Hyaluronic Acid and Its Composites as a Local Antimicrobial/Antiadhesive Barrier.

Living in biofilms is probably the most common condition for bacteria and fungi and biofilm-related infections account for the majority of bacterial infectious diseases worldwide. Among others biofilm-related infections, those associated with implanted biomaterials have an enormous and still largely underestimated impact in orthopaedics and trauma, cardio-surgery and several other surgical disciplines. Given the limited efficacy of existing antibiotics in the prevention and treatment of bacterial biofilms, new strategies are needed to protect implants and host tissues, overcoming the striking ability of the microorganisms to adhere on different surfaces and to immediately protect themselves by forming the biofilm matrix. Adhesion is a necessary first step in microbial colonization and pathogenesis and provides a potential target for new preventive and treatment approach. Among various polymers, tested as antibacterial coatings, hyaluronic acid and some of its composites do offer a well-established long-term safety profile and a proven ability to reduce bacterial adhesion and biofilm formation. Aim of the present review is to summarize the available evidence concerning the antiadhesion/antibiofilm activity of hyaluronic acid and some of its derivatives to reduce/prevent bacterial adhesion and biofilm formation in various experimental and clinical settings.

Leucocyte esterase, glucose and C-reactive protein in the diagnosis of prosthetic joint infections: a prospective study.

Analysis of joint fluid is of paramount importance for the diagnosis of prosthetic joint infections. Different markers of inflammation and/or infection in joint fluid have been proposed for diagnosis of these infections. In this study we evaluated the performance of leucocyte esterase, C-reactive protein (CRP) and glucose assays in synovial fluids from 129 patients with septic (n = 27) or aseptic (n = 102) prosthetic joint failure. Samples were collected in serum tubes and centrifuged to limit the presence of corpuscle interfering with the assays. Determinations of leucocyte esterase and glucose were carried out by means of enzymatic colorimetric reactions performed on strips for urine analysis. Tests were considered positive when graded + or ++ whereas traces or absence of colour were considered negative. CRP was measured using an automated turbidimetric method and considered suggestive for infections when >10 mg/L. Leucocyte esterase was positive in 25/27 infected patients and negative in 99/102 not infected patients (sensitivity 92.6%, specificity 97.0%). CRP was higher than the threshold in 22/27 infected patients and in 6/102 not infected patients (sensitivity: 81.5%; specificity: 94.1%) whereas glucose showed the lowest sensitivity (77.8%) and specificity (81.4%), being negative in 21/27 and 19/102 infected and not infected patients, respectively. CRP led to a correct diagnosis in 19 of 22 patients with discordant esterase and glucose results. In conclusion, evaluation of leucocyte esterase, glucose and CRP may represent a useful tool for rapid diagnosis of prosthetic joint infections.

Stereotactic body radiation therapy and intensity modulated radiation therapy induce different plasmatic cytokine changes in non-small cell lung cancer patients: a pilot study.

PURPOSE: To assess kinetics of plasmatic cytokines during radiation therapy (RT) for locally advanced and early-stage non-small cell lung cancer (NSCLC). METHODS: This prospective study was conducted on 15 early-stage NSCLC underwent to extreme hypofractionated regimen (52 Gy in 8 fractions) with stereotactic body RT (SBRT), and 13 locally advanced NSCLC underwent to radical moderated hypofractionated regimen (60 Gy in 25 fractions) with intensity modulated RT (IMRT). For patients undergoing SBRT, peripheral blood samples were collected on the first day of SBRT (TFd), the last day (TLd) and 45 days (T45d) after the end of SBRT. For patients undergoing IMRT, blood samples were collected at: TFd, 2 weeks (T2w), 4 weeks (T4w), TLd, and T45d. The following cytokines were measured: IL-1, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17A, EGF, FGF-2, INF-γ, MIP-1α, MIP-1ß, TGF-α, TNF-α, and VEGF. Cytokine levels measured in different RT time and compared. RESULTS: No difference in baseline levels of cytokines was documented between patient radiation approaches (except for MIP-1α). For SBRT patients, a mean reduction of IL-10 and IL-17 plasma level was documented between TLd and TFd, respectively (p < 0.05). For IMRT patients, a statistically significant (p < 0.05) mean plasma level reduction was documented between T4w and TFd for all the following cytokines: IL-1, IL-1ra, IL-2, IL-12, FGF-2, MIP-1α, MIP-1ß, TGF-α, TNF-α, VEGF. CONCLUSIONS: SBRT and IMRT induce different plasmatic cytokine changes in NSCLC patients, supporting hypothesis that RT regimes of dose schedules and techniques have different impacts on the host immune response.

