Antibodies Induced by Smallpox Vaccination after at Least 45 Years Cross-React with and In Vitro Neutralize Mpox Virus: A Role for Polyclonal B Cell Activation?

AIMS: To evaluate whether antibodies specific for the vaccinia virus (VV) are still detectable after at least 45 years from immunization. To confirm that VV-specific antibodies are endowed with the capacity to neutralize Mpox virus (MPXV) in vitro. To test a possible role of polyclonal non-specific activation in the maintenance of immunologic memory. METHODS: Sera were collected from the following groups: smallpox-vaccinated individuals with or without latent tuberculosis infection (LTBI), unvaccinated donors, and convalescent individuals after MPXV infection. Supernatant of VV- or MPXV-infected Vero cells were inactivated and used as antigens in ELISA or in Western blot (WB) analyses. An MPXV plaque reduction neutralization test (PRNT) was optimized and performed on study samples. VV- and PPD-specific memory T cells were measured by flow cytometry. RESULTS: None of the smallpox unvaccinated donors tested positive in ELISA or WB analysis and their sera were unable to neutralize MPXV in vitro. Sera from all the individuals convalescing from an MPXV infection tested positive for anti-VV or MPXV IgG with high titers and showed MPXV in vitro neutralization capacity. Sera from most of the vaccinated individuals showed IgG anti-VV and anti-MPXV at high titers. WB analyses showed that positive sera from vaccinated or convalescent individuals recognized both VV and MPXV antigens. Higher VV-specific IgG titer and specific T cells were observed in LTBI individuals. CONCLUSIONS: ELISA and WB performed using supernatant of VV- or MPXV-infected cells are suitable to identify individuals vaccinated against smallpox at more than 45 years from immunization and individuals convalescing from a recent MPXV infection. ELISA and WB results show a good correlation with PRNT. Data confirm that a smallpox vaccination induces a long-lasting memory in terms of specific IgG and that antibodies raised against VV may neutralize MPXV in vitro. Finally, higher titers of VV-specific antibodies and higher frequency of VV-specific memory T cells in LTBI individuals suggest a role of polyclonal non-specific activation in the maintenance of immunologic memory.

Effectiveness of Immunotherapy in Non-Small Cell Lung Cancer Patients with a Diagnosis of COPD: Is This a Hidden Prognosticator for Survival and a Risk Factor for Immune-Related Adverse Events?

The interplay between the immune system and chronic obstructive pulmonary disease (COPD) and non-small cell lung cancer (NSCLC) is complex and multifaceted. In COPD, chronic inflammation and oxidative stress can lead to immune dysfunction that can exacerbate lung damage, further worsening the respiratory symptoms. In NSCLC, immune cells can recognise and attack the cancer cells, which, however, can evade or suppress the immune response by various mechanisms, such as expressing immune checkpoint proteins or secreting immunosuppressive cytokines, thus creating an immunosuppressive tumour microenvironment that promotes cancer progression and metastasis. The interaction between COPD and NSCLC further complicates the immune response. In patients with both diseases, COPD can impair the immune response against cancer cells by reducing or suppressing the activity of immune cells, or altering their cytokine profile. Moreover, anti-cancer treatments can also affect the immune system and worsen COPD symptoms by causing lung inflammation and fibrosis. Immunotherapy itself can also cause immune-related adverse events that could worsen the respiratory symptoms in patients with COPD-compromised lungs. In the present review, we tried to understand the interplay between the two pathologies and how the efficacy of immunotherapy in NSCLC patients with COPD is affected in these patients.

[Management of the patient with extensive stage microcytoma. The importance of collaboration between oncology and radiotherapy.] / Gestione del paziente con microcitoma a stadio esteso. L'importanza della collaborazione tra oncologia e radioterapia.

Small cell lung cancer (SCLC) represents one of the most complex challenges in the oncological field, with a very slow advancement in research, contrary to the rapid evolutionary of the disease. For nearly two years, the mainstay of treatment for extensive-stage disease (ES-SCLC) has been the combination of platinum-based chemotherapy and immunotherapy, following the approval of atezolizumab and subsequently durvalumab, based on a modest, but significant improvement in overall survival compared to chemotherapy alone. The poor prognosis after the failure of first-line treatment explains the need to maximize the duration and efficacy of up-front systemic therapies, in particular, the emerging role of radiotherapy, also in ES-SCLC. On 10 November 2022, a meeting concerning the integrated treatment of patients with ES-SCLC was held in Rome and was attended by 12 specialists in oncology and radiotherapy from various centers in Lazio, under the direction of Federico Cappuzzo, Emilio Bria and Sara Ramella. The aim of the meeting was to share their clinical experience and to provide a series of practical indications in order to support physicians in the correct integration between first-line chemo-immunotherapy and radiotherapy treatments in ES-SCLC.

