Factors Influencing the Diagnostic Performance of Repeat Endoscopic Ultrasound-Guided Fine-Needle Aspiration/Biopsy after the First Inconclusive Diagnosis of Pancreatic Solid Lesions.

Background/Aims: Endoscopic ultrasound-guided fine-needle aspiration/biopsy (EUS-FNA/B) is essential in diagnosing solid pancreatic lesions (SPLs), but without rapid on-site evaluation (ROSE), a repeat EUS-FNA/B is crucial for clarifying an inconclusive diagnosis. We aimed to evaluate factors associated with improved diagnostic performance of repeat EUS-FNA/B for initially inconclusive SPL diagnoses without ROSE. Methods: Of 5,894 patients subjected to EUS-FNA/B, 237 (4.0%) with an initially inconclusive diagnosis of SPLs were retrospectively enrolled from five tertiary medical centers between January 2016 and June 2021. Diagnostic performance and procedural factors of EUS-FNA/B were analyzed. Results: The diagnostic accuracies of first and repeat EUS-FNA/B were 96.2% and 67.6%, respectively. Of 237 patients with an inconclusive diagnosis from initial EUS-FNA/B, 150 were pathologically diagnosed after repeat EUS-FNA/B. In multivariate analysis of repeat EUS-FNA/B, tumor location (body/tail vs head: odds ratio [OR], 3.74; 95% confidence interval [CI], 1.48 to 9.46), number of needle passes (≥4 vs ≤3: OR, 4.80; 95% CI, 1.44 to 15.99), needle type (FNB vs FNA: OR, 3.26; 95% CI, 1.44 to 7.36), needle size (22 gauge vs 19/20 gauge: OR, 2.35; 95% CI, 1.19 to 4.62), and suction method (suction vs others: OR, 5.19; 95% CI, 1.30 to 20.75) were associated with a significantly improved diagnostic performance. Conclusions: Repeat EUS-FNA/B is essential for patients with an inconclusive EUS-FNA/B without ROSE. To improve the diagnostic performance of repeated EUS-FNA/B, it is recommended that 22-gauge FNB needles, ≥4 needle passes, and suction methods are used.

Effect of NV gene deletion in the genome of hirame rhabdovirus (HIRRV) on viral replication and the type I interferon response of the host cell.

Hirame rhabdovirus (HIRRV), a member of the genus Novirhabdovirus, causes morbidity and mortality in farmed olive flounder (Paralichthys olivaceus). As no information is available on the role of the NV gene of HIRRV, we produced a recombinant HIRRV with the NV gene deleted (rHIRRV-ΔNV) using reverse genetic technology and investigated whether the NV gene knockout affected HIRRV replication and the type I interferon response of the host cell. The rescue of rHIRRV-ΔNV was successful only when IRF9-gene-knockout Epithelioma papulosum cyprini (ΔIRF9-EPC) cells were used, suggesting that the NV protein of HIRRV might be involved in inhibition of the type I interferon response of the host cell. This conclusion was also supported by the significantly higher level of Mx gene induction in EPC cells infected with rHIRRV-ΔNV than in cells infected with recombinant HIRRV without the deletion. When cells were coinfected with rHIRRV-ΔNV and either wild-type HIRRV or wild-type viral hemorrhagic septicemia virus (VHSV), there was a decrease in the growth rate of not only wild-type HIRRV but also wild-type VHSV in a concentration-dependent manner. Further studies are required to investigate the role of HIRRV NV in virulence and its possible importance for the development of attenuated vaccines.

Direct differentiation of bone marrow mononucleated cells into insulin producing cells using pancreatic ß-cell-derived components.

