The Maritime Declaration of Health (MDH) as a tool to detect maritime traffic-related health risks: analysis of MDH forms submitted to Spanish ports, October 2014 to March 2015.

The international maritime traffic of people and goods has often contributed to the spread of pathogens affecting public health. The Maritime Declaration of Health (MDH), according to the International Health Regulations (IHR) (2005), is a document containing data related to the state of health on board a ship during passage and on arrival at port. It is a useful tool for early detection of public health risks. The main objective of our study was to evaluate compliance with the model provided in the IHR, focusing on the format and degree of completion of MDH forms received at Spanish ports. We reviewed the content of 802 MDH forms submitted to nine Spanish ports between October 2014 and March 2015. Study results show that 22% of MDH forms presented did not comply with the recommended model and 39% were incomplete. The proportion of cargo ships with correct and complete MDH forms was lower than passenger ships; thus, the nine health questions were answered less frequently by cargo ships than passenger ships (63% vs 90%, p value < 0.001). The appropriate demand and usage of MDH forms by competent authorities should improve the quality of the document as a tool and improve risk assessment.

The efficacy versus toxicity profile of combination virotherapy and TLR immunotherapy highlights the danger of administering TLR agonists to oncolytic virus-treated mice.

Injection of oncolytic vesicular stomatitis virus (VSV) into established B16ova melanomas results in tumor regression, in large part by inducing innate immune reactivity against the viral infection, mediated by MyD88- and type III interferon (IFN)-, but not TLR-4-, signaling. We show here that intratumoral (IT) treatment with lipopolysaccharide (LPS), a TLR-4 agonist, significantly enhanced the local therapy induced by VSV by combining activation of different innate immune pathways. Therapy was further enhanced by co-recruiting a potent antitumor, adaptive T-cell response by using a VSV engineered to express the ovalbumin tumor-associated antigen ova, in combination with LPS. However, the combination of IT LPS with systemically delivered VSV resulted in rapid morbidity and mortality in the majority of mice. Decreasing the intravenous (IV) dose of VSV to levels at which toxicity was ameliorated did not enhance therapy compared with IT LPS alone. Toxicity of the systemic VSV + IT LPS regimen was associated with rapidly elevated levels of serum tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, which neither systemic VSV, nor IT LPS, alone induced. These data show that therapy associated with direct IT injections of oncolytic viruses can be significantly enhanced by combination with agonists of innate immune activation pathways, which are not themselves activated by the virus alone. Importantly, they also highlight possible, unforeseen dangers of combination therapies in which an immunotherapy, even delivered locally at the tumor site, may systemically sensitize the patient to a cytokine shock-like response triggered by IV delivery of oncolytic virus.

Role of PATJ in stroke prognosis by modulating endothelial to mesenchymal transition through the Hippo/Notch/PI3K axis.

Through GWAS studies we identified PATJ associated with functional outcome after ischemic stroke (IS). The aim of this study was to determine PATJ role in brain endothelial cells (ECs) in the context of stroke outcome. PATJ expression analyses in patient's blood revealed that: (i) the risk allele of rs76221407 induces higher expression of PATJ, (ii) PATJ is downregulated 24 h after IS, and (iii) its expression is significantly lower in those patients with functional independence, measured at 3 months with the modified Rankin scale ((mRS) ≤2), compared to those patients with marked disability (mRS = 4-5). In mice brains, PATJ was also downregulated in the injured hemisphere at 48 h after ischemia. Oxygen-glucose deprivation and hypoxia-dependent of Hypoxia Inducible Factor-1α also caused PATJ depletion in ECs. To study the effects of PATJ downregulation, we generated PATJ-knockdown human microvascular ECs. Their transcriptomic profile evidenced a complex cell reprogramming involving Notch, TGF-ß, PI3K/Akt, and Hippo signaling that translates in morphological and functional changes compatible with endothelial to mesenchymal transition (EndMT). PATJ depletion caused loss of cell-cell adhesion, upregulation of metalloproteases, actin cytoskeleton remodeling, cytoplasmic accumulation of the signal transducer C-terminal transmembrane Mucin 1 (MUC1-C) and downregulation of Notch and Hippo signaling. The EndMT phenotype of PATJ-depleted cells was associated with the nuclear recruitment of MUC1-C, YAP/TAZ, ß-catenin, and ZEB1. Our results suggest that PATJ downregulation 24 h after IS promotes EndMT, an initial step prior to secondary activation of a pro-angiogenic program. This effect is associated with functional independence suggesting that activation of EndMT shortly after stroke onset is beneficial for stroke recovery.

