Calcineurin inhibition rescues alloantigen-specific central memory T cell subsets that promote chronic GVHD.

Calcineurin inhibitors (CNIs) constitute the backbone of modern acute graft-versus-host disease (aGVHD) prophylaxis regimens but have limited efficacy in the prevention and treatment of chronic GVHD (cGVHD). We investigated the effect of CNIs on immune tolerance after stem cell transplantation with discovery-based single-cell gene expression and T cell receptor (TCR) assays of clonal immunity in tandem with traditional protein-based approaches and preclinical modeling. While cyclosporin and tacrolimus suppressed the clonal expansion of CD8+ T cells during GVHD, alloreactive CD4+ T cell clusters were preferentially expanded. Moreover, CNIs mediated reversible dose-dependent suppression of T cell activation and all stages of donor T cell exhaustion. Critically, CNIs promoted the expansion of both polyclonal and TCR-specific alloreactive central memory CD4+ T cells (TCM) with high self-renewal capacity that mediated cGVHD following drug withdrawal. In contrast to posttransplant cyclophosphamide (PT-Cy), CSA was ineffective in eliminating IL-17A-secreting alloreactive T cell clones that play an important role in the pathogenesis of cGVHD. Collectively, we have shown that, although CNIs attenuate aGVHD, they paradoxically rescue alloantigen-specific TCM, especially within the CD4+ compartment in lymphoid and GVHD target tissues, thus predisposing patients to cGVHD. These data provide further evidence to caution against CNI-based immune suppression without concurrent approaches that eliminate alloreactive T cell clones.

GVHD targets organoid-forming bile duct stem cells in a TGF-ß-dependent manner.

ABSTRACT: Graft-versus-host disease (GVHD) is a major life-threatening complication that occurs after allogeneic hematopoietic cell transplantation (HCT). Although adult tissue stem cells have been identified as targets of GVHD in the skin and gut, their role in hepatic GVHD is yet to be clarified. In the current study, we explored the fate of bile duct stem cells (BDSCs), capable of generating liver organoids in vitro, during hepatic GVHD after allogeneic HCT. We observed a significant expansion of biliary epithelial cells (BECs) on injury early after allogeneic HCT. Organoid-forming efficiency from the bile duct was also significantly increased early after allogeneic HCT. Subsequently, the organoid-forming efficiency from bile ducts was markedly decreased in association with the reduction of BECs and the elevation of plasma concentrations of bilirubin, suggesting that GVHD targets BDSCs and impairs the resilience of BECs. The growth of liver organoids in the presence of liver-infiltrating mononuclear cells from allogeneic recipients, but not from syngeneic recipients, was significantly reduced in a transforming growth factor-ß (TGF-ß)-dependent manner. Administration of SB-431542, an inhibitor of TGF-ß signaling, from day 14 to day 28, protected organoid-forming BDSCs against GVHD and mitigated biliary dysfunction after allogeneic HCT, suggesting that BDSCs are a promising therapeutic target for hepatic GVHD.

Microbiota dictate T cell clonal selection to augment graft-versus-host disease after stem cell transplantation.

Allogeneic T cell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a closed genetic system within which donor T cells interact with peptide-major histocompatibility complexes (MHCs), though clonal interrogation remains challenging due to the sparseness of the T cell repertoire. We developed a Bayesian model using donor and recipient T cell receptor (TCR) frequencies in murine stem cell transplant systems to define limited common expansion of T cell clones across genetically identical donor-recipient pairs. A subset of donor CD4+ T cell clonotypes differentially expanded in identical recipients and were microbiota dependent. Microbiota-specific T cells augmented GVHD lethality and could target microbial antigens presented by gastrointestinal epithelium during an alloreactive response. The microbiota serves as a source of cognate antigens that contribute to clonotypic T cell expansion and the induction of GVHD independent of donor-recipient genetics.

IL-6-mediated endothelial injury impairs antiviral humoral immunity after bone marrow transplantation.

Endothelial function and integrity are compromised after allogeneic bone marrow transplantation (BMT), but how this affects immune responses broadly remains unknown. Using a preclinical model of CMV reactivation after BMT, we found compromised antiviral humoral responses induced by IL-6 signaling. IL-6 signaling in T cells maintained Th1 cells, resulting in sustained IFN-γ secretion, which promoted endothelial cell (EC) injury, loss of the neonatal Fc receptor (FcRn) responsible for IgG recycling, and rapid IgG loss. T cell-specific deletion of IL-6R led to persistence of recipient-derived, CMV-specific IgG and inhibited CMV reactivation. Deletion of IFN-γ in donor T cells also eliminated EC injury and FcRn loss. In a phase III clinical trial, blockade of IL-6R with tocilizumab promoted CMV-specific IgG persistence and significantly attenuated early HCMV reactivation. In sum, IL-6 invoked IFN-γ-dependent EC injury and consequent IgG loss, leading to CMV reactivation. Hence, cytokine inhibition represents a logical strategy to prevent endothelial injury, thereby preserving humoral immunity after immunotherapy.

