JUN mediates the senescence associated secretory phenotype and immune cell recruitment to prevent prostate cancer progression.

BACKGROUND: Prostate cancer develops through malignant transformation of the prostate epithelium in a stepwise, mutation-driven process. Although activator protein-1 transcription factors such as JUN have been implicated as potential oncogenic drivers, the molecular programs contributing to prostate cancer progression are not fully understood. METHODS: We analyzed JUN expression in clinical prostate cancer samples across different stages and investigated its functional role in a Pten-deficient mouse model. We performed histopathological examinations, transcriptomic analyses and explored the senescence-associated secretory phenotype in the tumor microenvironment. RESULTS: Elevated JUN levels characterized early-stage prostate cancer and predicted improved survival in human and murine samples. Immune-phenotyping of Pten-deficient prostates revealed high accumulation of tumor-infiltrating leukocytes, particularly innate immune cells, neutrophils and macrophages as well as high levels of STAT3 activation and IL-1ß production. Jun depletion in a Pten-deficient background prevented immune cell attraction which was accompanied by significant reduction of active STAT3 and IL-1ß and accelerated prostate tumor growth. Comparative transcriptome profiling of prostate epithelial cells revealed a senescence-associated gene signature, upregulation of pro-inflammatory processes involved in immune cell attraction and of chemokines such as IL-1ß, TNF-α, CCL3 and CCL8 in Pten-deficient prostates. Strikingly, JUN depletion reversed both the senescence-associated secretory phenotype and senescence-associated immune cell infiltration but had no impact on cell cycle arrest. As a result, JUN depletion in Pten-deficient prostates interfered with the senescence-associated immune clearance and accelerated tumor growth. CONCLUSIONS: Our results suggest that JUN acts as tumor-suppressor and decelerates the progression of prostate cancer by transcriptional regulation of senescence- and inflammation-associated genes. This study opens avenues for novel treatment strategies that could impede disease progression and improve patient outcomes.

Development and In Vivo Evaluation of Small-Molecule Ligands for Positron Emission Tomography of Immune Checkpoint Modulation Targeting Programmed Cell Death 1 Ligand 1.

A substantial portion of patients do not benefit from programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) checkpoint inhibition therapies, necessitating a deeper understanding of predictive biomarkers. Immunohistochemistry (IHC) has played a pivotal role in assessing PD-L1 expression, but small-molecule positron emission tomography (PET) tracers could offer a promising avenue to address IHC-associated limitations, i.e., invasiveness and PD-L1 expression heterogeneity. PET tracers would allow for improved quantification of PD-L1 through noninvasive whole-body imaging, thereby enhancing patient stratification. Here, a large series of PD-L1 targeting small molecules were synthesized, leveraging advantageous substructures to achieve exceptionally low nanomolar affinities. Compound 5c emerged as a promising candidate (IC50 = 10.2 nM) and underwent successful carbon-11 radiolabeling. However, a lack of in vivo tracer uptake in xenografts and notable accumulation in excretory organs was observed, underscoring the challenges encountered in small-molecule PD-L1 PET tracer development. The findings, including structure-activity relationships and in vivo biodistribution data, stand to illuminate the path forward for refining small-molecule PD-L1 PET tracers.

Vaccine Based on Recombinant Fusion Protein Combining Hepatitis B Virus PreS with SARS-CoV-2 Wild-Type- and Omicron-Derived Receptor Binding Domain Strongly Induces Omicron-Neutralizing Antibodies in a Murine Model.

