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Background: Head and neck squamous cell carcinomas (HNSCCs) are heterogeneous in terms of origin and aetiology. In addition, there is uncertainty about the genetic evolution from initial diagnosis to recurrence after primary treatments and further disease progression following systemic treatment. Changes in the genetic profile have implications on the selection of appropriate treatments for patients, especially in the era of targeted therapies and immunotherapies., Methods: We analysed a cohort of nine HNSCC patients with metachronous recurrence. All patients had paired primary and recurrent samples suitable for whole-exome sequencing, while transcriptomic data from seven patients could be analysed (multiple recurrent samples collected at different time points were available for three patients)., Results: At the genomic level, the recurrences shared a fraction of the somatic single nucleotide variants (SNVs) with the index primary tumours, but they also acquired many additional mutations, while losing only a few others. A similar behaviour was also observed when examining the changes of mutational signatures between primary and recurrent samples. Overall, recurrences appeared thus more genetically diverse than the respective primary tumours. The transcriptomic analysis showed that recurrent samples had lower immune cell presence, which was also confirmed by the multiplex immunofluorescence (IF) histology assays performed on the PhenoCycler platform. Several genes related to immune response were significantly downregulated compared to the primary samples., Conclusions: Our results underline the importance of analysing multiple samples per patient to obtain a more complete picture of the patient's tumour and advocate a re-biopsy in the event of recurrence and treatment failure, in order to select the most appropriate therapeutic strategy., (© 2024. The Author(s).)

Purpose: Personalized liposome-formulated mRNA vaccines (RNA-LPX) are a powerful new tool in cancer immunotherapy. In preclinical tumor models, RNA-LPX vaccines are known to achieve potent results when combined with conventional X-ray radiation therapy (XRT). Densely ionizing radiation used in carbon ion radiation therapy (CIRT) may induce distinct effects in combination with immunotherapy compared with sparsely ionizing X-rays., Methods and Materials: Within this study, we investigate the potential of CIRT and isoeffective doses of XRT to mediate tumor growth inhibition and survival in murine colon adenocarcinoma models in conjunction with neoantigen (neoAg)-specific RNA-LPX vaccines encoding both major histocompatibility complex (MHC) class I- and class II-restricted tumor-specific neoantigens. We characterize tumor immune infiltrates and antigen-specific T cell responses by flow cytometry and interferon-γ enzyme-linked immunosorbent spot (ELISpot) analyses, respectively., Results: NeoAg RNA-LPX vaccines significantly potentiate radiation therapy-mediated tumor growth inhibition. CIRT and XRT alone marginally prime neoAg-specific T cell responses detected in the tumors but not in the blood or spleens of mice. Infiltration and cytotoxicity of neoAg-specific T cells is strongly driven by RNA-LPX vaccines and is accompanied by reduced expression of the inhibitory markers PD-1 and Tim-3 on these cells. The neoAg RNA-LPX vaccine shows similar overall therapeutic efficacy in combination with both CIRT and XRT, even if the physical radiation dose is lower for carbon ions than for X-rays., Conclusions: We hence conclude that the combination of CIRT and neoAg RNA-LPX vaccines is a promising strategy for the treatment of radioresistant tumors., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy-associated programs regulating T cell entrance, arrest, and activation in the tumor environment. Differential expression of cell adhesion and tissue architecture programs, particularly the presence of the membrane tetraspanin claudin (CLDN)18 as a signature gene, demarcated immune-infiltrated from immune-depleted mouse pancreatic tumors. In human pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer, CLDN18 expression positively correlated with more differentiated histology and favorable prognosis. CLDN18 on the cell surface promoted accrual of cytotoxic T lymphocytes (CTLs), facilitating direct CTL contacts with tumor cells by driving the mobilization of the adhesion protein ALCAM to the lipid rafts of the tumor cell membrane through actin. This process favored the formation of robust immunological synapses (ISs) between CTLs and CLDN18-positive cancer cells, resulting in increased T cell activation. Our data reveal an immune role for CLDN18 in orchestrating T cell infiltration and shaping the tumor immune contexture., Competing Interests: Declaration of interests V.B. reports past personal fees from Codiak BioSciences and IO Biotech ApS outside of the submitted work. U.S. and Ö.T. are co-founders and respectively Chief Executive Officer and Chief Medical Officer of BioNTech AG, Mainz Germany and shareholders in the privately owned BioNTech AG as mentioned in the affiliation. F.D.S., M.E., and V.B. hold proprietary rights on the patent application no. PCT/EP2023/058504: “Methods for predicting and improving the therapeutic efficacy of cancer treatments and methods for cancer prognosis.”, (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Major histocompatibility complex (MHC) class I antigen presentation deficiency is a common cancer immune escape mechanism, but the mechanistic implications and potential strategies to address this challenge remain poorly understood. Studying β2-microglobulin (B2M) deficient mouse tumor models, we find that MHC class I loss leads to a substantial immune desertification of the tumor microenvironment (TME) and broad resistance to immune-, chemo-, and radiotherapy. We show that treatment with long-lasting mRNA-encoded interleukin-2 (IL-2) restores an immune cell infiltrated, IFNγ-promoted, highly proinflammatory TME signature, and when combined with a tumor-targeting monoclonal antibody (mAB), can overcome therapeutic resistance. Unexpectedly, the effectiveness of this treatment is driven by IFNγ-releasing CD8 + T cells that recognize neoantigens cross-presented by TME-resident activated macrophages. These macrophages acquire augmented antigen presentation proficiency and other M1-phenotype-associated features under IL-2 treatment. Our findings highlight the importance of restoring neoantigen-specific immune responses in the treatment of cancers with MHC class I deficiencies., Competing Interests: Declaration of interests J.D.B., M.V., L.A., M.D., and S.K. are employees and Ö.T. and U.S. are cofounders and management board members at BioNTech SE (Mainz, Germany). J.D.B., M.V., L.A., M.D., S.K., Ö.T., and U.S. hold securities from BioNTech SE. J.D.B., M.V., M.D., S.K., and U.S. are inventors on patents or patent applications related to this study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

