Reconvalescent plasma/camostat mesylate in early SARS-CoV-2 Q-PCR positive high-risk individuals (RES-Q-HR): a structured summary of a study protocol for a randomized controlled trial.

OBJECTIVES: Currently, there are no approved treatments for early disease stages of COVID-19 and few strategies to prevent disease progression after infection with SARS-CoV-2. The objective of this study is to evaluate the safety and efficacy of convalescent plasma (CP) or camostat mesylate administered within 72 h of diagnosis of SARS-CoV-2 infection in adult individuals with pre-existing risk factors at higher risk of getting seriously ill with COVID-19. Camostat mesylate acts as an inhibitor of the host cell serine protease TMPRSS2 and prevents the virus from entering the cell. CP represents another antiviral strategy in terms of passive immunization. The working hypothesis to be tested in the RES-Q-HR study is that the early use of CP or camostat mesylate reduces the likelihood of disease progression to (modified) WHO stages 4b-8 in SARS-CoV-2-positive adult patients at high risk of moderate or severe COVID-19 progression. TRIAL DESIGN: This study is a 4-arm (parallel group), multicenter, randomized (2:2:1:1 ratio), partly double-blind, controlled trial to evaluate the safety and efficacy of convalescent plasma (CP) or camostat mesylate with control or placebo in adult patients diagnosed with SARS-CoV-2 infection and high risk for progression to moderate/severe COVID-19. Superiority of the intervention arms will be tested. PARTICIPANTS: The trial is conducted at 10-15 tertiary care centers in Germany. Individuals aged 18 years or above with ability to provide written informed consent with SARS-CoV-2 infection, confirmed by PCR within 3 days or less before enrolment and the presence of at least one SARS-CoV-2 symptom (such as fever, cough, shortness of breath, sore throat, headache, fatigue, smell/and or taste disorder, diarrhea, abdominal symptoms, exanthema) and symptom duration of not more than 3 days. Further inclusion criteria comprise: Presence of at least one of the following criteria indicating increased risk for severe COVID-19: Age > 75 years Chronic obstructive pulmonary disease (COPD) and/or pulmonary fibrosis BMI > 40 kg/m2 Age > 65 years with at least one other risk factor (BMI > 35 kg/m2, coronary artery disease (CAD), chronic kidney disease (CKD) with GFR < 60 ml/min but ≥ 30 ml/min, diabetes mellitus, active tumor disease) BMI > 35 kg/m2 with at least one other risk factor (CAD, CKD with GFR < 60 ml/min but ≥ 30 ml/min, diabetes mellitus, active tumor disease) Exclusion criteria: 1. Age < 18 years 2. Unable to give informed consent 3. Pregnant women or breastfeeding mothers 4. Previous transfusion reaction or other contraindication to a plasma transfusion 5. Known hypersensitivity to camostat mesylate and/or severe pancreatitis 6. Volume stress due to CP administration would be intolerable 7. Known IgA deficiency 8. Life expectancy < 6 months 9. Duration SARS-CoV-2 typical symptoms > 3 days 10. SARS-CoV-2 PCR detection older than 3 days 11. SARS-CoV-2 associated clinical condition ≥ WHO stage 3 (patients hospitalized for other reasons than COVID-19 may be included if they fulfill all inclusion and none of the exclusion criteria) 12. Previously or currently hospitalized due to SARS-CoV-2 13. Previous antiviral therapy for SARS-CoV-2 14. ALT or AST > 5 x ULN at screening 15. Liver cirrhosis > Child A (patients with Child B/C cirrhosis are excluded from the trial) 16. Chronic kidney disease with GFR < 30 ml/min 17. Concurrent or planned anticancer treatment during trial period 18. Accommodation in an institution due to legal orders (§40(4) AMG). 19. Any psycho-social condition hampering compliance with the study protocol. 20. Evidence of current drug or alcohol abuse 21. Use of other investigational treatment within 5 half-lives of enrolment is prohibited 22. Previous use of convalescent plasma for COVID-19 23. Concomitant proven influenza A infection 24. Patients with organ or bone marrow transplant in the three months prior to screening visit INTERVENTION AND COMPARATOR: Participants will be randomized to the following 4 groups: 1) Convalescent plasma (CP), 2 units at screening/baseline visit (day 0) or day 1; CP is defined by the presence of neutralizing anti-SARS-CoV-2 antibodies with titers ≥ 1:160; individuals with body weight ≥ 150 kg will receive a third unit of plasma on day 3 2) Camostat mesylate (200 mg per capsule, one capsule taken each in the morning, afternoon and evening on days 1-7) 3) Standard of care (SOC, control for CP) 4) Placebo (identical in appearance to camostat mesylate capsules, one capsule taken each morning, afternoon and evening on days 1-7; for camostat mesylate control group) Participants will be monitored after screening/baseline on day 3, day 5, day 8, and day 14. On day 28 and day 56, telephone visits and on day 90, another outpatient visit are scheduled. Adverse events and serious adverse events will be monitored and reported until the end of the study. An independent data safety monitoring committee will review trial progression and safety. MAIN OUTCOMES: The primary endpoint of the study is the cumulative number of individuals who progress to or beyond category 4b on the modified WHO COVID-19 ordinal scale (defined as hospitalization with COVID-19 pneumonia and additional oxygen demand via nasal cannula or mask) within 28 days after randomization. RANDOMIZATION: Participants will be randomized using the Alea-Tool ( aleaclinical.com ) in a 2:2:1:1 ratio to the treatment arms (1) CP, (2) camostat mesylate, (3) standard of care (SoC), and (4) placebo matching camostat mesylate. Randomization will be stratified by study center. BLINDING (MASKING): The camostat mesylate treatment arm and the respective placebo will be blinded for participants, caregivers, and those assessing outcomes. The treatment arms convalescent plasma and standard of care will not be blinded and thus are open-labeled, unblinded. NUMBERS TO BE RANDOMIZED (SAMPLE SIZE): Overall, n = 994 participants will be randomized to the following groups: n = 331 to convalescent plasma (CP), n = 331 to camostat mesylate, n = 166 to standard of care (SoC), and n = 166 to placebo matching camostat mesylate. TRIAL STATUS: The RES-Q-HR protocol (V04F) was approved on the 18 December 2020 by the local ethics committee and by the regulatory institutions PEI/BfARM on the 2 December 2020. The trial was opened for recruitment on 26 December 2020; the first patient was enrolled on 7 January 2021 and randomized on 8 January 2021. Recruitment shall be completed by June 2021. The current protocol version RES-Q HR V05F is from 4 January 2021, which was approved on the 18 January 2021. TRIAL REGISTRATION: EudraCT Number 2020-004695-18 . Registered on September 29, 2020. ClinicalTrial.gov NCT04681430 . Registered on December 23, 2020, prior to the start of the enrollment (which was opened on December 26, 2020). FULL PROTOCOL: The full protocol (V05F) is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2).

