Steroid use in measles: A retrospective cohort study during the 2017 outbreak in tertiary referral center, Rome and Latina, Italy.

INTRODUCTION: Since early January 2017, a new measles outbreak in Italy has been observed. The aim of the study was to compare features between adults and children measles cases and evaluate the effect of steroid treatment on the above parameters. METHODS: A retrospective multicenter, descriptive study was performed. We analyzed all patients admitted to the Department of Public Health and Infectious Diseases, Sapienza University, Rome and Latina, from January 2017 to December 2017 and discharged with diagnosis of measles. RESULTS: We identified 113 patients discharged with the diagnosis of measles infection cases of which 59 adults and 54 children (≤16 years). In adult population 32 patients (54 %) were males, with a median age of 30.5 years old and all unvaccinated (100 %). Keratoconjunctivitis 30 (50 %) was the most frequent complication. In pediatric population 27 (50 %) patients were males, with a median age of 3 years old. Information on measles vaccination status was available for only 21 (38.8 %) of cases. Keratoconjunctivitis 40 (74 %) was the most frequent complication. Analyzing the differences between adult and pediatric patients we found that children were significantly more likely to have keratoconjunctivitis and diarrhea as complications than adults in which the rate of thrombocytopenia and hepatitis was highest. Thirty-nine adult subjects (66 %) have been treated with systemic corticosteroids. CONCLUSIONS: Pediatric patients differ from adults in complications and liver involvement. Regarding steroids use, although there is no clear indication of steroid use during measles, there is no evidence of a worse outcome in our cases series.

TB specific intracellular cytokines production in Synovial liquid for diagnosis of tuberculous arthritis.

INTRODUCTION: Skeletal tuberculosis (TB) accounts for about 10 to 35% of extrapulmonary cases and the knee is the most frequent site after the spine and hip. The diagnosis is difficult and largely clinical. CASE PRESENTATION: This is a case of a young Pakistani man with a history of joint pain for about 4 years, who was diagnosed with chronic arthritis of the right knee. Microscopy of synovial fluid and conventional diagnostic tests to identify Mycobacterium tuberculosis were negative, while a non-classical method based on intracellular cytokine flow cytometry response of CD4 T-cells in synovial fluid helped us to address the diagnosis, which was subsequently confirmed by Polymerase Chain Reaction (PCR). CONCLUSIONS: Thanks to an innovative immunological approach, supported by PCR for detection of M. tuberculosis DNA, we were able to diagnose tuberculous arthritis of the knee, which allowed prompt initiation of treatment to reduce morbidity and mortality.

Impact of IFN-Free and IFN-Based Treatment on Blood Myeloid Dendritic Cell, Monocyte, Slan-DC, and Activated T Lymphocyte Dynamics during HCV Infection.

Chronic hepatitis C virus infection leads to the activation of innate immunity, a key component in HCV fibrosis. In the past, the use of IFN-based treatment regimens did not permit an adequate evaluation of the impact of HCV clearance on immune cells, because of their antiviral and immunomodulatory properties. The recent development of direct-acting antiviral (DAA) therapy, which is associated with high rates of sustained virological response, enables a more accurate analysis of the immunological modifications following HCV eradication. We studied the dynamics of blood myeloid dendritic cells, monocytes, slan-DCs, and T lymphocytes during IFN-free and IFN-based regimens in hepatitis C virus infection.

Diagnostic performance in active TB of QFT-Plus assay and co-expression of CD25/CD134 in response to new antigens of Mycobacterium tuberculosis.