Trap and ambush therapy using sequential primary and tumor escape-selective oncolytic viruses.

In multiple models of oncolytic virotherapy, it is common to see an early anti-tumor response followed by recurrence. We have previously shown that frontline treatment with oncolytic VSV-IFN-ß induces APOBEC proteins, promoting the selection of specific mutations that allow tumor escape. Of these mutations in B16 melanoma escape (ESC) cells, a C-T point mutation in the cold shock domain-containing E1 (CSDE1) gene was present at the highest frequency, which could be used to ambush ESC cells by vaccination with the mutant CSDE1 expressed within the virus. Here, we show that the evolution of viral ESC tumor cells harboring the escape-promoting CSDE1C-T mutation can also be exploited by a virological ambush. By sequential delivery of two oncolytic VSVs in vivo, tumors which would otherwise escape VSV-IFN-ß oncolytic virotherapy could be cured. This also facilitated the priming of anti-tumor T cell responses, which could be further exploited using immune checkpoint blockade with the CD200 activation receptor ligand (CD200AR-L) peptide. Our findings here are significant in that they offer the possibility to develop oncolytic viruses as highly specific, escape-targeting viro-immunotherapeutic agents to be used in conjunction with recurrence of tumors following multiple different types of frontline cancer therapies.

VSV oncolytic virotherapy in the B16 model depends upon intact MyD88 signaling.

We show here, for the first time to our knowledge, that the antitumor therapy of oncolytic vesicular stomatitis virus (VSV) in the B16ova model depends upon signaling through myeloid differentiation primary response gene 88 (MyD88) in host cells. VSV-mediated therapy of B16ova tumors was abolished in MyD88(-/-) mice despite generation of antigen-specific T cell responses similar to those in immune-competent mice. Mice defective in only toll-like receptor 4 (TLR4), TLR7, or interleukin 1 (IL-1) signaling retained VSV-induced therapy, suggesting that multiple, redundant pathways of innate immune activation by the virus contribute to antitumor immune reactivity. Lack of MyD88 signaling was associated with decreased expression of proinflammatory cytokines and neutrophil infiltration in response to intratumoral virus, as well as decreased infiltration of draining lymph nodes (LN) with plasmacytoid dendritic cells (pDCs) (CD11b(-)GR1(+)B220(+)) and myeloid-derived suppressor cells (CD11b(+)GR1(+)F4/80(+)). MyD88 signaling in response to VSV was also closely associated with a type I interferon (IFN) response. This inhibited virus replication within the tumor but also protected the host from viral dissemination from the tumor. Therefore, the innate immune response to oncolytic viruses can be, simultaneously, protherapeutic, antioncolytic, and systemically protective. These paradoxically conflicting roles need to be carefully considered in future strategies designed to improve the efficacy of oncolytic virotherapy.

Vesicular stomatitis virus-induced immune suppressor cells generate antagonism between intratumoral oncolytic virus and cyclophosphamide.

