Trained immunity in recurrent Staphylococcus aureus infection promotes bacterial persistence.

Bacterial persister cells, a sub-population of dormant phenotypic variants highly tolerant to antibiotics, present a significant challenge for infection control. Investigating the mechanisms of antibiotic persistence is crucial for developing effective treatment strategies. Here, we found a significant association between tolerance frequency and previous infection history in bovine mastitis. Previous S. aureus infection led to S. aureus tolerance to killing by rifampicin in subsequent infection in vivo and in vitro. Actually, the activation of trained immunity contributed to rifampicin persistence of S. aureus in secondary infection, where it reduced the effectiveness of antibiotic treatment and increased disease severity. Mechanically, we found that S. aureus persistence was mediated by the accumulation of fumarate provoked by trained immunity. Combination therapy with metformin and rifampicin promoted eradication of persisters and improved the severity of recurrent S. aureus infection. These findings provide mechanistic insight into the relationship between trained immunity and S. aureus persistence, while providing proof of concept that trained immunity is a therapeutic target in recurrent bacterial infections involving persistent pathogens.

Dihydroartemisinin is an inhibitor of trained immunity through Akt/mTOR/HIF1α signaling pathway.

Trained immunity is mechanistically defined as the metabolically and epigenetically mediated long-term functional adaptation of the innate immune system, characterized by a heightened response to a secondary stimulation. Given appropriate activation, trained immunity represents an attractive anti-infective therapeutic target. Nevertheless, excessive immune response and subsequent inflammatory cascades may contribute to pathological tissue damage, indicating that the negative impacts of trained immunity appear to be significant. In this study, we show that innate immune responses such as the production of extracellular traps, pro-inflammatory cytokines, and autophagy-related proteins were markedly augmented in trained BMDMs. Furthermore, heat-killed C. albicans priming promotes the activation of the AIM2 inflammasome, and AIM2-/- mice exhibit impaired memory response induced by heat-killed C. albicans. Therefore, we establish that the AIM2 inflammasome is involved in trained immunity and emerges as a promising therapeutic target for potentially deleterious effects. Dihydroartemisinin can inhibit the memory response induced by heat-killed C. albicans through modulation of mTOR signaling and the AIM2 inflammasome. The findings suggest that dihydroartemisinin can reduce the induction of trained immunity by heat-killed C. albicans in C57BL/6 mice. Dihydroartemisinin is one such therapeutic intervention that has the potential to treat of diseases characterized by excessive trained immunity.

Strong Yet Tough Transparent Paper with Superb Foldability.

Transparent paper manufactured from wood fibers is emerging as a promising, cost-effective, and carbon-neutral alternatives to plastics. However, fully exploring their mechanical properties is one of the most pressing challenges. In this work, a strong yet tough transparent paper with superior folding endurance is prepared by rationally altering the native fiber structure. Microwave-assisted choline chloride/lactic acid deep eutectic solvent (DES) pulping is first utilized to isolate wood fibers from spruce wood. During this process, the S1 layer within the fibers is partially disrupted, forming protruding microfibrils that play a crucial role in enhancing cellulose accessibility. Subsequently, carboxymethylation treatment is applied to yield uniformly swollen carboxymethylated wood fibers (CM fibers), which improves the interaction between CM fibers during papermaking. The as-prepared transparent paper not only shows a 90% light transmittance (550 nm) but also exhibits impressive mechanical properties, including a folding endurance of over 26 000, a tensile strength of 248.4 MPa, and a toughness of 15.6 MJ m-3. This work provides a promising route for manufacturing transparent paper with superior mechanical properties from wood fibers and can extend their use in areas normally dominated by high-performance nonrenewable plastics.

Identification and biological characteristics of Enterococcus casseliflavus TN-47 isolated from Monochamus alternatus.