The new QuantiFERON-TB Gold Plus employs modified peptides optimized to elicit an IFNγ response from CD8+ cytotoxic T lymphocytes in addition to CD4+ T cells. With a view to improve the difficult identification of TB cases, we assessed the combination of two specific immunological markers comprising IFNγ secretion and T cells co-expression of CD25 and CD134 in response to Mycobacterium tuberculosis-specific antigens. A total of 34 subjects with suspected TB and 10 age-matched HD were prospectively enrolled. Assessing the performance of QFT-Plus in terms of the TB1 and TB2 results, we found that in TB patients, the quantitative IFNγ value in TB2 was similar to that in TB1, and we did not find any differences irrespective of the disease (pulmonary or extra-pulmonary). The flow cytometric CD25/CD134 assay, allowed a more accurate differentiation between M. tuberculosis-infected and uninfected patients, with a better combination of sensitivity and specificity, especially by evaluation of CD4+ T-cell subset. All individuals with negative QFT-Plus results displayed a positive CD25/CD134 response. Overall, a positive correlation was found between T cells co-expressing CD25/CD134 and IFNγ levels in response to both QFT-Plus TB antigen tubes, as well as between the QFT-Plus TB1 and TB2 tubes. We demonstrated that both TB1 and TB2 induce a higher expression of CD25+CD134+ markers on CD4+ T cells among infected TB subjects, compared to the lower degree of CD8+ T cells, mainly induced to TB2 stimulation. We suggest that a combined use of classic QFT-Plus and specific CD25/CD134 response may be a useful means in the diagnostic workup for active TB.

Myeloid and lymphoid activation markers in AIDS and non-AIDS presenters.

HIV infection is characterized by a state of chronic activation of the immune system, which is not completely reversed by antiretroviral treatment (ART). The aim of this study was to assess myeloid and lymphoid activation markers during HIV infection, before and one year after ART initiation, in AIDS and non-AIDS presenters. Treatment naïve HIV positive patients were enrolled in this study. Myeloid dendritic cell (mDC), plasmacytoid dendritic cell (pDC), slanDC, monocyte and T-lymphocyte cell counts and activation status, were assessed by flow cytometry in peripheral blood samples. Soluble (s)CD14 and sCD163 were assessed in plasma samples using ELISA assays. Statistical analyses were performed using GraphPad Prism and Minitab Express. Thirty-four ART naïve HIV-1 infected subjects were enrolled in this study (22 non-AIDS and 12 AIDS presenters). Seventeen healthy donors (HD) were included as control group. Although circulating mDC levels resulted unchanged, HLA-DR expression was decreased on mDCs of HIV positive subjects compared to HD (p < 0,0001). AIDS presenters showed the lowest level of expression of HLA-DR on mDCs. Circulating levels of pDCs were decreased in HIV patients compared to HD (p < 0,001), without any changes in HLA-DR expression. SlanDC cell counts were extremely reduced in AIDS presenters, compared to non-AIDS presenters and HD (p < 0,01 and p < 0,0001, respectively) and showed higher HLA-DR expression in HIV patients compared to HD (p < 0,01). Intermediate monocyte (IM) cell counts were increased in AIDS and non-AIDS presenters compared to HD (p < 0,001 and p < 0,001 respectively). Furthermore, IM expansion was directly correlated to HIV viral load (p = 0,036) and independent from CD4 cell counts and activation levels. Plasma concentrations of sCD14 and sCD163 resulted increased in HIV infected subjects compared to HD (p < 0,0001 and p < 0,001), with the highest levels observed in AIDS presenters. After 1 year, ART was able to increase pDC and decrease IM absolute cell counts and modify HLA-DR expression on mDCs and slanDCs, approaching the levels observed in HD. ART reduced also CD4 and CD8 activation levels. In conclusion, in untreated HIV infected subjects circulating dendritic cells resulted altered either in numbers or in HLA-DR expression, especially in AIDS presenters. IM absolute counts were equally increased in AIDS and non-AIDS presenters. ART was able to reduce myeloid and lymphoid inflammation in both advanced and non-advanced HIV patients, confirming the role of ART in hampering disease progression and immune activation associated non-AIDS events.

Persistent high plasma levels of sCD163 and sCD14 in adult patients with measles virus infection.