EGFR/Ras-induced CCL20 production modulates the tumour microenvironment.

BACKGROUND: The activation of the EGFR/Ras-signalling pathway in tumour cells induces a distinct chemokine repertoire, which in turn modulates the tumour microenvironment. METHODS: The effects of EGFR/Ras on the expression and translation of CCL20 were analysed in a large set of epithelial cancer cell lines and tumour tissues by RT-qPCR and ELISA in vitro. CCL20 production was verified by immunohistochemistry in different tumour tissues and correlated with clinical data. The effects of CCL20 on endothelial cell migration and tumour-associated vascularisation were comprehensively analysed with chemotaxis assays in vitro and in CCR6-deficient mice in vivo. RESULTS: Tumours facilitate progression by the EGFR/Ras-induced production of CCL20. Expression of the chemokine CCL20 in tumours correlates with advanced tumour stage, increased lymph node metastasis and decreased survival in patients. Microvascular endothelial cells abundantly express the specific CCL20 receptor CCR6. CCR6 signalling in endothelial cells induces angiogenesis. CCR6-deficient mice show significantly decreased tumour growth and tumour-associated vascularisation. The observed phenotype is dependent on CCR6 deficiency in stromal cells but not within the immune system. CONCLUSION: We propose that the chemokine axis CCL20-CCR6 represents a novel and promising target to interfere with the tumour microenvironment, and opens an innovative multimodal strategy for cancer therapy.

Tspan8 is expressed in breast cancer and regulates E-cadherin/catenin signalling and metastasis accompanied by increased circulating extracellular vesicles.

Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8- tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up-regulation of E-cadherin and down-regulation of Twist, p120-catenin, and ß-catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal-epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell-cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several-fold increase in EV number in cell culture and the circulation of tumour-bearing animals. We observed increased protein levels of E-cadherin and p120-catenin in these EVs; furthermore, Tspan8 and p120-catenin were co-immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Resident microglia rather than peripheral macrophages promote vascularization in brain tumors and are source of alternative pro-angiogenic factors.

