The immunomodulatory ballet of tumour-derived extracellular vesicles and neutrophils orchestrating the dynamic CD73/PD-L1 pathway in cancer.

Head and neck squamous cell carcinoma (HNSCC) is a global cancer burden with a 5-year overall survival rate of around 50%, stagnant for decades. A tumour-induced immunosuppressive microenvironment contributes to HNSCC progression, with the adenosine (ADO) pathway and an upregulated expression of inhibitory immune checkpoint regulators playing a key role in this context. The correlation between high neutrophil-to-lymphocyte ratio (NLR) with advanced tumour staging suggests involvement of neutrophils (NØ) in cancer progression. Interestingly, we associated a high NLR with an increased intracellular PD-L1 localization in primary HNSCC samples, potentially mediating more aggressive tumour characteristics and therefore synergistically favouring tumour progression. Still, further research is needed to harness this knowledge for effective treatments and overcome resistance. Since it is hypothesized that the tumour microenvironment (TME) may be influenced by small extracellular vesicles (sEVs) secreted by tumours (TEX), this study aims to investigate the impact of HNSCC-derived TEX on NØ and blockade of ADO receptors as a potential strategy to reverse the pro-tumour phenotype of NØ. UMSCC47-TEX exhibited CD73 enzymatic activity involved in ADO signalling, as well as the immune checkpoint inhibitor PD-L1. Data revealed that TEX induce chemotaxis of NØ and the sustained interaction promotes a shift into a pro-tumour phenotype, dependent on ADO receptors (P1R), increasing CD170high subpopulation, CD73 and PD-L1 expression, followed by an immunosuppressive secretome. Blocking A3R reduced CD73 and PD-L1 expression. Co-culture experiments with HNSCC cells demonstrated that TEX-modulated NØ increase the CD73/PD-L1 axis, through Cyclin D-CDK4/6 signalling. To support these findings, the CAM model with primary tumour was treated with NØ supernatant. Moreover, these NØ promoted an increase in migration, invasion, and reduced cell death. Targeting P1R on NØ, particularly A3R, exhibited potential therapeutic strategy to counteract immunosuppression in HNSCC. Understanding the TEX-mediated crosstalk between tumours and NØ offers insights into immunomodulation for improving cancer therapies.

Comparison of the oncolytic activity of a replication-competent and a replication-deficient herpes simplex virus 1.

In 2015, the oncolytic herpes simplex virus 1 (HSV-1) T-VEC (talimogene laherparepvec) was approved for intratumoral injection in non-resectable malignant melanoma. To determine whether viral replication is required for oncolytic activity, we compared replication-deficient HSV-1 d106S with replication-competent T-VEC. High infectious doses of HSV-1 d106S killed melanoma (n = 10), head-and-neck squamous cell carcinoma (n = 11), and chondrosarcoma cell lines (n = 2) significantly faster than T-VEC as measured by MTT metabolic activity, while low doses of T-VEC were more effective over time. HSV-1 d106S and, to a lesser extent T-VEC, triggered caspase-dependent early apoptosis as shown by pan-caspase inhibition and specific induction of caspases 3/7, 8, and 9. HSV-1 d106S induced a higher ratio of apoptosis-inducing infected cell protein (ICP) 0 to apoptosis-blocking ICP6 than T-VEC. T-VEC was oncolytic for an extended period of time as viral replication continued, which could be partially blocked by the antiviral drug aciclovir. High doses of T-VEC, but not HSV-1 d106S, increased interferon-ß mRNA as part of the intrinsic immune response. When markers of immunogenic cell death were assessed, ATP was released more efficiently in the context of T-VEC than HSV-1 d106S infection, whereas HMGB1 was induced comparatively well. Overall, the early oncolytic effect on three different tumour entities was stronger with the non-replicative strain, while the replication-competent virus elicited a stronger innate immune response and more pronounced immunogenic cell death.

Subcellular localization of PD-L1 and cell-cycle-dependent expression of nuclear PD-L1 variants: implications for head and neck cancer cell functions and therapeutic efficacy.