Have Diagnostics, Therapies, and Vaccines Made the Difference in the Pandemic Evolution of COVID-19 in Comparison with "Spanish Flu"?

In 1918 many countries, but not Spain, were fighting World War I. Spanish press could report about the diffusion and severity of a new infection without censorship for the first-time, so that this pandemic is commonly defined as "Spanish flu", even though Spain was not its place of origin. "Spanish flu" was one of the deadliest pandemics in history and has been frequently compared with the coronavirus disease (COVID)-19 pandemic. These pandemics share similarities, being both caused by highly variable and transmissible respiratory RNA viruses, and diversity, represented by diagnostics, therapies, and especially vaccines, which were made rapidly available for COVID-19, but not for "Spanish flu". Most comparison studies have been carried out in the first period of COVID-19, when these resources were either not yet available or their use had not long started. Conversely, we wanted to analyze the role that the advanced diagnostics, anti-viral agents, including monoclonal antibodies, and innovative COVID-19 vaccines, may have had in the pandemic containment. Early diagnosis, therapies, and anti-COVID-19 vaccines have markedly reduced the pandemic severity and mortality, thus preventing the collapse of the public health services. However, their influence on the reduction of infections and re-infections, thus on the transition from pandemic to endemic condition, appears to be of minor relevance. The high viral variability of influenza and coronavirus may probably be contained by the development of universal vaccines, which are not easy to be obtained. The only effective weapon still remains the disease prevention, to be achieved with the reduction of promiscuity between the animal reservoirs of these zoonotic diseases and humans.

Different configurations of SARS-CoV-2 spike protein delivered by integrase-defective lentiviral vectors induce persistent functional immune responses, characterized by distinct immunogenicity profiles.

Several COVID-19 vaccine strategies utilizing new formulations for the induction of neutralizing antibodies (nAbs) and T cell immunity are still under evaluation in preclinical and clinical studies. Here we used Simian Immunodeficiency Virus (SIV)-based integrase defective lentiviral vector (IDLV) delivering different conformations of membrane-tethered Spike protein in the mouse immunogenicity model, with the aim of inducing persistent nAbs against multiple SARS-CoV-2 variants of concern (VoC). Spike modifications included prefusion-stabilizing double proline (2P) substitutions, mutations at the furin cleavage site (FCS), D614G mutation and truncation of the cytoplasmic tail (delta21) of ancestral and Beta (B.1.351) Spike, the latter mutation to markedly improve IDLV membrane-tethering. BALB/c mice were injected once with IDLV delivering the different forms of Spike or the recombinant trimeric Spike protein with 2P substitutions and FCS mutations in association with a squalene-based adjuvant. Anti-receptor binding domain (RBD) binding Abs, nAbs and T cell responses were detected up to six months from a single immunization with escalating doses of vaccines in all mice, but with different levels and kinetics. Results indicated that IDLV delivering the Spike protein with all the combined modifications, outperformed the other candidates in terms of T cell immunity and level of both binding Abs and nAbs soon after the single immunization and persistence over time, showing the best capacity to neutralize all formerly circulating VoC Alpha, Beta, Gamma and Delta. Although present, the lowest response was detected against Omicron variants (BA.1, BA.2 and BA.4/5), suggesting that the magnitude of immune evasion may be related to the higher genetic distance of Omicron as indicated by increased number of amino acid substitutions in Spike acquired during virus evolution.

New Monoclonal Antibodies Specific for Different Epitopes of the Spike Protein of SARS-CoV-2 and Its Major Variants: Additional Tools for a More Specific COVID-19 Diagnosis.

