Role of leucine-rich repeat kinase 2 in severe acute pancreatitis.

Introduction: Intrapancreatic activation of trypsinogen caused by alcohol or high-fat intake and the subsequent autodigestion of the pancreas tissues by trypsin are indispensable events in the development of acute pancreatitis. In addition to this trypsin-centered paradigm, recent studies provide evidence that innate immune responses triggered by translocation of intestinal bacteria to the pancreas due to intestinal barrier dysfunction underlie the immunopathogenesis of acute pancreatitis. Although severe acute pancreatitis is often associated with pancreatic colonization by fungi, the molecular mechanisms linking fungus-induced immune responses to the development of severe acute pancreatitis are poorly understood. Leucine-rich repeat kinase 2 (LRRK2) is a multifunctional protein that mediates innate immune responses to fungi and bacteria. Mutations in Lrrk2 is a risk factor for Parkinson's disease and Crohn's disease, both of which are driven by innate immune responses to gut organisms. Discussion: In this Minireview article, we discuss how activation of LRRK2 by the recognition of fungi induces severe acute pancreatitis.

Crosstalk between NOD2 and TLR2 suppresses the development of TLR2-mediated experimental colitis.

Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular sensor for muramyl dipeptide (MDP), a degradation product of bacterial cell wall peptidoglycan (PGN). PGN stimulates cell-surface Toll-like receptor 2 (TLR2) independently of NOD2, indicating the presence of crosstalk between extracellular TLR2 and intracellular NOD2 upon exposure to PGN. NOD2-deficient mice were sensitive, while TLR2-deficient mice were resistant to experimental colitis induced by intrarectal administration of PGN. Severe colitis in NOD2-deficient mice was accompanied by increased expression of nuclear factor-kappa B-dependent cytokines and decreased expression of autophagy-related 16-like 1 (ATG16L1). MDP activation of NOD2 enhanced autophagy mediated by TLR2 in human dendritic cells. mRNA expression of TLR2 tended to be higher in the colonic mucosa of patients with active ulcerative colitis compared to that of those in remission. Induction of remission was associated with increased mRNA expression of ATG16L1 in both ulcerative colitis and Crohn's disease patients. Conversely, mRNA expression of receptor-interacting serine/threonine-protein kinase 2 was higher in the inflammatory colonic mucosa of patients with active disease than in the non-inflamed mucosa of patients in remission, in both ulcerative colitis and Crohn's disease. These findings highlight the role of NOD2-TLR2 crosstalk in the immunopathogenesis of colitis.

Cytokine and chemokine profiles in ulcerative colitis relapse after coronavirus disease 2019 vaccination.

Coronavirus disease 2019 (COVID-19) vaccines are highly effective; however, vaccine-related adverse events, including autoimmunity, have been reported. Case reports describing relapse or new-onset of ulcerative colitis (UC) after COVID-19 mRNA vaccination are available. However, the molecular mechanisms underlying the development of colonic inflammation associated with COVID-19 mRNA vaccination are poorly understood. Furthermore, it is unclear whether the relapse of UC after COVID-19 vaccination is driven by unique cytokine responses that differ from those of UC not associated with vaccination. mRNAs derived from COVID-19 vaccines are potent inducers of type I IFN response. We encountered three cases of UC relapse after COVID-19 vaccination. mRNA expressions of IFN-α, IFN-ß, IL-1ß, and IL-12/23p40 showed higher tendency in the colonic mucosa of patients with UC associated with vaccination compared with those not associated with vaccination. In contrast, the expressions of C-X-C motif chemokine ligand 9 (CXCL9) and CXCL10 were comparable. Immunofluorescence analyses also showed higher expression of IFN-α in the colonic mucosa of patients with UC associated with COVID-19 vaccination than in those not associated with vaccination. Taken together, these data suggest that the colonic mucosa of patients with UC who relapsed after COVID-19 vaccination was characterized by enhanced type I IFN responses.

Biological Evaluation of Antibody-Drug Conjugates Produced by Tag-Free Lipoate Ligase A Modification.

