Fluorescence-Guided Laparoscopy after Oral Hypericin Administration for Staging of Locally Advanced Gastric Cancer-A Pilot Study.

(1) Background: Laparoscopic staging is essential in gastric cancer (GC) to rule out peritoneal metastasis (PM). Hypericin, a plant-derived fluorescent compound, has been suggested to improve laparoscopic visualization of PM from GC. This prospective, single-arm, open-label clinical trial aimed to assess the feasibility and safety of oral hypericin administration as well as the suitability of fluorescence-guided laparoscopy (FGL) for improving the sensitivity and specificity of staging in GC patients (EudraCT-Number: 2015-005277-21; clinicaltrials.gov identifier: NCT-02840331). (2) Methods: GC patients received Laif® 900, an approved hypericin-containing phytopharmaceutical, once orally two to four hours before white light and ultraviolet light laparoscopy. The peritoneal cancer index was evaluated, biopsies taken and hypericin concentrations in serum and peritoneal tissue were determined by mass spectrometry. (3) Results: Between 2017 and 2021, out of 63 patients screened for eligibility, 50 patients were enrolled and treated per protocol. The study intervention was shown to be feasible and safe in all patients. Standard laparoscopy revealed suspicious lesions in 27 patients (54%), among whom 16 (59%) were diagnosed with PM. FGL identified suspicious areas in 25 patients (50%), among whom PM was confirmed in 13 cases (52%). Although hypericin concentrations in serum reached up to 5.64 ng/mL, no hypericin was detectable in peritoneal tissue biopsies. (4) Conclusions: FGL in patients with GC was shown to be feasible but futile in this study. Sufficient levels of hypericin should be ensured in target tissue prior to reassessing FGL with hypericin.

Long-term recurrence-free survival following pancreatic surgery with HIPEC treatment for locally advanced pancreatic adenocarcinoma.

More than 5 years previous to this report, a female patient in her 60s underwent oncological left-sided pancreatic resection and adrenalectomy including splenectomy for locally advanced pancreatic adenocarcinoma (PDAC), recommended by a multidisciplinary tumour board (MDT). Additionally, she was treated with gemcitabine-containing hyperthermic intraperitoneal chemotherapy (HIPEC) for 60 minutes in the framework of a clinical trial (PanHIPEC), aiming to determine the safety and feasibility (not efficacy) of this approach. Following the postoperative MDT recommendation, she subsequently received adjuvant chemotherapy consisting of six cycles of gemcitabine and cisplatin for a histopathologically confirmed PDAC of the pancreatic tail with infiltration of the left-sided adrenal gland (pT3, pN1 (3/16), cM0, L0, V0, Pn1, R0, G2). Five years and five months after pancreatic surgery and HIPEC, the patient has no signs of tumour recurrence as determined by follow-up examination including CT scan and CA19-9 tumour marker serology.

Quantitative Metabolomics and Lipoprotein Analysis of PDAC Patients Suggests Serum Marker Categories for Pancreatic Function, Pancreatectomy, Cancer Metabolism, and Systemic Disturbances.

Pancreatic ductal adenocarcinoma (PDAC) is difficult to diagnose in the early stages and lacks reliable biomarkers. The scope of this project was to establish quantitative nuclear magnetic resonance (NMR) spectroscopy to comprehensively study blood serum alterations in PDAC patients. Serum samples from 34 PDAC patients obtained before and after pancreatectomy as well as 83 age- and sex-matched control samples from healthy donors were analyzed with in vitro diagnostics research (IVDr) proton NMR spectroscopy at 600 MHz. Uni- and multivariate statistics were applied to identify significant biofluid alterations. We identified 29 significantly changed metabolites and 98 lipoproteins when comparing serum from healthy controls with those of PDAC patients. The most prominent features were assigned to (i) markers of pancreatic function (e.g., glucose and blood triglycerides), (ii) markers related to surgery (e.g., ketone bodies and blood cholesterols), (iii) PDAC-associated markers (e.g., amino acids and creatine), and (iv) markers for systemic disturbances in PDAC (e.g., gut metabolites DMG, TMAO, DMSO2, and liver lipoproteins). Quantitative serum NMR spectroscopy is suited as a diagnostic tool to investigate PDAC. Remarkably, 2-hydroxybutyrate (2-HB) as a previously suggested marker for insulin resistance was found in extraordinarily high levels only after pancreatectomy, suggesting this metabolite is the strongest marker for pancreatic loss of function.