Myeloid cells are an essential part of the glioblastoma microenvironment. However, in brain tumors the function of these immune cells is not sufficiently clarified. In our study, we investigated differential pro-angiogenic activities of resident microglia and peripheral macrophages and their impact on glioma vascularization and progression. Our data demonstrate stable accumulation of microglia/macrophages during tumor growth. These cells often interact with tumor blood vessels correlating with vascular remodeling. Here, we identified resident microglia as well as peripheral macrophages as part of the perivascular niche, primarily contacting endothelial cells. We found overexpression of a variety of pro-angiogenic molecules within freshly isolated microglia/macrophages from glioma. CXCL2, until now a poorly described chemokine, was strongly up-regulated and showed better angiogenic activity than VEGF in vitro. Blocking the CXCL2-CXCR2 signaling pathway resulted in considerably diminished glioma sizes. Additionally, the importance of microglia/macrophages in tumor angiogenesis was confirmed by depletion of these cells in vivo. Vessel density decreased by 50% leading to significantly smaller tumor volumes. Remarkably, selective reduction of resident microglia affected tumoral vessel count comparable to ablation of the whole myeloid cell fraction. These results provide evidence that resident microglia are the crucial modulatory cell population playing a central role in regulation of vascular homeostasis and angiogenesis in brain tumors. Thus, resident microglia represent an alternative source of pro-angiogenic growth factors and cytokines.

Growth and metastasis of B16-F10 melanoma cells is not critically dependent on host CD73 expression in mice.

BACKGROUND: Recent studies have suggested that adenosine generated by ecto-5'-nucleotidase (CD73) in the tumor microenvironment plays a major role in promoting tumor growth by suppressing the immune response and stimulating angiogenesis via A2A and A2B receptors. However, adenosine has also been reported to inhibit tumor growth acting via A1 and A3 receptors. Therefore the aim of this study was to clarify the role of host CD73, which catalyzes the extracellular hydrolysis of AMP to adenosine, on tumor growth and metastasis of B16-F10 melanoma cells. METHODS: CD73 and alkaline phosphatase (AP) activity of B16-F10 melanoma cells were measured by HPLC. Tumor cells were injected either subcutaneously or intradermally in WT and CD73-/- mice and tumor growth was monitored by MRI at 9.4 T. Immune cell subpopulations within tumors were assessed by FACS after enzymatic digestion. An endothelium specific CD73-/- was created using Tie2-Cre+ mice and CD73flox/flox (loxP) mice. Chimeric mice lacking CD73-/- on hematopoietic cells was generated by bone marrow transplantation. Lung metastatic spread was measured after intravenous B16-F10 application. RESULTS: B16-F10 cells showed very little CD73 and negligible AP activity. Neither complete loss of host CD73 nor specific knockout of CD73 on endothelial cells or hematopoietic cells affected tumor growth after subcutaneous or intradermal tumor cell application. Only peritumoral edema formation was significantly attenuated in global CD73-/- mice in the intradermal model. Immune cell composition revealed no differences in the different transgenic mice models. Also lung metastasis after intravenous B16-F10 injection was not altered in CD73-/- mice. CONCLUSIONS: CD73 expression on host cells, particularly on endothelial and hematopoietic cells, does not modulate tumor growth and metastatic spread of B16-F10 melanoma cells most likely because of insufficient adenosine formation by the tumor itself.

Chemokines.

Tumor growth is restricted to approximately 2 microm diameters by simple dissociation of nutrients and oxygen. Hence, tumors require the formation of new blood vessels for further growth progression. This process is referred to as tumor neo-angiogenesis. The process of tumor neo-angiogenesis is directed by complex bidirectional interactions between the tumor and the vessels, and creates a favorable microenvironment for angiogenesis. The tumor vessel system not only facilitates tumor growth by providing nutrients and oxygen but also functions as a convenient route for metastasis. A group of small cytokine-like molecules called chemokines have been shown to participate in angiogenesis under homeostatic and neoplastic conditions. This review summarizes their role in tumor-associated angiogenesis.

Chemokines in tumor-associated angiogenesis.

Tumor growth is dependent on several key factors. Apart from immune escape and an efficient blockade of apoptotic signals, tumors require oxygen and nutrients to grow past a diameter of 2 microm. Therefore, it is of vital importance for the tumor to facilitate tumor-associated angiogenesis, e.g., the de novo formation of new blood vessels. In addition to established and key angiogenic factors, such as vascular endothelial growth factor, chemokines, a superfamily of cytokine-like proteins that bind to seven transmembrane-spanning G-protein-coupled receptors, have been associated with angiogenesis under homeostatic conditions. Chemokines were initially identified as key factors that control the directional migration of leukocytes, stem cells and cancer cells in vitro and which critically regulate their trafficking in vivo. Recently their role in establishing a favorable microenvironment for tumor-associated angiogenesis, a process that requires complex bidirectional interactions of the tumor and associated vessels, has been the focus of research. Chemokine-promoted angiogenesis not only facilitates tumor growth by supplying nutrients and oxygen but it is also a prerequisite to tumor metastasis. Hence, the pharmacologic control of tumor angiogenesis presents a promising strategy for novel anticancer therapeutics. Here, we discuss the current pathogenetic concepts of tumor-associated angiogenesis in the context of chemokines and their receptors and highlight promising therapeutic strategies.