The programmed cell death 1 ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis is primarily associated with immunosuppression in cytotoxic T lymphocytes (CTLs). However, mounting evidence is supporting the thesis that PD-L1 not only functions as a ligand but mediates additional cellular functions in tumor cells. Moreover, it has been demonstrated that PD-L1 is not exclusively localized at the cellular membrane. Subcellular fractionation revealed the presence of PD-L1 in various cellular compartments of six well-characterized head and neck cancer (HNC) cell lines, including the nucleus. Via Western blotting, we detected PD-L1 in its well-known glycosylated/deglycosylated state at 40-55 kDa. In addition, we detected previously unknown PD-L1 variants with a molecular weight at approximately 70 and > 150 kDa exclusively in nuclear protein fractions. These in vitro findings were confirmed with primary tumor samples from head and neck squamous cell carcinoma (HNSCC) patients. Furthermore, we demonstrated that nuclear PD-L1 variant expression is cell-cycle-dependent. Immunofluorescence staining of PD-L1 in different cell cycle phases of synchronized HNC cells supported these observations. Mechanisms of nuclear PD-L1 trafficking remain less understood; however, proximity ligation assays showed a cell-cycle-dependent interaction of the cytoskeletal protein vimentin with PD-L1, whereas vimentin could serve as a potential shuttle for nuclear PD-L1 transportation. Mass spectrometry after PD-L1 co-immunoprecipitation, followed by gene ontology analysis, indicated interaction of nuclear PD-L1 with proteins involved in DNA remodeling and messenger RNA (mRNA) splicing. Our results in HNC cells suggest a highly complex regulation of PD-L1 and multiple tumor cell-intrinsic functions, independent of immune regulation. These observations bear significant implications for the therapeutic efficacy of immune checkpoint inhibition.

BMSC-HNC Interaction: Exploring Effects on Bone Integrity and Head and Neck Cancer Progression.

In recent research, the tumor microenvironment has been shown to attract mesenchymal stromal cells (MSCs), which is of particular interest due to its implications for cancer progression. The study focused on understanding the interaction between bone marrow-derived MSCs (BMSCs) and head and neck cancer (HNC) cells. This interaction was found to activate specific markers, notably the osteogenic marker alkaline phosphatase and the oncogene Runx2. These activations corresponded with the release of collagenase enzymes, MMP9 and MMP2. To gain insights into bone resorption related to this interaction, bovine bone slices were used, supporting the growth of "heterogeneous spheroids" that contained both BMSCs and HNC cells. Through scanning electron microscopy and energy-dispersive X-ray (EDX) analysis, it was observed that these mixed spheroids were linked to a notable increase in bone degradation and collagen fiber exposure, more so than spheroids of just BMSCs or HNC cells. Furthermore, the EDX results highlighted increased nitrogen content on bone surfaces with these mixed clusters. Overall, the findings underscore the significant role of BMSCs in tumor growth, emphasizing the need for further exploration in potential cancer treatment strategies.

Extracellular Vesicles Derived from Osteogenic-Differentiated Human Bone Marrow-Derived Mesenchymal Cells Rescue Osteogenic Ability of Bone Marrow-Derived Mesenchymal Cells Impaired by Hypoxia.

In orthopedics, musculoskeletal disorders, i.e., non-union of bone fractures or osteoporosis, can have common histories and symptoms related to pathological hypoxic conditions induced by aging, trauma or metabolic disorders. Here, we observed that hypoxic conditions (2% O2) suppressed the osteogenic differentiation of human bone marrow-derived mesenchymal cells (hBMSC) in vitro and simultaneously increased reactive oxygen species (ROS) production. We assumed that cellular origin and cargo of extracellular vesicles (EVs) affect the osteogenic differentiation capacity of hBMSCs cultured under different oxygen pressures. Proteomic analysis revealed that EVs isolated from osteogenic differentiated hBMSC cultured under hypoxia (hypo-osteo EVs) or under normoxia (norm-osteo EVs) contained distinct protein profiles. Extracellular matrix (ECM) components, antioxidants and pro-osteogenic proteins were decreased in hypo-osteo EVs. The proteomic analysis in our previous study revealed that under normoxic culture conditions, pro-osteogenic proteins and ECM components have higher concentrations in norm-osteo EVs than in EVs derived from naïve hBMSCs (norm-naïve EVs). When selected for further analysis, five anti-hypoxic proteins were significantly upregulated (response to hypoxia) in norm-osteo EVs. Three of them are characterized as antioxidant proteins. We performed qRT-PCR to verify the corresponding gene expression levels in the norm-osteo EVs' and norm-naïve EVs' parent cells cultured under normoxia. Moreover, we observed that norm-osteo EVs rescued the osteogenic ability of naïve hBMSCs cultured under hypoxia and reduced hypoxia-induced elevation of ROS production in osteogenic differentiated hBMSCs, presumably by inducing expression of anti-hypoxic/ antioxidant and pro-osteogenic genes.