High-dimensional in situ proteomics imaging to assess γδ T cells in spatial biology.

This study presents a high-dimensional immunohistochemistry approach to assess human γδ T cell subsets in their native tissue microenvironments at spatial resolution, a hitherto unmet scientific goal due to the lack of established antibodies and required technology. We report an integrated approach based on multiplexed imaging and bioinformatic analysis to identify γδ T cells, characterize their phenotypes, and analyze the composition of their microenvironment. Twenty-eight γδ T cell microenvironments were identified in tissue samples from fresh frozen human colon and colorectal cancer where interaction partners of the immune system, but also cancer cells were discovered in close proximity to γδ T cells, visualizing their potential contributions to cancer immunosurveillance. While this proof-of-principle study demonstrates the potential of this cutting-edge technology to assess γδ T cell heterogeneity and to investigate their microenvironment, future comprehensive studies are warranted to associate phenotypes and microenvironment profiles with features such as relevant clinical characteristics.

Identification and relative abundance of naturally presented and cross-reactive influenza A virus MHC class I-restricted T cell epitopes.

Cytotoxic T lymphocytes are key for controlling viral infection. Unravelling CD8+ T cell-mediated immunity to distinct influenza virus strains and subtypes across prominent HLA types is relevant for combating seasonal infections and emerging new variants. Using an immunopeptidomics approach, naturally presented influenza A virus-derived ligands restricted to HLA-A*24:02, HLA-A*68:01, HLA-B*07:02, and HLA-B*51:01 molecules were identified. Functional characterization revealed multifunctional memory CD8+ T cell responses for nine out of sixteen peptides. Peptide presentation kinetics was optimal around 12 h post infection and presentation of immunodominant epitopes shortly after infection was not always persistent. Assessment of immunogenic epitopes revealed that they are highly conserved across the major zoonotic reservoirs and may contain a single substitution in the vicinity of the anchor residues. These findings demonstrate how the identified epitopes promote T cell pools, possibly cross-protective in individuals and can be potential targets for vaccination.

Efficacy of Different Oncolytic Vaccinia Virus Strains for the Treatment of Murine Peritoneal Mesothelioma.

Effective treatment options for peritoneal surface malignancies (PSMs) are scarce. Oncolytic virotherapy with recombinant vaccinia viruses might constitute a novel treatment option for PSM. We aimed to identify the most effective oncolytic vaccinia virus strain in two murine mesothelioma cell lines and the oncolytic potential in a murine model of peritoneal mesothelioma. Cell lines AB12 and AC29 were infected in vitro with vaccinia virus strains Lister (GLV-1h254), Western Reserve (GLV-0b347), and Copenhagen (GLV-4h463). The virus strain GLV-0b347 was shown most effective in vitro and was further investigated by intraperitoneal (i.p.) application to AB12 and AC29 mesothelioma-bearing mice. Feasibility, safety, and effectiveness of virotherapy were assessed by evaluating the peritoneal cancer index (PCI), virus detection in tumor tissues and ascites, virus growth curves, and comparison of overall survival. After i.p. injection of GLV-0b347, virus was detected in both tumor cells and ascites. In comparison to mock-treated mice, overall survival was significantly prolonged, ascites was less frequent and PCI values declined. However, effective treatment was only observed in animals with limited tumor burden at the time point of virus application. Nonetheless, intraperitoneal virotherapy with GLV-0b347 might constitute a novel therapeutic option for the treatment of peritoneal mesothelioma. Additional treatment modifications and combinational regimes will be investigated to further enhance treatment efficacy.

Phase I/II trial of a peptide-based COVID-19 T-cell activator in patients with B-cell deficiency.

T-cell immunity is central for control of COVID-19, particularly in patients incapable of mounting antibody responses. CoVac-1 is a peptide-based T-cell activator composed of SARS-CoV-2 epitopes with documented favorable safety profile and efficacy in terms of SARS-CoV-2-specific T-cell response. We here report a Phase I/II open-label trial (NCT04954469) in 54 patients with congenital or acquired B-cell deficiency receiving one subcutaneous CoVac-1 dose. Immunogenicity in terms of CoVac-1-induced T-cell responses and safety are the primary and secondary endpoints, respectively. No serious or grade 4 CoVac-1-related adverse events have been observed. Expected local granuloma formation has been observed in 94% of study subjects, whereas systemic reactogenicity has been mild or absent. SARS-CoV-2-specific T-cell responses have been induced in 86% of patients and are directed to multiple CoVac-1 peptides, not affected by any current Omicron variants and mediated by multifunctional T-helper 1 CD4+ T cells. CoVac-1-induced T-cell responses have exceeded those directed to the spike protein after mRNA-based vaccination of B-cell deficient patients and immunocompetent COVID-19 convalescents with and without seroconversion. Overall, our data show that CoVac-1 induces broad and potent T-cell responses in patients with B-cell/antibody deficiency with a favorable safety profile, which warrants advancement to pivotal Phase III safety and efficacy evaluation. ClinicalTrials.gov identifier NCT04954469.