Despite having potent oncolytic activity, in vitro, direct intratumoral injection of oncolytic vesicular stomatitis virus (VSV) into established AE17ova mesothelioma tumors in C57Bl/6 mice had no therapeutic effect. During studies to combine systemic cyclophosphamide (CPA) with VSV to suppress the innate immune reaction against VSV, we observed that CPA alone had highly significant antitumor effects in this model. However, against our expectations, the combination of CPA and VSV consistently reduced therapeutic efficacy compared to CPA alone, despite the fact that the combination increased intratumoral VSV titers. We show here that CPA-mediated therapy against AE17ova tumors was immune-mediated and dependent upon both CD4 T cells and natural killer (NK) cells. However, intratumoral VSV induced a transforming growth factor-ß (TGF-ß)-dependent suppressive activity, mediated by CD11b(+)GR-1(+) cells that significantly inhibited both antigen-specific T-cell activation, and CPA-activated, NK-dependent killing of AE17ova tumor cells. Overall, our results show that treatment with oncolytic viruses can induce a variety of immune-mediated consequences in vivo with both positive, or negative, effects on antitumor therapy. These underexplored immune consequences of treatment with oncolytic viruses may have significant, and possibly unexpected, impacts on how virotherapy interacts in combination with other agents which modulate antitumor immune effectors.

The spike protein of SARS-CoV-2 induces heme oxygenase-1: Pathophysiologic implications.

BACKGROUND: Acute kidney injury (AKI) is both a consequence and determinant of outcomes in COVID-19. The kidney is one of the major organs infected by the causative virus, SARS-CoV-2. Viral entry into cells requires the viral spike protein, and both the virus and its spike protein appear in the urine of COVID-19 patients with AKI. We examined the effects of transfecting the viral spike protein of SARS-CoV-2 in kidney cell lines. METHODS: HEK293, HEK293-ACE2+ (stably overexpressing ACE2), and Vero E6 cells having endogenous ACE2 were transfected with SARS-CoV-2 spike or control plasmid. Assessment of gene and protein expression, and syncytia formation was performed, and the effects of quercetin on syncytia formation examined. FINDINGS: Spike transfection in HEK293-ACE2+ cells caused syncytia formation, cellular sloughing, and focal denudation of the cell monolayer; transfection in Vero E6 cells also caused syncytia formation. Spike expression upregulated potentially nephrotoxic genes (TNF-α, MCP-1, and ICAM1). Spike upregulated the cytoprotective gene HO-1 and relevant signaling pathways (p-Akt, p-STAT3, and p-p38). Quercetin, an HO-1 inducer, reduced syncytia formation and spike protein expression. INTERPRETATION: The major conclusions of the study are: 1) Spike protein expression in kidney cells provides a relevant model for the study of maladaptive and adaptive responses germane to AKI in COVID-19; 2) such spike protein expression upregulates HO-1; and 3) quercetin, an HO-1 inducer, may provide a clinically relevant/feasible protective strategy in AKI occurring in the setting of COVID-19. FUNDING: R01-DK119167 (KAN), R01-AI100911 (JPG), P30-DK079337; R01-DK059600 (AA).

Oncolytic measles virus therapy enhances tumor antigen-specific T-cell responses in patients with multiple myeloma.

Oncolytic virus therapy leads to immunogenic death of virus-infected tumor cells and this has been shown in preclinical models to enhance the cytotoxic T-lymphocyte response against tumor-associated antigens (TAAs), leading to killing of uninfected tumor cells. To investigate whether oncolytic virotherapy can increase immune responses to tumor antigens in human subjects, we studied T-cell responses against a panel of known myeloma TAAs using PBMC samples obtained from ten myeloma patients before and after systemic administration of an oncolytic measles virus encoding sodium iodide symporter (MV-NIS). Despite their prior exposures to multiple immunosuppressive antimyeloma treatment regimens, T-cell responses to some of the TAAs were detectable even before measles virotherapy. Measurable baseline T-cell responses against MAGE-C1 and hTERT were present. Furthermore, MV-NIS treatment significantly (P < 0.05) increased T-cell responses against MAGE-C1 and MAGE-A3. Interestingly, one patient who achieved complete remission after MV-NIS therapy had strong baseline T-cell responses both to measles virus proteins and to eight of the ten tested TAAs. Our data demonstrate that oncolytic virotherapy can function as an antigen agnostic vaccine, increasing cytotoxic T-lymphocyte responses against TAAs in patients with multiple myeloma, providing a basis for continued exploration of this modality in combination with immune checkpoint blockade.