Prolonging culture to 15 days improves bacterial detection in bone and joint infections.

Since the optimal incubation period of cultures for diagnosis of bone and joint infections is still a matter of debate, the present study aimed to evaluate the effects of different incubation periods (5 and 15 days) on microbial isolation. Samples from 387 patients with bone and joint infections (including prosthetic ones) were analyzed from March 2012 to February 2014. In 197 patients (51 %) growth was obtained within 48 hrs, while in 124 (32 %) and 66 (17 %) patients cultures yielded positive results within and after 5 days of incubation, respectively. Of 449 microorganisms isolated, 247 grew within 48 hrs, 131 within the first 5 days of incubation while 71 were isolated after 5 days. Staphylococcus aureus was the most frequently isolated pathogen within 48 hrs, while Propionibacteria were prevalently isolated after 5 days of incubation. Interestingly, about 25 % of microorganisms isolated after 5 days of incubation were coagulase-negative staphylococci. Extending incubation period of broth cultures improves isolation rates of pathogens involved in bone and joint infections thus improving management of these infections.

Drowning.

Approximately 500,000 deaths due to drowning are reported annually, 30,000 of which are reported in Europe. Because of the relatively low incidence of drowning victims at emergency departments, most emergency physicians do not routinely handle drowning victims. Although confusion regarding the classification and pathophysiology of drowning could be reduced by following the Utstein style consensus, the application of therapeutic modalities and, most important, the estimation of probable prognostic outcomes remain difficult for emergency physicians. This article presents an overview of the classification, pathophysiology, emergency-department treatment and prognostic outcomes of drowning accidents.

Preservation of an injured vital tooth using ultrasonic device and mineral trioxide aggregate.

The purpose of this case report is to present a root fracture repair procedure for non devitalized injured tooth. One injured, non-endodontically treated maxillary anterior tooth in which an incomplete vertical root fracture involving only the buccal side was suspected, underwent an exploratory flap to visualize the pattern of bone loss and assess the type of root fracture. The pre-operative diagnosis was confirmed. A groove following fracture line was prepared using retro-tips driven by an ultrasonic device and sealed with Mineral Trioxide Aggregate (MTA), following filling of the bone defect with Calcium Sulphate. At 24 months follow up the case showed clinical and radiographic success. The present surgical approach showed preservation of function and vitality of tooth with a shallow incomplete vertical root fracture.

Is autologous platelet concentrate beneficial for post-extraction socket healing? A systematic review.

The aim of this systematic review was to assess if the use of autologous platelet concentrates may be beneficial to the healing of extraction sockets. Medline, Embase and Cochrane Central Register of Controlled Trials were searched using a combination of specific search terms. Hand searching of the relevant journals and of the bibliographies of reviews was also performed. Prospective comparative studies evaluating the effect of a platelet concentrate on fresh extraction sockets were included. Outcome variables related to hard and soft tissue healing, aesthetics and postoperative discomfort were considered. A methodological study quality assessment was made. The initial search yielded 425 articles, eight were finally included. 207 tooth extractions (104 tests and 103 controls) in 115 patients were evaluated. The articles provided a broad range of variable outcomes to assess the regenerative potential of platelet concentrate and its possible benefits to the treatment. Favourable effects on hard and soft tissue healing and postoperative discomfort reduction were often reported. A large heterogeneity was found regarding study design, sample size, surgical techniques and methods for preparing platelet concentrates. Standardization of experimental design is needed in order to detect the true effect of platelet concentrates in regenerative procedures of extraction sockets.

Plasma HHV-8 viral load in HHV-8-related lymphoproliferative disorders associated with HIV infection.

This is a mono-institutional analysis of the clinical features, immunological and virological findings, and prognostic factors of patients with HIV infection and HHV-8-lymphoproliferative disorders. Patients with Multicentric Castleman Disease and HHV-8-related lymphoma diagnosed and treated from April 1987 to June 2004 were included in the study. HHV-8 and HIV plasma viral load, CD4+ count, hematologic parameters, and general wellbeing (performance status) were assessed at the onset of the diseases and analyzed in order to identify possible prognostic factors. Nine patients with Multicentric Castleman disease, and 16 with HHV-8-related lymphomas (13 primary effusion lymphomas and 3 solid lymphomas), were diagnosed and treated out of 327 HIV-related non-Hodgkin's lymphomas. Four patients with Multicentric Castleman disease received only antiretroviral drugs; 5 HAART plus oral etoposide. Nine patients with primary effusion lymphoma were treated with a CHOP-like regimen (Cyclophosphamide, Prednisone anthracyclines, Vinca alkaloids, Bleomycin, Etoposide) and HAART; 1 with etoposide and HAART, 1 with HAART alone. The patients with solid lymphoma underwent CHOP-like chemotherapy. Patients with Multicentric Castleman disease showed lower median values of HHV-8 viral load and longer overall survival compared with HHV-8-related lymphomas. Patients with viral load of HHV-8, >40,000 cp/ml had a significant shorter overall survival. In the univariate analysis, HHV-8-related lymphoma, HHV-8 viral load >40,000 cp/ml and performance status >2 were associated with an increased risk of death. Multivariate analysis confirmed the diagnosis of lymphoma as an independent predictor of shorter survival.