Diversity of CRISPR-Cas type II-A systems in Streptococcus anginosus.

Streptococcus anginosus is a commensal Streptococcal species that is often associated with invasive bacterial infections. However, little is known about its molecular genetic background. Many Streptococcal species, including S. anginosus, harbor clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. A CRISPR-Cas type II-A system as well as a type II-C system have been reported for this species. To characterize the CRISPR-Cas type II systems of S. anginosus in more detail, we conducted a phylogenetic analysis of Cas9 sequences from CRISPR-Cas type II systems with a special focus on streptococci and S. anginosus. In addition, a phylogenetic analysis of S. anginosus strains based on housekeeping genes included in MLST analysis, was performed. All analyzed Cas9 sequences of S. anginosus clustered with the Cas9 sequences of CRISPR type II-A systems, including the Cas9 sequences of S. anginosus strains reported to harbor a type II-C system. The Cas9 genes of the CRISPR-Cas type II-C systems of other bacterial species separated into a different cluster. Moreover, analyzing the CRISPR loci found in S. anginosus, two distinct csn2 genes could be detected, a short form showing high similarity to the canonical form of the csn2 gene present in S. pyogenes. The second CRISPR type II locus of S. anginosus contained a longer variant of csn2 with close similarities to a csn2 gene that has previously been described in Streptococcus thermophilus. Since CRISPR-Cas type II-C systems do not contain a csn2 gene, the S. anginosus strains reported to have a CRISPR-Cas type II-C system appear to carry a variation of CRISPR-Cas type II-A harboring a long variant of csn2.

OSCC in Never-Smokers and Never-Drinkers Is Associated with Increased Expression of Tumor-Infiltrating Lymphocytes and Better Survival.

The aim of this study was to investigate the clinical, histopathologic, and immunologic differences of oral squamous cell carcinoma of never-smokers/never-drinkers and smokers/drinkers. Immunohistochemical staining for CD4, CD8, FoxP3, CD1a, and p16 was performed in 131 oral squamous cell carcinomas from smokers/drinkers and never-smokers/never-drinkers. Associations of smoking/drinking status with clinicopathologic data, immunohistochemical antibody expression, and survival were examined. Oral squamous cell carcinoma in never-smokers/never-drinkers was associated with the female gender (p < 0.001). Never-smokers/never-drinkers were older at diagnosis than smokers/drinkers (p < 0.001). Never-smokers/never-drinkers had more tumors in the maxilla, mandible, and tongue (p < 0.001). Pre-existing oral potentially malignant disorders appeared to be more common in never-smokers/never-drinkers (p < 0.001). Perineural invasion was more common in smokers/drinkers (p = 0.039). Never-smoking/never-drinking was associated with better overall survival (p = 0.004) and disease-specific survival (p = 0.029). High CD4+ T cell infiltration was associated with never-smoking/never-drinking (p = 0.008). Never-smokers/never-drinkers also showed increased CD8+ T cell infiltration (p = 0.001) and increased FoxP3+ Treg infiltration (p = 0.023). Furthermore, the total group of tumor-infiltrating lymphocytes was associated with never smoking/never drinking (p = 0.005). To conclude oral squamous cell carcinoma of the never-smokers/never-drinkers appears to be a distinct type of tumor, as it appears to have unique clinical and pathologic features and a more immunogenic microenvironment.

The C-terminal 32-mer fragment of hemoglobin alpha is an amyloidogenic peptide with antimicrobial properties.

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.

Opposing MMP-9 Expression in Mesenchymal Stromal Cells and Head and Neck Tumor Cells after Direct 2D and 3D Co-Culture.