Use of biological therapy to enhance both virotherapy and adoptive T-cell therapy for cancer.

To protect viral particles from neutralization, sequestration, nonspecific adhesion, and mislocalization following systemic delivery, we have previously exploited the natural tumor-homing properties of antigen-specific CD8+ T cells. Thus, OT-I T cells, preloaded in vitro with the oncolytic vesicular stomatitis virus (VSV), can deliver virus to established B16ova tumors to generate significantly better therapy than that achievable with OT-I T cells, or systemically delivered VSV, alone. Here, we demonstrate that preconditioning immune-competent mice with Treg depletion and interleukin-2 (IL-2), before adoptive T-cell therapy with OT-I T cells loaded with VSV, leads to further highly significant increases in antitumor therapy. Therapy was associated with antitumor immune memory, but with no detectable toxicities associated with IL-2, Treg depletion, or systemic dissemination of the oncolytic virus. Efficacy was contributed by multiple factors, including improved persistence of T cells; enhanced delivery of VSV to tumors; increased persistence of OT-I cells in vivo resulting from tumor oncolysis; and activation of NK cells, which acquire potent antitumor and proviral activities. By controlling the levels of virus loaded onto the OT-I cells, adoptive therapy was still effective in mice preimmune to the virus, indicating that therapy with virus-loaded T cells may be useful even in virus-immune patients. Taken together, our data show that it is possible to combine adoptive T-cell therapy, with biological therapy (Treg depletion+IL-2), and VSV virotherapy, to treat established tumors under conditions where none of the individual modalities alone is successful.

Cold panniculitis.

Cold panniculitis has been described in children and young women following cold exposure. Histopathologically, cold panniculitis shows a mostly lobular panniculitis, which consists of an infiltrate of lymphocytes and histiocytes in the fat lobules. Usually, the dermis shows a superficial and deep perivascular infiltrate mostly composed of lymphocytes, with no vasculitis. Inflammation is most intense at the dermal-subcutaneous junction. Differential diagnosis of cold panniculitis should be established with subcutaneous fat necrosis of the newborn, sclerema neonatorum, poststeroid panniculitis, chilblains, and frostbites.

Active immunotherapy with 1E10 anti-idiotype vaccine in patients with small cell lung cancer: report of a phase I trial.

1E10 is an anti-idiotype murine monoclonal antibody (Ab2 MAb) specific to an Ab1 MAb which reacts with NeuGc-containing gangliosides, sulfatides and with antigens expressed in some human tumors. Preparations containing this Ab2 were capable to induce a strong anti-metastatic effect in tumor-bearing mice. We conducted a Phase I clinical trial to evaluate the toxicity and humoral immune response elicited by 1E10 vaccine in patients with small cell lung cancer (SCLC). Eligible patients were those who after received chemotherapy and/or radiotherapy had partial or complete response to treatment. Patients received four biweekly injections with 2 mg of aluminum hydroxide-precipitated 1E10 MAb, then other six doses at 28-day intervals, and later the patients who maintained a good performance status were reimmunized. Six patients with limited-stage disease and three with extensive-stage disease were enrolled in the study. Most of the patients who received at least four doses of 1E10 vaccine developed strong specific antibody responses against 1E10 MAb and NeuGc-GM3 ganglioside. Antibodies able to react with lung carcinoma tissue sections were detected in sera from vaccinated patients. A prolonged survival was observed in several patients treated with the anti-idiotype vaccine. No evidence of serious adverse effects was found.

Definitive Management of Oligometastatic Melanoma in a Murine Model Using Combined Ablative Radiation Therapy and Viral Immunotherapy.