Regulatory T cells suppress myeloma-specific immunity during autologous stem cell mobilization and transplantation.

ABSTRACT: Autologous stem cell transplantation (ASCT) is the standard of care consolidation therapy for eligible patients with myeloma but most patients eventually progress, an event associated with features of immune escape. Novel approaches to enhance antimyeloma immunity after ASCT represent a major unmet need. Here, we demonstrate that patient-mobilized stem cell grafts contain high numbers of effector CD8 T cells and immunosuppressive regulatory T cells (Tregs). We showed that bone marrow (BM)-residing T cells are efficiently mobilized during stem cell mobilization (SCM) and hypothesized that mobilized and highly suppressive BM-derived Tregs might limit antimyeloma immunity during SCM. Thus, we performed ASCT in a preclinical myeloma model with or without stringent Treg depletion during SCM. Treg depletion generated SCM grafts containing polyfunctional CD8 T effector memory cells, which dramatically enhanced myeloma control after ASCT. Thus, we explored clinically tractable translational approaches to mimic this scenario. Antibody-based approaches resulted in only partial Treg depletion and were inadequate to recapitulate this effect. In contrast, a synthetic interleukin-2 (IL-2)/IL-15 mimetic that stimulates the IL-2 receptor on CD8 T cells without binding to the high-affinity IL-2Ra used by Tregs efficiently expanded polyfunctional CD8 T cells in mobilized grafts and protected recipients from myeloma progression after ASCT. We confirmed that Treg depletion during stem cell mobilization can mitigate constraints on tumor immunity and result in profound myeloma control after ASCT. Direct and selective cytokine signaling of CD8 T cells can recapitulate this effect and represent a clinically testable strategy to improve responses after ASCT.

Scratching and Stretching the Shrunken Cranial Dura for Primary Closure.

BACKGROUND: At craniotomy, the dura shrinks due to the drying effect of illumination and air exposure, rendering its primary closure difficult. When the opening is narrow, it can be patched with autologous grafts. However, tissue harvesting and meticulous suturing take time. As the cost-effectiveness of using dural substitutes is poor, we developed a new technique that involves dural scratching for the primary closure of dural openings. METHODS: We applied our technique because in 53 adults who underwent supratentorial craniotomy we encountered difficulties with the primary closure of a dural opening 5 mm or less in width. With a scalpel, we placed several fine scratches parallel to the edge of the opening on the surface of the dura, taking care not to perforate the deep layer. This relieved dural tension and the tissue was stretched enough to permit closing with sutures. RESULTS: Our technique achieved primary dural closure in 46 of 53 patients (87%) who primarily had undergone narrow supratentorial craniotomies. The other 7 additionally required small autologous grafts to patch narrow residual openings at the intersection of the durotomy. There were no procedure-related complications such as cerebrospinal fluid leakage and persistent headache due to tension of the closed dura. CONCLUSIONS: Dural scratching is simple, safe, requires no special instrumentation, facilitates primary closure of the shrunken dura by stretching, and reduces the need for patch grafting.

TIGIT inhibition and lenalidomide synergistically promote antimyeloma immune responses after stem cell transplantation in mice.

Autologous stem cell transplantation (ASCT) with subsequent lenalidomide maintenance is standard consolidation therapy for multiple myeloma, and a subset of patients achieve durable progression-free survival that is suggestive of long-term immune control. Nonetheless, most patients ultimately relapse, suggesting immune escape. TIGIT appears to be a potent inhibitor of myeloma-specific immunity and represents a promising new checkpoint target. Here we demonstrate high expression of TIGIT on activated CD8+ T cells in mobilized peripheral blood stem cell grafts from patients with myeloma. To guide clinical application of TIGIT inhibition, we evaluated identical anti-TIGIT antibodies that do or do not engage FcγR and demonstrated that anti-TIGIT activity is dependent on FcγR binding. We subsequently used CRBN mice to investigate the efficacy of anti-TIGIT in combination with lenalidomide maintenance after transplantation. Notably, the combination of anti-TIGIT with lenalidomide provided synergistic, CD8+ T cell-dependent, antimyeloma efficacy. Analysis of bone marrow (BM) CD8+ T cells demonstrated that combination therapy suppressed T cell exhaustion, enhanced effector function, and expanded central memory subsets. Importantly, these immune phenotypes were specific to the BM tumor microenvironment. Collectively, these data provide a logical rationale for combining TIGIT inhibition with immunomodulatory drugs to prevent myeloma progression after ASCT.