Establishment of a novel method to assess MEK1/2 inhibition in PBMCs for clinical drug development.

The Raf/MEK/ERK signaling pathway plays a key role in regulating cellular proliferation, differentiation, apoptosis, cytokine production, and immune responses. However, it is also involved in diseases such as cancer, and numerous viruses rely on an active Raf/MEK/ERK pathway for propagation. This pathway, and particularly MEK1/2, are therefore promising therapeutic targets. Assessment of target engagement is crucial to determine pharmacodynamics or the efficacy of a MEK1/2 inhibitor. In the field of infectious diseases, this is usually first determined in clinical trials with healthy volunteers. One method to detect MEK1/2 inhibitor target engagement is to assess the degree of ERK1/2 phosphorylation, as ERK1/2 is the only known substrate of MEK1/2. As healthy subjects, however, only feature a low baseline MEK1/2 activation and therefore low ERK1/2 phosphorylation in most tissues, assessing target engagement is challenging, and robust methods are urgently needed. We hence developed a method using PBMCs isolated from whole blood of healthy blood donors, followed by ex vivo treatment with the MEK1/2 inhibitor zapnometinib and stimulation with PMA to first inhibit and then induce MEK1/2 activation. As PMA cannot activate MEK1/2 upon MEK1/2 inhibition, MEK1/2 inhibition results in impaired MEK1/2 activation. In contrast, PMA stimulation without MEK1/2 inhibition results in high MEK1/2 activation. We demonstrated that, without MEK1/2 inhibitor treatment, MEK1/2 stimulation with PMA induces high MEK1/2 activation, which is clearly distinguishable from baseline MEK1/2 activation in human PBMCs. Furthermore, we showed that treatment with the MEK1/2 inhibitor zapnometinib maintains the MEK1/2 activation at approximately baseline level despite subsequent stimulation with PMA. As our protocol is easy to follow and preserves the cells in an in vivo-like condition throughout the whole handling process, this approach can be a major advance for the easy assessment of MEK1/2 inhibitor target engagement in healthy probands for clinical drug development.

T cells of colorectal cancer patients' stimulated by neoantigenic and cryptic peptides better recognize autologous tumor cells.

BACKGROUND: Patients with cancers that exhibit extraordinarily high somatic mutation numbers are ideal candidates for immunotherapy and enable identifying tumor-specific peptides through stimulation of tumor-reactive T cells (Tc). METHODS: Colorectal cancers (CRC) HROC113 and HROC285 were selected based on high TMB, microsatellite instability and HLA class I expression. Their HLA ligandome was characterized using mass spectrometry, compared with the HLA ligand atlas and HLA class I-binding affinity was predicted. Cryptic peptides were identified using Peptide-PRISM. Patients' Tc were isolated from either peripheral blood (pTc) or tumor material (tumor-infiltrating Tc, TiTc) and expanded. In addition, B-lymphoblastoid cells (B-LCL) were generated and used as antigen-presenting cells. pTc and TiTc were stimulated twice for 7 days using peptide pool-loaded B-LCL. Subsequently, interferon gamma (IFNγ) release was quantified by ELISpot. Finally, cytotoxicity against autologous tumor cells was assessed in a degranulation assay. RESULTS: 100 tumor-specific candidate peptides-97 cryptic peptides and 3 classically mutated neoantigens-were selected. The neoantigens originated from single nucleotide substitutions in the genes IQGAP1, CTNNB1, and TRIT1. Cryptic and neoantigenic peptides inducing IFNγ secretion of Tc were further investigated. Stimulation of pTc and TiTc with neoantigens and selected cryptic peptides resulted in increased release of cytotoxic granules in the presence of autologous tumor cells, substantiating their improved tumor cell recognition. Tetramer staining showed an enhanced number of pTc and TiTc specific for the IQGAP1 neoantigen. Subpopulation analysis prior to peptide stimulation revealed that pTc mainly consisted of memory Tc, whereas TiTc constituted primarily of effector and effector memory Tc. This allows to infer that TiTc reacting to neoantigens and cryptic peptides must be present within the tumor microenvironment. CONCLUSION: These results prove that the analyzed CRC present both mutated neoantigenic and cryptic peptides on their HLA class I molecules. Moreover, stimulation with these peptides significantly strengthened tumor cell recognition by Tc. Since the overall number of neoantigenic peptides identifiable by HLA ligandome analysis hitherto is small, our data emphasize the relevance of increasing the target scope for cancer vaccines by the cryptic peptide category.