The emergence of the new pathogen SARS-CoV-2 determined a rapid need for monoclonal antibodies (mAbs) to detect the virus in biological fluids as a rapid tool to identify infected individuals to be treated or quarantined. The majority of commercially available antigenic tests for SARS-CoV-2 rely on the detection of N antigen in biologic fluid using anti-N antibodies, and their capacity to specifically identify subjects infected by SARS-CoV-2 is questionable due to several structural analogies among the N proteins of different coronaviruses. In order to produce new specific antibodies, BALB/c mice were immunized three times at 20-day intervals with a recombinant spike (S) protein. The procedure used was highly efficient, and 40 different specific mAbs were isolated, purified and characterized, with 13 ultimately being selected for their specificity and lack of cross reactivity with other human coronaviruses. The specific epitopes recognized by the selected mAbs were identified through a peptide library and/or by recombinant fragments of the S protein. In particular, the selected mAbs recognized different linear epitopes along the S1, excluding the receptor binding domain, and along the S2 subunits of the S protein of SARS-CoV-2 and its major variants of concern. We identified combinations of anti-S mAbs suitable for use in ELISA or rapid diagnostic tests, with the highest sensitivity and specificity coming from proof-of-concept tests using recombinant antigens, SARS-CoV-2 or biological fluids from infected individuals, that represent important additional tools for the diagnosis of COVID-19.

Isolation and Characterization of Mouse Monoclonal Antibodies That Neutralize SARS-CoV-2 and Its Variants of Concern Alpha, Beta, Gamma and Delta by Binding Conformational Epitopes of Glycosylated RBD With High Potency.

Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.

Melanoma Cell Resistance to Vemurafenib Modifies Inter-Cellular Communication Signals.

The therapeutic success of BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi) in BRAF-mutant melanoma is limited by the emergence of drug resistance, and several lines of evidence suggest that changes in the tumor microenvironment can play a pivotal role in acquired resistance. The present study focused on secretome profiling of melanoma cells sensitive or resistant to the BRAFi vemurafenib. Proteomic and cytokine/chemokine secretion analyses were performed in order to better understand the interplay between vemurafenib-resistant melanoma cells and the tumor microenvironment. We found that vemurafenib-resistant melanoma cells can influence dendritic cell (DC) maturation by modulating their activation and cytokine production. In particular, human DCs exposed to conditioned medium (CM) from vemurafenib-resistant melanoma cells produced higher levels of pro-inflammatory cytokines-that potentially facilitate melanoma growth-than DCs exposed to CM derived from parental drug-sensitive cells. Bioinformatic analysis performed on proteins identified by mass spectrometry in the culture medium from vemurafenib-sensitive and vemurafenib-resistant melanoma cells suggests a possible involvement of the proteasome pathway. Moreover, our data confirm that BRAFi-resistant cells display a more aggressive phenotype compared to parental ones, with a significantly increased production of interferon-γ, interleukin-8, vascular-endothelial growth factor, CD147/basigin, and metalloproteinase 2 (MMP-2). Plasma levels of CD147/basigin and MMP-2 were also measured before the start of therapy and at disease progression in a small group of melanoma patients treated with vemurafenib or vemurafenib plus cobimetinib. A significant increment in CD147/basigin and MMP-2 was observed in all patients at the time of treatment failure, strengthening the hypothesis that CD147/basigin might play a role in BRAFi resistance.

Immunogenicity of Viral Vaccines in the Italian Military.

Military personnel of all armed forces receive multiple vaccinations and have been doing so since long ago, but relatively few studies have investigated the possible negative or positive interference of simultaneous vaccinations. As a contribution to fill this gap, we analyzed the response to the live trivalent measles/mumps/rubella (MMR), the inactivated hepatitis A virus (HAV), the inactivated trivalent polio, and the trivalent subunits influenza vaccines in two cohorts of Italian military personnel. The first cohort was represented by 108 students from military schools and the second by 72 soldiers engaged in a nine-month mission abroad. MMR and HAV vaccines had never been administered before, whereas inactivated polio was administered to adults primed at infancy with a live trivalent oral polio vaccine. Accordingly, nearly all subjects had baseline antibodies to polio types 1 and 3, but unexpectedly, anti-measles/-mumps/-rubella antibodies were present in 82%, 82%, and 73.5% of subjects, respectively (43% for all of the antigens). Finally, anti-HAV antibodies were detectable in 14% and anti-influenza (H1/H3/B) in 18% of the study population. At mine months post-vaccination, 92% of subjects had protective antibody levels for all MMR antigens, 96% for HAV, 69% for the three influenza antigens, and 100% for polio types 1 and 3. An inverse relationship between baseline and post-vaccination antibody levels was noticed with all the vaccines. An excellent vaccine immunogenicity, a calculated long antibody persistence, and apparent lack of vaccine interference were observed.