PURPOSE: The oligometastatic state is an intermediate state between a malignancy that can be completely eradicated with conventional modalities and one in which a palliative approach is undertaken. Clinically, high rates of local tumor control are possible with stereotactic ablative radiation therapy (SABR), using precisely targeted, high-dose, low-fraction radiation therapy. However, in oligometastatic melanoma, virtually all patients develop progression systemically at sites not initially treated with ablative radiation therapy that cannot be managed with conventional chemotherapy and immunotherapy. We have demonstrated in mice that intravenous administration of vesicular stomatitis virus (VSV) expressing defined tumor-associated antigens (TAAs) generates systemic immune responses capable of clearing established tumors. Therefore, in the present preclinical study, we tested whether the combination of systemic VSV-mediated antigen delivery and SABR would be effective against oligometastatic disease. METHODS AND MATERIALS: We generated a model of oligometastatic melanoma in C57BL/6 immunocompetent mice and then used a combination of SABR and systemically administered VSV-TAA viral immunotherapy to treat both local and systemic disease. RESULTS: Our data showed that SABR generates excellent control or cure of local, clinically detectable, and accessible tumor through direct cell ablation. Also, the immunotherapeutic activity of systemically administered VSV-TAA generated T-cell responses that cleared subclinical metastatic tumors. We also showed that SABR induced weak T-cell-mediated tumor responses, which, particularly if boosted by VSV-TAA, might contribute to control of local and systemic disease. In addition, VSV-TAA therapy alone had significant effects on control of both local and metastatic tumors. CONCLUSIONS: We have shown in the present preliminary murine study using a single tumor model that this approach represents an effective, complementary combination therapy model that addresses the need for both systemic and local control in oligometastatic melanoma.

Diagnosis of bacteraemia and growth times.

OBJECTIVE: The objective of this study was to predict the diagnosis of bacteraemia as a function of the time at which the automated BacT/Alert system continuously detects microorganism growth. METHODS: A retrospective study of a database of 1334 patients with a positive blood culture between January 2011 and June 2013 was conducted. Together with the final blood culture results and the patient's history, growth was then analysed to assess whether it represented true bacteraemia or bacterial contamination. The earliest detection times of bacterial growth in each batch of blood cultures were analysed in a blinded fashion after classification. RESULTS: In total, 590 batches of blood cultures corresponded to true bacteraemia and 744 to bacterial contamination. In the bacteraemia group, the median growth time was 12.72 h (interquartile range (IQR) 10.08-17.58 h). In the contaminated blood culture group, the median growth time was 20.6h (IQR 17.04-32.16 h) (p<0.001). Analysis of the receiver operating characteristics (ROC) curve (area under the curve 0.80, 95% confidence interval 0.771-0.826) showed that 90% of the contaminants grew after 14.7h (sensitivity 90.5%, specificity 63.4%, positive predictive value (PPV) 65.9%, negative predictive value (NPV) 90.7%). Forty-five percent of the bacteraemia organisms grew in under 12h (sensitivity 45.3%, specificity 95%, PPV 87.8%, NPV 68.7%). Microorganisms such as Candida sp and Bacteroides sp presented median growth times significantly longer than those of the other microorganisms. The administration of antibiotics in the week prior to bacteraemia was found to delay the growth time of microorganisms (p<0.001). CONCLUSIONS: Knowing the time to detection of microorganism growth can help to distinguish between true bacteraemia and bacterial contamination, thus allowing more timely clinical decisions to be made, before definitive microorganism identification.

CD45 y leucemia linfoide aguda pediátrica / CD45 and childhood acute lymphoblastic leukemia