BACKGROUND AND AIMS: Measles is an infectious disease that represents a serious public health problem worldwide, being associated with increased susceptibility to secondary infections, especially in the respiratory and gastrointestinal tracts. The aim of this study was to evaluate sCD163 and sCD14 levels in measles virus (MV) infected patients, as markers of immune activation, in order to better understand their role in the pathogenesis of the disease. TNF-α plasma levels were also evaluated. METHODS: sCD163, sCD14 and TNF-α were measured by ELISA in plasma samples of 27 MV infected patients and 27 healthy donors (HD) included as controls. RESULTS: At the time of hospital admission, sCD163 and sCD14 levels were significantly higher in MV infected patients than in HD, while a decrease in TNF-α levels were found even if without statistical significance. sCD163 and sCD14 levels were significantly decreased after two months from acute infection compared to hospital admission although they remained significantly higher compared to HD. TNF-α levels increased significantly during the follow-up period. Considering clinical parameters, sCD163 levels positively correlated with aspartate aminotransferase, white blood cell count and neutrophils rate, while negatively correlated with the lymphocyte percentage. sCD14 levels positively correlated with the neutrophil and lymphocyte percentages. CONCLUSIONS: These results indicate that, despite the resolution of symptoms, an important macrophage/monocyte activation persists in measles patients, even after two months from infection.

Changes in inflammatory biomarkers in HCV-infected patients undergoing direct acting antiviral-containing regimens with or without interferon.

BACKGROUND AND AIMS: Increased levels of chemokine interferon-gamma (IFN-γ)-inducible protein-10 (CXCL10), soluble CD163 (sCD163) and soluble CD14 (sCD14) have been reported in HCV infection. The aim of this study was to compare, sCD163 and sCD14 levels in HCV-infected patients undergoing direct acting antiviral (DAA)-containing regimens with or without interferon (IFN). METHODS: sCD163, sCD14 and CXCL10 were longitudinally measured by ELISA in 159 plasma samples from 25 HCV-infected patients undergoing IFN-based treatment plus telaprevir or boceprevir and 28 HCV infected subjects treated with DAA IFN-free regimens. Twenty-five healthy donors (HD) were included as controls. RESULTS: At baseline CXCL10, sCD163 and sCD14 levels were higher in HCV-infected patients than in HD. CXCL10 and sCD163 levels were significantly decreased in responder (R) patients who achieved sustained virological response (SVR), with both IFN-based and IFN-free regimens, while they were persistently elevated in non-responders (NR) patients who stopped IFN-based treatments because of failure or adverse events. Conversely, sCD14 levels were apparently unchanged during therapy, but at the end of treatment the levels reached normal ranges. Comparing the two regimens, the extent of CXCL10 reduction was more pronounced in patients undergoing DAA IFN-free therapies, whereas sCD163 and sCD14 reduction was similar in the two groups. Interestingly, only in IFN-based regimens baseline sCD163 levels were significantly higher in NR than in R patients, while in the IFN-free treatment group also patients with high sCD163 plasma levels obtained SVR. At the end of therapy, even if the biomarkers were largely decreased, their levels remained significantly higher compared to HD. Only in the early fibrosis stages, sCD163 values tended to normalize. CONCLUSIONS: These results indicate that IFN-free regimens including newer DAA induce an early and marked decrease in circulating inflammatory biomarkers. However, the full normalization of biomarkers was not obtained, especially in patients with advanced fibrosis, thus underlying the need for a treatment in the early stages of HCV infection.

Active HCV infection is associated with increased circulating levels of interferon-gamma (IFN-γ)-inducible protein-10 (IP-10), soluble CD163 and inflammatory monocytes regardless of liver fibrosis and HIV coinfection.

BACKGROUND AND OBJECTIVE: Interferon-gamma (IFN-γ)-inducible protein-10 (IP-10), soluble (s) CD163 and sCD14 play an important role in the pathogenesis of HCV and HIV infection and are involved in inflammation and liver fibrosis. The aim of the present study was to evaluate at a single time point, plasma soluble biomarkers and inflammatory monocytes subsets in different groups of subjects: (i) HIV monoinfected patients on suppressive ART; (ii) HIV/HCV coinfected patients on ART, with undetectable HIV viremia (including either subjects who had active HCV replication or those who cleared HCV); (iii) HCV monoinfected individual with active viral replication. METHODS: Hundred and twenty-nine plasma samples were analyzed including HCV and HIV monoinfected patients, HIV/HCV coinfected patients, with active HCV infection (AHI) or with HCV viral clearance (VHC) and healthy donors (HD). Levels of IP-10, sCD163 and sCD14 were measured by ELISA. Absolute cell counts of monocyte subpopulations were enumerated in whole blood by using flow cytometric analyses. RESULTS: IP-10 and sCD163 plasma levels were higher in HCV monoinfected and in AHI coinfected pts compared to HIV monoinfected and HD, whereas sCD14 levels were higher only in HIV monoinfected patients. Considering the degree of fibrosis, sCD163 and sCD14 levels positively correlated with kPa values (as assessed by fibroscan) and FIB-4 in HCV monoinfected group. On the other hand, IP-10 did not correlate with the fibrosis stage and it was found increased also in patients with low fibrosis. Moreover, we found an increase of the inflammatory NCM subset, in non-cirrhotic HCV subjects, while no alterations were observed in HIV, AHI and VHC. CONCLUSIONS: Our study suggests a scenario in which active HCV infection is associated with a strong pro-inflammatory state, even in the initial stage of liver fibrosis, regardless the presence of HIV coinfection, thus underlying the need of an early anti-HCV treatment.