The concept of tag-free protein modification has attracted considerable interest in chemical biology because of its flexible and straightforward reaction process. In 2021, a groundbreaking approach using lipoate ligase A (LplA) for tag-free enzymatic modification of antibodies was unveiled, demonstrating its potential for the generation of precise antibody conjugates. In this study, to further explore LplA-mediated antibody-drug conjugate (ADC) synthesis, we performed initial biological evaluations of ADCs synthesized using LplA. Using the anti-HER2 antibody trastuzumab, we introduced octanoic acid azide using LplA and subsequently obtained an ADC using click chemistry with the drug DBCO-VC-PAB-MMAE. The bioactivity of the synthesized anti-HER2-ADC was evaluated using HER2-positive SKBR-3 and HER2-negative MCF7 cells. Its toxicity and selectivity were found to be comparable to those of the FDA-approved Kadcyla. In addition, a stability study involving rat and human plasma demonstrated the stability of the LplA-mediated ADC. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. These preliminary in vitro evaluations suggested that LplA-derived ADCs can have considerable pharmaceutical potential. Our results can set the stage for further in vivo evaluations and safety assessments. We suggest that the integration of tag-free LplA methods into the production of ADCs can offer a novel and promising approach for biopharmaceutical manufacturing.

A Comparison of the Efficacies of OK-432 and Talc Slurry for Pleurodesis in Patients with Prolonged Air Leak after Pulmonary Resection.

PURPOSE: A prolonged air leak (PAL) is one of the common postoperative complications of pulmonary resection. The aim of this study was to evaluate the efficacy and safety of pleurodesis with sterile talc or OK-432 for postoperative air leak. METHODS: Patients with postoperative air leak who received chemical pleurodesis using sterile talc or OK-432 were retrospectively identified from medical records data. For pleurodesis with either agent, prior assessment and approval by the hospital safety department were carried out for each case, in addition to individual consent. RESULTS: Between February 2016 and June 2022, 39 patients had PALs and underwent chemical pleurodesis. Among them, 24 patients received pleurodesis with talc (Talc group) and 15 with OK-432 (OK-432 group). The leak resolved after less than two pleurodesis treatments in 22 patients (91.7%) in the Talc group compared with 14 patients (93.3%) in the OK-432 group. Pleurodesis significantly increased white blood cell counts, C-reactive protein concentration, and body temperature in the OK-432 group compared with that in the Talc group (p <0.001, p = 0.003, and p <0.001, respectively). CONCLUSIONS: Pleurodesis with talc may be an effective treatment option for postoperative air leak. Our findings suggest that talc was as effective as OK-432 and resulted in a milder systemic inflammatory response.

Bispecific Antibodies Produced via Chemical Site-Specific Conjugation Technology: AJICAP Second-Generation.

Bispecific antibodies (BisAbs) are biotherapeutics that amalgamate the specificities of two distinct antibodies into one molecule, however, their engineering requires genetic modification and remains time-consuming. Therefore, we used AJICAP second-generation technology, which drives the production of site-specific conjugation without genetic modification requirements, to generate BisAbs. Using haloketone chemistry as an alternative to maleimide chemistry, we successfully produced site-specific antibody conjugates. Pharmacokinetic studies revealed that the haloketone-based antibody conjugate was stable in the rat plasma. The resultant BisAbs were rigorously evaluated, and surface plasmon resonance measurements and flow cytometry analyses confirmed that the antigen binding remained intact. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. Further cytotoxicity evaluation emphasized the pronounced activity of the generated BisAbs. This novel approach introduces a fully chemical, site-specific strategy capable of producing BisAbs, heralding a new era in the field of biotherapeutics.

Visualization of Gastrointestinal Bezoar Movement Causing and Releasing Small Bowel Obstruction on Computed Tomography in a Patient With Diabetes Mellitus.

Although delayed gastric emptying promotes gastrointestinal bezoar formation in patients with diabetes mellitus (DM), the association between movement of gastrointestinal bezoars and glycemic status remains unclear. We report a case of small bowel obstruction (SBO) caused by impaction of the migrated gastric bezoar into the small bowel in a patient with DM. Correction of hyperglycemia and lactic acidosis led to normalization of gastrointestinal motility, followed by expulsion of the impacted bezoar and resolution of SBO. This case suggests a link between hyperglycemia, metabolic acidosis, and gastrointestinal motility based on visualization of gastrointestinal bezoar movement in the gastrointestinal tract using computed tomography.

Highly potent, orally active novel small-molecule HPK1 inhibitor DS21150768 induces anti-tumor responses in multiple syngeneic tumor mouse models.