Safety of Multiple Vaccinations and Durability of Vaccine-Induced Antibodies in an Italian Military Cohort 5 Years after Immunization.

We previously examined the safety and immunogenicity of multiple vaccines administered to a military cohort, divided into two groups, the first composed of students at military schools, thus operating inside the national borders for at least 3 years, and the other formed of soldiers periodically engaged in a 9-month-long mission abroad (Lebanon). In the current study, we analyzed 112 individuals of this cohort, 50 pertaining to the first group and 62 to the second group, in order to examine the possible late appearance of side effects and to calculate the half-life of the induced antibodies. Moreover, the possible involvement of B-cell polyclonal activation as a pathogenetic mechanism for long term antibody persistence has even been explored. No late side effects, as far as autoimmunity and/or lymphoproliferation appearance, have been noticed. The long duration of the vaccine induced anti-HAV antibodies has been confirmed, whereas the antibodies induced by tetravalent meningococcal polysaccharide vaccine have been found to persist above the threshold for putative protection for a longer time, and anti-tetanus, diphtheria, and polio 1 and 3 for a shorter time than previously estimated. No signs of polyclonal B-cell activation have been found, as a possible mechanism to understand the long antibody persistence.

Virus like particles of GII.4 norovirus bind Toll Like Receptors 2 and 5.

Norovirus (NoV) is now recognized as a major cause of gastroenteritis outbreaks, worldwide. Norovirus replication mechanisms are still poorly understood, mainly because a reliable cell culture system is still lacking. The present study aims at understanding some aspects of the immune response against norovirus, and particularly the capacity of virus like particles (VLPs) from an Italian strain, belonging to the GII.4 genotype predominating worldwide, to interact with target cells via Toll Like Receptors (TLRs). The capacity of GII.4 NoV VLPs to interact and cause the activation of TLR2, 4 and 5 was studied in recombinant HEK cells. The results obtained show the ability of GII.4 NoV VLPs to induce activation of TLR2 and 5. The results on TLRs activation confirm that GII.4 NoV VLPs interact with TLR2 and 5, that may represent putative receptors and play a role in NoV infection of intestinal cells.

Tetanus-diphtheria vaccination in adults: the long-term persistence of antibodies is not dependent on polyclonal B-cell activation and the defective response to diphtheria toxoid re-vaccination is associated to HLADRB1∗01.

Cellular and humoral immune responses to tetanus-diphtheria vaccine (Td) were assessed in human leukocyte antigen (HLA)-typed Italian military personnel who received multiple concomitant vaccines. Td-specific antibodies and T-lymphocytes were measured in individuals with one (group-1) and more than one (group-2) Td boosters. A third group (group-3), who received several vaccines, but not Td, was studied to verify the hypothesis of the polyclonal B-cell activation as mechanism for antibody persistence. The antibody response to Td toxoids was higher in group-1, who showed lower baseline antibody levels, than in group-2 subjects. The antibody response to tetanus was higher than to diphtheria toxoid in both groups. No correlation between antibody and cellular response, and no interference in the response to Td by co-administration of different vaccines were observed. HLA-DRB1∗01 allele was detected at significant higher frequency in subjects unable to double the baseline anti-diphtheria antibody levels after the vaccination. Anti-tetanus and diphtheria antibodies half-lives were assessed and the long-lasting persistence above the threshold for protection (0.1 IU/ml) was estimated in over 65 and 20 years, respectively. No significant increase of anti-diphtheria antibodies was observed in consequence of polyclonal B-cell activation. This study emphasizes the duration of Td vaccination-induced seroprotection, suggesting that re-vaccination should probably be performed at intervals longer than 10 years. No reciprocal interference by concomitantly administered vaccines has been observed. HLA-DRB1∗01 allele was significantly associated with anti-diphtheria defective response. Finally, this study does not confirm that anti-diphtheria antibody levels are maintained by polyclonal B-cell activation. Clinical trial registry: The study was registered with NCT01807780.

Anti-polysaccharide and anti-diphtheria protective antibodies after 13-valent pneumococcal conjugate vaccination in rheumatoid arthritis patients under immunosuppressive therapy.