Transplantation of stem cell-derived insulin producing cells (IPCs) has been proposed as an alternative to islet transplantation for the treatment of diabetes mellitus. However, current IPC differentiation protocols are focused on generating functional cells from the pluripotent stem cells and tend to rely on multistep, long-term exposure to various exogenous factors. In this study, we addressed the observation that under stress, pancreatic ß-cells release essential components that direct the differentiation of the bone marrow nucleated cells (BMNCs) into IPCs. Without any supplementation with known differentiation-inducing factors, IPCs can be generated from BMNCs by in vitro priming for 6 days with conditioned media (CM) from the ß-cells. In vitro primed BMNCs expressed the ß-cell-specific transcription factors, as well as insulin, and improved hyperglycemia and glucose intolerance after transplantation into the streptozotocin-induced diabetic mice. Furthermore, we have found that components of the CM which trigger the differentiation were enclosed by or integrated into micro particles (MPs), rather than being secreted as soluble factors. Identification of these differentiation-directing factors might enable us to develop novel technologies required for the production of clinically applicable IPCs.

Selection of Thymocytes Expressing Transgenic TCR Specific for a Minor Histocompatibility Antigen, H60.

Minor histocompatibility antigens are MHC-bound peptides and contribute to the generation of allo-responses after allogeneic transplantation. H60 is a dominant minor H antigen that induces a strong CD8 T-cell response in MHC-matched allogeneic transplantation settings. Here, we report establishment of a TCR transgenic mouse line named J15, wherein T cells express TCRs specific for H60 in complex with H-2K(b), and different fates of the thymocytes expressing J15 TCRs in various thymic antigenic environments. Thymocytes expressing the J15 TCRs were positively selected and differentiated into CD8(+) single positive (SP) cells in the thymus of C57BL/6 mice, wherein the cognate antigen H60 is not expressed. However, thymocytes were negatively selected in thymus tissue where H60 was transgenically expressed under the control of the actin promoter, with double-positive stages of cells being deleted. Despite the ability of the H60H peptide (LTFHYRNL) variant to induce cytotoxic activity from H60-specific CTL lines at ~50% of the activity induced by normal H60 peptides (LTFNYRNL), J15-expressing thymocytes were positively selected in the thymus where the variant H60H was transgenically expressed. These results demonstrate that a single amino-acid change in the H60 epitope peptide influences the fate of thymocytes expressing the cognate TCR.

Subdominant H60 antigen-specific CD8 T-cell response precedes dominant H4 antigen-specific response during the initial phase of allogenic skin graft rejection.

In allogeneic transplantation, including the B6 anti-BALB.B settings, H60 and H4 are two representative dominant minor histocompatibility antigens that induce strong CD8 T-cell responses. With different distribution patterns, H60 expression is restricted to hematopoietic cells, whereas H4 is ubiquitously expressed. H60-specific CD8 T-cell response has been known to be dominant in most cases of B6 anti-BALB.B allo-responses, except in the case of skin transplantation. To understand the mechanism underlying the subdominance of H60 during allogeneic skin transplantation, we investigated the dynamics of the H60-specific CD8 T cells in B6 mice transplanted with allogeneic BALB.B tail skin. Unexpectedly, longitudinal bioluminescence imaging and flow cytometric analyses revealed that H60-specific CD8 T cells were not always subdominant to H4-specific cells but instead showed a brief dominance before the H4 response became predominant. H60-specific CD8 T cells could expand in the draining lymph node and migrate to the BALB.B allografts, indicating their active participation in the anti-BALB.B allo-response. Enhancing the frequencies of H60-reactive CD8 T cells prior to skin transplantation reversed the immune hierarchy between H60 and H4. Additionally, H60 became predominant when antigen presentation was limited to the direct pathway. However, when antigen presentation was restricted to the indirect pathway, the expansion of H60-specific CD8 T cells was limited, whereas H4-specific CD8 T cells expanded significantly, suggesting that the temporary immunodominance and eventual subdominance of H60 could be due to their reliance on the direct antigen presentation pathway. These results enhance our understanding of the immunodominance phenomenon following allogeneic tissue transplantation.