With the widespread use of antibiotics, the incidence of antibiotic resistance in microorganisms has increased. Monochamus alternatus is a trunk borer of pine trees. This study aimed to investigate the in vitro antimicrobial and biological characteristics of Enterococcus casseliflavus TN-47 (PP411196), isolated from the gastrointestinal tract of M. alternatus in Jilin Province, PR China. Among 13 isolates obtained from the insects, five were preliminarily screened for antimicrobial activity. E. casseliflavus TN-47, which exhibited the strongest antimicrobial activity, was identified. E. casseliflavus TN-47 possessed antimicrobial activity against Staphylococcus aureus USA300 and Salmonella enterica serovar Pullorum ATCC 19945. Furthermore, E. casseliflavus TN-47 was sensitive to tetracyclines, penicillins (ampicillin, carbenicillin, and piperacillin), quinolones and nitrofuran antibiotics, and resistant to certain beta-lactam antibiotics (oxacillin, cefradine and cephalexin), macrolide antibiotics, sulfonamides and aminoglycosides. E. casseliflavus TN-47 could tolerate low pH and pepsin-rich conditions in the stomach and grow in the presence of bile acids. E. casseliflavus TN-47 retained its strong auto-aggregating ability and hydrophobicity. This strain did not exhibit any haemolytic activity. These results indicate that E. casseliflavus TN-47 has potential as a probiotic. This study provides a theoretical foundation for the future applications of E. casseliflavus TN-47 and its secondary metabolites in animal nutrition and feed.

STING-dependent trained immunity contributes to host defense against Clostridium perfringens infection via mTOR signaling.

Clostridium perfringens (C. perfringens) infection is recognized as one of the most challenging issues threatening food safety and perplexing agricultural development. To date, the molecular mechanisms of the interactions between C. perfringens and the host remain poorly understood. Here, we show that stimulator of interferon genes (STING)-dependent trained immunity protected against C. perfringens infection through mTOR signaling. Heat-killed Candida albicans (HKCA) training elicited elevated TNF-α and IL-6 production after LPS restimulation in mouse peritoneal macrophages (PM). Although HKCA-trained PM produced decreased levels of TNF-α and IL-6, the importance of trained immunity was demonstrated by the fact that HKCA training resulted in enhanced bacterial phagocytic ability and clearance in vivo and in vitro during C. perfringens infection. Interestingly, HKCA training resulted in the activation of STING signaling. We further demonstrate that STING agonist DMXAA is a strong inducer of trained immunity and conferred host resistance to C. perfringens infection in PM. Importantly, corresponding to higher bacterial burden, reduction in cytokine secretion, phagocytosis, and bacterial killing were shown in the absence of STING after HKCA training. Meanwhile, the high expression levels of AKT/mTOR/HIF1α were indeed accompanied by an activated STING signaling under HKCA or DMXAA training. Moreover, inhibiting mTOR signaling with rapamycin dampened the trained response to LPS and C. perfringens challenge in wild-type (WT) PM after HKCA training. Furthermore, STING­deficient PM presented decreased levels of mTOR signaling-related proteins. Altogether, these results support STING involvement in trained immunity which protects against C. perfringens infection via mTOR signaling.

IFI204 protects host defense against Staphylococcus aureus-induced pneumonia by promoting extracellular traps formation.

Interferon-inducible protein 204 (IFI204) is an intracellular DNA receptor that can recognize DNA viruses and intracellular bacteria. Extracellular traps (ETs) have been recognized as an indispensable antimicrobial barrier that play an indispensable role in bacterial, fungal, parasitic, and viral infections. However, how ETs form and the mechanisms by which IFI204 function in Staphylococcus aureus pneumonia are still unclear. Moreover, by in vitro experiments, we proved that IFI204 deficiency decreases the formation of ETs induced by Staphylococcus aureus in a NOX-independent manner. More importantly, Deoxyribonuclease I (DNase I) treatment significantly inhibited the formation of ETs. IFI204 contributed to ETs formation by promoting citrullination of histone H3 and the expression of PAD4 (peptidylarginine deiminase 4). Altogether, these findings highlight the potential importance of IFI204 for host defense against S. aureus USA300-TCH1516 infection.