[Current status of surgical treatment of peritoneal metastases from colorectal cancer]. / Aktueller Stand der chirurgischen Therapie peritonealer Metastasen bei kolorektalen Karzinomen.

Cytoreductive surgery, often in combination with hyperthermic intraperitoneal chemotherapy (HIPEC), has been instrumental in improving the survival of patients with peritoneal metastases from colorectal cancer. Recent studies have highlighted the benefits of complete cytoreduction, while the role of the HIPEC treatment remains unclear. An oxaliplatin-based HIPEC over 30 min could not achieve any clear benefits in studies on colorectal cancer, neither in the therapeutic nor in the prophylactic setting, but caused relevant side effects and increased the morbidity. The negative results of these studies with respect to oxaliplatin-based HIPEC require critical appraisal; however, they should by no means be regarded as a general setback for surgical treatment of peritoneal metastases and be misunderstood as a general failure of this treatment. While HIPEC after complete surgical cytoreduction of peritoneal metastases from colorectal cancer requires further research, cytoreductive surgery should still be regarded as a highly effective treatment for suitable patients with limited abdominal tumor dissemination.

Assay validity of point-of-care platelet function tests in thrombocytopenic blood samples.

Introduction: Point-of-care (POC) platelet function tests are faster and easier to perform than in-depth assessment by flow cytometry. At low platelet counts, however, POC tests are prone to assess platelet function incorrectly. Lower limits of platelet count required to obtain valid test results were defined and a testing method to facilitate comparability between different tests was established. Materials and methods: We assessed platelet function in whole blood samples of healthy volunteers at decreasing platelet counts (> 100, 80-100, 50-80, 30-50 and < 30 x109/L) using two POC tests: impedance aggregometry and in-vitro bleeding time. Flow cytometry served as the gold standard. The number of platelets needed to reach 50% of the maximum function (ED50) and the lower reference limit (EDref) were calculated to define limits of test validity. Results: The minimal platelet count required for reliable test results was 100 x109/L for impedance aggregometry and in-vitro bleeding time but only 30 x109/L for flow cytometry. Comparison of ED50 and EDref showed significantly lower values for flow cytometry than either POC test (P value < 0.05) but no difference between POC tests nor between the used platelet agonists within a test method. Conclusion: Calculating the ED50 and EDref provides an effective way to compare values from different platelet function assays. Flow cytometry enables correct platelet function testing as long as platelet count is > 30 x109/L whereas impedance aggregometry and in-vitro bleeding time are inconsistent unless platelet count is > 100 x109/L.

A Baseline Cellular Antiviral State Is Maintained by cGAS and Its Most Frequent Naturally Occurring Variant rs610913.

Upon recognition of aberrantly located DNA, the innate immune sensor cyclic GMP-AMP synthase (cGAS) activates stimulator of IFN genes (STING)/IFN regulatory factor (IRF)3-driven antiviral responses. In this study, we characterized the ability of a specific variant of the human cGAS-encoding gene MB21D1, rs610913, to alter cGAS-mediated DNA sensing and viral infection. rs610913 is a frequent G>T polymorphism resulting in a P261H exchange in the cGAS protein. Data from the International Collaboration for the Genomics of HIV suggested that rs610913 nominally associates with HIV-1 acquisition in vivo. Molecular modeling of cGAS(P261H) hinted toward the possibility for an additional binding site for a potential cellular cofactor in cGAS dimers. However, cGAS(wild-type [WT]) or cGAS(P261H)-reconstituted THP-1 cGAS knockout cells shared steady-state expression of IFN-stimulated genes, as opposed to cells expressing the enzymatically inactive cGAS(G212A/S213A). Accordingly, cGAS(WT) and cGAS(P261H) cells were less susceptible to lentiviral transduction and infection with HIV-1, HSV-1, and Chikungunya virus as compared with cGAS knockout or cGAS(G212A/S213A) cells. Upon DNA challenge, innate immune activation appeared to be mildly reduced upon expression of cGAS(P261H) compared with cGAS(WT). Finally, DNA challenge of PBMCs from donors homozygously expressing rs610913 provoked a trend toward a slightly reduced type I IFN response as compared with PBMCs from GG donors. Taken together, the steady-state activity of cGAS maintains a baseline antiviral state rendering cells more refractory to IFN-stimulated gene-sensitive viral infections. rs610913 failed to grossly differ phenotypically from the WT gene, suggesting that cGAS(P261H) and WT cGAS share a similar ability to sense viral infections in vivo.