Recent thymic emigrants in lymphoma patients with and without human immunodeficiency virus infection candidates for autologous peripheral stem cell transplantation.

Signal joint T cell receptor excision circles (sjTRECs) have been reported as a clinical marker to measure the potential for recovery of the immune system after immunosuppressive treatments. The aim of this study was to investigate the thymic regenerative potential in 55 human immunodeficiency virus (HIV)-1 infected (HIV(+)) and non-infected (HIV(-)) lymphoma patients, candidates for autologous stem cell transplantation (ASCT). Moreover, the possible associations between sjTRECs and other immunological and clinical parameters were examined. SjTRECs levels in peripheral blood mononuclear cells (PBMCs) were quantified by real-time polymerase chain reaction and T lymphocyte subsets were analysed by flow cytometry. Our data showed that sjTRECs were reduced in lymphoma patients compared to healthy controls, although a weak significant association between low sjTRECs levels and increasing age was maintained [odds ratio (OR) = 4.00; 95% confidence interval (CI) 1.09-17.17]. We found that different chemotherapeutic treatments seem to induce similar effects on the thymic reservoir, independently from their intensity (type and number of cycles of previous chemotherapy). Results from multivariate models including adjustment for patients' sex, type of lymphoma and type of chemotherapy showed that thymic output was independent from HIV infection (OR, 0.95; 95% CI 0.20-4.48). SjTRECs levels correlated with naive T cell subsets in overall lymphoma patients and after stratification by HIV infection (r > 0.37). HIV replication should be maximally suppressed to properly evaluate thymic output by sjTREC markers. Our results suggested that de novo T cell generation is maintained partially in pretreated recurrent lymphoma patients, candidates for ASCT, and could contribute to restore the immune function after transplantation.

Ca, Pi, and F in the fluid of biofilm formed under sucrose.

Calcium (Ca), inorganic phosphorus (P(i)), and fluoride (F) concentrations are low in the whole plaque biofilm formed under exposure to sucrose. It was hypothesized that this would be reflected in the biofilm fluid, where these low values should greatly influence the de/remineralization process. Dental biofilms were formed in situ over enamel blocks mounted in palatal appliances and exposed 8 times/day to distilled water, glucose+fructose, or sucrose solutions for 14 days. While Ca, P(i), and F concentrations in the whole biofilms were significantly lower in the glucose+fructose and sucrose groups, no effect on biofilm fluid was observed, even after a cariogenic challenge. An increase in whole biofilm mineral ions was observed 24 hrs after the carbohydrate treatments were suspended, but this effect was also not observed in the fluid. These results suggest that there is a homeostatic mechanism that maintains biofilm fluid mineral ion concentration, regardless of its total concentration in the whole biofilm.

Small non-coding RNAs and genomic imprinting.

Experimental and computer-assisted approaches have led to the identification of hundreds of imprinted small RNA genes, mainly clustered in two chromosomal domains (human 15q11-->q13 and 14q32 loci). The genes are only detected in placental mammals and belong to the C/D RNA and microRNA gene families. These are small non-coding RNAs involved in RNA-guided post-transcriptional RNA modifications and RNA-mediated gene silencing, respectively. Here, we discuss their potential functions and report the identification of novel small RNA genes lying within (or nearby) known imprinted chromosomal domains.

Box C/D RNA guides for the ribose methylation of archaeal tRNAs. The tRNATrp intron guides the formation of two ribose-methylated nucleosides in the mature tRNATrp.