Characteristic morphology and immunohistochemical patterns of clear cell papillary renal cell tumours may be observed in renal cell carcinomas, a critical pitfall in renal biopsy cytopathology.

BACKGROUND: Clear cell papillary renal cell tumour (CCPRCT) was renamed from previous clear cell papillary renal cell carcinoma (CCPRCC) in the latest WHO Classification of Tumours. It is essential to differentiate RCC from CCPRCT in renal mass biopsies (RMB). DESIGN: RMB cases with subsequent resections were reviewed. The pathology reports and pertinent clinical information were recorded. RESULTS: Fifteen cases displaying either CCPRCT morphology (20% diffuse, 67% focal) or immunohistochemical patterns (cup-like CA9: 20% diffuse, 47% focal; CK7: 33% diffuse, 40% focal) were identified. One case was positive for TFE3. TSC mutation was identified in one case. Both cases exhibited both CCPRCT morphology and immunohistochemical patterns for CA9 and CK7, with focal high-grade nuclei. RMB diagnoses were as follows: 6 (40%) as CCRCC, 2 (13%) as CCPRCT, 2 (13%) as CCRCC versus CCPRCT, 2 (13%) as CCRCC versus PRCC, 1 (7%) as RCC with TSC mutation versus CCPRCT, 1 (7%) as TFE3-rearranged RCC versus PRCC, and 1 (7%) as cyst with low-grade atypia. 71% of patients underwent nephrectomy, 21% received systemic treatment for stage 4 RCCs, and 7% with ablation for small renal mass (1.6 cm) with low-grade CCRCC. CONCLUSIONS: Our study highlights that morphologic and immunochemical features of CCPRCT may be present in RCCs, including RCC-TFE3 expression and TSC-associated RCC, a critical pitfall to misdiagnose aggressive RCC as indolent CCPRCT and result in undertreatment. Careful examination of morphology and immunostains for CA9, CK7, and TFE3, as well as molecular tests, is crucial for distinguishing aggressive RCC from indolent CCPRCT.

Novo pathogenic variations of NLRP7 increasing the risk of gestational trophoblastic neoplasia.

Identification of novo mutations of NLRP7 in HM patients. NLRP7 mutations increasing the risk of HM progression.

Culturing the Chicken Intestinal Microbiota and Potential Application as Probiotics Development.

Pure cultures of chicken intestinal microbial species may still be crucial and imperative to expound on the function of gut microbiota, and also contribute to the development of potential probiotics and novel bioactive metabolites from gut microbiota. In this study, we isolated and identified 507 chicken intestinal bacterial isolates, including 89 previously uncultured isolates. Among these, a total of 63 Lactobacillus strains, belonging to L. vaginalis, L. crispatus, L. gallinarum, L. reuteri, L. salivarius, and L. saerimneri, exhibited antibacterial activity against S. Pullorum. Acid tolerance tests showed Limosilactobacillus reuteri strain YPG14 (L. reuteri strain YPG14) has a particularly strong tolerance to acid. We further characterized other probiotic properties of L. reuteri strain YPG14. In simulated intestinal fluid, the growth of L. reuteri strain YPG14 remained stable after incubation for 4 h. The auto-aggregation test showed the auto-aggregation percentage of L. reuteri strain YPG14 was recorded as 15.0 ± 0.38%, 48.3 ± 2.51%, and 75.1 ± 4.44% at 3, 12, and 24 h, respectively. In addition, the mucin binding assay showed L. reuteri strain YPG14 exhibited 12.07 ± 0.02% adhesion to mucin. Antibiotic sensitivity testing showed that L. reuteri strain YPG14 was sensitive to the majority of the tested antibiotics. The anti-Salmonella Pullorum (S. Pullorum) infection effect in vivo revealed that the consumption of L. reuteri strain YPG14 could significantly improve body weight loss and survival rate of chicks infected by S. Pullorum; reduce the loads of S. Pullorum in the jejunum, liver, spleen, and feces; and alleviate the jejunum villi morphological structure damage, crypt loss, and inflammatory cell infiltration caused by S. Pullorum. Overall, this study may help us to understand the diversity of chicken intestinal microflora and provide some insights for potential probiotic development from gut microbiota and may find application in the poultry industry.