Immunological diagnosis as an adjunctive tool for an early diagnosis of tuberculous meningitis of an immune competent child in a low tuberculosis endemic country: a case report.

BACKGROUND: Pediatric tuberculous meningitis is a highly morbid, often fatal disease. Its prompt diagnosis and treatment saves lives, in fact delays in the initiation of therapy have been associated with high mortality rates. CASE PRESENTATION: This is a case of an Italian child who was diagnosed with tuberculous meningitis after a history of a month of headache, fatigue and weight loss. Cerebrospinal fluid analysis revealed a lymphocytic pleocytosis with predominance and decreased glucose concentration. Microscopy and conventional diagnostic tests to identify Mycobacterium tuberculosis were negative, while a non classical method based on intracellular cytokine flow cytometry response of CD4 cells in cerebral spinal fluid helped us to address the diagnosis, that was subsequently confirmed by a nested polymerase chain reaction amplifying a 123 base pair fragment of the M. tuberculosis DNA. CONCLUSIONS: We diagnosed tuberculous meningitis at an early stage through an innovative immunological approach, supported by a nested polymerase chain reaction for detection of M. tuberculosis DNA. An early diagnosis is required in order to promptly initiate a therapy and to increase the patient's survival.

Brief Report: Soluble CD163 in CMV-Infected and CMV-Uninfected Subjects on Virologically Suppressive Antiretroviral Therapy in the ICONA Cohort.

AIMS: To contribute to the understanding of the role played by cytomegalovirus (CMV) in sustaining monocyte/macrophage-mediated immune activation in antiretroviral therapy treated HIV-infected subjects. DESIGN AND METHODS: We selected 23 CMV-uninfected and 46 CMV-infected HIV+ subjects, matched for age, CD4 nadir, HIV infection duration, and viral hepatitis serostatus. All subjects were on successful antiretroviral therapy since at least 1 year. A group of 16 healthy donors with similar age and sex was also included. Plasma levels of tumor necrosis factor-alpha, interleukin-6, sCD163, sCD14, and CMV immunoglobulin G levels were measured in duplicate with human enzyme-linked immunosorbent assay kits. RESULTS: We found significantly higher sCD163 plasma levels in HIV+CMV+ compared with HIV+CMV- subjects and healthy donors. This augmentation was confirmed also when subjects positive for hepatitis C virus-Ab were excluded from analysis. Interestingly, a correlation between anti-CMV immunoglobulin G levels and sCD163, tumor necrosis factor-alpha, interleukin-6, and sCD14 in HIV+CMV+ subjects was found. CONCLUSIONS: CMV coinfection could be a major driver of monocyte/macrophage activation in virally suppressed HIV+ individuals and might explain the increased risk of non-AIDS morbidity/mortality in HIV/CMV-coinfected subjects.

Liver Fibrosis in HCV Monoinfected and HIV/HCV Coinfected Patients: Dysregulation of Matrix Metalloproteinases (MMPs) and Their Tissue Inhibitors TIMPs and Effect of HCV Protease Inhibitors.