Bone marrow-derived mesenchymal stromal cells (BMSCs) respond to a variety of tumor cell-derived signals, such as inflammatory cytokines and growth factors. As a result, the inflammatory tumor microenvironment may lead to the recruitment of BMSCs. Whether BMSCs in the tumor environment are more likely to promote tumor growth or tumor suppression is still controversial. In our experiments, direct 3D co-culture of BMSCs with tumor cells from the head and neck region (HNSCC) results in strong expression and secretion of MMP-9. The observed MMP-9 secretion mainly originates from BMSCs, leading to increased invasiveness. In addition to our in vitro data, we show in vivo data based on the chorioallantoic membrane (CAM) model. Our results demonstrate that MMP-9 induces hemorrhage and increased perfusion in BMSC/HNSCC co-culture. While we had previously outlined that MMP-9 expression and secretion originate from BMSCs, our data showed a strong downregulation of MMP-9 promoter activity in HNSCC cells upon direct contact with BMSCs using the luciferase activity assay. Interestingly, the 2D and 3D models of direct co-culture suggest different drivers for the downregulation of MMP-9 promoter activity. Whereas the 3D model depicts a BMSC-dependent downregulation, the 2D model shows cell density-dependent downregulation. In summary, our data suggest that the direct interaction of HNSCC cells and BMSCs promotes tumor progression by significantly facilitating angiogenesis via MMP-9 expression. On the other hand, data from 3D and 2D co-culture models indicate opposing regulation of the MMP-9 promoter in tumor cells once stromal cells are involved.

[99m-Technetium-pertechnetate- and 99m-Technetium-sestamibi-scintigraphy for visualization of hypofunctioning thyroid tissue and staging in a dog with thyroid carcinoma]. / 99m-Technetium-Pertechnetat- und 99m-Technetium-Sestamibi-Szintigrafie zur Darstellung von hypofunktionellem Schilddrüsengewebe und Staging bei einem Hund mit Schilddrüsenkarzinom.

A 10-year-old female mixed breed dog was presented for thyroid scintigraphy due to a cervical mass. Apart from 99m-Technetium-pertechnetate (Tc-pertechnetate) scintigraphy, a second scintigraphy using 99m-Technetium sestamibi (Tc-MIBI) was performed because of additional hypothyroidism suspective for a "cold" nodule and as screening for metastases.Twenty minutes following intravenous injection of 38 MBq Tc-pertechnetate, a "hot" cervical as well as a "hot" intrathoracal nodule were seen with an uptake of 8.40 and 0.25 %, respectively. The second scintigraphy was performed 20 minutes after intravenous injection of 364 MBq Tc-MIBI and 70 minutes following the first. After subtraction of pertechnetate activity and decay correction, both nodules showed an uptake of 0.99 and 0.03 %. Additionally, both thyroid lobes were visible in the thyroid loge with a weak MIBI-uptake. For both lesions, the ratio Tc-uptake/Tc-MIBI-uptake was 8.48 and 8.33, respectively.Following the extirpation of the cervical mass, histopathology revealed atrophied healthy thyroid tissue almost completely displaced by a well-differentiated follicular thyroid carcinoma.This case report describes performance, utility and calculative correction of consecutive pertechnetate- and MIBI-scan, that enable a visualization of hypofunctional thyroid tissue.Therefore and because of their similar MIBI metabolic activity, both nodules were considered to be dystopic tissue/metastases so that this dog had to be classified as prognostically less favorable WHO stabe IV. Different from human patients, both scintigraphies should be performed shortly after another in dogs in order to avoid the necessity of a second anesthetic procedure. A reliable qualitative/visual evaluation of the MIBI-scan is therefore not possible, so that a quantitative assessment using the uptake after calculative correction of the pertechnetate activity is recommended.

TGFß+ small extracellular vesicles from head and neck squamous cell carcinoma cells reprogram macrophages towards a pro-angiogenic phenotype.