Genome mining reveals polysaccharide-degrading potential and new antimicrobial gene clusters of novel intestinal bacterium Paenibacillus jilinensis sp. nov.

BACKGROUND: Drug-resistant bacteria have posed a great threat to animal breeding and human health. It is obviously urgent to develop new antibiotics that can effectively combat drug-resistant bacteria. The commensal flora inhabited in the intestines become potential candidates owing to the production of a wide range of antimicrobial substances. In addition, host genomes do not encode most of the enzymes needed to degrade dietary structural polysaccharides. The decomposition of these polysaccharides mainly depends on gut commensal-derived CAZymes. RESULTS: We report a novel species isolated from the chicken intestine, designated as Paenibacillus jilinensis sp. nov. and with YPG26T (= CCTCC M2020899T) as the type strain. The complete genome of P. jilinensis YPG26T is made up of a single circular chromosome measuring 3.97 Mb in length and containing 49.34% (mol%) G + C. It carries 33 rRNA genes, 89 tRNA genes, and 3871 protein-coding genes, among which abundant carbohydrate-degrading enzymes (CAZymes) are encoded. Moreover, this strain has the capability to antagonize multiple pathogens in vitro. We identified putative 6 BGCs encoding bacteriocin, NRPs, PKs, terpenes, and protcusin by genome mining. In addition, antibiotic susceptibility testing showed sensitivity to all antibiotics tested. CONCLUSIONS: This study highlights the varieties of CAZymes genes and BGCs in the genome of Paenibacillus jilinensis. These findings confirm the beneficial function of the gut microbiota and also provide a promising candidate for the development of new carbohydrate degrading enzymes and antibacterial agents.

Novel bi-allelic variants in KASH5 are associated with meiotic arrest and non-obstructive azoospermia.

KASH5 is an essential component of the LINC (linker of the nucleoskeleton and cytoskeleton) complex that regulates chromosome movements and nuclear envelope (NE) remodeling in mouse spermatocytes during meiosis prophase I, but its expression and function in human cells, as well as its association with male infertility are largely unknown. In this study, a novel heterozygous copy number variation (CNV) (seq [GRCh37] del(19) (19q13.33) chr19: g.49894043-49903011del) and a heterozygous loss of function variant (NM_144688: c.979_980del: p.R327Sfs*21) in human KASH5 were identified in a non-obstructive azoospermia (NOA)-affected patient and in his infertile sister by whole-exome sequencing and CNV array. Spermatogenesis in the proband was arrested at zygotene-like stage with a deficiency in homolog pairing and synapsis. KASH5 protein expression in human spermatocytes was evaluated and reported first in this study. Single-cell RNA sequencing demonstrated that the LINC complex and associated genes in human and mouse shared a similar expression pattern, indicating a conserved mechanism in the regulation of chromosome movements and NE remodeling. Kash5 knockout mouse displayed similar phenotypes, including a meiotic arrest at a zygotene-like stage and impaired pairing and synapsis. Collectively, we have identified novel rare variants within human KASH5 in patients with NOA and meiosis arrest. Our study expands the knowledge of KASH5 and associated proteins in regulating human meiosis prophase I progress and provides new insight into the genetic etiology of NOA.

A Theoretical Study of the Sensing Mechanism of a Schiff-Based Sensor for Fluoride.