Gilteritinib enhances graft-versus-leukemia effects against FLT3-ITD mutant leukemia after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is a potentially curative therapy for FLT3 internal tandem duplication mutant (FLT3-ITD+) acute myeloid leukemia, but relapse rate is high. A recent study showed that sorafenib, a first generation FLT3 and multikinase inhibitor, enhanced graft-versus-leukemia (GVL) effects against FLT3-ITD+ leukemia via interleukin-15 (IL-15) production. However, it remains to be clarified whether this effect could be mediated by selective FLT3 inhibition. We investigated whether gilteritinib, a selective FLT3 inhibitor, could enhance GVL effects against FLT3-ITD transfected Ba/F3 leukemia (Ba/F3-FLT3-ITD) in mice. Oral administration of gilteritinib from day +5 to +14 after allo-SCT reduced expression of the co-inhibitory receptors PD-1 and TIGIT on donor CD8+ T cells and enhanced IL-15 expression in Ba/F3-FLT3-ITD. Bioluminescent imaging using luciferase-transfected Ba/F3-FLT3-ITD demonstrated that gilteritinib significantly suppressed leukemia expansion after allo-SCT, whereas it did not impact the morbidity or mortality of graft-versus-host disease (GVHD), resulting in significant improvement of overall survival. In conclusion, short-term administration of gilteritinib after allo-SCT enhanced GVL effects against FLT3-ITD+ leukemia without exacerbating GVHD.

Clinical Application of Apheresis in Very Small Dogs Weighing <8 kg to Pediatric Patients.

Apheresis in low body weight children and adolescents is challenging due to a variety of technical and clinical issues including vascular access, low total blood volume, and hypotension. Although dogs have been a valuable preclinical model for apheresis, the procedure can be challenging since many pure-bred dogs are extremely small. Therefore, apheresis in these very small breeds presents very similar challenges as seen when performing the procedure in very low body weight people. We describe apheresis of four very small dogs, weighing from 4.6 to 7.6 kg, using either a COBESpectra and Spectra Optia apheresis system (Terumo BCT, Lakewood, CO, USA). Two dogs underwent large volume leukapheresis to collect mononuclear cells in preparation for hematopoietic stem cell transplantation and two dogs underwent therapeutic plasma exchange to treat an immune-mediated disease. In all cases, a dual-lumen hemodialysis catheter placed in the jugular vein provided adequate machine inlet and return flow rates. Machine priming was necessary to maintain hemodynamic stability during the beginning of the procedure, and rinseback was avoided for the same reason. Anticoagulant citrate dextrose solution, solution A was used for the large volume leukapheresis procedures and a combination of anticoagulant citrate dextrose solution, solution A and heparin was used for the therapeutic plasma exchange procedures. As such, serum iCa levels were regularly monitored and 10% calcium gluconate constant rate infusions were used to prevent citrate toxicity. All dogs completed the aphereses with no life-threatening adverse events. We conclude that aphereses in very small dogs is feasible if close attention is paid to hemodynamic stability and citrate toxicity.

Intracranial Response to Nivolumab in a Patient with PD-L1-negative Lung Adenocarcinoma.

We herein report the case of a 52-year-old man with stage IV lung adenocarcinoma. The patient was negative for epidermal growth factor receptor (EGFR) mutations and echinoderm microtubule-associated protein-like 4 (EML4) /anaplastic lymphoma kinase (ALK) rearrangement. He was treated with nivolumab as a third-line chemotherapy. After four cycles of nivolumab treatment, a partial response was observed in the brain and at the primary tumor site. Nivolumab treatment has been continued for 11 months without progression. Immunohistochemistry revealed that the programmed death-ligand 1 (PD-L1) expression was 0% (according to the tumor proportion score). Our case indicates that the efficacy of programmed cell death 1 inhibitors is not solely predicted by the PD-L1 status, and that immune checkpoint inhibitors might be effective for the treatment of central nervous system metastasis.

[Extendable Cords to Prevent Tumbling of a Suction Device during Craniotomy].

OBJECTIVE: Suction is necessary during craniotomy, and intraoperative tumbling of the suction device interrupts operative procedures. To avoid this, we developed a technique that would fasten the device to an extendable cord as is used to secure cell phones. SURGICAL TECHNIQUE: We used this technique in more than 300 craniotomies at the specific point of time when the suction device tends to tumble, i. e., during the opening and closure of a wound, which requires frequent instrument exchanges. Extendable cords fastened to the tip of the suction hose using a gift tie were attached to the drapes to secure the suction device next to the operative field. During the operation, the extendable cord followed the suction device manipulations. Consequently, although there was some tension in the cord during its extension, the maneuverability of the suction device was maintained. As the hanging suction device was closer to the operative field than devices stored in conventional pockets, its manipulation was easier and quicker. Upon release, the suction device automatically returned to its original position without distracting the surgeon. Tumbling of the device was prevented, and there were no procedure-related complications. CONCLUSIONS: Our simple modification using extendable cords prevented tumbling, avoided unnecessary replacements, and eased the manipulation of a suction device.