Requirement of CD4 help for induction of CD8 T cell response specific for virally derived h60.

CD40-CD40L-mediated help from CD4 T cells is essential to induce primary CD8 T cell responses specific to the non-inflammatory cell-based antigen H60. In this study, using H60 as a model antigen, we generated recombinant vaccinia viruses (rVVs) expressing the H60 CD8 epitope and investigated whether CD4 help was required to activate the CD8 T cell response specific to the virally expressed H60. The immune response after infection with rVVs expressing H60 was similar to that after immunization with H60 congenic splenocytes, with a peak frequency of H60-specific CD8 T cells detected in the blood on day 10 post-infection. A CD8 T cell response specific for virally derived H60 was not induced in CD4-depleted mice, but was in CD40-deficient mice. These results provide insights into the characterization of the CD8 T cell response specifically for antigens originating from cellular sources compared to viral sources.

A single-amino-acid variant of the H60 CD8 epitope generates specific immunity with diverse TCR recruitment.

TCR of CD8 T cells recognizes peptides of 8-9 amino acids in length (epitope) complexed with MHC class I. Peptide ligands differing from an epitope by one or two amino acids are thought to modulate the immune response specific to that epitope. H60 is a minor histocompatibility antigen for which the specific CD8 T-cell response dominates during alloresponse after MHC-matched allogeneic transplantation. In the present study, we developed a transgenic mouse (designated H60H Tg) expressing a variant of H60, designated H60H, in which the arginine residue at position 4 of the H60 epitope sequence (LTFNYRNL) is replaced by a histidine residue (LTFHYRNL). Immunization of female C57BL/6 mice with splenocytes from male H60H Tg induced a CD8 T cell primary response and memory response after re-challenge. The response was CD4 help-dependent, demonstrating the potency of H60H as a cellular antigen. The response induced by the H60H cellular antigen was comparable to that induced by H60 in its peak magnitude and overall immune kinetics. H60H challenge recruited broadly diverse TCRs to the specific response, shaping a TCR repertoire different from that of the natural H60 epitope. However, some of the TCRs did overlap between the H60H- and H60-specific CD8 T cells, suggesting that H60H might modulate the H60-specific response. These results may provide a basis for the modulation of the H60-specific CD8 T-cell response.

The effect of intravitreal bevacizumab in patients with acute central serous chorioretinopathy.

PURPOSE: To evaluate the effect of intravitreal bevacizumab injection (IVBI) in acute central serous chorioretinopathy (CSC) patients. METHODS: Patients with acute CSC received IVBI (1.25 mg/0.05 mL) or observation by randomization. Twelve eyes in each group completed 6 months of regular follow-up and were ultimately included in this study. Each patient was assessed using best corrected visual acuity measurements, fluorescein angiography, and optical coherence tomography at baseline and had regular follow-ups after treatment. RESULTS: All patients showed improvements in visual acuity and fluorescein angiographic leakage and had resolution of their neurosensory detachment following treatment. There were no significant differences in visual acuity, central retinal thickness, or remission duration between the IVBI group and the control group at baseline or after treatment (p>0.05). CONCLUSIONS: Intravitreal bevacizumab showed no positive effect in acute CSC patients compared to the observation group, and there were no adverse effects of treatment. Further investigation will be helpful to understand this therapy in patients with CSC.

TCR diversity of H60-specific CD8 T cells during the response evolution and influence of CD4 help.