[Impact of the SARS-CoV-2 Pandemic on Liver Surgery and Liver Transplantations in Germany]. / Einfluss der Coronapandemie auf die Leberchirurgie und Lebertransplantationen in Deutschland.

BACKGROUND: The SARS-CoV-2 pandemic has led to restrictions in surgical care worldwide and therefore also posed new challenges to liver surgery. The respective procedures often entail high perioperative risks and resource requirements. However, the indication for liver surgery is frequently without alternatives. To date, there is little knowledge about the impact of the pandemic on liver surgery in Germany. METHODS: A retrospective data analysis of liver surgery procedures in Germany as well as transplantations was conducted. Evaluations were based on procedure codes recorded between 2010 and 2020 according to diagnosis-related groups (DRG) by the Federal Statistical Office of Germany (Destatis) and data from the German Organ Procurement Organization (Deutsche Stiftung Organtransplantation; DSO). RESULTS: According to DRG procedure codes relating to liver surgery recorded between 2010 and 2020 in Germany, the annual fluctuation for the first year of the pandemic 2020 remained comparable to previous years. Furthermore, the development of post-mortem liver transplantations as well as living liver donations remained stable in Germany in 2020 and 2021. CONCLUSIONS: The number of liver surgery procedures in Germany was subject to a dynamic development until 2020, without apparent changes in the first year of the SARS-CoV-2 pandemic. The most frequently performed liver procedures, as well as liver transplantations, remained stable with respect to their annually recorded numbers. Publication of data regarding procedures in liver surgery and transplantation in 2021 need to be awaited and analyzed to evaluate whether the observations presented in this article prove stable any further.

Orf Virus-Based Vectors Preferentially Target Professional Antigen-Presenting Cells, Activate the STING Pathway and Induce Strong Antigen-Specific T Cell Responses.

Background: Orf virus (ORFV)-based vectors are attractive for vaccine development as they enable the induction of potent immune responses against specific transgenes. Nevertheless, the precise mechanisms of immune activation remain unknown. This study therefore aimed to characterize underlying mechanisms in human immune cells. Methods: Peripheral blood mononuclear cells were infected with attenuated ORFV strain D1701-VrV and analyzed for ORFV infection and activation markers. ORFV entry in susceptible cells was examined using established pharmacological inhibitors. Using the THP1-Dual™ reporter cell line, activation of nuclear factor-κB and interferon regulatory factor pathways were simultaneously evaluated. Infection with an ORFV recombinant encoding immunogenic peptides (PepTrio-ORFV) was used to assess the induction of antigen-specific CD8+ T cells. Results: ORFV was found to preferentially target professional antigen-presenting cells (APCs) in vitro, with ORFV uptake mediated primarily by macropinocytosis. ORFV-infected APCs exhibited an activated phenotype, required for subsequent lymphocyte activation. Reporter cells revealed that the stimulator of interferon genes pathway is a prerequisite for ORFV-mediated cellular activation. PepTrio-ORFV efficiently induced antigen-specific CD8+ T cell recall responses in a dose-dependent manner. Further, activation and expansion of naïve antigen-specific CD8+ T cells was observed in response. Discussion: Our findings confirm that ORFV induces a strong antigen-specific immune response dependent on APC uptake and activation. These data support the notion that ORFV D1701-VrV is a promising vector for vaccine development and the design of innovative immunotherapeutic applications.

Phase I/II Multicenter Trial of a Novel Therapeutic Cancer Vaccine, HepaVac-101, for Hepatocellular Carcinoma.