mRNA based infectious disease vaccines have opened the venue for development of novel nucleic acids-based therapeutics. For all mRNA therapeutics dedicated delivery systems are required, where different functionalities and targeting abilities need to be optimized for the respective applications. One option for advanced formulations with tailored properties are lipid-polymer hybrid nanoparticles with complex nanostructure, which allow to combine features of several already well described nucleic acid delivery systems. Here, we explored hyaluronic acid (HA) as coating of liposome-mRNA complexes (LRCs) to investigate effects of the coating on surface charge, physicochemical characteristics and biological activity. HA was electrostatically attached to positively charged complexes, forming hybrid LRCs (HLRCs). At different N/P ratios, physico-chemical characterization of the two sets of particles showed similarity in size (around 200 nm) and mRNA binding abilities, while the presence of the HA shell conferred a negative surface charge to otherwise positive complexes. High transfection efficiency of LRCs and HLRCs in vitro has been obtained in THP-1 and human monocytes derived from PBMC, an interesting target cell population for cancer and immune related pathologies. In mice, quantitative biodistribution of radiolabeled LRC and HLRC particles, coupled with bioluminescence studies to detect the protein translation sites, hinted towards both particles' accumulation in the hepatic reticuloendothelial system (RES). mRNA translated proteins though was found mainly in the spleen, a major source for immune cells, with preference for expression in macrophages. The results showed that surface modifications of liposome-mRNA complexes can be used to fine-tune nanoparticle physico-chemical characteristics. This provides a tool for assembly of stable and optimized nanoparticles, which are prerequisite for future therapeutic interventions using mRNA-based nanomedicines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Antibody-dependent cellular phagocytosis (ADCP) is a process through which myeloid cells are able to exert their phagocytic function after recognition of opsonized bacteria, viruses, infected cells or any cells targeted by a specific antibody. ADCP of tumor cells represents a potent effector mechanism of monoclonal antibody therapy mediated by tumor associated macrophages (TAM) and other phagocytic cells as an in situ anti-tumor activity. Here we described a protocol based on flow cytometry and immunofluorescence assays enabling extensive comparative studies to address whether a monoclonal antibody engaging Fcγ receptors on macrophages can mediate in vitro ADCP of tumor cells., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Human papilloma virus (HPV) infection is a causative agent for several cancers types (genital, anal and head and neck region). The HPV E6 and E7 proteins are oncogenic drivers and thus are ideal candidates for therapeutic vaccination. We recently reported that a novel ribonucleic acid lipoplex (RNA-LPX)-based HPV16 vaccine, E7 RNA-LPX, mediates regression of mouse HPV16 + tumors and establishes protective T cell memory. An HPV16 E6/E7 RNA-LPX vaccine is currently being investigated in two phase I and II clinical trials in various HPV-driven cancer types; however, it remains a high unmet medical need for treatments for patients with radiosensitive HPV16 + tumors. Therefore, we set out to investigate the therapeutic efficacy of E7 RNA-LPX vaccine combined with standard-of-care local radiotherapy (LRT). We demonstrate that E7 RNA-LPX synergizes with LRT in HPV16 + mouse tumors, with potent therapeutic effects exceeding those of either monotherapy. Mode of action studies revealed that the E7 RNA-LPX vaccine induced high numbers of intratumoral-E7-specific CD8 + T cells, rendering cold tumors immunologically hot, whereas LRT primarily acted as a cytotoxic therapy, reducing tumor mass and intratumor hypoxia by predisposing tumor cells to antigen-specific T cell-mediated killing. Overall, LRT enhanced the effector function of E7 RNA-LPX-primed T cell responses. The therapeutic synergy was dependent on total radiation dose, rather than radiation dose-fractionation. Together, these results show that LRT synergizes with E7 RNA-LPX and enhances its anti-tumor activity against HPV16 + cancer models. This work paves into a new translational therapy for HPV16 + cancer patients., (© 2021. The Author(s).)

Background: Despite the preclinical promise of CD40 and 4-1BB as immuno-oncology targets, clinical efforts evaluating CD40 and 4-1BB agonists as monotherapy have found limited success. DuoBody-CD40×4-1BB (GEN1042/BNT312) is a novel investigational Fc-inert bispecific antibody for dual targeting and conditional stimulation of CD40 and 4-1BB to enhance priming and reactivation of tumor-specific immunity in patients with cancer., Methods: Characterization of DuoBody-CD40×4-1BB in vitro was performed in a broad range of functional immune cell assays, including cell-based reporter assays, T-cell proliferation assays, mixed-lymphocyte reactions and tumor-infiltrating lymphocyte assays, as well as live-cell imaging. The in vivo activity of DuoBody-CD40×4-1BB was assessed in blood samples from patients with advanced solid tumors that were treated with DuoBody-CD40×4-1BB in the dose-escalation phase of the first-in-human clinical trial (NCT04083599)., Results: DuoBody-CD40×4-1BB exhibited conditional CD40 and 4-1BB agonist activity that was strictly dependent on crosslinking of both targets. Thereby, DuoBody-CD40×4-1BB strengthened the dendritic cell (DC)/T-cell immunological synapse, induced DC maturation, enhanced T-cell proliferation and effector functions in vitro and enhanced expansion of patient-derived tumor-infiltrating lymphocytes ex vivo. The addition of PD-1 blocking antibodies resulted in potentiation of T-cell activation and effector functions in vitro compared with either monotherapy, providing combination rationale. Furthermore, in a first-in-human clinical trial, DuoBody-CD40×4-1BB mediated clear immune modulation of peripheral antigen presenting cells and T cells in patients with advanced solid tumors., Conclusion: DuoBody-CD40×4-1BB is capable of enhancing antitumor immunity by modulating DC and T-cell functions and shows biological activity in patients with advanced solid tumors. These findings demonstrate that targeting of these two pathways with an Fc-inert bispecific antibody may be an efficacious approach to (re)activate tumor-specific immunity and support the clinical investigation of DuoBody-CD40×4-1BB for the treatment of cancer., Competing Interests: Competing interests: US and ÖT are management board members and employees at BioNTech (Mainz, Germany). AM, FG, KS, AT, BS, JQ, MD and SK are employees at BioNTech. Some of the authors have securities from BioNTech. ES and FV are employees at TRON. HCA III, IA, JMB, SMB, DV, VMS, MF, DPES, SK, TA, ECWB and MJ-K are employees at Genmab and own stock and/or stock options. HCA III, IA, DPES, US, AM and FG are inventors on patents and patent applications related to CD40×4-1BB bispecific antibodies., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Checkpoint inhibitors (CPI) have revolutionized the treatment paradigm for advanced solid tumors; however, there remains an opportunity to improve response rates and outcomes. In preclinical models, 4-1BB costimulation synergizes with CPIs targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis by activating cytotoxic T-cell-mediated antitumor immunity. DuoBody-PD-L1×4-1BB (GEN1046) is an investigational, first-in-class bispecific immunotherapy agent designed to act on both pathways by combining simultaneous and complementary PD-L1 blockade and conditional 4-1BB stimulation in one molecule. GEN1046 induced T-cell proliferation, cytokine production, and antigen-specific T-cell-mediated cytotoxicity superior to clinically approved PD-(L)1 antibodies in human T-cell cultures and exerted potent antitumor activity in transplantable mouse tumor models. In dose escalation of the ongoing first-in-human study in heavily pretreated patients with advanced refractory solid tumors (NCT03917381), GEN1046 demonstrated pharmacodynamic immune effects in peripheral blood consistent with its mechanism of action, manageable safety, and early clinical activity [disease control rate: 65.6% (40/61)], including patients resistant to prior PD-(L)1 immunotherapy., Significance: DuoBody-PD-L1×4-1BB (GEN1046) is a first-in-class bispecific immunotherapy with a manageable safety profile and encouraging preclinical and early clinical activity. With its ability to confer clinical benefit in tumors typically less sensitive to CPIs, GEN1046 may fill a clinical gap in CPI-relapsed or refractory disease or as a combination therapy with CPIs. See related commentary by Li et al., p. 1184. This article is highlighted in the In This Issue feature, p. 1171., (©2022 The Authors; Published by the American Association for Cancer Research.)