Introducción: El CD45 se expresa en las células hematopoyéticas, su determinación es indispensable para la clasificación inmunofenotípica de la leucemia linfoide aguda (LLA). Objetivo: Evaluar la expresión del antígeno CD45 en los blastos de pacientes pediátricos con LLA y su relación con las características biológicas, morfológicas y clínicas al inicio de la enfermedad, la respuesta al tratamiento y la supervivencia global (SG) de los enfermos. Métodos: Se estudiaron 160 pacientes con LLA entre diciembre del 2012 y diciembre del 2017, tratados con el protocolo ALL-IC BFM-SG 2009. El inmunofenotipaje celular de la médula ósea se realizó por citometría de flujo. Resultados: El fenotipo B CD45+ predominó en los menores de seis años de edad y en los mayores de diez, el fenotipo T CD45+. Se encontró diferencia significativa entre la ausencia de adenopatías mediastínicas, el fenotipo leucémico y la ausencia de CD45 (p=0.004); entre la respuesta a la prednisona en sangre periférica al día ocho, el fenotipo leucémico y la ausencia de CD45 (p=0.001). Se encontraron diferencias significativas entre la respuesta a la prednisona en sangre periférica el día ocho y la respuesta en médula ósea el día 33, según fenotipo leucémico (p=0.009) y la presencia en los blastos del antígeno CD45 (p=0.02). Se encontró diferencia significativa entre la SG de los enfermos, según fenotipo leucémico y la ausencia del antígeno CD45 (p=0.017). Conclusión: La expresión o ausencia del antígeno de CD45 en los blastos tiene relación con la respuesta al tratamiento y la SG de pacientes pediátricos con LLA(AU)

Introduction: CD45 is expressed in hematopoietic cells, its determination is essential for the immunophenotypic classification of acute lymphoid leukemia (ALL). Objective: To evaluate the expression of the CD45 antigen in the blasts of pediatric patients with ALL and its relationship with the biological, morphological and clinical characteristics at the onset of the disease, the response to treatment and the overall survival (OS) of the patients. Methods: 160 patients with ALL were studied between December 2012 and December 2017, treated with the ALL-IC BFM-SG 2009 protocol. Bone marrow cellular immunophenotyping was performed by flow cytometry. Results: Patients with the CD45 + B phenotype predominated in those under six years of age, while those with a CD45 + T phenotype in those older than ten. A significant difference was found between the absence of mediastinal lymph nodes, the leukemic phenotype and the absence of CD45 (p = 0.004). A significant difference was found between the response to prednisone in peripheral blood at day eight, the leukemic phenotype and the absence of CD45, p = 0.001. Significant differences were found between the response to prednisone in peripheral blood on day eight and the response in bone marrow on day 33, according to leukemic phenotype and the presence in blasts of the CD45 antigen (p = 0.009 and p = 0.02, respectively). A significant difference was found between the OS of patients, according to leukemic phenotype and the absence of the CD45 antigen, p = 0.017. Conclusion: The expression or absence of the CD45 antigen in blasts is related to the response to treatment and OS of pediatric patients with ALL(AU)

CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors.

A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CAR(v2)) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CAR(v2) fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.Molecular Therapy-Nucleic Acids (2013) 2, e93; doi:10.1038/mtna.2013.19; published online 21 May 2013.

Systemic combination virotherapy for melanoma with tumor antigen-expressing vesicular stomatitis virus and adoptive T-cell transfer.

Oncolytic virotherapy offers the potential to treat tumors both as a single agent and in combination with traditional modalities such as chemotherapy and radiotherapy. Here we describe an effective, fully systemic treatment regimen, which combines virotherapy, acting essentially as an adjuvant immunotherapy, with adoptive cell transfer (ACT). The combination of ACT with systemic administration of a vesicular stomatitis virus (VSV) engineered to express the endogenous melanocyte antigen glycoprotein 100 (gp100) resulted in regression of established melanomas and generation of antitumor immunity. Tumor response was associated with in vivo T-cell persistence and activation as well as treatment-related vitiligo. However, in a proportion of treated mice, initial tumor regressions were followed by recurrences. Therapy was further enhanced by targeting an additional tumor antigen with the VSV-antigen + ACT combination strategy, leading to sustained response in 100% of mice. Together, our findings suggest that systemic virotherapy combined with antigen-expressing VSV could be used to support and enhance clinical immunotherapy protocols with adoptive T-cell transfer, which are already used in the clinic.