PURPOSE: Immunotherapy for hepatocellular carcinoma (HCC) shows considerable promise in improving clinical outcomes. HepaVac-101 represents a single-arm, first-in-human phase I/II multicenter cancer vaccine trial for HCC (NCT03203005). It combines multipeptide antigens (IMA970A) with the TLR7/8/RIG I agonist CV8102. IMA970A includes 5 HLA-A*24 and 7 HLA-A*02 as well as 4 HLA-DR restricted peptides selected after mass spectrometric identification in human HCC tissues or cell lines. CV8102 is an RNA-based immunostimulator inducing a balanced Th1/Th2 immune response. PATIENTS AND METHODS: A total of 82 patients with very early- to intermediate-stage HCCs were enrolled and screened for suitable HLA haplotypes and 22 put on study treatment. This consisted in a single infusion of low-dose cyclophosphamide followed by nine intradermal coadministrations of IMA970A and CV8102. Only patients with no disease relapse after standard-of-care treatments were vaccinated. The primary endpoints of the HepaVac-101 clinical trial were safety, tolerability, and antigen-specific T-cell responses. Secondary or exploratory endpoints included additional immunologic parameters and survival endpoints. RESULTS: The vaccination showed a good safety profile. Transient mild-to-moderate injection-site reactions were the most frequent IMA970A/CV8102-related side effects. Immune responses against ≥1 vaccinated HLA class I tumor-associated peptide (TAA) and ≥1 vaccinated HLA class II TAA were respectively induced in 37% and 53% of the vaccinees. CONCLUSIONS: Immunotherapy may provide a great improvement in treatment options for HCC. HepaVac-101 is a first-in-human clinical vaccine trial with multiple novel HLA class I- and class II-restricted TAAs against HCC. The results are initial evidence for the safety and immunogenicity of the vaccine. Further clinical evaluations are warranted.

Prolonged Exposure to Oxaliplatin during HIPEC Improves Effectiveness in a Preclinical Micrometastasis Model.

Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) was considered a promising treatment for patients with peritoneal metastasis from colorectal cancer. However, the recently published randomized controlled PRODIGE 7 trial failed to demonstrate survival benefits through the addition of short-term oxaliplatin-based HIPEC. Constituting a complex multifactorial treatment, we investigated HIPEC in a preclinical model concerning the elimination of minimal tumor residues, thereby aiming to better understand the size of effects and respective clinical trial results. Patient samples of peritoneal perfusates obtained during HIPEC treatments and oxaliplatin-containing solutions at clinically relevant dosages, conforming with established HIPEC protocols, were assessed regarding their ability to eliminate modelled ~100 µm thickness cancer cell layers. Impedance-based real-time cell analysis and classical end-point assays were used. Flow cytometry was employed to determine the effect of different HIPEC drug solvents on tumor cell properties. Effectiveness of peritoneal perfusate patient samples and defined oxaliplatin-containing solutions proved limited but reproducible. HIPEC simulations for 30 min reduced the normalized cell index below 50% with peritoneal perfusates from merely 3 out of 9 patients within 72 h, indicating full-thickness cytotoxic effects. Instead, prolonging HIPEC to 1 h enhanced these effects and comprised 7 patients' samples, while continuous drug exposure invariably resulted in complete cell death. Further, frequently used drug diluents caused approximately 25% cell size reduction within 30 min. Prolonging oxaliplatin exposure improved effectiveness of HIPEC to eliminate micrometastases in our preclinical model. Accordingly, insufficient penetration depth, short exposure time, and the physicochemical impact of drug solvents may constitute critical factors.

Integrin activation enables rapid detection of functional Vδ1+ and Vδ2+ γδ T cells.

Conformational change of the ß2 integrin lymphocyte function-associated antigen 1 (LFA-1) is an early marker of T cell activation. A protocol using the mAb clone m24 recognizing the active, extended high-affinity conformation has been previously described for the assessment of functional CD4+ and CD8+ T cells in response to MHC-peptide stimulation. We investigated the applicability of the m24 mAb to detect the activation of γδ T cells in response to different soluble and immobilized stimuli. m24 mAb staining was associated with the expression of cytokines and was detectable as early as 10 min after stimulation, but with different kinetics depending on the nature of the stimulus. Hence, we conclude that this assay is suitable for the detection of functional γδ T cells and allows the assessment of activation more rapidly than alternative methods such as cytokine detection. Intracellular staining, protein trafficking inhibitors, or prior knowledge of the stimulating moiety recognized are no longer required for monitoring γδ T cell activation.