Tumor immune escape by the loss of homeostatic chemokine expression.

The novel keratinocyte-specific chemokine CCL27 plays a critical role in the organization of skin-associated immune responses by regulating T cell homing under homeostatic and inflammatory conditions. Here we demonstrate that human keratinocyte-derived skin tumors may evade T cell-mediated antitumor immune responses by down-regulating the expression of CCL27 through the activation of epidermal growth factor receptor (EGFR)-Ras-MAPK-signaling pathways. Compared with healthy skin, CCL27 mRNA and protein expression was progressively lost in transformed keratinocytes of actinic keratoses and basal and squamous cell carcinomas. In vivo, precancerous skin lesions as well as cutaneous carcinomas showed significantly elevated levels of phosphorylated ERK compared with normal skin, suggesting the activation of EGFR-Ras signaling pathways in keratinocyte-derived malignancies. In vitro, exogenous stimulation of the EGFR-Ras signaling pathway through EGF or transfection of the dominant-active form of the Ras oncogene (H-RasV12) suppressed whereas an EGFR tyrosine kinase inhibitor increased CCL27 mRNA and protein production in keratinocytes. In mice, neutralization of CCL27 led to decreased leukocyte recruitment to cutaneous tumor sites and significantly enhanced primary tumor growth. Collectively, our data identify a mechanism of skin tumors to evade host antitumor immune responses.

IL-25 augments type 2 immune responses by enhancing the expansion and functions of TSLP-DC-activated Th2 memory cells.

Interleukin (IL) 25 (IL-17E), a distinct member of the IL-17 cytokine family, plays important roles in evoking T helper type 2 (Th2) cell-mediated inflammation that features the infiltrations of eosinophils and Th2 memory cells. However, the cellular sources, target cells, and underlying mechanisms remain elusive in humans. We demonstrate that human Th2 memory cells expressing distinctive levels of IL-25 receptor (R) are one of the responding cell types. IL-25 promotes cell expansion and Th2 cytokine production when Th2 central memory cells are stimulated with thymic stromal lymphopoietin (TSLP)-activated dendritic cells (DCs), homeostatic cytokines, or T cell receptor for antigen triggering. The enhanced functions of Th2 memory cells induced by IL-25 are associated with sustained expression of GATA-3, c-MAF, and JunB in an IL-4-independent manner. Although keratinocytes, mast cells, eosinophils, and basophils express IL-25 transcripts, activated eosinophils and basophils from normal and atopic subjects were found to secrete bioactive IL-25 protein, which augments the functions of Th2 memory cells. Elevated expression of IL-25 and IL-25R transcripts was observed in asthmatic lung tissues and atopic dermatitis skin lesions, linking their possible roles with exacerbated allergic disorders. Our results provide a plausible explanation that IL-25 produced by innate effector eosinophils and basophils may augment the allergic inflammation by enhancing the maintenance and functions of adaptive Th2 memory cells.

Expression and tissue distribution of mouse Hax1.

HAX1 is an ubiquitously expressed human gene. Though a number of cellular and viral proteins are known to interact with HAX1, its function is still not completely understood. On the basis of these identified interaction partners, HAX1 seems to play a role in apoptosis and the organization of the cytoskeleton. The cDNAs for human and mouse Hax1 share 86% identity and 80% identity at the protein level, suggesting a similar functional importance. To date, no conclusive data on the tissue specific expression of the murine Hax1 are available and only one interaction partner has been identified. Here, we show a detailed expression analysis for the murine ortholog by RT-PCR, Northern and Western blot. Furthermore, the distribution of Hax1 within different mouse tissues was studied by immunohistochemistry (IHC). In general, we found a good correlation between the results obtained from different detection techniques. Similar to its human counterpart, mouse Hax1 seems to be ubiquitously expressed. At the RNA level, we found the highest expression of Hax1 in liver, kidney and testis. In sharp contrast to the human HAX1 which is highly expressed in skeletal muscle, the mouse ortholog was detected only at very low levels. Using a specific antibody, we detected Hax1 in the majority of mouse tissues by IHC. Interestingly, the most prominent expression of Hax1 was found in epithelial, endothelial and muscle cells. Surprisingly, thymus, spleen and pancreas did not show detectable immunostaining. Furthermore, we have studied the subcellular localisation of Hax1 in a keratinocyte and a neuronal cell line by immunofluorescence. We found Hax1 to be localised mainly in the cytoplasm and detected a partial colocalisation with mitochondria. The results presented here summarize for the first time the expression of the murine Hax1 in different tissues and two cell lines. Further studies will elucidate the functional importance of this protein in individual cell types with respect to structural aspects, cell mobility and apoptosis.