Cancer therapy is an emergent application for mRNA therapeutics. While in tumor immunotherapy, mRNA encoding for tumor-associated antigens is delivered to antigen-presenting cells in spleen and lymph nodes, other therapeutic options benefit from immediate delivery of mRNA nanomedicines directly to the tumor. However, tumor targeting of mRNA therapeutics is still a challenge, since, in addition to delivery of the cargo to the tumor, specifics of the targeted cell type as well as its interplay with the tumor microenvironment are crucial for successful intervention. This study investigated lipoplex nanoparticle-mediated mRNA delivery to spheroid cell culture models of melanoma. Insights into cell-type specific targeting, non-cell-autonomous effects, and penetration capacity in tumor and stroma cells of the mRNA lipoplex nanoparticles were obtained. It was shown that both coculture of different cell types as well as three-dimensional cell growth characteristics can modulate distribution and transfection efficiency of mRNA lipoplex formulations. The results demonstrate that three-dimensional coculture spheroids can provide a valuable surplus of information in comparison to adherent cells. Thus, they may represent in vitro models with enhanced predictivity for the in vivo activity of cancer nanotherapeutics.

Objectives: We aimed to identify clinical, anamnestic, and sociodemographic characteristics associated with a positive swab for SARS-CoV2, and to provide a predictive score to identify at risk population in children aged 2-14 years attending school and tested for clinical symptoms of COVID-19., Design: Cross sectional study., Setting: Outpatient clinic of the IRCCS Burlo Garofolo, a maternal and child health tertiary care hospital and research centre in Italy., Data Collection and Analysis: Data were collected through a predefined form, filled out by parents, and gathered information on sociodemographic characteristics, and specific symptoms, which were analysed to determine their association with a positive SARS-CoV-2 swab. The regression coefficients of the variables included in the multivariate analysis were further used in the calculation of a predictive score of the positive or negative test., Results: Between September 20th and December 23rd 2020, from 1484 children included in the study, 127 (8.6%) tested positive. In the multivariate analysis, the variables retained by the model were the presence of contact with a cohabiting, non-cohabiting or unspecified symptomatic case (respectively OR 37.2, 95% CI 20.1-68.7; 5.1, 95% CI 2.7-9.6; 15.6, 95% CI 7.3-33.2); female sex (OR 1.49, 95% CI 1.0-2.3); age (6-10 years old: OR 3.2, 95% CI 1.7-6.1 p<0.001; >10 years old: OR 4.8, 95% CI 2.7-8.8 p<0.001); fever (OR 3.9, 95% CI 2.3-6.4); chills (OR 1.9, 95% CI 1.1-3.3); headache (OR 1.45, 95% CI 0.9-2.4); ageusia (OR 1.3, 95% CI 0.5-4.0); sore throat (OR 0.48, 95% CI 0.3-0.8); earache (OR 0.4, 95% CI 0.1-1.3); rhinorrhoea (OR 0.8, 95% CI 0.5-1.3); and diarrhoea (OR 0.52, 95% CI 0.2-1.1). The predictive score based on these variables generated 93% sensitivity and 99% negative predictive value., Conclusions: The timely identification of SARS-CoV2 cases among children is useful to reduce the dissemination of the disease and its related burden. The predictive score may be adopted in a public health perspective to rapidly identify at risk children., Competing Interests: The authors have declared that no competing interests exist.

Synchronous primary malignancies occur in a small proportion of head and neck squamous cell carcinoma (HNSCC) patients. Here, we analysed three synchronous primaries and a recurrence from one patient by comparing the genomic and transcriptomic profiles among the tumour samples and determining the recurrence origin. We found remarkable levels of heterogeneity among the primary tumours, and through the patterns of shared mutations, we traced the origin of the recurrence. Interestingly, the patient carried germline variants that might have predisposed him to carcinogenesis, together with a history of alcohol and tobacco consumption. The mutational signature analysis confirmed the impact of alcohol exposure, with Signature 16 present in all tumour samples. Characterisation of immune cell infiltration highlighted an immunosuppressive environment in all samples, which exceeded the potential activity of T cells. Studies such as the one described here have important clinical value and contribute to personalised treatment decisions for patients with synchronous primaries and matched recurrences.

A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD-foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD-foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD 'down', one-RBD 'up' state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4 + and IFNγ + CD8 + T cell responses. Prime-boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2-18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA 1-3 , and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728).

Antigen-encoding, lipoplex-formulated RNA (RNA-LPX) enables systemic delivery to lymphoid compartments and selective expression in resident antigen-presenting cells. We report here that the rejection of CT26 tumors, mediated by local radiotherapy (LRT), is further augmented in a CD8 + T cell-dependent manner by an RNA-LPX vaccine that encodes CD4 + T cell-recognized neoantigens (CD4 neoantigen vaccine). Whereas CD8 + T cells induced by LRT alone were primarily directed against the immunodominant gp70 antigen, mice treated with LRT plus the CD4 neoantigen vaccine rejected gp70-negative tumors and were protected from rechallenge with these tumors, indicating a potent poly-antigenic CD8 + T cell response and T cell memory. In the spleens of CD4 neoantigen-vaccinated mice, we found a high number of activated, poly-functional, Th1-like CD4 + T cells against ME1, the immunodominant CD4 neoantigen within the poly-neoantigen vaccine. LRT itself strongly increased CD8 + T cell numbers and clonal expansion. However, tumor infiltrates of mice treated with CD4 neoantigen vaccine/LRT, as compared to LRT alone, displayed a higher fraction of activated gp70-specific CD8 + T cells, lower PD-1/LAG-3 expression and contained ME1-specific IFNγ + CD4 + T cells capable of providing cognate help. CD4 neoantigen vaccine/LRT treatment followed by anti-CTLA-4 antibody therapy further enhanced the efficacy with complete remission of gp70-negative CT26 tumors and survival of all mice. Our data highlight the power of combining synergistic modes of action and warrants further exploration of the presented treatment schema., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Objective: The human body is colonized by bacteria, fungi and viruses. Resident commensal bacteria are a fundamental line of resistance to colonization by exogenous microbes. They actively regulate the production of nutrients by the host through a negative feedback mechanism, in order to prevent the availability of nutrients for potential pathogens. While only a small fraction of these microorganism may be pathogenic, the relationship between host and commensal microbiome is now studied as a whole, impacting several aspects of the host biology. Some studies have made clear the progresses in examining the role of microbiome on transplants and graft versus host disease (GVHD) severity and its pathogenesis: the risk of complications from allogenic hematopoietic stem cells transplantation (HSCT) is greater with the highest mortality if a patient has a lower bacterial diversity in the gut prior to the transplantation process beginning. Microbiota-associated molecular patterns are directly recognized by pathogen recognition receptors. The development of molecular methods has greatly expanded our knowledge of the composition and function of the microbiome in health and disease, shortening the response times vs. microbiological culture tests. The gut flora can make the difference when it comes to allo-HSCT. The aim of the study was to monitor microbiome of 10 children during allo-HSCT., Methods: Oral specimens and gut faecal microbiome (100 grams) samples were collected at 2, 16, 24 days. The samples were analysed by polymerase chain reaction and primary sequencing was done. To calculate the biodiversity of microbiome the Shannon index and the Observed species index were chosen., Results: Our study suggests some differences in the diversity indices (DIs) in 5 children affected by GVHD vs. not affected. The DIs in oral and faecal specimens show in all patients a diminution in the post-transplant phase with an improvement in species diversity after 16 days from the transplant. The Observed species index in faeces specimens after 16 days was higher in patients which had not GVHD; moreover, patients with GVHD showed a deterioration at 24 days. Oral specimens after 24 days showed a parallel trend in the two groups. The Shannon index shows a downward trend in faeces specimens of the children with GVHD at 24 days; the children without GVHD recover a good trend of entropy. Oral specimens at 24 days show low entropy in the two groups. Very aggressive bacterial species as Cronobacter and Routella in the faeces specimens of a child had not serious consequences for disease status: Cronobacter were not present 24 days after transplantation., Conclusions: The data show the microbial metabolome could have an impact on patients with GVHD vs. no GVHD. A better understanding of the role of the oral and gut microbiome in GVHD can give directions to move towards the development of innovative approaches for preventing GVHD following allo-HCT, reducing also antibiotic therapy.