Augmenting T-cell activity is a promising approach to enhance the efficacy of cancer immunotherapy treatment. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in immune cells and negatively regulates T-cell receptor signaling. It is reported that inhibition of the kinase function of HPK1 results in tumor growth suppression by enhancing cancer immunity. Thus, developing HPK1 inhibitors has attracted considerable attention as a future cancer immunotherapy approach. However, despite recent progress in HPK1 biology and pharmacology, various challenges still remain, such as developing HPK1 inhibitors with favorable pharmacological profiles and identifying tumor characteristics that can be applied to define susceptibility to HPK1 inhibition. Here, we present the identification and pharmacological evaluation of DS21150768, a potent small-molecule HPK1 inhibitor with a novel chemical scaffold. DS21150768 shows remarkable inhibition of HPK1 kinase activity, and in vitro studies demonstrated its potent activity to enhance T-cell function. DS21150768 is orally bioavailable and shows sustained plasma exposure, which leads to enhanced cytokine responses in vivo. We conducted a comparison of the anti-tumor efficacy of DS21150768 alone or in combination with anti-PD-1 antibody in 12 different mouse cancer cell models, and observed that the treatments suppressed tumor growth in multiple models. Furthermore, Gene Set Enrichment Analysis demonstrated significant enrichment of immune-related gene signatures in the tumor models responsive to DS21150768 treatment. Our results provide a path forward for the future development of HPK1 inhibitors and fundamental insights into biomarkers of HPK1-targeted therapy.

Desulfoferula mesophilus gen. nov. sp. nov., a mesophilic sulfate-reducing bacterium isolated from a brackish lake sediment.

A novel sulfate-reducing bacterium (strain 12FAKT) was isolated from sediment sampled from a brackish lake in Japan. Respiratory growth was observed with formate and pyruvate as an electron donor. Sulfate, thiosulfate, elemental sulfur and dimethyl sulfoxide were utilized as an electron acceptor. The isolate grew over a temperature range of 18-42 °C (optimum 35-37 °C), a NaCl concentration range of 50-450 mM (optimum 150-300 mM) and a pH range of 6.6-7.5. The 12FAKT genome consists of a circular chromosome with a length of 4.5 Mbp and G + C content of 63.6%. Based on 16S rRNA gene sequence similarity, the closest cultured relative was Desulfarculus baarsii 2st14T (92.2%). Genome-based phylogenetic analysis placed strain 12FAKT within the family Desulfarculaceae but did not affiliate the strain with any existing genus. Taken together, we propose a novel species of a novel genus, Desulfoferula mesophilus gen. nov. sp. nov. with the type strain 12FAKT (= DSM 115219T = JCM 39399T).

Leucine-rich repeat kinase 2 promotes the development of experimental severe acute pancreatitis.

Translocation of gut bacteria into the pancreas promotes the development of severe acute pancreatitis (SAP). Recent clinical studies have also highlighted the association between fungal infections and SAP. The sensing of gut bacteria by pattern recognition receptors promotes the development of SAP via the production of proinflammatory cytokines; however, the mechanism by which gut fungi mediate SAP remains largely unknown. Leucine-rich repeat kinase 2 (LRRK2) is a multifunctional protein that regulates innate immunity against fungi via Dectin-1 activation. Here, we investigated the role of LRRK2 in SAP development and observed that administration of LRRK2 inhibitors attenuated SAP development. The degree of SAP was greater in Lrrk2 transgenic (Tg) mice than in control mice and was accompanied by an increased production of nuclear factor-kappaB-dependent proinflammatory cytokines. Ablation of the fungal mycobiome by anti-fungal drugs inhibited SAP development in Lrrk2 Tg mice, whereas the degree of SAP was comparable in Lrrk2 Tg mice with or without gut sterilization by a broad range of antibiotics. Pancreatic mononuclear cells from Lrrk2 Tg mice produced large amounts of IL-6 and TNF-α upon stimulation with Dectin-1 ligands, and inhibition of the Dectin-1 pathway by a spleen tyrosine kinase inhibitor protected Lrrk2 Tg mice from SAP. These data indicate that LRRK2 activation is involved in the development of SAP through proinflammatory cytokine responses upon fungal exposure.

Circumferential Stenosis of the Second Part of the Duodenum Caused by Eosinophilic Gastroenteritis.

Isolated eosinophilic gastroenteritis (EGE) of the second part of the duodenum is rare. We herein report a case of EGE limited to the second part of the duodenum that caused circumferential stenosis due to massive wall thickening. A boring biopsy was useful to verify the accumulation of eosinophils. Induction of remission by prednisolone was accompanied by a marked reduction in the mRNA expression of IL-6, C-C motif chemokine ligand 17 (CCL17), and CCL26 without any reduction in prototypical EGE-associated T helper type 2 cytokines (IL-5, IL-13). Thus, the enhanced expression of IL-6, CCL17, and CCL26 might be involved in the development of EGE in this case.