A survey among physicians in surgery and anesthesiology departments after the first surge of SARS-CoV-2 infections in Germany : Preparing for further challenges ahead.

BACKGROUND: The SARS-CoV­2 pandemic has extensively challenged healthcare systems all over the world. Many elective operations were postponed or cancelled, changing priorities and workflows in surgery departments. AIMS: The primary aim of this cross-sectional study was to assess the workload and psychosocial burden of surgeons and anesthesiologists, working in German hospitals during the first wave of SARS-CoV­2 infections in 2020. METHODS: Quantitative online survey on the workplace situation including psychosocial and work-related stress factors among resident and board-certified surgeons and anesthesiologists. Physicians in German hospitals across all levels of healthcare were contacted via departments, professional associations and social media posts. RESULTS: Among 154 total study participants, 54% of respondents stated a lack of personal protective equipment in their own wards and 56% reported increased staff shortages since the onset of the pandemic. While routine practice was reported as fully resumed in 71% of surgery departments at the time of the survey, work-related dissatisfaction among responding surgeons and anesthesiologists increased from 24% before the pandemic to 36% after the first wave of infections. As a countermeasure, 94% of participants deemed the establishment of action plans to increase pandemic preparedness and strengthening German public health systems a useful measure to respond to current challenges. CONCLUSION: The aftermath of the first wave of SARS-CoV­2 infections in Germany has left the surgical staff strained, despite temporarily decreased workloads. Overall, a critical review of the altered conditions is indispensable to identify and promote effective solutions and prudent action plans required to address imminent challenges.

A COVID-19 peptide vaccine for the induction of SARS-CoV-2 T cell immunity.

T cell immunity is central for the control of viral infections. CoVac-1 is a peptide-based vaccine candidate, composed of SARS-CoV-2 T cell epitopes derived from various viral proteins1,2, combined with the Toll-like receptor 1/2 agonist XS15 emulsified in Montanide ISA51 VG, aiming to induce profound SARS-CoV-2 T cell immunity to combat COVID-19. Here we conducted a phase I open-label trial, recruiting 36 participants aged 18-80 years, who received a single subcutaneous CoVac-1 vaccination. The primary end point was safety analysed until day 56. Immunogenicity in terms of CoVac-1-induced T cell response was analysed as the main secondary end point until day 28 and in the follow-up until month 3. No serious adverse events and no grade 4 adverse events were observed. Expected local granuloma formation was observed in all study participants, whereas systemic reactogenicity was absent or mild. SARS-CoV-2-specific T cell responses targeting multiple vaccine peptides were induced in all study participants, mediated by multifunctional T helper 1 CD4+ and CD8+ T cells. CoVac-1-induced IFNγ T cell responses persisted in the follow-up analyses and surpassed those detected after SARS-CoV-2 infection as well as after vaccination with approved vaccines. Furthermore, vaccine-induced T cell responses were unaffected by current SARS-CoV-2 variants of concern. Together, CoVac-1 showed a favourable safety profile and induced broad, potent and variant of concern-independent T cell responses, supporting the presently ongoing evaluation in a phase II trial for patients with B cell or antibody deficiency.

Can Any Drug Be Repurposed for Cancer Treatment? A Systematic Assessment of the Scientific Literature.

Drug repurposing is a complementary pathway for introducing new drugs against cancer. Broad systematic assessments of ongoing repurposing efforts in oncology are lacking, but may be helpful to critically appraise current and future efforts. Hence, we conducted a systematic PubMed search encompassing 100 frequently prescribed and 100 randomly selected drugs, and assessed the published preclinical anti-cancer effects. Furthermore, we evaluated all the identified original articles for methodological quality. We found reports indicating anti-cancer effects for 138/200 drugs, especially among frequently prescribed drugs (81/100). Most were reports suggesting single-agent activity of the drugs (61%). Basic information, such as the cell line used or control treatments utilized, were reported consistently, while more detailed information (e.g., excluded data) was mostly missing. The majority (56%) of in vivo studies reported randomizing animals, while only few articles stated that the experiments were conducted in a blinded fashion. In conclusion, we found promising reports of anti-cancer effects for the majority of the assessed drugs, but speculate that many of them are false-positive findings. Reward systems should be adjusted to encourage the widespread usage of high reporting quality and bias-reducing methodologies, aiming to decrease the rate of false-positive results, and thereby increasing the trust in the findings.