BACKGROUND: H60 is a hematopoietic cell-specific dominant minor histocompatibility antigen that is considered to be ideal for modeling leukemia treatment after bone-marrow transplantation. We characterized the H60-specific CD8 T-cell response as CD4 help dependent. This study investigated the T-cell receptor (TCR) repertoires during the evolution of H60-specific CD8 T-cell responses and influence of CD4 help on the diversity. METHODS: Ex vivo TCR V beta and complementarity-determining region 3 length spectratypic and clonotypic analyses were performed using H60-tetramer-binding CD8 T cells purified from the mice undergoing the primary, secondary, and tertiary responses with cognate help, and the secondary response with noncognate separate CD4 help. RESULTS: Involvement of a broad spectrum of TCRs was observed in the H60-specific primary response. With the involvement of diverse V beta families in the secondary and tertiary responses, complementarity-determining region 3 length and clonotypic diversities within the V beta subfamilies gradually decreased throughout the response evolution. In tertiary repertoires, the usage of V beta 8.3 and focused clonal usage within each V beta subfamily were prominent. When noncognate separate CD4 help was provided during the induction of H60-specific secondary responses, extremely limited TCRs constituted the repertoire of reactive CD8 T cells, and most of these TCRs coincided with those observed in the secondary or tertiary repertoires provided with cognate help. CONCLUSIONS: This study is the first to characterize the diversity of TCRs specific for hematopoietic cell-specific mouse minor H antigens and demonstrate the effect of CD4 help on CD8 TCR repertoire diversity. Our data provide a basis for modeling therapeutic applications.

Cognate CD4 help is essential for the reactivation and expansion of CD8 memory T cells directed against the hematopoietic cell-specific dominant minor histocompatibility antigen, H60.

In contrast to previous notions of the help-independency of memory CD8 T cells during secondary expansion, here we show that CD4 help is indispensable for the re-expansion of once-helped memory CD8 T cells, using a hematopoietic cell-specific dominant minor histocompatibility (H) antigen, H60, as a model antigen. H60-specific memory CD8 T cells generated during a helped primary response vigorously expanded only when rechallenged under helped conditions. The help requirement for an optimal secondary response was confirmed by a reduction in peak size by CD4 depletion, and was reproduced after skin transplantation. Helpless conditions or noncognate separate help during the secondary response resulted in a significant reduction in the peak size and different response kinetics. Providing CD4 help again during a tertiary challenge restored robust memory expansion; however, the repeated deprivation of help further reduced clonal expansion. Adoptively transferred memory CD8 T cells did not proliferate in CD40L(-/-) hosts. In the CD40(-/-) hosts, marginal memory expansion was detected after priming with male H60 cells but was completely abolished by priming with peptide-loaded CD40(-/-) cells, suggesting the essential role of CD40 and CD40L in memory responses. These results provide insight into the control of minor H antigen-specific CD8 T-cell responses, to maximize the graft-versus-leukemia response.

Use of transesophageal atrial pacing for documentation of arrhythmias suspected in infants and children.

Transesophageal atrial pacing study was used to document arrhythmias in 67 infants and children age 2 months to 16 years (mean, 8.3 years), who had palpitations or symptoms suggesting tachyarrhythmias but had no electrocardiographic documentation of cardiac dysrhythmias. The transesophageal pacing and medical records were reviewed retrospectively. In 47 of 67 (70%) of the infants and children with suspected tachyarrhythmias, transesophageal atrial pacing induced various tachycardias, which may be the cause of symptoms. In 10 of 67 patients, tachycardia was induced during infusion of isoproterenol. During the study, tachycardia was initiated in 14 of 15 patients less than < 6 years-old and in 33 of 52 patients > or = 6 years-old (P < 0.05). Of these induced tachycardias, 25 of 47 were atrioventricular reciprocating tachycardia, 16 atrioventricular nodal reentrant tachycardia, and 6 idiopathic left ventricular tachycardia. Both transesophageal study and invasive electrophysiologic study were performed in 10 patients. The mechanisms of tachycardia in the invasive study and transesophageal study were identical except for one patient. In conclusion, transesophageal atrial pacing and recording was less invasive, safe and useful for documenting arrhythmias in infants and children who had symptoms suggesting tachyarrhythmias, especially in patients < 6 years of age. Evaluation of the mechanism of induced tachycardia provided useful information regarding the prognosis and therapeutic options in infants and children.