BACKGROUND: COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a recurrent endemic disease affecting the whole world. Since November 2021, Omicron and its subvariants have dominated in the spread of the disease. In order to prevent severe courses of disease, vaccines are needed to boost and maintain antibody levels capable of neutralizing Omicron. Recently, we produced and characterized a SARS-CoV-2 vaccine based on a recombinant fusion protein consisting of hepatitis B virus (HBV)-derived PreS and two SARS-CoV-2 wild-type RBDs. OBJECTIVES: To develop a PreS-RBD vaccine which induces high levels of Omicron-specific neutralizing antibodies. METHODS: We designed, produced, characterized and compared strain-specific (wild-type: W-PreS-W; Omicron: O-PreS-O), bivalent (mix of W-PreS-W and O-PreS-O) and chimeric (i.e., W-PreS-O) SARS-CoV-2 protein subunit vaccines. Immunogens were characterized in vitro using protein chemical methods, mass spectrometry, and circular dichroism in combination with thermal denaturation and immunological methods. In addition, BALB/c mice were immunized with aluminum-hydroxide-adsorbed proteins and aluminum hydroxide alone (i.e., placebo) to study the specific antibody and cytokine responses, safety and Omicron neutralization. RESULTS: Defined and pure immunogens could be produced in significant quantities as secreted and folded proteins in mammalian cells. The antibodies induced after vaccination with different doses of strain-specific, bivalent and chimeric PreS-RBD fusion proteins reacted with wild-type and Omicron RBD in a dose-dependent manner and resulted in a mixed Th1/Th2 immune response. Interestingly, the RBD-specific IgG levels induced with the different vaccines were comparable, but the W-PreS-O-induced virus neutralization titers against Omicron (median VNT50: 5000) were seven- and twofold higher than the W-PreS-W- and O-PreS-O-specific ones, respectively, and they were six-fold higher than those of the bivalent vaccine. CONCLUSION: Among the tested immunogens, the chimeric PreS-RBD subunit vaccine, W-PreS-O, induced the highest neutralizing antibody titers against Omicron. Thus, W-PreS-O seems to be a highly promising COVID-19 vaccine candidate for further preclinical and clinical evaluation.

A ventricular fibrillation cardiac arrest model with extracorporeal cardiopulmonary resuscitation in rats: 8 minutes arrest time leads to increased myocardial damage but does not increase neuronal damage compared to 6 minutes.

Introduction: Extracorporeal cardiopulmonary resuscitation (ECPR) is an emerging strategy in highly selected patients with refractory cardiac arrest (CA). Animal models can help to identify new therapeutic strategies to improve neurological outcome and cardiac function after global ischemia in CA. Aim of the study was to establish a reproducible ECPR rat model of ventricular fibrillation CA (VFCA) that leads to consistent neuronal damage with acceptable long-term survival rates, which can be used for future research. Materials and methods: Male Sprague Dawley rats were resuscitated with ECPR from 6 min (n = 15) and 8 min (n = 16) VFCA. Animals surviving for 14 days after return of spontaneous resuscitation (ROSC) were compared with sham operated animals (n = 10); neurological outcome was assessed daily until day 14. In the hippocampal cornu ammonis 1 region viable neurons were counted. Microglia and astrocyte reaction was assessed by Iba1 and GFAP immunohistochemistry, and collagen fibers in the myocardium were detected in Azan staining. QuPath was applied for quantification. Results: Of the 15 rats included in the 6 min CA group, all achieved ROSC (100%) and 10 (67%) survived to 14 days; in the 8 min CA group, 15 (94%) achieved ROSC and 5 (31%) reached the endpoint. All sham animals (n = 10) survived 2 weeks. The quantity of viable neurons was significantly decreased, while the area displaying Iba1 and GFAP positive pixels was significantly increased in the hippocampus across both groups that experienced CA. Interestingly, there was no difference between the two CA groups regarding these changes. The myocardium in the 8 min CA group exhibited significantly more collagen fibers compared to the sham animals, without differences between 6- and 8-min CA groups. However, this significant increase was not observed in the 6 min CA group. Conclusion: Our findings indicate a uniform occurrence of neuronal damage in the hippocampus across both CA groups. However, there was a decrease in survival following an 8-min CA. Consequently, a 6-min duration of CA resulted in predictable neurological damage without significant cardiac damage and ensured adequate survival rates up to 14 days. This appears to offer a reliable model for investigating neuroprotective therapies.