An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) may contribute to liver fibrosis in patients with hepatitis C (HCV) infection. We measured the circulating levels of different MMPs and TIMPs in HCV monoinfected and HIV/HCV coinfected patients and evaluated the potential for anti-HCV therapy to modulate MMP and TIMP levels in HCV subjects. We analyzed 83 plasma samples from 16 HCV monoinfected patients undergoing dual or triple anti-HCV therapy, 15 HIV/HCV coinfected patients with undetectable HIV load, and 10 healthy donors (HD). Levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, TIMP-1, and TIMP-2 were measured by a SearchLight Multiplex Immunoassay Kit. MMP-2 and MMP-9 were the highest expressed MMPs among all the analyzed samples and their levels significantly increased in HCV monoinfected and HIV/HCV coinfected subjects compared to HD. TIMP-1 levels were significantly higher in HCV and HIV/HCV subjects compared to HD and were correlated with liver stiffness. These findings raise the possibility of using circulating TIMP-1 as a non-invasive marker of liver fibrosis in HCV infection. A longitudinal study demonstrated that MMP-9 levels significantly decreased (40% reduction from baseline) in patients receiving dual as well as triple direct-acting antivirals (DAA) anti-HCV therapy, which had no effect on MMP-2, TIMP-1, and TIMP-2. As the dysregulation of MMP-2 and MMP-9 may reflect inflammatory processes in the liver, the decrease of MMP-9 following HCV protease inhibitor treatment suggests a positive effect on the reduction of liver inflammation.

Blood Myeloid Dendritic Cells and slanDC in Antiretroviral Therapy- Suppressed HIV-Infected Patients.

Myeloid dendritic cells (mDCs) play a complex role in HIV infection regardless of viral replication. The aim of our study was to analyse mDCs in long term antiretroviral therapy (ART)- suppressed HIV-infected patients. We evaluated the numbers of mDCs and slanDC in the context of different degree of CD4+ T cell recovery, persistent T cell activation (as HLA-DR+/CD3+ expression) and monocyte-macrophage activation assessed in terms of circulating levels of both sCD14 and sCD163. We enrolled 72 aviremic patients under effective ART and 34 healthy donors (HD). Patients were divided into two groups on the bases of ΔCD4, indicating the difference between the value of CD4 at the time of sampling and CD4 nadir. Higher levels of mDCs and slanDC were found in patients with ΔCD4>200/mmc in comparison to HD. In those patients also an increased level of sCD14 was found, whereas sCD163 seemed to be at normal levels. An augmentation of activated CD4 T lymphocytes was found, although less pronounced in patients with ΔCD4<200/mmc. In conclusion, our findings showed an expansion of mDCs with a shift to inflammatory slanDC that could sustain both microbial translocation and HIV latency in CD4 T cells.

Treatment of latent tuberculosis infection induces changes in multifunctional Mycobacterium tuberculosis-specific CD4+ T cells.

To ascertain whether multiparametric flow cytometry assessment of multifunctional Mycobacterium tuberculosis (Mtb)-specific CD4(+) and CD8(+) T cells can distinguish between untreated and treated patients with latent tuberculosis infection (LTBI), we enrolled 14 LTBI subjects treated with isoniazid (INH) therapy, 16 untreated LTBI patients, and 25 healthy controls. The analysis of mono-functional CD4(+) and CD8(+) T cells producing single cytokines showed significant differences only between uninfected and infected LTBI subjects (both treated and untreated). Conversely, the analysis of multifunctional CD4(+) T cells revealed a significant reduction in the frequency of two CD4(+) T cells subsets, those producing IFN-γ, IL-2, and TNF-α simultaneously (triple positive; p = 0.005) and those producing IL-2 alone (p = 0.0359), as well as a shift towards T cells producing only one cytokine in treated as compared to untreated LTBI subjects. Assigning a triple-positive CD4(+) T cells a cut-off >0.082 %, 94 % of untreated LTBI patients were scored as positive, as compared to only 28 % of treated LTBI patients and none of the healthy controls. No significant differences between untreated and treated LTBI subjects in terms of Mtb-specific CD8(+) T cell cytokine profiles (p > 0.05) were identified. The significant changes in the cytokine profiles of Mtb-specific T cells after INH therapy suggest that analysis of multifunctional T cells may be a promising means for the monitoring of LTBI treatment success.

Multifunctional Analysis of CD4+ T-Cell Response as Immune-Based Model for Tuberculosis Detection.