Objectives: Therapeutic drug monitoring is becoming increasingly important in clinical decision-making in children with inflammatory bowel disease (IBD). However, enzyme-linked immunosorbent assay (ELISA) assays do not allow results to be provided in real-time. We sought to compare 2 point-of-care (POC) devices for quantification of serum infliximab concentration with 2 validated ELISA assays in children with IBD., Methods: We studied 32 serum samples from 19 children with IBD treated with infliximab. Serum samples were collected immediately before drug infusion (trough level). Infliximab was measured using 2 POC infliximab assays, Quantum Blue (POC IFX/QB) and Rida Quick (POC IFX/RQ), and 2 ELISA assays: Lisa-Tracker (used as primary reference), and Promonitor (used as second control). Intraclass correlation coefficient (ICC) was assessed for quantitative comparison. Qualitative analysis was also performed to evaluate whether POC assays would correctly classify infliximab serum according to a target window (between 3 and 7 μg/mL)., Results: ICC was 0.82 and 0.87 for POC IFX/QB and POC IFX/RQ with the primary reference ELISA assay, respectively; ICC between the 2 ELISA assays was 0.87. Classification of results according to therapeutic intervals showed good agreement between pairs of assays, with kappa of 0.67 and 0.80 for POC IFX/QB and POC IFX/RQ, respectively, with reference ELISA, and 0.81 between the 2 ELISAs. Accuracy of POC assays was better for drug levels <3 μg/mL., Conclusions: POC infliximab assays showed good agreement with traditional ELISA assays. POC devices may represent a viable option for real-time therapeutic drug monitoring in children treated with infliximab.

HPV16 infections are associated with a variety of cancers and there is compelling evidence that the transforming activity of HPV16 critically depends on the expression of the viral oncoproteins E6 and E7. Therapeutic cancer vaccines capable of generating durable and specific immunity against these HPV16 antigens hold great promise to achieve long-term disease control. Here we show in mice that HPV16 E7 RNA-LPX, an intravenously administered cancer vaccine based on immuno-pharmacologically optimized antigen-encoding mRNA, efficiently primes and expands antigen-specific effector and memory CD8 + T cells. HPV-positive TC-1 and C3 tumors of immunized mice are heavily infiltrated with activated immune cells and HPV16-specific T cells and are polarized towards a proinflammatory, cytotoxic and less immune-suppressive contexture. E7 RNA-LPX immunization mediates complete and durable remission of progressing tumors. Circulating memory T cells are highly cytotoxic and protect from tumor rechallenge. Moreover, E7 RNA-LPX immunization sensitizes anti-PD-L1 refractory tumors to checkpoint blockade. In conclusion, our data highlight the potential of HPV16 RNA-LPX for the treatment of HPV-driven cancers.

TLR7 agonists are considered promising drugs for cancer therapy. The currently available compounds are not well tolerated when administered intravenously and therefore are restricted to disease settings amenable for topical application. Here we present the preclinical characterization of SC1, a novel synthetic agonist with exquisite specificity for TLR7. We found that intravenously administered SC1 mediates systemic release of type I interferon, but not of proinflammatory cytokines such as TNFα and IL6, and results in activation of circulating immune cells. Tumors of SC1-treated mice have brisk immune cell infiltrates and are polarized towards a Th1 type signature. Intratumoral CD8 + T cells and CD11b + conventional dendritic cells (cDCs) are significantly increased, plasmacytoid dendritic cells (pDCs) are strongly activated and macrophages are M1 phenotype polarized, whereas myeloid-derived suppressor cells (MDSCs) are decreased. We further show that treatment of mice with SC1 profoundly inhibits the growth of established syngeneic tumors and results in significantly prolonged survival. Mice, which are tumor-free after SC1 treatment are protected from subsequent tumor rechallenge. The antitumor effect of SC1 depends on antigen-specific CD8 + T cells, which we found to be strongly enriched in the tumors of SC1-treated mice. In conclusion, this study shows that systemically administered SC1 mobilizes innate and adaptive immunity and is highly potent as single agent in mice and thereby provides a rationale for clinical testing of this compound.

Hyaluronic acid (HA) is a glycosaminoglycan of extracellular matrix related to cell surface which interacts with various cell types. To understand the role of HA during hepatocarcinogenesis, we assessed the effect of the inhibition of HA deposition and its association with heterogeneous hepatocellular carcinoma (HCC) cells. In this study, we used transgenic mice C57BL/6J-Tg(Alb1HBV)44Bri/J (HBV-TG) and normal C57BL/6 J (WT) for in vivo study, while HCC cells Huh7 and JHH6 as in vitro models. Both models were treated with an HA inhibitor 4-methylumbelliferone (4MU). We observed that 4MU treatments in animal model down-regulated the mRNA expressions of HA-related genes Has3 and Hyal2 only in HBV-TG but not in normal WT. As observed in vivo, in HCC cell lines, the HAS2 mRNA expression was down-regulated in Huh7 while HAS3 in JHH6, both with or without the presence of extrinsic HA. Interestingly, in both models, the expressions of various cancer stem cells (CD44, CD90, CD133, and EpCAM) were also decreased. Further, histological analysis showed that 4MU treatment with dose 25 mg/kg/day reduced fibrosis, inflammation, and steatosis in vivo, in addition to be pro-apoptotic. We concluded that the inhibition of HA reduced the expressions of HA-related genes and stem cells markers in both models, indicating a possible modulation of cells-to-cells and cells-to-matrix interaction.