Transforming growth factor ß (TGFß) is a major component of tumor-derived small extracellular vesicles (TEX) in cancer patients. Mechanisms utilized by TGFß+ TEX to promote tumor growth and pro-tumor activities in the tumor microenvironment (TME) are largely unknown. TEX produced by head and neck squamous cell carcinoma (HNSCC) cell lines carried TGFß and angiogenesis-promoting proteins. TGFß+ TEX stimulated macrophage chemotaxis without a notable M1/M2 phenotype shift and reprogrammed primary human macrophages to a pro-angiogenic phenotype characterized by the upregulation of pro-angiogenic factors and functions. In a murine basement membrane extract plug model, TGFß+ TEX promoted macrophage infiltration and vascularization (p < 0.001), which was blocked by using the TGFß ligand trap mRER (p < 0.001). TGFß+ TEX injected into mice undergoing the 4-nitroquinoline-1-oxide (4-NQO)-driven oral carcinogenesis promoted tumor angiogenesis (p < 0.05), infiltration of M2-like macrophages in the TME (p < 0.05) and ultimately tumor progression (p < 0.05). Inhibition of TGFß signaling in TEX with mRER ameliorated these pro-tumor activities. Silencing of TGFß emerges as a critical step in suppressing pro-angiogenic functions of TEX in HNSCC.

Checkpoint Inhibitors in Cancer Therapy: Clinical Benefits for Head and Neck Cancers.

Recently, considerable progress has been achieved in cancer immunotherapy. Targeted immune checkpoint therapies have been established for several forms of cancers, which resulted in a tremendous positive impact on patient survival, even in more advanced tumor stages. With a better understanding of cellular responses to immune checkpoint therapies, it will soon be feasible to find targeted compounds which will make personalized medicine practicable. This is a great opportunity, but it also sets tremendous challenges on both the scientific and clinical aspects. Head and neck tumors evade immune surveillance through various mechanisms. They contain fewer lymphocytes (natural killer cells) than normal tissue with an accumulation of immunosuppressive regulatory T cells. Standard therapies for HNSCC, such as surgery, radiation, and chemotherapy, are becoming more advantageous by targeting immune checkpoints and employing combination therapies. The purpose of this review is to provide an overview of the expanded therapeutic options, particularly the combination of immune checkpoint inhibition with various conventional and novel therapeutics for head and neck tumor patients.

Current trends and outcomes of non-elective neurosurgical care in Central Europe during the second year of the COVID-19 pandemic.

Reflecting the first wave COVID-19 pandemic in Central Europe (i.e. March 16th-April 15th, 2020) the neurosurgical community witnessed a general diminution in the incidence of emergency neurosurgical cases, which was impelled by a reduced number of traumatic brain injuries (TBI), spine conditions, and chronic subdural hematomas (CSDH). This appeared to be associated with restrictions imposed on mobility within countries but also to possible delayed patient introduction and interdisciplinary medical counseling. In response to one year of COVID-19 experience, also mapping the third wave of COVID-19 in 2021 (i.e. March 16 to April 15, 2021), we aimed to reevaluate the current prevalence and outcomes for emergency non-elective neurosurgical cases in COVID-19-negative patients across Austria and the Czech Republic. The primary analysis was focused on incidence and 30-day mortality in emergency neurosurgical cases compared to four preceding years (2017-2020). A total of 5077 neurosurgical emergency cases were reviewed. The year 2021 compared to the years 2017-2019 was not significantly related to any increased odds of 30 day mortality in Austria or in the Czech Republic. Recently, there was a significant propensity toward increased incidence rates of emergency non-elective neurosurgical cases during the third COVID-19 pandemic wave in Austria, driven by their lower incidence during the first COVID-19 wave in 2020. Selected neurosurgical conditions commonly associated with traumatic etiologies including TBI, and CSDH roughly reverted to similar incidence rates from the previous non-COVID-19 years. Further resisting the major deleterious effects of the continuing COVID-19 pandemic, it is edifying to notice that the neurosurgical community´s demeanor to the recent third pandemic culmination keeps the very high standards of non-elective neurosurgical care alongside with low periprocedural morbidity. This also reflects the current state of health care quality in the Czech Republic and Austria.

Loss of MMP-27 Predicts Mandibular Bone Invasion in Oral Squamous Cell Carcinoma.