In the current work, we studied the sensing process of the sensor (E)-2-((quinolin-8ylimino) methyl) phenol (QP) for fluoride anion (F-) with a "turn on" fluorescent response by density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations. The proton transfer process and the twisted intramolecular charge transfer (TICT) process of QP have been explored by using potential energy curves as functions of the distance of N-H and dihedral angle C-N=C-C both in the ground and the excited states. According to the calculated results, the fluorescence quenching mechanism of QP and the fluorescent response for F- have been fully explored. These results indicate that the current calculations completely reproduce the experimental results and provide compelling evidence for the sensing mechanism of QP for F-.

Akkermansia muciniphila Suppresses High-Fat Diet-Induced Obesity and Related Metabolic Disorders in Beagles.

Obesity is one of the prevalent chronic diseases in human and companion animals usually associated with several metabolic disorders. The gut commensal bacterium Akkermansia muciniphila (A. muciniphila) is known for its therapeutic effects on metabolic disorders and inflammations. Here, we isolated the A. muciniphila AKK2 strain from the feces of interferon-inducible protein 204-/- (IFI204-/-) mice and further evaluated its anti-obesity effects on high-fat diet (HFD)-fed C57BL/6J mice and beagles. The results showed that it effectively controlled weight gain. Microbiome analysis using 16S rRNA gene sequencing revealed that HFD alters gut microbiota composition and A. muciniphila AKK2 increases the Firmicutes/Bacteroidetes (F/B) ratio in beagles. Furthermore, we prepared microcapsules containing A. muciniphila AKK2, and tolerance tests showed the encapsulation maintained high viability and stability in an aerobic environment and simulated the secretion of gastrointestinal fluids. Overall, this study widens the spectrum of A. muciniphila applications to prevent obesity.

GSDMD contributes to host defence against Staphylococcus aureus skin infection by suppressing the Cxcl1-Cxcr2 axis.

Gasdermin D (GSDMD), a member of the gasdermin protein family, is a caspase substrate, and its cleavage is required for pyroptosis and IL-1ß secretion. To date, the role and regulatory mechanism of GSDMD during cutaneous microbial infection remain unclear. Here, we showed that GSDMD protected against Staphylococcus aureus skin infection by suppressing Cxcl1-Cxcr2 signalling. GSDMD deficiency resulted in larger abscesses, more bacterial colonization, exacerbated skin damage, and increased inflammatory cell infiltration. Although GSDMD deficiency resulted in defective IL-1ß production, the critical role of IL-1ß was counteracted by the fact that Caspase-1/11 deficiency also resulted in less IL-1ß production but did not aggravate disease severity during S. aureus skin infection. Interestingly, GSDMD-deficient mice had increased Cxcl1 secretion accompanied by increased recruitment of neutrophils, whereas Caspase-1/11-deficient mice presented similar levels of Cxcl1 and neutrophils as wild-type mice. Moreover, the absence of GSDMD promoted Cxcl1 secretion in bone marrow-derived macrophages induced by live, dead, or different strains of S. aureus. Corresponding to higher transcription and secretion of Cxcl1, enhanced NF-κB activation was shown in vitro and in vivo in the absence of GSDMD. Importantly, inhibiting the Cxcl1-Cxcr2 axis with a Cxcr2 inhibitor or anti-Cxcl1 blocking antibody rescued host defence defects in the GSDMD-deficient mice. Hence, these results revealed an important role of GSDMD in suppressing the Cxcl1-Cxcr2 axis to facilitate pathogen control and prevent tissue damage during cutaneous S. aureus infection.

Quantitative magnetic resonance imaging for determining bone marrow fat fraction at 1.5 T and 3.0 T: a technique to noninvasively assess cellularity and potential malignancy of the bone marrow.