Cancer-associated mutations, mostly single nucleotide variations, can act as neoepitopes and prime targets for effective anti-cancer T-cell immunity. T cells recognizing cancer mutations are critical for the clinical activity of immune checkpoint blockade (ICB) and they are potent vaccine antigens. High frequencies of mutation-specific T cells are rarely spontaneously induced. Hence, therapies that broaden the tumor specific T-cell response are of interest. Here, we analyzed neoepitope-specific CD8 + T-cell responses mounted either spontaneously or after immunotherapy regimens, which induce local tumor inflammation and cell death, in mice bearing tumors of the widely used colon carcinoma cell line CT26. A comprehensive immune reactivity screening of 2474 peptides covering 628 transcribed CT26 point mutations was conducted. All tested treatment regimens were found to induce a single significant CD8 + T-cell response against a non-synonymous D733A point mutation in the Smc3 gene. Surprisingly, even though Smc3 D733A turned out to be the immune-dominant neoepitope in CT26 tumor bearing mice, neither T cells specific for this neoepitope nor their T cell receptors (TCRs) were able to recognize or lyse tumor cells. Moreover, vaccination with the D733A neoepitope did not result in anti-tumoral activity despite induction of specific T cells. This is to our knowledge the first report that neoepitope specific CD8 + T cells primed by tumor-released antigen exposure in vivo can be functionally irrelevant.

Immunosuppression is a hallmark of tumor progression, and treatments that inhibit or deplete monocytic myeloid-derived suppressive cells could promote anti-tumor immunity. c-FLIP is a central regulator of caspase-8-mediated apoptosis and necroptosis. Here we show that low-dose cytotoxic chemotherapy agents cause apoptosis linked to c-FLIP down-regulation selectively in monocytes. Enforced expression of c-FLIP or viral FLIP rescues monocytes from cytotoxicity and concurrently induces potent immunosuppressive activity, in T cell cultures and in vivo models of tumor progression and immunotherapy. FLIP-transduced human blood monocytes can suppress graft versus host disease. Neither expression of FLIP in granulocytes nor expression of other anti-apoptotic genes in monocytes conferred immunosuppression, suggesting that FLIP effects on immunosuppression are specific to monocytic lineage and distinct from death inhibition. Mechanistically, FLIP controls a broad transcriptional program, partially by NF-κB activation. Therefore, modulation of FLIP in monocytes offers a means to elicit or block immunosuppressive myeloid cells.

Targeting mRNA to eukaryotic cells is an emerging technology for basic research and provides broad applications in cancer immunotherapy, vaccine development, protein replacement, and in vivo genome editing. Although a plethora of nanoparticles for efficient mRNA delivery exists, in vivo mRNA targeting to specific organs, tissue compartments, and cells remains a major challenge. For this reason, methods for reporting the in vivo targeting specificity of different mRNA nanoparticle formats will be crucial. Here, we describe a straightforward method for monitoring the in vivo targeting efficiency of mRNA-loaded nanoparticles in mice. To achieve accurate mRNA delivery readouts, we loaded lipoplex nanoparticles with Cre-recombinase-encoding mRNA and injected these into commonly used Cre reporter mouse strains. Our results show that this approach provides readouts that accurately report the targeting efficacy of mRNA into organs, tissue structures, and single cells as a function of the used mRNA delivery system. The method described here establishes a versatile basis for determining in vivo mRNA targeting profiles and can be systematically applied for testing and improving mRNA packaging formats.

Intradermal administration of antigen-encoding RNA has entered clinical testing for cancer vaccination. However, insight into the underlying mechanism of RNA uptake, translation and antigen presentation is still limited. Utilizing pharmacologically optimized naked RNA, the dose-response kinetics revealed a rise in reporter signal with increasing RNA amounts and a prolonged RNA translation of reporter protein up to 30 days after intradermal injection. Dendritic cells (DCs) in the dermis were shown to engulf RNA, and the signal arising from the reporter RNA was significantly diminished after DC depletion. Macropinocytosis was relevant for intradermal RNA uptake and translation in vitro and in vivo. By combining intradermal RNA vaccination and inhibition of macropinocytosis, we show that effective priming of antigen-specific CD8(+) T-cells also relies on this uptake mechanism. This report demonstrates that direct antigen translation by dermal DCs after intradermal naked RNA vaccination is relevant for efficient priming of antigen-specific T-cells.

Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses. However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encoded antigen by DC populations and macrophages in various lymphoid compartments. RNA-LPX triggers interferon-α (IFNα) release by plasmacytoid DCs and macrophages. Consequently, DC maturation in situ and inflammatory immune mechanisms reminiscent of those in the early systemic phase of viral infection are activated. We show that RNA-LPX encoding viral or mutant neo-antigens or endogenous self-antigens induce strong effector and memory T-cell responses, and mediate potent IFNα-dependent rejection of progressive tumours. A phase I dose-escalation trial testing RNA-LPX that encode shared tumour antigens is ongoing. In the first three melanoma patients treated at a low-dose level, IFNα and strong antigen-specific T-cell responses were induced, supporting the identified mode of action and potency. As any polypeptide-based antigen can be encoded as RNA, RNA-LPX represent a universally applicable vaccine class for systemic DC targeting and synchronized induction of both highly potent adaptive as well as type-I-IFN-mediated innate immune mechanisms for cancer immunotherapy.

Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient's tumour possesses a unique set of mutations ('the mutanome') that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient's individual tumour-specific mutations. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4(+) T cells. Vaccination with such CD4(+) immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4(+) T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient's tumour with vaccines produced 'just in time'.

Background: Working in health care carries the risk of transmission of infected blood to patients by hospital workers and to other health personnel in the form of occupational infections. Conscientious application of the standard precautions is the main method used to avoid needle stick injuries, contamination of skin and mucous membranes, cuts with sharp tools, and inadequate disposal and recapping of needles. The aim of this work was to investigate in Friuli Venezia Giulia, a region in north-east Italy, the enhancement carried out to prevent situations of biologic risk for health care workers, and to verify the related laboratory analyses., Methods: Biological accidents occurring during the years 2012-2013 in the departments of oncology and pediatric-obstetric surgery, and in the intensive care unit at Burlo Garofolo Children's Hospital in Trieste (a large town in Friuli Venezia Giulia) were reviewed, and a new panel of tests was introduced for patients and health care workers, to also detect human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), and aspartate transaminase and immunoglobulin G. All tests were submitted for external quality assessment., Results: In total, 230 nosocomial events were reported by health care workers in the above-mentioned hospital departments in 2012-2013. There were 158 accidents in 2012, including 55 accidental needle stick injuries (34.81%), 59 blood splashes (37.34%), and 44 cuts with infected instruments (27.84%). The risk of sustaining a cut was related to movement error during surgery when the appropriate procedure was not followed or when devices were being assembled and passed between doctors and nurses. Most accidents happened among physicians compared to nurses; the high percentage of needle stick injuries (34.81%) versus nurses (25.94%) were due to incorrect recapping of needles after use. No cases of health care workers being infected with HCV, HBV, or HIV were identified. In 2013, the number of biological accidents decreased to 61, comprising two needle stick injuries (3.27%), 35 blood splashes (57.37%), and 25 cuts with contaminated instruments (40.98%). The number of subcutaneous abscesses with scarring resulting from cuts with sharp instruments decreased from three in 2012 (one of which was the subject of medicolegal proceedings) to none in 2013. Although our study population was relatively small, we did detect a statistically significant decrease in the number of needle stick injuries (P<0.05, χ(2) test)., Conclusion: In this early experience at a maternal and child health institution in the Friuli Venezia Giulia region, application of a safety protocol, centralized organization of HIV tests, improved external quality assessment, and introduction of internal quality control for immunoglobulin G contributed to a decrease in the number of work-related biological accidents and their complications, which have the potential to result in medicolegal problems.