Preclinical and Clinical Observations Implying Combination Therapy to Enhance the Efficacy of the Her-2/neu B-Cell Peptide-Based Vaccine HER-Vaxx and to Prevent Immune Evasion.

Her-2/neu-targeting therapy by passive application with trastuzumab is associated with acquired resistance and subsequent metastasis development, which is attributed to the upregulation of tumoral PD-L1 expression and the downregulation of Her-2/neu. We aimed to investigate this association, following active immunization with our recently constructed B-cell peptide-based Her-2/neu vaccines in both preclinical and clinical settings. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and combined positive score (CPS) were applied to evaluate Her-2/neu and PD-L1 expression using a murine syngeneic tumor model for Her-2/neu lung metastases and tumor biopsies from a gastric cancer patient with disease progression. A significant and concomitant reduction in Her-2/neu and the upregulation of PD-L1 expression was observed in vaccinated mice after 45 days, but not after 30 days, of metastases development. A significant increase in tumor-infiltrating B lymphocytes was observed at both time points. The downregulation of Her-2/neu and the upregulation of PD-L1 were observed in a patient's primary tumor at the disease progression time point but not prior to vaccination (Her-2/neu IHC: 3 to 0, FISH: 4.98 to 1.63; PD-L1 CPS: 0% to 5%). Our results further underline the need for combination therapy by targeting PD-L1 to prevent metastasis formation and immune evasion of Her-2/neu-positive and PD-L1-negative tumor cells.

Decoding molecular programs in melanoma brain metastases.

Melanoma brain metastases (MBM) variably respond to therapeutic interventions; thus determining patient's prognosis. However, the mechanisms that govern therapy response are poorly understood. Here, we use a multi-OMICS approach and targeted sequencing (TargetSeq) to unravel the programs that potentially control the development of progressive intracranial disease. Molecularly, the expression of E-cadherin (Ecad) or NGFR, the BRAF mutation state and level of immune cell infiltration subdivides tumors into proliferative/pigmented and invasive/stem-like/therapy-resistant irrespective of the intracranial location. The analysis of MAPK inhibitor-naive and refractory MBM reveals switching from Ecad-associated into NGFR-associated programs during progression. NGFR-associated programs control cell migration and proliferation via downstream transcription factors such as SOX4. Moreover, global methylome profiling uncovers 46 differentially methylated regions that discriminate BRAFmut and wildtype MBM. In summary, we propose that the expression of Ecad and NGFR sub- classifies MBM and suggest that the Ecad-to-NGFR phenotype switch is a rate-limiting process which potentially indicates drug-response and intracranial progression states in melanoma patients.

Active immunization with a Her-2/neu-targeting Multi-peptide B cell vaccine prevents lung metastases formation from Her-2/neu breast cancer in a mouse model.

In pre-clinical and clinical settings, active immunization with a Her-2/neu vaccine (HerVaxx), comprising B-cell peptide from Trastuzumab binding site, has been shown to reduce primary tumor growth via induction of polyclonal anti-tumor immune responses and immunological memory. Here, we tested the combination of HerVaxx and the recently identified B-cell epitope/mimotope of Pertuzumab, i.e. a multi-peptide B-cell vaccine, for preventing Her-2/neu lung metastases formation in a mouse model. Active immunization with the multi-peptide vaccine was associated with decreased lung weights, and histological evaluation of the lungs showed that the significant reduction of lung metastases was associated with increased CD4+ and CD8+ T cell infiltration. Notably, along with the overall reduction of lungs weights and Her-2 positive metastases, a formation of Her-2/neu-negative tumors but with increased PD-L1 expression was observed. Our results might pave the way to a multi-peptide B-cell Her-2/neu vaccine serving as a secondary intervention in adjuvant settings to prevent tumor recurrence and spread. Moreover, combination therapy targeting PD-L1 may result in total remission of metastases. Such a therapy may be used clinically to alternately target Her-2/neu and PD-L1 in metastatic breast cancer.