Mono- and multifunctional specific CD4(+) and CD8(+) T-cell responses were evaluated to improve the immune-based detection of active tuberculosis (TB) and latent infection (LTBI). We applied flow cytometry to investigate cytokines profile (IFN-γ, TNF-α, and IL-2) of T cells after stimulation with TB antigens in 28 TB-infected subjects (18 active TB and 10 LTBI) and 10 uninfected controls. Cytokines production by CD4(+) T cells at single-cell levels was higher in TB-infected subjects than uninfected controls (P < 0.0001). Assigning to activated CD4(+) T cells, producing any of the three cytokines, a cut-off >0.45%, it was possible to differentiate TB-infected (>0.45%) by uninfected subjects (<0.45%). Among TB-infected subjects, the frequencies of multifunctional CD4(+) T cells, simultaneously producing all 3 cytokines, are lower in active TB than LTBI subjects (P = 0.003). Thus, assigning to triple-positive CD4(+) T cells a cut-off <0.182%, TB-infected individuals could be classified as active TB subjects (<0.182%) or LTBI subjects (>0.182%). The magnitude of CD8(+) T-cell responses showed no differences between active TB and LTBI. Multifunctional CD4(+) T-cell responses could have the potential to identify at single time point subjects without TB infection and patients having active or latent TB.

Long-term IFN-γ and IL-2 response for detection of latent tuberculosis infection in healthcare workers with discordant immunologic results.

Discordant results between the interferon-gamma release assays (IGRAs) and tuberculin skin test (TST) are common in latent tuberculosis infection (LTBI). We evaluated whether the measurement of IFN-γ and interleukin (IL)-2T-cell responses, after prolonged Mycobacterium tuberculosis-specific antigen stimulation, can be used as adjunctive biomarker for LTBI detection in subjects with discordant results between TST and QuantiFERON-Gold In-Tube (QFT). 196 healthcare workers were screened for LTBI and in 90 of those participants, the QFT was repeated after 18 h, and IFN-γ/IL-2 immune response was measured after 72 h long-term stimulation. Of the 196 patients, 34 had positive, 155 negative, and 7 indeterminate QFT results. Discordant TST+/QFT- results were found in 29 (14.7%) patients, of whom 6 (20.6%) were Bacillus Calmette-Guerin (BCG) vaccinated. None of 23 non-BCG vaccinated subjects showed a specific IFN-γ immune response after 18 h nor 72 h of incubation, whereas 3/23 (13.04%) discordant subjects produced a specific long-term IL-2 response, which might reflect a LTBI status. In LTBI group (TST+/QFT+) both cytokine levels were increased after long-term in comparison to short-term stimulation. No significant long-term IFN-γ/IL-2 secretion was detected in control group (TST-/QFT-). Taken together, our data showed that the 87% of discordant patients who did not respond to the long-term assay, as controls subjects, were judged LTBI negative. The use of classic QFT and long-term IL-2 response may have a potential role to clarify the LTBI status in individuals in whom the diagnosis of LTBI is uncertain due to the discordance of the available diagnostic tests, such as TST and IGRA.

Molecular mechanisms of liver fibrosis in HIV/HCV coinfection.

Chronic hepatitis C virus (HCV) infection is an important cause of morbidity and mortality in people coinfected with human immunodeficiency virus (HIV). Several studies have shown that HIV infection promotes accelerated HCV hepatic fibrosis progression, even with HIV replication under full antiretroviral control. The pathogenesis of accelerated hepatic fibrosis among HIV/HCV coinfected individuals is complex and multifactorial. The most relevant mechanisms involved include direct viral effects, immune/cytokine dysregulation, altered levels of matrix metalloproteinases and fibrosis biomarkers, increased oxidative stress and hepatocyte apoptosis, HIV-associated gut depletion of CD4 cells, and microbial translocation. In addition, metabolic alterations, heavy alcohol use, as well drug use, may have a potential role in liver disease progression. Understanding the pathophysiology and regulation of liver fibrosis in HIV/HCV co-infection may lead to the development of therapeutic strategies for the management of all patients with ongoing liver disease. In this review, we therefore discuss the evidence and potential molecular mechanisms involved in the accelerated liver fibrosis seen in patients coinfected with HIV and HCV.

Dendritic cells in blood and urine samples from bladder cancer patients undergoing BCG immunotherapy.