Mixed cryoglobulinemia is a lymphoproliferative disorder associated with hepatitis C virus (HCV). In patients chronically affected by HCV the prevalence of mixed cryoglobulinemia is variable ranging from 0% to 56%. To verify whether polyomaviruses (PyV) play a role in this disorder a total of 222 blood samples from 63 HCV chronic patients, 43 with mixed cryoglobulinemia, 59 chronic lymphocytic leukemia, 50 polytransfused patients, and 50 blood donors were evaluated for Merkel (MCPyV), BKV, JCV, and SV40. EBV was additionally included in the analysis since association with this disorder has been reported. Mixed cryoglobulinemia patients infected chronically with HCV resulted negative for both PyV and EBV. MCPyV was found in 1 subject with Merkel Cell Carcinoma, in 10% of polytransfused and in 10% of blood donors while EBV was detected in 22% of polytransfused, 10% of B-cell lymphatic leukemia patients and 4% of blood donors (P < 0.01). Taken together, the absence of PyV and EBV in HCV-mixed cryoglobulinemia patients seems to exclude a direct involvement of these viruses in the pathogenesis of this disease while the presence of MCPyV in healthy individuals, at the same rate as in polytransfused patients, may reinforce data on a minimal role of this virus in other human pathologies., (© 2013 Wiley Periodicals, Inc.)

Involvement of health personnel in a medical audit can reduce the number of errors in laboratory medicine. The checked control of point of care testing (POCT) could be an answer to developing a better medical service in the emergency department and decreasing the time taken to report tests. The performance of sanitary personnel from different disciplines was studied over an 18-month period in a children's hospital. Clinical errors in the emergency and laboratory departments were monitored by: nursing instruction using specific courses, POCT, and external quality control; improvement of test results and procedural accuracy; and reduction of hemolyzed and nonprotocol-conforming samples sent to the laboratory department. In January 2012, point of care testing (POCT) was instituted in three medical units (neonatology, resuscitation, delivery room) at the Children's Hospital in Trieste, northeast Italy, for analysis of hematochemical samples. In the same period, during the months of January 2012 and June 2013, 1,600 samples sent to central laboratory and their related preanalytical errors were examined for accuracy. External quality control for POCT was also monitored in the emergency department; three meetings were held with physicians, nurses, and laboratory technicians to highlight problems, ie, preanalytical errors and analytical methodologies associated with POCT. During the study, there was an improvement in external quality control for POCT from -3 or -2 standard deviations or more to one standard deviation for all parameters. Of 800 samples examined in the laboratory in January 2012, we identified 64 preanalytical errors (8.0%); in June 2013, there were 17 preanalytical errors (2.1%), representing a significant decrease (P<0.05, χ(2) test). Multidisciplinary management and clinical audit can be used as tools to detect errors caused by organizational problems outside the laboratory and improve clinical and economic outcomes.

Vaccination with in vitro transcribed RNA encoding tumor antigens is an emerging approach in cancer immunotherapy. Attempting to further improve RNA vaccine efficacy, we have explored combining RNA with immunomodulators such as rapamycin. Rapamycin, the inhibitor of mTOR, was used originally for immunosuppression. Recent reports in mouse systems, however, suggest that mTOR inhibition may enhance the formation and differentiation of the memory CD8(+) T-cell pool. Because memory T-cell formation is critical to the outcome of vaccination approaches, we studied the impact of rapamycin on the in vivo primed RNA vaccine-induced immune response using the chicken ovalbumin-expressing B16 melanoma model in C57BL/6 mice. Our data show that treatment with rapamycin at the effector-to-memory transition phase skews the vaccine-induced immune response toward the formation of a quantitatively and qualitatively superior memory pool and results in a better recall response. Tumor-infiltrating immune cells from these mice display a favorable ratio of effector versus suppressor cell populations. Survival of mice treated with the combined regimen of RNA vaccination with rapamycin is significantly longer (91.5 days) than that in the control groups receiving only one of these compounds (32 and 46 days, respectively). Our findings indicate that rapamycin enhances therapeutic efficacy of antigen-specific CD8(+) T cells induced by RNA vaccination, and we propose further clinical exploration of rapamycin as a component of immunotherapeutic regimens., (©2013 AACR.)

Background: In northeastern Italy, according to Italian legislation, authorized public facilities can accept the donation and preservation of cord blood stem cells (CB-SC). Attitudes and knowledge in pregnant women differs between the local and immigrant (non-European Union [EU]) population. In this study we assessed the choices that pregnant women have with respect to the public and private harvesting system and the main reasons driving their decisions. We examined the ethnic origin of the families and compared tests for syphilis screening and leukocyte (WBC) counts in the CB-SC bags that are required for validation of the collection., Methods: Out of a population of 3450 pregnant patients at the Institute for Maternal and Child Health of Trieste, northeast Italy, 772 women agreed to cord blood harvesting and the associated lab tests. Of these, 221 women (28.6%) were from immigrant families of non-EU countries. Their ethnic affiliation was recorded, and tests were performed for syphilis screening and for nucleated red blood cell (NRBC) interference with the WBC count in CB-SC bags to assess cellularity and to determine if storage was appropriate., Results: Of the 772 pregnant women, 648 (84.0%) accessed the public collection system, which is free of charge, and 124 (15.0%) accessed the private fee-based system. One woman from the non-EU group opted for the private fee-based system. Of the 3450 pregnant women screened for syphilis at the Institute for Maternal and Child Health, the Treponema pallidum hemagglutination (TPHA) and Venereal Disease Research Laboratory (VDRL) tests were the main tests performed (66.0% of total cases) because many gynecologists in the public harvesting system apply the Italian regulations of the 1988 Decree, while the private system requires tests on syphilis and leaves the option to the lab physicians to select the best determination method. We found that the chemiluminescence method was more specific (97.0%) than the TPHA (83.0%) and nontreponemal rapid plasma reagin VDRL (75.0%) tests (P < 0.05, χ(2) test). The specificity link between the two automatic methods versus microscopes for WBC dosing and NRBC interference was r(2) = 0.08 (ADVIA 120) and r(2) = 0.94 (XE-2100). The public system does not include human T-cell lymphotropic virus testing; this is reserved for the population from endemic zones., Conclusion: In northeastern Italy current legislation prevents the establishment of private fee-based banks for storage of CB-SC. The cryopreservation, for future autologous personal or family use, is possible only by sending to foreign private banks, with a further fee of €300. These regulations confirm that Italian legislation tries to increase the anonymous allogenic donations and the number of CB-CS bags stored in the free-cost public system, that are available to anyone with therapeutic needs. Private banking is used almost exclusively by the wealthier local population. In the public system, many physicians continue to use older Italian laws regarding syphilis diagnosis, and NRBC interference on WBC count may have an impact on cord blood harvesting. Our findings suggest that in the EU there is no consensus policy on donor management. The value of storage for potential use within the family is useful only with collaboration between the public and the private systems.

