nude mice (T-cell-deficient) also did not mount an inflammatory response to SEB indicated the T-cell dependency of the response. The V beta specificity of the SEB effect was demonstrated by the fact that SJL/J mice, which lack V beta 8+ T cells (the major SEB-reactive T cell population in mice), exhibited much weaker responses. Deletion or tolerization of SEB-reactive V beta T cells was not observed after a single intradermal injection of such minute amounts of SEB.

Spectrum of inclusion body myositis.

The clinical, laboratory, and biopsy features are described for a large group of patients with inclusion body myositis (IBM) (15 men and four women; mean age, 63 years). A quantitative histopathologic analysis of muscle biopsy specimens revealed less fiber necrosis and endomysial and perivascular inflammation in IBM than in polymyositis (PM) and dermatomyositis, but a more frequent occurrence of dark-angular and hypertrophied fibers. Rimmed vacuoles were present in 3.4% of all fibers and 15- to 18-nm filaments were identified in the biopsy specimens of nine of 11 patients. A panel of monoclonal antibodies immunoreactive with lymphocytes and cells of monocyte/macrophage lineage suggested that the inflammatory reaction in IBM was similar to that in PM (but not dermatomyositis) and mediated by cellular immune responses. These studies confirm the clinical and histopathologic distinctions between IBM and chronic PM, and that differentiation between these disorders is often difficult.

Simultaneous in situ detection of IgE receptors and cytoplasmic granules in murine cutaneous mast cells.

A method is described for the simultaneous in situ detection of surface receptors and cytoplasmic granules in mast cells of frozen sections of mouse skin. Surface IgE receptors are detected after saturation of the receptors with a murine monoclonal antibody of IgE isotype. The latter is subsequently detected by monospecific rabbit anti-mouse IgE (purified on protein A-Sepharose) followed by FITC-conjugated goat anti-rabbit IgG. Cytoplasmic granules are localized by staining with TRITC-avidin conjugate. Normal cutaneous mast cells show green surface fluorescence and red intracellular granules. The method is specific for mast cells; other cells with Fc receptors for IgE are not seen. This method should be useful in the study of situations in which mast cells may have become degranulated.

Characterization of mononuclear cells in cytocentrifuge and imprint preparations using monoclonal antibodies and an avidin--biotin immunoperoxidase staining system.

The application of an avidin--biotin immunoperoxidase staining system was investigated for the characterization of mononuclear cells in cytocentrifuge and imprint preparations with monoclonal antibodies. The system was found to be very sensitive, permitting the use of high dilutions of monoclonal antibody. The system also permitted the study of single cell morphology. Its utilization should be complementary to the study of patterns of cellular proliferation in frozen section material. Furthermore, it may lend itself to automation for quantitative analysis of cell populations.

Acid alpha-naphthyl acetate esterase activity in stored buffy coat smears.

The stability of acid alpha-naphthyl acetate esterase activity in smears of buffy coats was studied. Fixed smears may be stored up to 20 days at 25 degrees C. Unfixed smears may be stored up to 24 hr at 25 degrees C before there is a significant decrease in activity. In constant, fixed or unfixed smears held at -10 degrees C begin to lose activity within 24 hr.

Liver T cell subsets and adhesion molecules in murine graft-versus-host disease.

Murine GVHD across multiple minor histocompatibility barriers (B10.D2 into irradiated BALB/c) results in cell-mediated destruction of bile ducts inside the liver. Similar changes are characteristic of hepatic GVHD in humans following BMT. We have defined the phenotypes of inflammatory cells and the accessory/adhesion molecules expressed in the liver between day 7-14 of murine GVHD. T cells (CD3+) comprised 65% of hepatic inflammatory cells. alpha-beta and gamma-delta cells accounted for 92 and 8%, respectively of hepatic T cells. The percentage of CD4+ cells (29%) was 3 times that of CD8+ cells (11%). Lymphocyte function-associated antigen-1 (LFA-1) was expressed by the majority of inflammatory cells. Thirty per cent of the cells were positive for Mac-1, a differentiation marker of macrophages, large granular lymphocytes, and natural killer cells. Expression of intercellular adhesion molecule-1 and major histocompatibility complex class II (IAd) molecules on bile duct epithelial and portal vein endothelial cells was induced during GVHD. These results suggest that hepatic GVHD is induced by donor alpha-beta T cells through mechanisms that may involve CD4:1Ad and LFA-1:ICAM-1 interactions.

Candida albicans induces selective expansion of human T lymphocytes expressing the T-cell receptor variable region V beta 5.1.