Immunogenicity of 13-valent pneumococcal polysaccharide (PnPS) conjugate vaccine (PCV13) was evaluated in 38 rheumatoid arthritis patients under immunosuppressive treatment and 20 healthy controls (HC). Antibodies to all PnPS and diphtheria-toxin analogue conjugate protein were measured pre- (T0), 1 (T1), 6 (T2), 12 (T3) months post-immunization. Patients and HC had similar response to individual PnPS. Mean antibody levels to all PnPS but one doubled at T1 compared with T0, with T3 persistence for only 8-7/13 PnPS. Baseline antibody levels was inversely associated with the rate of responders at T1 (T1/T0≥2) to 11/13 PnPS. Few subjects reached protective IgG levels against some serotypes frequently isolated in Italian patients with invasive pneumococcal disease. Antibody response was not influenced by therapy, except the one to PS7F, which was reduced by tumor necrosis factor-α-inhibitors. Vaccination increased also anti-diphtheria IgG. Despite this study substantially confirmed the PCV13 immunogenicity in immunocompromised patients, it also revealed some limitations.

The Multirole of Liposomes in Therapy and Prevention of Infectious Diseases.

Liposomes are closed bilayer structures spontaneously formed by hydrated phospholipids that are widely used as efficient delivery systems for drugs or antigens, due to their capability to encapsulate bioactive hydrophilic, amphipathic, and lipophilic molecules into inner water phase or within lipid leaflets. The efficacy of liposomes as drug or antigen carriers has been improved in the last years to ameliorate pharmacokinetics and capacity to release their cargo in selected target organs or cells. Moreover, different formulations and variations in liposome composition have been often proposed to include immunostimulatory molecules, ligands for specific receptors, or stimuli responsive compounds. Intriguingly, independent research has unveiled the capacity of several phospholipids to play critical roles as intracellular messengers in modulating both innate and adaptive immune responses through various mechanisms, including (i) activation of different antimicrobial enzymatic pathways, (ii) driving the fusion-fission events between endosomes with direct consequences to phagosome maturation and/or to antigen presentation pathway, and (iii) modulation of the inflammatory response. These features can be exploited by including selected bioactive phospholipids in the bilayer scaffold of liposomes. This would represent an important step forward since drug or antigen carrying liposomes could be engineered to simultaneously activate different signal transduction pathways and target specific cells or tissues to induce antigen-specific T and/or B cell response. This lipid-based host-directed strategy can provide a focused antimicrobial innate and adaptive immune response against specific pathogens and offer a novel prophylactic or therapeutic option against chronic, recurrent, or drug-resistant infections.

A method permissive to fixation and permeabilization for the multiparametric analysis of apoptotic and necrotic cell phenotype by flow cytometry.

Annexin-V/propidium iodide method (A-V/PI) is a common flow cytometric method for the multiparametric analysis of cells in apoptosis. However, A-V/PI does not permit fixation and/or permeabilization of cells making impossible evaluation of intracellular markers, restricting the analysis in a narrow time frame after staining and excluding the possibility to study pathogen-infected cells. We developed a method permitting fixation and permeabilization of stained cells: Fixed Apoptotic/Necrotic (FAN) cells test. FAN relies on the same principle of A-V/PI, but uses reagents that maintain their binding and fluorescence characteristics after fixation/permeabilization: a fluorochrome-labeled anti-phosphatidylserine antibody and fluorescent amine-binding dyes. FAN was tested to discriminate apoptotic and necrotic cells using different stimuli on several cell types and results were always comparable to those obtained using A-V/PI. FAN, unlike A-V/PI, permitted to correlate cell death with intracellular and surface markers expression and to perform cytometry even two weeks after sample preparation. As fixation of stained cells inactivates infective pathogens, we used FAN in an in vitro model of Mycobacterium tuberculosis (Mtb) infection of macrophages to monitor cellular infection and cell death induction. Using a red-fluorescent Mtb, fluorochrome labeled anti-TNF-α and anti-MHC class II monoclonal antibodies, FAN permitted to establish that the extent of macrophage death correlates with intracellular Mtb content and that dying cells accumulate TNF-α and down-modulate MHC class II molecules. Results suggest that FAN may represent an additional tool to study programmed cell death particularly useful when fixation procedures are required for a safe infected sample analysis or to comparatively analyze multiple samples. © 2017 International Society for Advancement of Cytometry.

Liposomes loaded with bioactive lipids enhance antibacterial innate immunity irrespective of drug resistance.