BACKGROUND: Pediatric bone marrow assessment by MRI is challenging and primarily experiential and qualitative, with a paucity of clinically useful quantitative imaging techniques. OBJECTIVE: MRI fat fraction (MRI-FF) is a technique used to quantify the degree of fat in other organ systems. The purpose of this study was to assess whether MRI-FF accurately measures bone marrow composition. MATERIALS AND METHODS: This two-part study included a validation phase, followed by an application phase. For the validation phase, the MRI-FF of piglet bones (6 long bones, 8 axial bones) was performed at 1.5 tesla (T) and 3.0 T, and correlated to the histological fat fraction (H-FF). We used Bland-Altman plots to compare MRI-FF at 1.5 tesla T and 3.0 T. For the application phase, five children with malignant marrow disease were recruited along with seven age- and gender-matched control subjects. The MRI-FF in the children was correlated to the H-FF. Boxplots were used to compare the MRI-FF of patients and control subjects. RESULTS: For the validation animal study, the MRI-FF of piglet bones at both 1.5 T and 3.0 T demonstrated moderate positive correlation to H-FF (r=0.41 and 0.42, respectively). MRI-FF at 1.5 T and 3.0 T were in good agreement, on average 7.7% apart. For the application phase, we included 5 children (4 with leukemia, 1 rhabdomyosarcoma) with median age 7 years, range (3-10 years). All children had MRI-FF and H-FF below 10%. The MRI-FF in patients (3.8±1.2) was significantly lower than that of control subjects (46.1±12.3%) (P<0.01). CONCLUSION: MRI-FF is a valid technique to assess bone marrow fat fraction at both 1.5 T and 3.0 T. The MRI-FF in children with malignant marrow processes is significantly lower than in control subjects with normal marrow.

Chinese public's knowledge, perceived severity, and perceived controllability of COVID-19 and their associations with emotional and behavioural reactions, social participation, and precautionary behaviour: a national survey.

BACKGROUND: The outbreak of the coronavirus disease-19 (COVID-19) has caused enormous stress among the public in China. Intellectual input from various aspects is needed to fight against COVID-19, including understanding of the public's emotion and behaviour and their antecedents from the psychological perspectives. Drawing upon the cognitive appraisal theory, this study examined three cognitive appraisals (i.e., perceived severity, perceived controllability, and knowledge of COVID-19) and their associations with a wide range of emotional and behavioural outcomes among the Chinese public. METHODS: Participants were 4607 citizens (age range: 17-90 years, Mage = 23.71 years) from 31 provinces in China and they took part in a cross-sectional survey online. RESULTS: The results showed that the public's emotional and behavioural reactions were slightly affected by the outbreak of COVID-19. Moreover, the public had limited participation in the events regarding COVID-19 but actively engaged in precautionary behaviour. In addition, results of structural equation model with latent variables revealed that the three appraisals were differentially related to the outcome variables (i.e., negative emotion, positive emotion, sleep problems, aggression, substance use, mobile phone use, social participation, and precautionary behaviour). CONCLUSIONS: The findings highlight the utility of cognitive appraisal, as a core process of coping stress, in explaining the public's emotion and behaviour in the encounter of public health concern. Practically, the findings facilitate the government and practitioners to design and deliver targeted intervention programs to the public.

A Case of Metastatic Biliary Tract Cancer Diagnosed Through Identification of an IDH1 Mutation.

The role of next-generation sequencing from either circulating tumor DNA (ctDNA) or formalin-fixed paraffin-embedded (FFPE) tissue to identify therapeutically targetable genomic alterations has been well established. Genomic profiling may also have untapped potential as a diagnostic tool in cases in which traditional immunohistochemistry assays cannot establish a clear histologic diagnosis. Expanding the number of histologies with unique genomic signatures or alterations is critical in this setting. Here we describe a case of a 73-year-old man who presented with a duodenal mass extending to the liver and peritoneal carcinomatosis, initially thought to be metastatic duodenal adenocarcinoma. Subsequent genomic profiling of ctDNA and FFPE tissue revealed an IDH1 mutation, which is rare in duodenal adenocarcinoma but common in biliary tract cancers (BTCs). This finding prompted a second biopsy, which revealed pancreaticobiliary adenocarcinoma. The clinical significance of IDH mutations in terms of their molecular specificity to certain histologies is reviewed. Recent and ongoing investigations into IDH inhibitors for advanced and metastatic BTCs are also discussed. KEY POINTS: This case demonstrates a novel use of next-generation sequencing as a diagnostic tool to modify a primary cancer diagnosis, leading to important changes in therapy.Isocitrate dehydrogenase mutations are rare in solid organ malignancies and are highly specific for biliary tract cancers (BTCs) within the gastrointestinal malignancies.IDH inhibition is an active area of investigation in metastatic BTCs; early results have been promising.