Regulation of type I IFN responses by deubiquitinating enzyme A in inflammatory bowel diseases.

The development of Inflammatory bowel disease (IBD) is driven by excessive production of pro-inflammatory cytokines including TNF-α, IL-12, and IL-23. This notion is supported by the remarkable clinical success of biologics targeting these cytokines. Recognition of cell wall components derived from intestinal bacteria by Toll-like receptors (TLRs) induces the production of these pro-inflammatory cytokines by macrophages and dendritic cells in human IBD and experimental colitis model. Although sensing of bacterial nucleic acids by endosomal TLRs, specifically TLR3, TLR7, and TLR9 leads to robust production of type I IFNs, it remains debatable whether TLR-mediated type I IFN responses are pathogenic or protective in IBD patients. Additionally, recent studies identified deubiquitinating enzyme A (DUBA) as a novel negative regulator of TLR-mediated type I IFN responses. In light of these observations and their potential applications, in this review, we summarize recent findings on the roles of type I IFN responses and DUBA-mediated negative regulation of these responses in human IBD and experimental colitis model.

Low-dose gemcitabine plus nab-paclitaxel versus standard-dose gemcitabine plus nab-paclitaxel in elderly patients with metastatic pancreatic cancer: A randomized Phase II trial.

Background and Aim: A multicenter, open-label randomized Phase II trial was conducted to determine whether low-dose gemcitabine plus nab-paclitaxel (GnP) could improve tolerability and show equivalent efficacy to the standard-dose GnP for elderly patients with metastatic pancreatic cancer. Methods: Consecutive patients aged ≥65 years with metastatic pancreatic cancer who presented at one of four Japanese referral centers between November 2016 and January 2021 were enrolled. The 60 patients were randomly assigned to low- or standard-dose groups with a 1:1 ratio. Patients in the low-dose GnP group received gemcitabine at a dose of 250 mg/m2 and nab-paclitaxel at 125 mg/m2. Results: Low-dose GnP significantly decreased the rate of cases requiring dose reduction (16.7% vs 63.3%). The response rate (36.7% vs 33.3%) and progression-free survival (7.3 vs 8 months) were comparable between the low- and standard-dose groups as determined by independent review. The difference in the median overall survival between the two groups was not significant (7.9 vs 12 months). The proportion of patients with hematologic and non-hematologic treatment-related adverse events was comparable between the two groups. Conclusion: Low-dose GnP had an equivalent efficacy to conventional therapy; however, it did not reduce adverse events.

Impact of Smad4 and p53 mutations on the prognosis of patients with pancreatic ductal adenocarcinoma undergoing chemotherapy.

BACKGROUND: This prospective cohort study evaluated the feasibility of using endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) samples for comprehensive mutational analysis of cancer-related genes using microtissues. METHODS: Fifty patients with suspected pancreatic cancer presenting consecutively at the Kindai University Hospital between January 2018 and January 2019 were enrolled. Cancerous tissues from EUS-FNB were obtained from each tumor and subjected to histological examination and mutational analysis. The primary endpoint was the collection rate of EUS-FNB specimens suitable for comprehensive cancer panels using deep sequencing. Clinical history and genetic variations between the disease control and progressive disease groups of patients on chemotherapy were evaluated as secondary endpoints. RESULTS: The collection rate of EUS-FNB specimens suitable for comprehensive cancer panels using deep sequencing was 93.6%. The cancer panel was sequenced for 25 patients with pancreatic cancer treated initially with systemic chemotherapy. Mutation in p53 and Smad4 were positively and negatively associated, respectively, with disease control at the initial evaluation. The median time to progression in 15 patients with p53 and without Smad4 mutations was 182.0 days; whereas, it was 92.5 days in other 10 patients; this difference was significant (p = 0.020). CONCLUSIONS: Tissue samples from EUS-FNB were suitable for mutational analysis. Pancreatic cancers with p53 and without Smad4 mutations responded better to chemotherapy and had a better prognosis than those others.

Oral administration of ovalbumin protects mice from concanavalin A-induced hepatitis through suppression of interferon-gamma responses.

