Human Bronchial Epithelial Cell Transcriptome Changes in Response to Serum from Patients with Different Status of Inflammation.

PURPOSE: To investigate the transcriptome of human bronchial epithelial cells (HBEC) in response to serum from patients with different degrees of inflammation. METHODS: Serum from 19 COVID-19 patients obtained from the Hannover Unified Biobank was used. At the time of sampling, 5 patients had a WHO Clinical Progression Scale (WHO-CPS) score of 9 (severe illness). The remaining 14 patients had a WHO-CPS of below 9 (range 1-7), and lower illness. Multiplex immunoassay was used to assess serum inflammatory markers. The culture medium of HBEC was supplemented with 2% of the patient's serum, and the cells were cultured at 37 °C, 5% CO2 for 18 h. Subsequently, cellular RNA was used for RNA-Seq. RESULTS: Patients with scores below 9 had significantly lower albumin and serum levels of E-selectin, IL-8, and MCP-1 than patients with scores of 9. Principal component analysis based on 500 "core genes" of RNA-seq segregated cells into two subsets: exposed to serum from 4 (I) and 15 (II) patients. Cells from a subset (I) treated with serum from 4 patients with a score of 9 showed 5566 differentially expressed genes of which 2793 were up- and 2773 downregulated in comparison with cells of subset II treated with serum from 14 patients with scores between 1 and 7 and one with score = 9. In subset I cells, a higher expression of TLR4 and CXCL8 but a lower CDH1, ACE2, and HMOX1, and greater effects on genes involved in metabolic regulation, cytoskeletal organization, and kinase activity pathways were observed. CONCLUSION: This simple model could be useful to characterize patient serum and epithelial cell properties.

Humoral immune response following prime and boost BNT162b2 vaccination in people living with HIV on antiretroviral therapy.

OBJECTIVES: People living with HIV (PLWH) with low CD4 T-cell counts may be at a higher risk for severe coronavirus disease 2019 (COVID-19) outcomes and in need of efficient vaccination. The World Health Organization (WHO) now recommends prioritizing PLHIV for COVID-19 vaccination. Data on immune responses after messenger RNA (mRNA) vaccination in PLHIV in relation to CD4 counts are scarce. We aimed at assessing the humoral immune response in PLHIV after mRNA vaccination against COVID-19. METHODS: We examined a cohort of PLHIV after prime (n = 88) and boost (n = 52) vaccination with BNT162b2. We assessed levels of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein-specific immunoglobulin G (IgG)/IgA and circulating neutralizing antibodies in plasma and correlated results to the cellular immune status. BNT162b2-vaccinated health care workers served as controls. RESULTS: All PLWH had a viral load of ≤ 200 HIV-1 RNA copies/mL and 96.5% had a viral load of < 50 copies/mL. Anti-S IgG and neutralizing antibody responses after BNT162b2 priming were significantly lower in PLHIV having a CD4:CD8 T-cell ratio of < 0.5. However, we observed robust humoral immunity in the majority of PLWH receiving antiretroviral therapy (ART) irrespective of CD4 T-cell nadir, current CD4 count or CD4:CD8 ratio after full BNT162b2 vaccination. Nevertheless, HIV-negative controls produced significantly higher mean anti-S IgG concentrations with less variability. CONCLUSIONS: The majority of PLWH mounted robust responses after complete BNT162b2 vaccination but overall amounts of antibodies directed against the SARS-CoV-2 receptor-binding domain were variable. The impact on clinical efficacy remains unclear.

α-1-antitrypsin inhibits acute liver failure in mice.

UNLABELLED: Acute liver failure remains a critical clinical condition, with high mortality rates, and increased apoptosis of hepatocytes represents a key event in the cause of liver failure. Alpha-1-antitrypsin (AAT) is synthesized and secreted mainly by hepatocytes, and plasma purified AAT is used for augmentation therapy in patients with AAT deficiency. Because AAT therapy exerts antiinflammatory and immune modulatory activities in various experimental models, and it was recently suggested that AAT exerts antiapoptotic activities, we aimed to explore whether administration of AAT may represent a therapeutic strategy to treat acute liver failure in mice. Well-established preclinical models of acute liver failure such as the Jo2 FAS/CD95 activating model and models of acetaminophen and α-amanitin poisoning were used. Therapeutic effects of AAT were evaluated by monitoring animal survival, histopathological changes, measurement of caspase activity, and serum cytokine levels. Systemic treatment with AAT significantly decreased Jo2-induced liver cell apoptosis and prolonged survival of mice. Native and oxidized (lacking elastase inhibitory activity) forms of AAT were equally effective in preventing acute liver injury and showed direct inhibition of active caspase-3 and -8 in liver homogenates and in a cell-free system in vitro. Concomitantly, mice treated with AAT showed significantly lower serum levels of tumor necrosis factor alpha (TNF-α), which also paralleled the reduced activity of ADAM17 (TACE). Noticeably, the increased survival and a reduction of apoptotic hepatocytes were also observed in the α-amanitin and acetaminophen-induced liver injury mouse models. CONCLUSION: Our data suggest that systemic administration of AAT can be a promising therapy to treat acute liver failure and clinical studies to explore this treatment in humans should be initiated.