Background: The Friuli Venezia Giulia (FVG), a region of North Eastern Italy, has passed legislation (Decree No 2324/2010) to regulate the banking of umbilical cord blood samples for personal, autologous, or family-directed use, and to implement the Agreement of the State-Regions Permanent Conference (Decree No 62/CSR/2010). This paper aims to identify the formalities and the reasons why families collect and bank their cord blood in foreign banks for both personal and private use., Methods: To this end, at the Institute for Maternal and Child Health of Trieste (the regional capital city of the FVG), Italy, which assists about 1800 pregnant women a year, 129 questionnaires, drafted from January 2010 to December 2011 and concerning the granting of authorization to export samples, were examined., Results: The collected data showed that 75% of involved families had resorted to anonymous public collection, which is available to anyone with therapeutic needs, and provided compatibility and hematologic protocols recognized by the scientific and international community (main indications: leukemia, hemoglobinopaties, and inherited hematologic and immunologic disorders). Conversely, 25.0% requested private storage at a foreign bank for personal or family-dedicated use. The principal motivation by disease was for treatment for diabetes (22.4%) and celiac disease (19.7%) (a multiorgan disease for which the FVG region has provided safeguards by approving a specific law granting support to families; Decree No 561/2007). For these two types of disease we found that information was received from the internet and not from general medical physicians, with a significant difference found using the χ(2) test (P < 0.01)., Conclusion: The indication of treating these diseases with cord blood stem cell transplantation appears to be well grounded and encouraging, and has recently been corroborated by the international literature; however, the economic and social motivations promoting cord blood storage, for a fee, in the event of diseases that are still under study, require accurate information through general medical physicians on the actual possibilities of treatment.

Background: The immature (or reticulated) platelet fraction (IPF) is rich in nucleic acids, especially RNA, and can be used as a predictive factor for platelet recovery in platelet immunomediated consumption or in postchemotherapy myelosuppression. Our aim was to determine if transfusions with IPF-rich solutions, during autologous peripheral blood stem cell transplantation, reduce the occurrence of bleeding and hemorrhagic complications., Patients and Methods: Transfusions were administered to 40 children, affected with hematological pathologies, who underwent autologous peripheral hematopoietic progenitor cell transplantation. There were two groups of 20 patients, one group treated with IPF-poor and the other with IPF-rich solutions. In the two groups, the conditioning regimen was the same for the same pathology (hematological pathologies: 14 acute lymphoblastic leukemia; twelve acute myelocytic leukemia; four non-Hodgkin's lymphoma; two Hodgkin's lymphoma; eight solid tumors). A new automated analyzer was used to quantify the IPF: the XE2100 (Sysmex, Kobe, Japan) blood cell counter with upgraded software., Results: The 20 patients who received solutions with a high percentage of IPF (3%-9% of total number of infused platelets) required fewer transfusions than the 20 patients who received transfusions with a low percentage of IPF (0%-1% of total number of infused platelets): 83 versus 129 (mean of number of transfusions 4.15 versus 6.45) and a significant difference was found between the two groups by using the Mann-Whitney test (P < 0.001). The prophylactic transfusions decreased from three to two per week. There was only one case of massive hemorrhage., Conclusion: The use of IPF solutions reduces the number of transfusions and bleedings after peripheral blood stem cell transplantation in pediatric patients.

Engagement of the B cell receptor (BCR) by surface-tethered antigens (Ag) leads to formation of a synapse that promotes Ag uptake for presentation onto major histocompatibility complex class II (MHCII) molecules. We have highlighted the membrane trafficking events and associated molecular mechanisms involved in Ag extraction and processing at the B cell synapse. MHCII-containing lysosomes are recruited to the synapse where they locally undergo exocytosis, allowing synapse acidification and the extracellular release of hydrolases that promote the extraction of the immobilized Ag. Lysosome recruitment and secretion results from the polarization of the microtubule-organizing center (MTOC), which relies on the cell division cycle (Cdc42)-downstream effector, atypical protein kinase C (aPKCζ). aPKCζ is phosphorylated upon BCR engagement, associates to lysosomal vesicles, and is required for their polarized secretion at the B cell synapse. Regulation of B lymphocyte polarity therefore emerges as a central mechanism that couples Ag extraction to Ag processing and presentation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: The anti-Müllerian hormone (AMH) is a dimeric protein secreted by the female ovaries and has two fundamental roles in follicle genesis. It delays the entrance of the primordial follicle into the pool of follicles in growth and diminishes the sensitivity of the ovarian follicle towards follicle-stimulating hormone (FSH). The purpose of this work was to study the AMH (nv 2.0-6.8 ng/mL) as a marker during assisted reproductive technology (ART), in order to identify cases of infertility due to polycystic ovarian syndrome (PCOS). This syndrome affects 10% of women with infertility problems, and a new biological marker could be useful to general practitioners of internal medicine to help generate the suspicion of PCOS so that they can refer the patient to the gynecologist for confirmation., Methods: This study enrolled 236 patients aged 26-46 years undergoing assisted reproductive technology at the Institute for Maternal and Child Health, Trieste, Italy. On the third day of the ovarian cycle, the patients were given doses of AMH, FSH, and luteinizing hormone (LH, in cases of AMH < 2.0-6.8 ng/mL). A control pelvic ultrasound was also carried out., Results: We identified 57 patients who were starting in vitro fertilization or embryo transfer with AMH values within the normal range (3.64 ± 1.51 ng/mL), 77 with values below normal (1.38 ± 0.32 ng/mL), and 96 cases with undetectable values of AMH. Six patients had very high AMH levels (10.0 ± 2.28 ng/mL) and, of these, five were found to have PCOS on pelvic ultrasound examination (P < 0.05). We also found inverse correlations between AMH levels and age (r = -0.52) and between AMH and FSH levels (r = -0.32)., Conclusion: In clinical practice it is common to encounter patients who turn to medicine in search of a cure for female infertility. In our experience, AMH two or three times the normal amount (10 ± 2.28 ng/mL), is a good indication of PCOS and infertility.