Following a search of the Pyrococcus genomes for homologs of eukaryotic methylation guide small nucleolar RNAs, we have experimentally identified in Pyrococcus abyssi four novel box C/D small RNAs predicted to direct 2'-O-ribose methylations onto the first position of the anticodon in tRNALeu(CAA), tRNALeu(UAA), elongator tRNAMet and tRNATrp, respectively. Remarkably, one of them corresponds to the intron of its presumptive target, pre-tRNATrp. This intron is predicted to direct in cis two distinct ribose methylations within the unspliced tRNA precursor, not only onto the first position of the anticodon in the 5' exon but also onto position 39 (universal tRNA numbering) in the 3' exon. The two intramolecular RNA duplexes expected to direct methylation, which both span an exon-intron junction in pre-tRNATrp, are phylogenetically conserved in euryarchaeotes. We have experimentally confirmed the predicted guide function of the box C/D intron in halophile Haloferax volcanii by mutagenesis analysis, using an in vitro splicing/RNA modification assay in which the two cognate ribose methylations of pre-tRNATrp are faithfully reproduced. Euryarchaeal pre-tRNATrp should provide a unique system to further investigate the molecular mechanisms of RNA-guided ribose methylation and gain new insights into the origin and evolution of the complex family of archaeal and eukaryotic box C/D small RNAs.

Changes in thymic function in HIV-positive patients treated with highly active antiretroviral therapy and interleukin-2.

Despite its potent antiviral activity, highly active antiretroviral therapy (HAART) only exerts a marginal effect on CD4+ T-cell regeneration in HIV-infected subjects. Combination therapies aimed at boosting T-cell activity and maturation may provide an important contribution to the restoration of immune function. Here, we report the results obtained by a two-year follow-up of a cohort of HIV-infected patients treated with a combination of HAART and interleukin-2 (IL-2). In these patients, in addition to a series of quantitative virological and immunological parameters, we investigated T-cell regeneration by an immunophenotypic assay monitoring CD4+ naïve T cells, and by analysis of thymic function, through the quantification of the excision DNA products of T-cell receptor rearrangement (TRECs) in lymphocytes. Compared with HAART alone, we found that the IL-2 combination therapy was equally effective in reducing the levels of viremia and marginally more effective in decreasing proviral DNA load. Strikingly, the IL-2 combination produced a marked increase in the number of CD4+ T cells bearing a naïve phenotype (CD45RA+, CD62L+), which was apparent for over 96 weeks after therapy. To assess whether these cells were the product of improved T-cell generation, we exploited a competitive quantitative molecular assay to quantify TRECs in peripheral blood lymphocytes. Surprisingly, we found that the levels of these molecules were unchanged in these patients. These findings indicate that improved thymic function does not account for the early rise of CD4 naïve cells in HIV-positive patients treated with IL-2, and suggest that alternative mechanisms of T-cell maturation and differentiation are responsible for this event.

Effects of interleukin-2 therapy on the proliferation and differentiation of CD4/CD25 positive and CD4/CD25 negative cells in HIV+ patients.

Interleukin-2 has been widely used in HIV-1+ subjects as an immunoactivating agent. In this study, we investigated cytokine production, Ki67 antigen expression and the modulation of the surface phenotype of the CD4/CD25+ subset as compared to the reciprocal CD4/CD25- subset in IL-2-treated HIV+ patients. Our findings suggest that CD4 T cells are heterogeneous in responding to IL-2, because CD4/CD25+ cells sharply increased their "memory" phenotype, their Ki67 antigen expression and were the main in vivo targets for IL-2-dependent proliferation during therapy, while the percentages of IFN-gamma+ (terminally differentiated) cells remained unchanged at the end of therapy. Conversely, the CD4+/CD25- subpopulation showed an expansion of differentiated cells and a slight increase in the proliferation rate. The use of anti-retroviral therapy alone (HAART) reduced the proliferation and increased the differentiation of both CD4 subsets. Our data suggest that IL-2 has a moderate capacity to activate resting T cells in vivo and is probably unable to boost HIV-1 from latency to the replicative state.

Different rates of CD4+ and CD8+ T-cell proliferation in interleukin-2-treated human immunodeficiency virus-positive subjects.

BACKGROUND: Interleukin-2 (IL-2) has been used successfully to increase CD4 cell counts in patients who are human immunodeficiency virus (HIV) positive. The mechanisms involved in this phenomenon are unknown. We hypothesized that a differential proliferation rate of CD4+ compared with CD8+ lymphocytes could be related to the increase of CD4 counts and of CD4/CD8 ratios that occur in HIV+ patients during IL-2 treatment. METHODS: We enrolled in our study 14 HIV+ patients treated with IL-2 or with highly active antiretroviral therapy (HAART) during a 96-week observation period. Using flow cytometry, we measured longitudinally the expression of the Ki67 antigen in peripheral blood CD4+ and CD8+ lymphocyte subsets. RESULTS: Compared with HAART alone, IL-2 produced a rapid increase of Ki67+ proliferating CD4 cells and a concomitant increase of the CD4/CD8 ratios, whereas the corresponding CD8 proliferation increased slightly. On the contrary, HAART alone was effective in suppressing equally both CD4 and CD8 proliferation. CONCLUSIONS: Our results suggest a selective activity of IL-2 on CD4 T-cell proliferation; on the contrary, CD8-specific proliferation is affected minimally during treatment. This information may offer the potential to plan correctly immune activating regimens.