Exceptional response to chemotherapy followed by concurrent radiotherapy and immunotherapy in a male with primary retroperitoneal serous Adenocarcinoma: a case report and literature review.

BACKGROUND: Primary retroperitoneal serous adenocarcinoma (PRSA) is an extremely uncommon malignancy exclusively reported in females. Due to the rarity of the disease, it is difficult to establish a standardized treatment. CASE PRESENTATION: We describe a unique case of PRSA in a 71-year-old male who presented with right-sided lower back pain and numbness. Magnetic resonance imaging identified a mass invading the adjacent psoas muscle and twelfth rib. Tissue biopsy confirmed poorly differentiated PRSA. Patient was initially treated with neoadjuvant carboplatin and paclitaxel chemotherapy regimen. This resulted in complete radiological resolution of the tumor. However, 12 weeks later, rapid recurrence was noted on follow-up CT scan. The patient was then treated with external radiotherapy with concurrent nivolumab, an anti-PD-1 antibody. The patient displayed a positive response to treatment with reduction in primary tumor and metastases and had a sustained disease control. CONCLUSION: Treatment with radiotherapy in combination with anti-PD-1 antibody could be an effective modality of management for PRSA.

Suboptimal Agreement Among Cytopathologists in Diagnosis of Malignancy Based on Endoscopic Ultrasound Needle Aspirates of Solid Pancreatic Lesions: A Validation Study.

BACKGROUND & AIMS: Despite the widespread use of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) to sample pancreatic lesions and the standardization of pancreaticobiliary cytopathologic nomenclature, there are few data on inter-observer agreement among cytopathologists evaluating pancreatic cytologic specimens obtained by EUS-FNA. We developed a scoring system to assess agreement among cytopathologists in overall diagnosis and quantitative and qualitative parameters, and evaluated factors associated with agreement. METHODS: We performed a prospective study to validate results from our pilot study that demonstrated moderate to substantial inter-observer agreement among cytopathologists for the final cytologic diagnosis. In the first phase, 3 cytopathologists refined criteria for assessment of quantity and quality measures. During phase 2, EUS-FNA specimens of solid pancreatic lesions from 46 patients were evaluated by 11 cytopathologists at 5 tertiary care centers using a standardized scoring tool. Individual quantitative and qualitative measures were scored and an overall cytologic diagnosis was determined. Clinical and EUS parameters were assessed as predictors of unanimous agreement. Inter-observer agreement (IOA) was calculated using multi-rater kappa (κ) statistics and a logistic regression model was created to identify factors associated with unanimous agreement. RESULTS: The IOA for final diagnoses, based on cytologic analysis, was moderate (κ = 0.56; 95% CI, 0.43-0.70). Kappa values did not increase when categories of suspicious for malignancy, malignant, and neoplasm were combined. IOA was slight to moderate for individual quantitative (κ = 0.007; 95% CI, -0.03 to -0.04) and qualitative parameters (κ = 0.5; 95% CI, 0.47-0.53). Jaundice was the only factor associated with agreement among all cytopathologists on multivariate analysis (odds ratio for unanimous agreement, 5.3; 95% CI, 1.1-26.89). CONCLUSIONS: There is a suboptimal level of agreement among cytopathologists in the diagnosis of malignancy based on analysis of EUS-FNA specimens obtained from solid pancreatic masses. Strategies are needed to refine the cytologic criteria for diagnosis of malignancy and enhance tissue acquisition techniques to improve diagnostic reproducibility among cytopathologists.