Many investigators consider children's drawings to be an important test in the evaluation of stress and anxiety, but few studies have examined the reliability and validity of indicators of emotional distress in children's projective drawings. In this report, we describe screening tests in children coming to the Friuli Venezia Giulia region in Northeastern Italy from non-European Union regions and suspected to have celiac disease, the problems involved in diagnosis of the disease, and the "drawn double nostalgia" of Romanian children for both Italian food and traditional Romanian foods. Of 3150 Western European cases, we found 712 with positive antibodies for IgA/IgG antitransglutaminase, 174 with a positive antiendomysium antibody confirmation test, and 20 with an IgA deficit. Of the children examined, 93% were children native to Western Europe, 4% were immigrants from Eastern Europe, and 1.6% originated from Africa. Among these, four Romanian children with celiac disease brought in their drawings, as requested in a hospital questionnaire. The prevalence of celiac disease is destined to increase among immigrants. Economic problems are common, and the twin nostalgia of immigrant children for foods and tastes that are "cozonac" (from the native country) and "panettone" (Italian cake flavor) represents a problem that will be difficult to resolve. Only some children's hospitals in Italy, ie, Burlo Garofolo and Gaslini, public and private foundations, or volunteer associations would be able to deal with this problem.

Dendritic cells (DCs) sample peripheral tissues of the body in search of antigens to present to T cells. This requires two processes, antigen processing and cell motility, originally thought to occur independently. We found that the major histocompatibility complex II-associated invariant chain (Ii or CD74), a known regulator of antigen processing, negatively regulates DC motility in vivo. By using microfabricated channels to mimic the confined environment of peripheral tissues, we found that wild-type DCs alternate between high and low motility, whereas Ii-deficient cells moved in a faster and more uniform manner. The regulation of cell motility by Ii depended on the actin-based motor protein myosin II. Coupling antigen processing and cell motility may enable DCs to more efficiently detect and process antigens within a defined space.

This study aimed at evaluating the relationships among pathologies and methods employed to detect transglutaminase (tTG) and carbohydrate-deficient transferrin (CDT) in 33 celiac subjects presenting either high or low levels of tTG. Elevated tTG values were associated with increased CDT levels. A significant correlation was found between CDT and tTG values (r=0.57; p>0.0001). In the absence of wider studies and assessments on the interferences between methods used for these dosages and in presence of pathologies as carbohydrate-deficient glycoprotein syndrome (CDGs), this account assumes a possible correlation with celiac disease and suggests that the CDT test should not be used in celiac patients to authorize the return of the driving license after its withdrawal due to alcohol abuse [1, 2, 3].

Antigen binding to the B-cell receptor (BCR) induces multiple signaling cascades that ultimately lead to B lymphocyte activation. In addition, the BCR regulates the key trafficking events that allow the antigen to reach endocytic compartments devoted to antigen processing, i.e., that are enriched for major histocompatibility factor class II (MHC II) and accessory molecules such as H2-DM. Here, we analyze the role in antigen processing and presentation of the tyrosine kinase Syk, which is activated upon BCR engagement. We show that convergence of MHC II- and H2-DM-containing compartments with the vesicles that transport BCR-uptaken antigens is impaired in cells lacking Syk activity. This defect in endocytic trafficking compromises the ability of Syk-deficient cells to form MHC II-peptide complexes from BCR-internalized antigens. Altered endocytic trafficking is associated to a failure of Syk-deficient cells to properly reorganize their actin cytoskeleton in response to BCR engagement. We propose that, by modulating the actin dynamics induced upon BCR stimulation, Syk regulates the positioning and transport of the vesicles that carry the molecules required for antigen processing and presentation.

Antigen (Ag) capture and presentation onto major histocompatibility complex (MHC) class II molecules by B lymphocytes is mediated by their surface Ag receptor (B cell receptor [BCR]). Therefore, the transport of vesicles that carry MHC class II and BCR-Ag complexes must be coordinated for them to converge for processing. In this study, we identify the actin-associated motor protein myosin II as being essential for this process. Myosin II is activated upon BCR engagement and associates with MHC class II-invariant chain complexes. Myosin II inhibition or depletion compromises the convergence and concentration of MHC class II and BCR-Ag complexes into lysosomes devoted to Ag processing. Accordingly, the formation of MHC class II-peptides and subsequent CD4 T cell activation are impaired in cells lacking myosin II activity. Therefore, myosin II emerges as a key motor protein in BCR-driven Ag processing and presentation.

Antigen capture and presentation onto MHC class II molecules by B lymphocytes is mediated by their surface antigen receptor - the B-cell receptor (BCR). The BCR must therefore coordinate the transport of MHC class II- and antigen-containing vesicles for them to converge and ensure efficient processing. Recently, progress has been made in understanding which and how these vesicular transport events are molecularly linked to BCR signaling. In particular, recent studies have emphasized the key roles of membrane microdomains and the actin cytoskeleton in regulation of membrane trafficking upon BCR engagement.

Intracellular antibodies or intrabodies have great potential in protein knockout strategies for intracellular antigens. We applied the Intracellular Antibody Capture Technology for the direct selection in yeast of a mouse scFv library (V(L)-V(H) format) constructed from animals immunised with recombinant non-structural protein NSP5 of Rotavirus. We selected five different intracellular antibodies (ICAbs), which specifically recognize Delta2, an NSP5 deletion mutant used as bait. The anti-NSP5 ICAbs were well expressed both in yeast and mammalian cells as cytoplasmic or nuclear-tagged forms. By immunofluorescence and co-immunoprecipitation assays we characterised the intracellular interaction of the five anti-NSP5 ICAbs with the co-expressed antigens.

Rotavirus genomes contain 11 double-stranded (ds) RNA segments. Genome segment 11 encodes the non-structural protein NSP5 and, in some strains, also NSP6. NSP5 is produced soon after viral infection and localizes in cytoplasmic viroplasms, where virus replication takes place. RNA interference by small interfering (si) RNAs targeted to genome segment 11 mRNA of two different strains blocked production of NSP5 in a strain-specific manner, with a strong effect on the overall replicative cycle: inhibition of viroplasm formation, decreased production of other structural and non-structural proteins, synthesis of viral genomic dsRNA and production of infectious particles. These effects were shown not to be due to inhibition of NSP6. The results obtained strengthen the importance of secondary transcription/translation in rotavirus replication and demonstrate that NSP5 is essential for the assembly of viroplasms and virus replication.

Intracellular antibodies or intrabodies (ICAbs) have great potential in protein knockout strategies for intracellular antigens. In this study, they have been used to investigate the role of the rotavirus non-structural protein NSP5 in the virus replication cycle. Intracellular antibody-capture technology was used to select single-chain Fv format (scFv) ICAbs against an NSP5 mutant. Five different specific ICAbs were selected and expressed in MA104 cells, in the scFv format, as cytoplasmic- and nuclear-tagged forms. By confocal microscopy, it was found that three of these ICAbs recognized the full-length wild-type NSP5 specifically, forming antigen-specific aggresomes in the cytoplasm of cotransfected cells. Expression of the ICAbs in rotavirus-infected cells largely reduced the assembly of viroplasms and cellular cytopathic effect. Replication of dsRNA was partially inhibited, despite there being no reduction in virus titre. These results demonstrate for the first time a key role for NSP5 during the virus replicative cycle.