Phagocytosis is a key mechanism of innate immunity, and promotion of phagosome maturation may represent a therapeutic target to enhance antibacterial host response. Phagosome maturation is favored by the timely and coordinated intervention of lipids and may be altered in infections. Here we used apoptotic body-like liposomes (ABL) to selectively deliver bioactive lipids to innate cells, and then tested their function in models of pathogen-inhibited and host-impaired phagosome maturation. Stimulation of macrophages with ABLs carrying phosphatidic acid (PA), phosphatidylinositol 3-phosphate (PI3P) or PI5P increased intracellular killing of BCG, by inducing phagosome acidification and ROS generation. Moreover, ABLs carrying PA or PI5P enhanced ROS-mediated intracellular killing of Pseudomonas aeruginosa, in macrophages expressing a pharmacologically-inhibited or a naturally-mutated cystic fibrosis transmembrane conductance regulator. Finally, we show that bronchoalveolar lavage cells from patients with drug-resistant pulmonary infections increased significantly their capacity to kill in vivo acquired bacterial pathogens when ex vivo stimulated with PA- or PI5P-loaded ABLs. Altogether, these results provide the proof of concept of the efficacy of bioactive lipids delivered by ABL to enhance phagosome maturation dependent antimicrobial response, as an additional host-directed strategy aimed at the control of chronic, recurrent or drug-resistant infections.

Functional impairment of activated protein C in breast cancer - relationship to survival outcomes.

An impairment of the activated protein C (APC) system has been occasionally reported in breast cancer (BC). However, the clinical significance and prognostic value of an impaired APC functionality in BC patients is still poorly understood. Thus, the present study was aimed at investigating the prognostic value of altered APC functionality for progression-free (PFS) and overall survival (OS) in a cohort study of BC patients. APC functionality was retrospectively analyzed by a coagulation inhibition assay (ThromboPath) in 290 consecutive patients with primary (n=246) or relapsing/recurrent (n=44) BC. All patients were prospectively followed for a median time of 3.5 years (14% recurrence rate). As control group, 145 age-matched healthy women were also investigated. The results obtained demonstrated that APC function was impaired in roughly 20% of all BC at baseline. BC women with stage I/II had a significantly lower rate of APC impairment (13%) than women with stage III (22%) or distant metastases (44%, p=0.001). At univariate analyses, an impairment of APC function had a negative prognostic impact in terms of PFS (5-year PFS rates 53% vs. 70%; HR=2.5; p<0.001) and OS (5-year OS rates 79% vs. 93%; HR=3.9; p=0.005). However, prognostic significance was retained in multivariate models only for PFS (HR=2.0; p=0.017). We may, thus, conclude that BC patients are in a prothrombotic condition, which could play a role in the progression of the disease. Monitoring coagulation changes in BC women could provide important prognostic information especially in patients with advanced stages.

Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses.

Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-ß-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated ß-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.

Novel T-cell assays for the discrimination of active and latent tuberculosis infection: the diagnostic value of PPE family.

OBJECTIVE: The diagnosis of active and latent tuberculosis remains a challenge. Although a new approach based on detecting Mycobacterium tuberculosis-specific T-cells has been introduced, it cannot distinguish between latent infection and active disease. The aim of this study was to evaluate the diagnostic potential of interleukin-2 (IL-2) as biomarker after specific antigen stimulation with PE35 and PPE68 for the discrimination of active and latent tuberculosis infection (LTBI). METHOD: The production of IL-2 was measured in the antigen-stimulated whole-blood supernatants following stimulation with recombinant PE35 and PPE68. RESULTS: The discrimination performance (assessed by the area under ROC curve) for IL-2 following stimulation with recombinant PE35 and PPE68 between LTBI and patients with active TB were 0.837 [95 % confidence interval (CI) 0.72-0.97] for LTBI diagnosis and 0.75 (95 % CI 0.63-0.89) for active TB diagnosis, respectively. Applying the 6.4 pg/mL cut-off for IL-2 induced by PE35 in the present study population resulted in sensitivity of 78 %, specificity of 83 %, PPV of 83 % and NPV of 78 % for the discrimination of active TB and LTBI. In addition, a sensitivity of 81 %, specificity of 71 %, PPV of 68 and 83 % of NPV was reported based on the 4.4 pg/mL cut-off for IL-2 induced by PPE68. CONCLUSION: This study confirms IL-2 induced by PE35 and PPE68 as a sensitive and specific biomarker and highlights IL-2 as new promising adjunct markers for discriminating of LTBI and active TB disease.