Invasion of the mandibular bone is frequent in oral squamous cell carcinoma (OSCC), which often results in extensive ablative and reconstructive procedures for the patient. The purpose of this single-center, retrospective study was to identify and evaluate potential biomarkers and risk factors for bone invasion in OSCC. Initially, in silico gene expression analysis was performed for different HNSCC tumor T-stages to find factors associated with invasive (T4a) tumor growth. Afterwards, the protein expression of bone-metabolizing MMP-27, TNFRSF11B (Osteoprotegerin, OPG), and TNFSF11 (RANKL) was investigated via Tissue Microarrays (TMAs) for their impact on mandibular bone invasion. TMAs were assembled from the bone-tumor interface of primary OSCCs of the floor of the mouth and gingiva from 119 patients. Sixty-four carcinomas with patho-histological jaw invasion (pT4a) were compared to 55 carcinomas growing along the mandible without invasion (pT2, pT3). Tissue samples were additionally evaluated for patterns of invasion using the WPOI grading system. Statistical analysis of in silico data revealed decreased MMP-27 mRNA expression to be strongly associated with the pT4a-stage in OSCC, indicating invasive tumor growth with infiltration of adjacent anatomical structures. Our own clinico-pathological data on OSCCs presented a significant decrease of MMP-27 in tumors invading the nearby mandible (pT4a), compared to pT2 and pT3 tumors without bone invasion. Loss of MMP27 evolved as the strongest predictor of mandibular bone invasion in binary logistic regression analysis. To our knowledge, this is the first study investigating the role of MMP-27 expression in OSCC and demonstrating the importance of the loss of MMP-27 in mandibular bone invasion.

The Renaissance of Cyclin Dependent Kinase Inhibitors.

Cyclin-dependent kinases (CDK) regulate cell cycle progression. During tumor development, altered expression and availability of CDKs strongly contribute to impaired cell proliferation, a hallmark of cancer. In recent years, targeted inhibition of CDKs has shown considerable therapeutic benefit in a variety of tumor entities. Their success is reflected in clinical approvals of specific CDK4/6 inhibitors for breast cancer. This review provides a detailed insight into the molecular mechanisms of CDKs as well as a general overview of CDK inhibition. It also summarizes the latest research approaches and current advances in the treatment of head and neck cancer with CDK inhibitors. Instead of monotherapies, combination therapies with CDK inhibitors may especially provide promising results in tumor therapy. Indeed, recent studies have shown a synergistic effect of CDK inhibition together with chemo- and radio- and immunotherapy in cancer treatment to overcome tumor evasion, which may lead to a renaissance of CDK inhibitors.

Angicin, a novel bacteriocin of Streptococcus anginosus.

As a conserved defense mechanism, many bacteria produce antimicrobial peptides, called bacteriocins, which provide a colonization advantage in a multispecies environment. Here the first bacteriocin of Streptococcus anginosus, designated Angicin, is described. S. anginosus is commonly described as a commensal, however it also possesses a high pathogenic potential. Therefore, understanding factors contributing to its host colonization and persistence are important. A radial diffusion assay was used to identify S. anginosus BSU 1211 as a potent bacteriocin producer. By genetic mutagenesis the background of bacteriocin production and the bacteriocin gene itself were identified. Synthetic Angicin shows high activity against closely related streptococci, listeria and vancomycin resistant enterococci. It has a fast mechanism of action and causes a membrane disruption in target cells. Angicin, present in cell free supernatant, is insensitive to changes in temperature from - 70 to 90 °C and pH values from 2 to 10, suggesting that it represents an interesting compound for potential applications in food preservation or clinical settings.

Differential Expression of PD-L1 during Cell Cycle Progression of Head and Neck Squamous Cell Carcinoma.

The expression of PD-L1 by tumor cells is mainly associated with its immunosuppressive effect. In fact, PD-1/PD-L1 immune checkpoint inhibitors demonstrated remarkable effects in advanced cancer patients including HNSCC. In this context, irradiation is currently being investigated as a synergistic treatment modality to immunotherapy. However, the majority of HNSCC patients still show little improvement or even hyperprogression. Interestingly, there is increasing evidence for additional cell-intrinsic functions of PD-L1 in tumor cells. In previous studies, we showed that PD-L1 has a strong influence on proliferation, migration, invasion, and survival after irradiation. We demonstrated that cellular expression and localization of PD-L1 differed depending on sensitivity to irradiation. Here, we show that PD-L1 is also differentially expressed during cell cycle progression of HNSCC. Furthermore, cellular localization of PD-L1 also changes depending on a particular cell cycle phase. Moreover, distinct observations occurred depending on the general differentiation status. Overall, the function of PD-L1 cannot be generalized. Rather, it depends on the differentiation status and microenvironment. PD-L1 expression and localization are variable, depending on different factors. These findings may provide insight into why differential response to PD-1/PD-L1 antibody therapy can occur. Detailed understanding of cell-intrinsic PD-L1 functions will further allow antibody-based immunotherapy to be optimized.