The liver is a tolerogenic organ that exhibits hypo-responsiveness to antigens circulating in the portal vein. Antigens that are orally administered at high doses reach the liver. In our previous study, we demonstrated that administering ovalbumin (OVA) orally at high doses generates unique CD4+ T cells and tolerogenic dendritic cells, both of which can suppress T helper type 1 (Th1) responses, in the livers of two groups of mice: DO11.10 mice with transgenic CD4+ T cell receptors for OVA and BALB/c mice that received OVA-specific CD4+ T cells through adoptive transfer. This study aimed to investigate whether oral administration of OVA at high doses inhibits the development of hepatitis in the presence of OVA-specific CD4+ T cells. Oral administration of OVA at high doses inhibited the development of OVA-specific and concanavalin A (Con A)-induced hepatitis in DO11.10 mice, and these effects were associated with the downregulation of Th1 responses. Furthermore, the adoptive transfer of CD4+ T cells from the liver of OVA-fed DO11.10 mice inhibited the development of Con A-induced hepatitis in recipient BALB/c mice through the downregulation of Th1 responses. Finally, oral administration of OVA at high doses inhibited the development of Con A-induced hepatitis in BALB/c mice bearing naïve OVA-specific CD4+ T cells. These results suggest that the oral administration of antigens at high doses suppresses Th1-mediated hepatitis in an antigen-non-specific manner in the presence of antigen-specific CD4+ T cells.

Activation of the aryl hydrocarbon receptor inhibits the development of experimental autoimmune pancreatitis through IL-22-mediated signaling pathways.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor expressed in hematopoietic and non-hematopoietic cells. Activation of the AhR by xenobiotics, microbial metabolites, and natural substances induces immunoregulatory responses. Autoimmune pancreatitis (AIP) is a chronic fibroinflammatory disorder of the pancreas driven by autoimmunity. Although AhR activation generally suppresses pathogenic autoimmune responses, the roles played by the AhR in AIP have been poorly defined. In this study, we examined how AhR activation affected the development of experimental AIP caused by the activation of plasmacytoid dendritic cells producing IFN-α and IL-33. Experimental AIP was induced in MRL/MpJ mice by repeated injections of polyinosinic-polycytidylic acid. Activation of the AhR by indole-3-pyruvic acid and indigo naturalis, which were supplemented in the diet, inhibited the development of experimental AIP, and these effects were independent of the activation of plasmacytoid dendritic cells producing IFN-α and IL-33. Interaction of indole-3-pyruvic acid and indigo naturalis with AhRs robustly augmented the production of IL-22 by pancreatic islet α cells. The blockade of IL-22 signaling pathways completely canceled the beneficial effects of AhR ligands on experimental AIP. Serum IL-22 concentrations were elevated in patients with type 1 AIP after the induction of remission with prednisolone. These data suggest that AhR activation suppresses chronic fibroinflammatory reactions that characterize AIP via IL-22 produced by pancreatic islet α cells.

AJICAP Second Generation: Improved Chemical Site-Specific Conjugation Technology for Antibody-Drug Conjugate Production.

The site-directed chemical conjugation of antibodies remains an area of great interest and active efforts within the antibody-drug conjugate (ADC) community. We previously reported a unique site modification using a class of immunoglobulin-G (IgG) Fc-affinity reagents to establish a versatile, streamlined, and site-selective conjugation of native antibodies to enhance the therapeutic index of the resultant ADCs. This methodology, termed "AJICAP", successfully modified Lys248 of native antibodies to produce site-specific ADC with a wider therapeutic index than the Food and Drug Administration-approved ADC, Kadcyla. However, the long reaction sequences, including the reduction-oxidation (redox) treatment, increased the aggregation level. In this manuscript, we aimed to present an updated Fc-affinity-mediated site-specific conjugation technology named "AJICAP second generation" without redox treatment utilizing a "one-pot" antibody modification reaction. The stability of Fc affinity reagents was improved owing to structural optimization, enabling the production of various ADCs without aggregation. In addition to Lys248 conjugation, Lys288 conjugated ADCs with homogeneous drug-to-antibody ratio of 2 were produced using different Fc affinity peptide reagent possessing a proper spacer linkage. These two conjugation technologies were used to produce over 20 ADCs from several combinations of antibodies and drug linkers. The in vivo profile of Lys248 and Lys288 conjugated ADCs was also compared. Furthermore, nontraditional ADC production, such as antibody-protein conjugates and antibody-oligonucleotide conjugates, were achieved. These results strongly indicate that this Fc affinity conjugation approach is a promising strategy for manufacturing site-specific antibody conjugates without antibody engineering.