Elucidating the mechanism behind and investigating the efficacy of Traditional Chinese Medicine and Traditional Tibetan Medicine in combination with standard therapeutics in hepatocellular carcinoma and cholangiocarcinoma in vitro.

In this study, we investigated compounds of plant and mushroom origin belonging to Traditional Chinese Medicine (TCM) and to Traditional Tibetan Medicine (TTM): a sandy beige mushroom Trametes robiniophila Murr, commonly known as Huaier/TCM as well as Ershiwuwei Songshi Wan and Qiwei Honghua Shusheng Wan, which both belong to TTM. We aimed to study the efficacy of TTM and TCM in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) in vitro. TCM and TTM were tested either as a monotherapy, or in combination with standard therapeutics: sorafenib for HCC treatment and gemcitabine for CCA. We also discovered a protective mechanism behind the most successful therapeutic combinations. The results demonstrated that TCM and TTM inhibited the proliferation of cancer cells in a time- and dose-dependent manner. The results were compared to classical chemotherapeutics currently used in the clinic: sorafenib for HCC and gemcitabine for CCA. In HCC settings, a combination of Huaier (16 mg/ml) with half of the human plasma concentration of sorafenib, Qiwei Honghua Shusheng Wan (1 mg/ml) monotherapy as well as its combination with half or even a quarter dose of the human plasma concentration of sorafenib represented the most efficient treatments, inhibiting the growth of HCC cells more effectively than the standard therapy. The inhibitory mechanism relied on a strong induction of apoptosis. In CCA settings, Ershiwuwei Songshi Wan and Qiwei Honghua Shusheng Wan as monotherapies or in combination with very low doses of gemcitabine inhibited the growth of CCA cells more efficiently than the standard therapy. Importantly, Ershiwuwei Songshi Wan at the 8 and 16 mg/ml concentrations and Qiwei Honghua Shusheng Wan at the 4 mg/ml concentration were efficacious with gemcitabine applied at massively reduced concentrations. The protective mechanism in CCA relied on a strong induction of early and late apoptosis. Cellular senescence and necroptosis were not associated with protection against HCC/CCA. Combination therapy with TCM or TTM allowed for a dose reduction of standard chemotherapeutics. This is especially important as both chemotherapeutic drugs show strong side effects in patients. The reduction of chemotherapeutics and the synergistic effect observed while applying them in combination with TCM and TTM has strong perspectives for the clinic and patients suffering from HCC and CCA.

High-Molecular-Weight Fractions of Spruce and Eucalyptus Lignin as a Perspective Nanoparticle-Based Platform for a Therapy Delivery in Liver Cancer.

The natural polymer, lignin, possesses unique biodegradable and biocompatible properties, making it highly attractive for the generation of nanoparticles for targeted cancer therapy. In this study, we investigated spruce and eucalyptus lignin nanoparticles (designated as S-and E-LNPs, respectively). Both LNP types were generated from high-molecular-weight (M w ) kraft lignin obtained as insoluble residues after a five-step solvent fractionation approach, which included ethyl acetate, ethanol, methanol, and acetone. The resulting S-and E-LNPs ranged in size from 16 to 60 nm with uniform spherical shape regardless of the type of lignin. The preparation of LNPs from an acetone-insoluble lignin fraction is attractive because of the use of high-M w lignin that is otherwise not suitable for most polymeric applications, its potential scalability, and the consistent size of the LNPs, which was independent of increased lignin concentrations. Due to the potential of LNPs to serve as delivery platforms in liver cancer treatment, we tested, for the first time, the efficacy of newly generated E-LNPs and S-LNPs in two types of primary liver cancer, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), in vitro. Both S-LNPs and E-LNPs inhibited the proliferation of HCC cells in a dose-dependent manner and did not affect CCA cell line growth. The inhibitory effect toward HCC was more pronounced in the E-LNP-treated group and was comparable to the standard therapy, sorafenib. Also, E-LNPs induced late apoptosis and necroptosis while inhibiting the HCC cell line. This study demonstrated that an elevated number of carbohydrates on the surface of the LNPs, as shown by NMR, seem to play an important role in mediating the interaction between LNPs and eukaryotic cells. The latter effect was most pronounced in E-LNPs. The novel S- and E-LNPs generated in this work are promising materials for biomedicine with advantageous properties such as small particle size and tailored surface functionality, making them an attractive and potentially biodegradable delivery tool for combination therapy in liver cancer, which still has to be verified in vivo using HCC and CCA models.

Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs.