KMT2C methyltransferase domain regulated INK4A expression suppresses prostate cancer metastasis.

BACKGROUND: Frequent truncation mutations of the histone lysine N-methyltransferase KMT2C have been detected by whole exome sequencing studies in various cancers, including malignancies of the prostate. However, the biological consequences of these alterations in prostate cancer have not yet been elucidated. METHODS: To investigate the functional effects of these mutations, we deleted the C-terminal catalytic core motif of Kmt2c specifically in mouse prostate epithelium. We analysed the effect of Kmt2c SET domain deletion in a Pten-deficient PCa mouse model in vivo and of truncation mutations of KMT2C in a large number of prostate cancer patients. RESULTS: We show here for the first time that impaired KMT2C methyltransferase activity drives proliferation and PIN formation and, when combined with loss of the tumour suppressor PTEN, triggers loss of senescence, metastatic dissemination and dramatically reduces life expectancy. In Kmt2c-mutated tumours we show enrichment of proliferative MYC gene signatures and loss of expression of the cell cycle repressor p16INK4A. In addition, we observe a striking reduction in disease-free survival of patients with KMT2C-mutated prostate cancer. CONCLUSIONS: We identified truncating events of KMT2C as drivers of proliferation and PIN formation. Loss of PTEN and KMT2C in prostate cancer results in loss of senescence, metastatic dissemination and reduced life expectancy. Our data demonstrate the prognostic significance of KMT2C mutation status in prostate cancer patients. Inhibition of the MYC signalling axis may be a viable treatment option for patients with KMT2C truncations and therefore poor prognosis.

New Emergence of the Novel Pestivirus Linda Virus in a Pig Farm in Carinthia, Austria.

Linda virus (LindaV) was first identified in a pig farm in Styria, Austria in 2015 and associated with congenital tremor (CT) type A-II in newborn piglets. Since then, only one more LindaV affected farm was retrospectively discovered 10 km away from the initially affected farm. Here, we report the recent outbreak of a novel LindaV strain in a farrow-to-finish farm in the federal state Carinthia, Austria. No connection between this farm and the previously affected farms could be discovered. The outbreak was characterized by severe CT cases in several litters and high preweaning mortality. A herd visit two months after the onset of clinical symptoms followed by a diagnostic workup revealed the presence of several viremic six-week-old nursery pigs. These animals shed large amounts of virus via feces and saliva, implying an important epidemiological role for within- and between-herd virus transmission. The novel LindaV strain was isolated and genetically characterized. The findings underline a low prevalence of LindaV in the Austrian pig population and highlight the threat when introduced into a pig herd. Furthermore, the results urge the need to better understand the routes of persistence and transmission of this enigmatic pestivirus in the pig population.

Deletion of the SELENOP gene leads to CNS atrophy with cerebellar ataxia in dogs.

We investigated a hereditary cerebellar ataxia in Belgian Shepherd dogs. Affected dogs developed uncoordinated movements and intention tremor at two weeks of age. The severity of clinical signs was highly variable. Histopathology demonstrated atrophy of the CNS, particularly in the cerebellum. Combined linkage and homozygosity mapping in a family with four affected puppies delineated a 52 Mb critical interval. The comparison of whole genome sequence data of one affected dog to 735 control genomes revealed a private homozygous structural variant in the critical interval, Chr4:66,946,539_66,963,863del17,325. This deletion includes the entire protein coding sequence of SELENOP and is predicted to result in complete absence of the encoded selenoprotein P required for selenium transport into the CNS. Genotypes at the deletion showed the expected co-segregation with the phenotype in the investigated family. Total selenium levels in the blood of homozygous mutant puppies of the investigated litter were reduced to about 30% of the value of a homozygous wildtype littermate. Genotyping >600 Belgian Shepherd dogs revealed an additional homozygous mutant dog. This dog also suffered from pronounced ataxia, but reached an age of 10 years. Selenop-/- knock-out mice were reported to develop ataxia, but their histopathological changes were less severe than in the investigated dogs. Our results demonstrate that deletion of the SELENOP gene in dogs cause a defect in selenium transport associated with CNS atrophy and cerebellar ataxia (CACA). The affected dogs represent a valuable spontaneous animal model to gain further insights into the pathophysiological consequences of CNS selenium deficiency.