The effects of violet and blue light irradiation on ESKAPE pathogens and human cells in presence of cell culture media.

Bacteria belonging to the group of ESKAPE pathogens are responsible for the majority of nosocomial infections. Due to the increase of antibiotic resistance, alternative treatment strategies are of high clinical relevance. In this context visible light as disinfection technique represents an interesting option as microbial pathogens can be inactivated without adjuvants. However cytotoxic effects of visible light on host cells have also been reported. We compared the cytotoxicity of violet and blue light irradiation on monocytic THP-1 and alveolar epithelium A549 cells with the inactivation effect on ESKAPE pathogens. THP-1 cells displayed a higher susceptibility to irradiation than A549 cells with first cytotoxic effects occurring at 300 J cm-2 (405 nm) and 400 J cm-2 (450 nm) in comparison to 300 J cm-2 and 1000 J cm-2, respectively. We could define conditions in which a significant reduction of colony forming units for all ESKAPE pathogens, except Enterococcus faecium, was achieved at 405 nm while avoiding cytotoxicity. Irradiation at 450 nm demonstrated a more variable effect which was species and medium dependent. In summary a significant reduction of viable bacteria could be achieved at subtoxic irradiation doses, supporting a potential use of visible light as an antimicrobial agent in clinical settings.

Substance P and Alpha-Calcitonin Gene-Related Peptide Differentially Affect Human Osteoarthritic and Healthy Chondrocytes.

Osteoarthritis (OA) is a degenerative joint disease that not only causes cartilage loss but also structural damage in all joint tissues. Joints are innervated by alpha-calcitonin gene-related peptide (αCGRP) and substance P (SP)-positive sensory nerve fibers. Alteration of sensory joint innervation could be partly responsible for degenerative changes in joints that contribute to the development of OA. Therefore, our aim was to analyze and compare the molecular effects of SP and αCGRP on the metabolism of articular chondrocytes from OA patients and non-OA cartilage donors. We treated the cells with SP or αCGRP and analysed the influence of these neuropeptides on chondrocyte metabolism and modulation of signaling pathways. In chondrocytes from healthy cartilage, SP had minimal effects compared with its effects on OA chondrocytes, where it induced inflammatory mediators, inhibited chondrogenic markers and promoted apoptosis and senescence. Treatment with αCGRP also increased apoptosis and senescence and reduced chondrogenic marker expression in OA chondrocytes, but stimulated an anabolic and protective response in healthy chondrocytes. The catabolic influence of SP and αCGRP might be due to activation of ERK signaling that could be counteracted by an increased cAMP response. We suggest that a switch between the G-subunits of the corresponding receptors after binding their ligands SP or αCGRP plays a central role in mediating the observed effects of sensory neuropeptides on chondrocytes.

Microbial Photoinactivation by Visible Light Results in Limited Loss of Membrane Integrity.

Interest in visible light irradiation as a microbial inactivation method has widely increased due to multiple possible applications. Resistance development is considered unlikely, because of the multi-target mechanism, based on the induction of reactive oxygen species by wavelength specific photosensitizers. However, the affected targets are still not completely identified. We investigated membrane integrity with the fluorescence staining kit LIVE/DEAD® BacLight™ on a Gram positive and a Gram negative bacterial species, irradiating Staphylococcus carnosus and Pseudomonas fluorescens with 405 nm and 450 nm. To exclude the generation of viable but nonculturable (VBNC) bacterial cells, we applied an ATP test, measuring the loss of vitality. Pronounced uptake of propidium iodide was only observed in Pseudomonas fluorescens at 405 nm. Transmission electron micrographs revealed no obvious differences between irradiated samples and controls, especially no indication of an increased bacterial cell lysis could be observed. Based on our results and previous literature, we suggest that visible light photoinactivation does not lead to rapid bacterial cell lysis or disruption. However, functional loss of membrane integrity due to depolarization or inactivation of membrane proteins may occur. Decomposition of the bacterial envelope following cell death might be responsible for observations of intracellular component leakage.