Candida albicans is a common pathogen which can present major problems as an opportunistic skin pathogen in patients with immunodeficiency. The exact nature of the T cell responses to C. albicans is poorly understood. The purpose of this study was to determine whether C. albicans could stimulate the selective expansion of T lymphocytes expressing particular V beta gene segments. Human T lymphocytes stimulated in vitro with an extract of C. albicans were analyzed for T cell receptor V beta gene expression by using a quantitative PCR technique. We found that stimulation of peripheral blood mononuclear cells (PBMC) produced a selective increase in the expression of V beta 5.1 and 5.2 gene transcripts. Using cytofluorographic analysis with available anti-V beta monoclonal antibodies, we verified that there was a significant selective expansion (P = 0.035) of V beta 5.1 positive T lymphocytes in PBMC from six subjects stimulated in vitro with C. albicans. PCR analysis of V beta 5.1 expansion in 10 subjects showed increases in V beta 5.1 gene transcripts in 7/10 subjects. More importantly, analysis of the T cell infiltrate 48 h after intradermal injections with C. albicans also showed significant expression of V beta 5.1 in the infiltrates, along with the infiltration of V beta 8.1 + T cells. The selective expansion of V beta 5.1 bearing T lymphocytes in PBMC stimulated with C. albicans and in skin test reactions to C. albicans suggests that a restricted population of T cells react to C. albicans. Furthermore, our present data raise the provocative possibility that one or more antigens in C. albicans can act as a superantigen, producing selective expansion of a population of T lymphocytes bearing a particular V beta specificity.

Evaluation of a tissue transport medium for immunological characterization of benign and malignant lymphoid tissues.

A tissue transport medium was evaluated for its usefulness in immunologic characterization of lymphocytes in tissue sections. A variety of lymphoid tissues were studied, including normal tissue, hyperplastic tissue and malignant lymphoma. Approximately equal parts of biopsy samples were taken and one part was immediately snap-frozen while the other was held in transport medium for up to one week before processing for frozen section. Sections were stained with fluorescein-conjugated anti-immunoglobulin antisera and with a variety of murine monoclonal antibodies to T-cell antigens or Ia antigen followed by staining with fluorescein-conjugated goat-antimouse gamma globulin. In all cases studied, there was complete agreement in the staining patterns of tissues which were either immediately snap-frozen or held in transport medium.

Hematology reference values. Analysis by different statistical technics and variations with age and sex.

The following hematologic parameters in 1,744 healthy men and women aged 16 to 89 years were studied: leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration. The frequency distributions of all parameters were statistically significantly deviated from Gaussian distributions. Standard Gaussian statistical methods as well as nonparametric methods were used to establish 95% reference intervals for each parameter. The effects of sex and age upon these parameters were also studied. Significant age differences, which were most striking for leukocyte count, erythrocyte count, MCV, and MCH, were detected.

Mononuclear cells in malignant and benign human breast tissue.

The mononuclear cell infiltrate in benign and malignant breast tissue was studied using monoclonal antibodies with an immunoenzymatic technique. Infiltrating duct carcinomas contained a mononuclear cell infiltrate made up mostly of T lymphocytes, with minor B-cell and macrophage populations. The most preponderant T-cell subset was the T8, cytotoxic-suppressor cell. Some tumors contained T6-positive cells, possibly immature T cells. In contrast, stromal lymphocytes in benign fibrous mastopathy were largely T cells, but the T4, helper-inducer cell was the most preponderant, and T6 cells were not detected. Intraepithelial lymphocytes were identified in benign breast tissue. Intraepithelial lymphocytes are T cells, with a great preponderance of the T8 phenotype. The T6-positive cells were also identified within the epithelium, but they seemed to represent myoepithelial cells rather than immature T cells.

Open comparison study of oxidative stress markers between patients with chronic renal failure in conservative therapy and patients in haemodialysis.

AIMS: Oxidative stress is defined as tissue damage caused by an imbalance between the excessive production of the oxidant components and an insufficient defence mechanism. It has been observed, as in patients with chronic kidney failure, that there exists a pro-oxidant state characterised by a higher level of reactive oxygen species (ROS), and that oxidative stress in dialysis patients can be aggravated by the activation of neutrophils associated with the production of free radicals. In patients undergoing dialysis even the molecules other than those of cytokines can accumulate and provoke an inflammatory response. This study proposes an analysis based on the total antioxidant capacity (TAC), thiol concentration (TC) and pro-oxidant capacity (POC) in the serum of various groups of patients: one group of dialysis subjects who had been undergoing substitutive treatment for more than ten years at the time of the study; one group of subjects with chronic renal insufficiency in its pre-terminal stage and subjected to conservative therapy; and the control group consisting of healthy volunteers. MATERIALS AND METHODS: Three types of tests were employed to assess the level of oxidative stress: oxy-adsorbent test, d-ROMS test, and SHp- test. Thirty-three subjects were selected: 11 undergoing haemodialysis for over then years; 14 patients with chronic kidney failure in its pre-terminal stage, and 8 normal subjects. In patients undergoing renal substitutive treatment, the serum levels (mean±sd) of TAC were 272.98±20.54; TC, 249.19±92.48, and POC, 95.06±15.70. In patients with chronic renal insufficiency in its pre-terminal stage and undergoing conservative treatment, the value of TAC was 226.5±27.89; TC, 336.42±102.08; and POC, 80.78±15.69. The levels of TAC in the serum of the controls were 335.62±46.35; TC, 434.09±22.23; and POC, 56.31±7.41. CONCLUSION: The analysed data suggest that in dialysis the patients with chronic kidney failure, whether undergoing conservative therapy during its pre-terminal stage or in substitution treatment during its terminal stage, there is a reduction in the antioxidant defence (in terms of TAC and thiolic barrier) and an increase in POC compared to the healthy subjects in the control group. Uraemia and haemodialysis increase the inflammatory response: an initial signal provokes the inflammatory state with the production of cytokines and free radicals or reactive oxygen, so that the lack of an antioxidant defence mechanism can bring about a vicious circle with the continual production of other free radicals.