Expression levels of CX3CR1 (C-X3-C motif chemokine receptor 1) on immune cells have significant importance in maintaining tissue homeostasis under physiological and pathological conditions. The factors implicated in the regulation of CX3CR1 and its specific ligand CX3CL1 (fractalkine) expression remain largely unknown. Recent studies provide evidence that host's misfolded proteins occurring in the forms of polymers or amyloid fibrils can regulate CX3CR1 expression. Herein, a novel example demonstrates that polymers of human ZZ alpha-1 antitrypsin (Z-AAT) protein, resulting from its conformational misfolding due to the Z (Glu342Lys) mutation in SERPINA1 gene, strongly lower CX3CR1 mRNA expression in human peripheral blood mononuclear cells (PBMCs). This parallels with increase of intracellular levels of CX3CR1 and Z-AAT proteins. Presented data indicate the involvement of the CX3CR1 pathway in the Z-AAT-related disorders and further support the role of misfolded proteins in CX3CR1 regulation.

Proteins can lose their structure and form polymers because of mutations or changes in their immediate environment which can lead to cell damage and disease. Interestingly, polymers formed by a variety of proteins can reduce the levels of CX3C chemokine receptor 1 (CX3CR1 for short) that controls the behaviour of immune cells and is implicated in a range of illnesses. Inherited ZZ alpha-1 antitrypsin deficiency is a rare genetic condition that highly increases the risk of liver and lung diseases. This disorder is characterised by mutant alpha-1 antitrypsin proteins (AAT for short) reacting together to form polymers; yet it remains unclear how the polymers affect different cells or organs, and lead to diseases. To investigate this question, Tumpara et al. examined whether polymers of mutant AAT influence the level of the CX3CR1 protein in specific classes of immune cells. Experiments revealed that in people with AAT deficiency, certain blood immune cells express lower levels of CX3CR1. Regardless of age, clinical diagnosis, or treatment regimen, all individuals with ZZ alpha-1 antitrypsin deficiency had AAT polymers circulating in their blood: the higher the levels of polymers measured, the lower the expression of CX3CR1 recorded in the specific immune cells. When Tumpara et al. added polymers of mutant AAT to the immune cells of healthy donors, the expression of CX3CR1 dropped in a manner dependent on the polymer concentration. According to microscopy data, AAT polymers occurred inside cells alongside the CX3CR1 protein, suggesting that the two molecular actors interact. In the future, new drugs that remove these polymers, either from inside cells or as they circulate in the body, could help patients suffering from conditions associated with this abnormal protein aggregation.

Cancer vaccines and immunotherapeutic approaches in hepatobiliary and pancreatic cancers.

Hepatobiliary and pancreatic cancers along with other gastrointestinal malignancies remain the leading cause of cancer-related deaths worldwide. Strategies developed in the recent years on immunotherapy and cancer vaccines in the setting of primary liver cancer as well as in pancreatic cancer are the scope of this review. Significance of orthotopic and autochthonous animal models which mimic and/or closely reflect human malignancies allowing for a prompt and trustworthy analysis of new therapeutics is underlined. Combinational approaches that on one hand, specifically target a defined cancer-driving pathway, and on the other hand, restore the functions of immune cells, which effector functions are often suppressed by a tumor milieu, are shown to have the strongest perspectives and future directions. Among combinational immunotherapeutic approaches a personalized- and individual cancer case-based therapy is of special importance.

Safety and efficacy of chemosaturation in patients with primary and secondary liver tumors.

BACKGROUND: Chemosaturation with percutaneous hepatic perfusion (CS-PHP; hepatic CHEMOSAT® delivery system; Delcath Systems Inc, USA) is a novel medical device, which delivers high doses of melphalan directly to the liver in patients with primary and secondary liver tumors while limiting systemic toxicity through hemofiltration of the hepatic venous blood. The aim of this study was to analyze the safety and efficacy of the second-generation CS-PHP after 54 treatments at Hannover Medical School, Germany. METHODS: Overall response rates (ORR) were assessed according to response evaluation criteria in solid tumors (RECIST1.1). Overall survival (OS), progression-free survival (PFS), and hepatic PFS (hPFS) were analyzed using the Kaplan-Meier estimation. RESULTS: 29 patients were treated with CS-PHP as last-line therapy up to five sessions. 19 patients had unresectable hepatic metastases from solid tumors [ocular melanoma (OM) n = 11; colorectal carcinoma n = 2; pancreatic adenocarcinoma n = 2; periampular carcinoma n = 2; breast and endometrial cancer each n = 1] and 10 patients were diagnosed with hepatocellular or cholangiocarcinoma (HCC/CCA). ORR was 19.2%. Patients with OM had the highest ORR (33.3%). Similar to patients with OM, patients with hepatobiliary tumors had durable disease stabilization (40%). Median OS, PFS, and hPFS were 261, 117, and 135 days, respectively. Tumor volume negatively correlated with OS. Complications and toxicities included thrombocytopenia, cardiovascular events, ulcerous bleeding, and edema. CONCLUSION: Second-generation CS-PHP seems to be effective and tolerable. Patient selection based on tumor volume and entity is of importance. Particularly, patients with OM and hepatobiliary tumors represent promising candidates for CS-PHP.