YAP/TAZ inhibition reduces metastatic potential of Ewing sarcoma cells.

Ewing sarcoma (EwS) is a highly metastatic bone cancer characterized by the ETS fusion oncoprotein EWS-FLI1. EwS cells are phenotypically highly plastic and switch between functionally distinct cell states dependent on EWS-FLI1 fluctuations. Whereas EWS-FLI1high cells proliferate, EWS-FLI1low cells are migratory and invasive. Recently, we reported activation of MRTFB and TEAD, effectors of RhoA and Hippo signalling, upon low EWS-FLI1, orchestrating key steps of the EwS migratory gene expression program. TEAD and its co-activators YAP and TAZ are commonly overexpressed in cancer, providing attractive therapeutic targets. We find TAZ levels to increase in the migratory EWS-FLI1low state and to associate with adverse prognosis in EwS patients. We tested the effects of the potent YAP/TAZ/TEAD complex inhibitor verteporfin on EwS cell migration in vitro and on metastasis in vivo. Verteporfin suppressed expression of EWS-FLI1 regulated cytoskeletal genes involved in actin signalling to the extracellular matrix, effectively blocked F-actin and focal-adhesion assembly and inhibited EwS cell migration at submicromolar concentrations. In a mouse EwS xenograft model, verteporfin treatment reduced relapses at the surgical site and delayed lung metastasis. These data suggest that YAP/TAZ pathway inhibition may prevent EwS cell dissemination and metastasis, justifying further preclinical development of YAP/TAZ inhibitors for EwS treatment.

Whole Exome Sequencing reveals NOTCH1 mutations in anaplastic large cell lymphoma and points to Notch both as a key pathway and a potential therapeutic target.

Patients diagnosed with Anaplastic Large Cell Lymphoma (ALCL) are still treated with toxic multi-agent chemotherapy and as many as 25-50% of patients relapse. To understand disease pathology and to uncover novel targets for therapy, Whole-Exome Sequencing (WES) of Anaplastic Lymphoma Kinase (ALK)+ ALCL was performed as well as Gene-Set Enrichment Analysis. This revealed that the T-cell receptor (TCR) and Notch pathways were the most enriched in mutations. In particular, variant T349P of NOTCH1, which confers a growth advantage to cells in which it is expressed, was detected in 12% of ALK+ and ALK- ALCL patient samples. Furthermore, we demonstrate that NPM-ALK promotes NOTCH1 expression through binding of STAT3 upstream of NOTCH1. Moreover, inhibition of NOTCH1 with γ-secretase inhibitors (GSIs) or silencing by shRNA leads to apoptosis; co-treatment in vitro with the ALK inhibitor Crizotinib led to additive/synergistic anti-tumour activity suggesting this may be an appropriate combination therapy for future use in the circumvention of ALK inhibitor resistance. Indeed, Crizotinib-resistant and sensitive ALCL were equally sensitive to GSIs. In conclusion, we show a variant in the extracellular domain of NOTCH1 that provides a growth advantage to cells and confirm the suitability of the Notch pathway as a second-line druggable target in ALK+ ALCL.

The Tyrosine Kinase Tec Regulates Effector Th17 Differentiation, Pathogenicity, and Plasticity in T-Cell-Driven Intestinal Inflammation.