Lymphocyte subsets and viral load in patients with HIV-associated non-Hodgkin's lymphoma treated with anti-CD20 monoclonal antibody and chemotherapy.

The anti-CD20 monoclonal antibody Rituximab is a novel antitumor agent used in association with chemotherapy (CT) for the treatment of high-grade/intermediate non-Hodgkin's lymphomas (NHL) in HIV-negative populations. This therapeutic combination is currently also being explored in HIV-positive patients with NHL (HIV-NHL). The objective of our study was to determine CD4 and CD8T cell counts, HIV plasma viremia and proviral load in patients with CD20-positive HIV-NHL treated with Rituximab plus CT and highly active antiretroviral therapy (HAART). We studied eight patients with HIV-NHL treated by anti-CD20 and CT before, after three, and after six cycles of therapy; CD4, CD8 and CD19 lymphocyte subsets were measured by monoclonal antibodies and flow cytometry. HIV plasma viremia was determined by the b-DNA assay, and proviral load by a quantitative competitive PCR. CD4T cell counts remained stable after three cycles of therapy, while a significant reduction of this subset was present at the end of therapy. HIV plasma viremia was significantly reduced after the third cycle, but returned to pretreatment levels at the end of therapy; we also observed individual fluctuations of proviral load during therapy, this marker being increased in two out of three patients at the end of therapy. These observations suggest that Rituximab plus CT accelerated the rate of CD4 depletion and of HIV replication in the peripheral blood of HIV-NHL patients and that HAART may be able to delay these effects.

Effects of therapy with highly active anti-retroviral therapy (HAART) and IL-2 on CD4+ and CD8+ lymphocyte apoptosis in HIV+ patients.

The kinetics and effects of in vivo spontaneous apoptosis and activation-induced cell death (AICD) upon CD4+ and CD8+ lymphocyte subsets and CD4 naive cell numbers were studied in HIV+ subjects with CD4 pretreatment values > 200/mm3, who were subsequently treated for 48 weeks with HAART alone or in combination with six cycles of subcutaneous IL-2. Irrespective of the type of treatment, patients showed a statistically significant increase in CD4 cell counts after 4 weeks, although the CD4 naive subset only increased significantly in the IL-2-treated subjects at the end of treatment. The percentage of CD4 cells undergoing spontaneous apoptosis and AICD was significantly reduced in all patients after 4 weeks and this reduction was maintained until the end of therapy; however, the level always remained significantly higher in comparison with healthy subjects. A statistically significant reduction in CD8 apoptosis levels required at least 24 weeks of therapy. Together these data suggest that a reduction in the level of apoptosis may contribute to the early rise in CD4 numbers measured after HAART, but that later on HAART is unable to improve further this biological parameter. Although the use of IL-2 had no additional effects on spontaneous apoptosis and AICD, it may be beneficial by stimulating a late increase in the numbers of CD4 naive cells in HIV-treated subjects.

Kinetics of lymphokine production in HIV+ patients treated with highly active antiretroviral therapy and interleukin 2.

This study presents the kinetics of CD4/CD25 cell numbers, serum sCD25 levels, and intracellular production and release of interleukin-2 (IL-2) and interleukin-16 (IL-16) in 11 HIV+ patients treated with six cycles of highly active antiretroviral therapy (HAART) plus six MUI of subcutaneous IL-2 compared to 10 HIV+ patients treated with HAART alone. IL-2 therapy induced moderate effects on CD4 T cell recovery and increased CD4/CD25+ cells and sCD25 levels after 2 weeks, while intracellular and secreted IL-2 was reduced and IL-16 was increased at the same time point. After 24 weeks, while HAART-treated patients had increased IL-2 production, in IL-2 treated patients, cytokine production was unaltered compared to pretreatment values. Decreased in vitro IL-2 production may depend on a feedback inhibition by IL-2 infusion. Because of its known antiviral effects, the increased IL-16 production seen after 2 weeks in IL-2-treated individuals may produce beneficial effects on HIV disease. The kinetics of cytokine production may serve to define better the use IL-2 in clinical trials.