OBJECTIVES: Immunotherapy with BCG (Bacille Calmette-Guérin) after transurethral resection of the bladder tumor represents a highly effective primary treatment for intermediate and high-risk superficial bladder cancer. The effectiveness of this therapy has been documented, but its mechanism of action is not clear yet. In the present study, we investigated the changes of dendritic cells (DC) numbers in peripheral blood and urine of patients with superficial bladder cancer undergoing BCG intravescical therapy. MATERIAL AND METHOD: We have enumerated plasmacytoid and myeloid DCs in the peripheral blood and in the urine of patients with bladder cancer in order to clarify the role of these cells in the evolution of the disease and the effect of therapy. DCs in blood and urine samples were assessed using the single-platform TruCOUNT assay with monoclonal antibodies. The study population included 37 healthy donors and 13 patients with diagnosis of primitive superficial bladder cancer. RESULTS: At the time of diagnosis a reduction of blood DCs was found in patients as opposed to healthy donors, while DCs were not found in the urine in the same way as in healthy subjects. Six of these patients were followed before and after weekly and monthly instillations of BCG. In the peripheral blood, we observed an immunological recovery of DCs from the third weekly instillation up to the sixth. In the urine of patients, we didn't find mDCs or pDCs at T0, but we found a statistically significant change from the third instillation up to the sixth. On the contrary, we didn't find mDCs in urine during monthly instillation. CONCLUSIONS: DC Count could be used in the monitoring of patients undergoing BCG therapy. Immunological restoration of mDC numbers in peripheral blood and the efflux in urine could be important for confirming the effectiveness of BCG instillation.

Extraction and characterization of a natural rubber from Euphorbia characias latex.

A natural rubber was identified and characterized for the first time in the latex of the perennial Mediterranean shrub Euphorbia characias. Four different methods, i.e., acetone, acetic acid, trichloroacetic acid, and Triton® X-100, followed by successive treatments with cyclohexane/ethanol, were employed to extract the natural rubber. The rubber content was shown to be 14% (w/v) of the E. characias latex, a low content compared with that of Hevea brasiliensis (30-35%) but a similar content to other rubber producing plants. E. characias rubber showed a molecular weight of 93,000 with a M(w) /M(n) of 2.9. (1) H NMR, (13) C NMR, and FTIR analysis revealed the characteristic of the cis-1,4-polyisoprene typical of natural rubber. These results provided novel insight into latex components and will ultimately benefit the broader understanding of E. characias latex composition.

Severe and persistent depletion of circulating plasmacytoid dendritic cells in patients with 2009 pandemic H1N1 infection.

BACKGROUND: Dysregulation of host immune responses plays a critical role in the pathogenesis of severe 2009 pandemic H1N1 infection. Whether H1N1 virus could escape innate immune defense in vivo remains to be investigated. The aim of this study was to evaluate the pattern of innate immune response during human 2009 H1N1 infection. We performed the enumeration of circulating myeloid dendritic cells (mDC) and plasmacytoid DC (pDC) in blood from patients with H1N1 pneumonia shortly after the onset of symptoms and during follow-up at different intervals of time. The analysis of CD4 and CD8 count, CD38 T-cell activation marker and serum cytokine/chemokine plasma levels was also done. METHODOLOGY/PRINCIPAL FINDINGS: Blood samples were collected from 13 hospitalized patients with confirmed H1N1-related pneumonia at time of admission and at weeks 1, 4, and 16 of follow-up. 13 healthy donors were enrolled as controls. In the acute phase of the disease, H1N1-infected patients exhibited a significant depletion in both circulating pDC and mDC in conjunction with a decrease of CD4 and CD8 T cell count. In addition, we found plasmatic hyperproduction of IP-10 and RANTES, whereas increase in T-cell immune activation was found at all time points. When we assessed the changes in DC count over time, we observed a progressive normalization of mDC number. On the contrary, H1N1-infected patients did not achieve a complete recovery of pDC count as values remained lower than healthy controls even after 16 weeks of follow-up. CONCLUSIONS: H1N1 disease is associated with a profound depletion of DC subsets. The persistence of pDC deficit for several weeks after disease recovery could be due to H1N1 virus itself or to a preexisting impairment of innate immunity.