T helper (Th) 17 cells are not only key in controlling infections mediated by extracellular bacteria and fungi but are also triggering autoimmune responses. Th17 cells comprise heterogeneous subsets, some with pathogenic functions. They can cease to secrete their hallmark cytokine IL-17A and even convert to other T helper lineages, a process known as transdifferentiation relying on plasticity. Both pathogenicity and plasticity are tightly linked to IL-23 signaling. Here, we show that the protein tyrosine kinase Tec is highly induced in Th17 cells. Th17 differentiation was enhanced at low interleukin-6 (IL-6) concentrations in absence of Tec, which correlates with increased STAT3 phosphorylation and higher Il23r expression. Therefore, we uncovered a function for Tec in the IL-6 sensing via STAT3 by CD4+ T cells, defining Tec as a fine-tuning negative regulator of Th17 differentiation. Subsequently, by using the IL-17A fate mapping mouse combined with in vivo adoptive transfer models, we demonstrated that Tec not only restrained effector Th17 differentiation but also pathogenicity and plasticity in a T-cell intrinsic manner. Our data further suggest that Tec regulates inflammatory Th17-driven immune responses directly impacting disease severity in a T-cell-driven colitis model. Notably, consistent with the in vitro findings, elevated levels of the IL-23 receptor (IL-23R) were observed on intestinal pre- and postconversion Th17 cells isolated from diseased Tec-/- mice subjected to adoptive transfer colitis, highlighting a fundamental role of Tec in restraining IL-23R expression, likely via the IL-6-STAT3 signaling axis. Taken together, these findings identify Tec as a negative regulator of Th17 differentiation, pathogenicity, and plasticity, contributing to the mechanisms which help T cells to orchestrate optimal immune protection and to restrain immunopathology.

Motor Cortex and Hippocampus Display Decreased Heme Oxygenase Activity 2 Weeks After Ventricular Fibrillation Cardiac Arrest in Rats.

Heme oxygenase (HO) and biliverdin reductase (BVR) activities are important for neuronal function and redox homeostasis. Resuscitation from cardiac arrest (CA) frequently results in neuronal injury and delayed neurodegeneration that typically affect vulnerable brain regions, primarily hippocampus (Hc) and motor cortex (mC), but occasionally also striatum and cerebellum. We questioned whether these delayed effects are associated with changes of the HO/BVR system. We therefore analyzed the activities of HO and BVR in the brain regions Hc, mC, striatum and cerebellum of rats subjected to ventricular fibrillation CA (6 min or 8 min) after 2 weeks following resuscitation, or sham operation. From all investigated regions, only Hc and mC showed significantly decreased HO activities, while BVR activity was not affected. In order to find an explanation for the changed HO activity, we analyzed protein abundance and mRNA expression levels of HO-1, the inducible, and HO-2, the constitutively expressed isoform, in the affected regions. In both regions we found a tendency for a decreased immunoreactivity of HO-2 using immunoblots and immunohistochemistry. Additionally, we investigated the histological appearance and the expression of markers indicative for activation of microglia [tumor necrosis factor receptor type I (TNFR1) mRNA and immunoreactivity for ionized calcium-binding adapter molecule 1 (Iba1])], and activation of astrocytes [immunoreactivity for glial fibrillary acidic protein (GFAP)] in Hc and mC. Morphological changes were detected only in Hc displaying loss of neurons in the cornu ammonis 1 (CA1) region, which was most pronounced in the 8 min CA group. In this region also markers indicating inflammation and activation of pro-death pathways (expression of HO-1 and TNFR1 mRNA, as well as Iba1 and GFAP immunoreactivity) were upregulated. Since HO products are relevant for maintaining neuronal function, our data suggest that neurodegenerative processes following CA may be associated with a decreased capacity to convert heme into HO products in particularly vulnerable brain regions.

CA1 Hippocampal Pyramidal Cells in Rats, Resuscitated From 8 Minutes of Ventricular Fibrillation Cardiac Arrest, Recover After 20 Weeks of Survival: A Retrospective Pilot Study.