Assessment of the oxidative stress markers in patients with chronic renal insufficiency undergoing dialysis treatment.

AIMS: Uraemia is a disease characterised by a significant oxidative stress, and it is a wide agreement that oxidative stress which accompanies uraemia, increases the inflammatory state and promotes the alterations of tiny molecules such as amino acids, proteins, lipids, and carbohydrates. There are numerous records of how ROS are connected to the pathology of end stage renal disease (ESRD). The aim of this study is to assess the Total Antioxidant Capacity (TAC), the Thiolic Capacity (TC) and the Pro-Oxidant Capacity (POC) in the serum of patients undergoing dialysis treatment. MATERIALS AND METHODS: Forthy-six patients have been recruited (32 men, 14 women; mean age 68.5±15.8) who received hemodialytic treatment triweekly. Three methods have been used: oxy adsorbent test (mmol/l) to determine TAC values; d-ROM test (mg/100 mg/H2O2) to determine POC; SHp-test (mmol/l) to determine TC. RESULTS: In patients who underwent hemodialysis, TAC levels were: pre-dialysis, 265.9±30.5; post-dialysis, 300.0±40.6; TC levels: pre-dialysis, 267.4±59.1; post-dialysis, 303.2±116.7; POC levels: predialysis, 86.2±16.9; post-dialysis, 98.6±17.0; NS: TAC, 335.6±46.3; TC, 434.0±22.2; POC, 56.3±7.4. TAC in both pre- and post-dialysis is reduced compared to the NS (p < 0.05); moreover TAC increases after dialysis (p < 0.05). Pre- and post-dialysis TC is reduced compared to NS (p < 0.05); available TC increases after dialysis, although not statistically significant. Pre- and post-dialysis POC in patients undergoing dialysis is increased compared to the NS (p < 0.05); moreover, POC tends to increase after dialysis ( p < 0.05). The data obtained from our study also show that the TAC is reduced in the patients subjected to hemodialysis compared to the NS, both before and after dialysis treatment; TAC increased after dialysis, even though it did not reach the level of the control group. CONCLUSION: Our study has demonstrated that exists a profound imbalance between antioxidants and the production of ROS in ESRD patients, which determines oxidative stress and eventually leads to atherosclerosis and cardiovascular complications. This, in turn, represents the major cause of morbidity and mortality in these patients.

Oxidant/antioxidant status in patients with chronic HIV infection.

The infection caused by HIV leads to an activation of the immune system, which involves local and systemic oxidative stress. In HIV-positive (HIV+) patients, oxidative damage is the result of HIV infection and its progression through the replication of the virus. We have examined 52 subjects: 26 HIV+ patients, and 26 healthy subjects (NC). Analysis of the parameters of the oxidant/antioxidant status (total antioxidant capacity (TAC), hydroperoxides (free radicals, PRO), thiols as thiolic capacity, TC) was carried out by means of the OXY-Absorbent test, the d-Rom test, and the -SHp test, respectively. Healthy subjects presented the following values: TAC (micromol/ml) 259.5+/-40.5; TC (micromol/l) 434.09+/-18.31; PRO (mg/dl) 54.09+/-7.3; CD4+ cells (cells/ml) 850+/-333. Values of HIV+ patients were the following: TAC 218.73+/-18.55 (ns vs NC; TC 250.88+/-93.11 (p 0.001 vs NC); PRO 110.5+/-23.61 (p 0.0005 vs NC); CD4+ cells 354+/-323.35 (p 0.0005 vs NC). The statistical analysis shows a direct correlation between TAC vs CD4+ cells; an indirect correlation between hydroperoxides vs CD4+ cells; not significant result between thiolic capacity vs CD4+ cells; finally, good correlations between TAC, hydroperoxides, and thiolic capacity vs HIV-RNA. The data obtained have proven that HIV+ patients present a condition of important oxidative stress. We may affi rm that this disease concurs with an increase of extreme stress; a condition in which the antioxidant defences are present, but are insufficient in neutralising the damaging actions of reactive species of oxygen, thus contributing to an acceleration in the natural history of HIV infections.