PURPOSE: The cornu ammonis 1 (CA1) region of the hippocampus is specifically vulnerable to global ischemia. We hypothesized that histopathological outcome in a ventricular fibrillation cardiac arrest (VFCA) rat model depends on the time point of the examination. METHODS: Male Sprague-Dawley rats were put into VFCA for 8 min, received chest compressions for 2 min, and were defibrillated to achieve return of spontaneous circulation. Animals surviving for 80 min, 14 days and 140 days were compared with controls. Viable neurons were counted in a 500 µm sector of the CA1 region and layer thickness measured. Microglia cells and astrocytes were counted in a 250×300 µm aspect. RESULTS: Control and 80 min surviving animals had similar numbers of pyramidal neurons in the CA1 region. In 14 days and 140 days survivors neuron numbers and layer thickness were severely diminished compared with controls (P < 0.001). Two-thirds of the 140 days survivors showed significantly more viable neurons than the last third. Microglia was increased in 14 days survivors compared with controls and 140 days survivors, while astrocytes increased in 14 days and 140 days survivors compared with controls (P < 0.001). 140 days survivors had significantly higher astrocyte counts compared with 14 days survivors. CONCLUSIONS: The amount and type of brain lesions present after global ischemia depend on the survival time. A consistent reduction in pyramidal cells in the CA1 region was present in all animals 14 days after VFCA, but in two-thirds of animals a repopulation of pyramidal cells seems to have taken place after 140 days.

A New Strategy Toward B Cell-Based Cancer Vaccines by Active Immunization With Mimotopes of Immune Checkpoint Inhibitors.

Therapeutic monoclonal antibodies (mAbs), targeting tumor antigens, or immune checkpoints, have demonstrated a remarkable anti-tumor effect against various malignancies. However, high costs for mono- or combination therapies, associated with adverse effects or possible development of resistance in some patients, warrant further development and modification to gain more flexibility for this immunotherapy approach. An attractive alternative to passive immunization with therapeutic antibodies might be active immunization with mimotopes (B-cell peptides) representing the mAbs' binding epitopes, to activate the patient's own anti-tumor immune response following immunization. Here, we identified and examined the feasibility of inducing anti-tumor effects in vivo following active immunization with a mimotope of the immune checkpoint programmed cell death 1 (PD1), alone or in combination with a Her-2/neu B-cell peptide vaccine. Overlapping peptides spanning the extracellular domains of human PD1 (hPD1) were used to identify hPD1-derived mimotopes, using the therapeutic mAb Nivolumab as a proof of concept. Additionally, for in vivo evaluation in a tumor mouse model, a mouse PD1 (mPD1)-derived mimotope was identified using an anti-mPD1 mAb with mPD1/mPDL-1 blocking capacity. The identified mimotopes were characterized by in vitro assays, including a reporter cell-based assay, and their anti-tumor effects were evaluated in a syngeneic tumor mouse model stably expressing human Her-2/neu. The identified PD1-derived mimotopes were shown to significantly block the mAbs' capacity in inhibiting the respective PD1/PD-L1 interactions. A significant reduction in tumor growth in vivo was observed following active immunization with the mPD1-derived mimotope, associated with a significant reduction in proliferation and increased apoptotic rates in the tumors. Particularly, combined vaccination with the mPD1-derived mimotope and a multiple B-cell epitope Her-2/neu vaccine potentiated the vaccine's anti-tumor effect. Our results suggest active immunization with mimotopes of immune checkpoint inhibitors either as monotherapy or as combination therapy with tumor-specific vaccines, as a new strategy for cancer treatment.

STAT3-dependent analysis reveals PDK4 as independent predictor of recurrence in prostate cancer.

Prostate cancer (PCa) has a broad spectrum of clinical behavior; hence, biomarkers are urgently needed for risk stratification. Here, we aim to find potential biomarkers for risk stratification, by utilizing a gene co-expression network of transcriptomics data in addition to laser-microdissected proteomics from human and murine prostate FFPE samples. We show up-regulation of oxidative phosphorylation (OXPHOS) in PCa on the transcriptomic level and up-regulation of the TCA cycle/OXPHOS on the proteomic level, which is inversely correlated to STAT3 expression. We hereby identify gene expression of pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of the TCA cycle, as a promising independent prognostic marker in PCa. PDK4 predicts disease recurrence independent of diagnostic risk factors such as grading, staging, and PSA level. Therefore, low PDK4 is a promising marker for PCa with dismal prognosis.

The targetable kinase PIM1 drives ALK inhibitor resistance in high-risk neuroblastoma independent of MYCN status.

Resistance to anaplastic lymphoma kinase (ALK)-targeted therapy in ALK-positive non-small cell lung cancer has been reported, with the majority of acquired resistance mechanisms relying on bypass signaling. To proactively identify resistance mechanisms in ALK-positive neuroblastoma (NB), we herein employ genome-wide CRISPR activation screens of NB cell lines treated with brigatinib or ceritinib, identifying PIM1 as a putative resistance gene, whose high expression is associated with high-risk disease and poor survival. Knockdown of PIM1 sensitizes cells of differing MYCN status to ALK inhibitors, and in patient-derived xenografts of high-risk NB harboring ALK mutations, the combination of the ALK inhibitor ceritinib and PIM1 inhibitor AZD1208 shows significantly enhanced anti-tumor efficacy relative to single agents. These data confirm that PIM1 overexpression decreases sensitivity to ALK inhibitors in NB, and suggests that combined front-line inhibition of ALK and PIM1 is a viable strategy for the treatment of ALK-positive NB independent of MYCN status.

Clinical and Serological Evaluation of LINDA Virus Infections in Post-Weaning Piglets.

The novel pestivirus species known as lateral-shaking inducing neuro-degenerative agent (LINDA) virus emerged in 2015 in a piglet-producing farm in Austria. Affected piglets showed strong congenital tremor as a result of severe lesions in the central nervous system. Here, we report the results of a controlled animal infection experiment. Post-weaning piglets were infected with LINDA to determine the susceptibility of pigs, the clinical consequences of infection and the humoral immune response against LINDA. No clinically overt disease signs were observed in the piglets. Viremia was hardly detectable, but LINDA was present in the spleen and several lymphatic organs until the end of the experiment on day 28 post-infection. Oronasal virus shedding together with the infection of one sentinel animal provided additional evidence for the successful replication and spread of LINDA in the piglets. Starting on day 14 post-infection, all infected animals showed a strong humoral immune response with high titers of neutralizing antibodies against LINDA. No cross-neutralizing activity of these sera with other pestiviral species was observed. According to these data, following postnatal infection, LINDA is a rather benign virus that can be controlled by the pig's immune system. However, further studies are needed to investigate the effects of LINDA on the fetus after intrauterine infection.

Intracranial abscess formation in an adult alpaca: a case report.

BACKGROUND: Intracranial abscess formation is an extremely rare and sporadically documented disease in South American Camelids (SACs). Herein we report the first case of otogenic brain abscess formation in this species. CASE PRESENTATION: A 4 years old female alpaca was presented to our veterinary hospital with a 6 month history of neurologic disorder symptoms, mainly head tilt to the right and emaciation. A comprehensive workup (ultrasound and computed tomography) revealed irreversible cranial nerve abnormalities, extensive lesions in the region of external, middle and internal right ear including destruction of bony structures (tympanic bulla, parts of temporal bone) and severe brain deformation caused by an intracranial abscess. The lesion was up to 6x7x4 cm and occupying almost 40% of the cranial cavity. No pathological findings were evident in other organs or structures. The late referral of the alpaca at this advanced stage of destructive disease precluded surgical intervention. CONCLUSIONS: This case report describes the clinical signs, diagnostic procedures and pathological findings in an adult female alpaca suffering from cranial nerve abnormalities caused by a massive otogenic brain abscess. Camelids suffering from otitis may not present with clinical signs until the pathology is severe. The importance of considering intracranial abscess formation as differential diagnosis in SACs showing the merest hint of nerve deficits cannot be emphasized enough in order to diagnose such pathological processes at an early and treatable stage.