Modified One-piece Supraorbital Approach for Orbital Tumors: Widely Preserved Orbital Roof in a Self-fitting Flap.

Objectives The one-piece supraorbital approach is a rational approach for the removal of orbital tumors. However, cutting the roof through the orbit is often difficult. We modified the technique to facilitate the osteotomy and improve the cosmetic effect. Design Three burr holes are made: the first, the MacCarty keyhole (burr hole 1), exposes the anterior cranial fossa and orbit; the second is placed above the supraorbital nerve (burr hole 2); and the third on the superior temporal line. Through burr hole 2, a small hole is created on the roof, 10 mm in depth. Next the roof is rongeured through burr hole 1 toward the preexisting small hole. Seamless osteotomies using a diamond-coated threadwire saw and the preexisting four holes are performed. Lastly the flap is removed. On closure, sutures are passed through holes in the cuts made with the threadwire saw, and tied. Results We applied our technique to address orbital tumors in two adult patients. The osteotomies in the roof were easy, and most parts of the roof were repositioned. Conclusions Our modification results in orbital osteotomies with greater preservation of the roof. Because the self-fitting flap does not require the use of fixation devices, the reconstruction is cosmetically satisfactory.

Moist-condition Training for Cerebrovascular Anastomosis: A Practical Step after Mastering Basic Manipulations.

As cerebrovascular anastomosis is performed in moist conditions that may impede precise manipulations, surgeons must undergo extensive preoperative training. We developed a simple moist-condition training method. It involves placing a free-floating inner platform hosting an artery from a chicken wing in an outer container filled with tap water to just below the specimen. Trainees performed anastomosis under magnification. Training sessions mimicked difficulties encountered during operations such as poor visibility of the lumen and problems handling the sutures. A retrospective comparison of 100 wet- and 100 dry-condition training sessions for end-to-side anastomoses with 8 stitches showed that under moist condition the time required for the entire procedure was significantly longer (17.8 ± 2.1 vs. 15.3 ± 2.1 min, p < 0.01) and the incidence of wrong stitching was greater (0.38 vs. 0%, p = 0.04). In 8 cases after introducing moist-condition training, the time required in superficial temporal artery to middle cerebral artery bypass surgery was significantly shorter than 8 cases before introducing the training (32.3 ± 5.6 min vs. 48.3 ± 15.9 min, p = 0.01). Incidence of wrong stitches was less in cases after introducing moist-condition training (2.7 vs. 7.4%, p = 0.10). Those indicate that moist-condition training is a useful and practical step and a bridge between training for basic manipulations under dry conditions and actual surgery.

[Modified Technique for Primary Dural Closure in the Lateral Suboccipital Approach:Dural Moisturizing with Fibrin Glue Coating].

OBJECTIVE: When employing the lateral suboccipital approach, the thin dura shrinks due to the drying effect of illumination and air exposure, and dural substitutes are often needed for closure. We developed a new technique involving dural moisturizing with fibrin glue coating that facilitates primary dural closure. PATIENTS AND METHODS: We used this technique in 12 adults who underwent the lateral suboccipital approach for 5 hemifacial spasms, 3 trigeminal neuralgias, 2 cerebellopontine meningiomas, 1 vestibular schwannoma, and 1 vertebral artery aneurysm. Fibrin glue was sprayed on the outer surface before opening the dura, and additionally sprayed on the inner surface of the reflected dural flap after opening the dura. After the intradural procedures the dura was closed with the usual knotted sutures. RESULTS: Dural closure was performed 65-340 minutes (mean: 161.9 minutes) post-durotomy. This technique resulted in primary dural closure with a sufficient area of preserved dura in all but one patient. In this patient, the dura shrank due to coagulation of the dural attachment to the meningioma for which a small autologous substitute was required. There were no procedure-related complications such as cerebrospinal fluid leakage and meningitis. CONCLUSIONS: Dural moisturizing with fibrin glue coating is simple, protects the dura from drying and shrinkage, and facilitates primary dural closure in patients undergoing the lateral suboccipital approach.

Rapidly progressive dementia caused by a superior sagittal sinus dural arteriovenous fistula: a case report.

We describe the case of a 72-year-old man who presented with dementia that had progressed rapidly over a few months. Laboratory analysis of blood and cerebrospinal fluid (CSF) showed no abnormalities, with the exception of a slightly increased CSF protein level. Results of routine magnetic resonance imaging (MRI) including diffusion-weighted imaging (DWI) and magnetic resonance angiography (MRA) were unremarkable. However, detailed neuroimaging studies including contrast-enhanced T1-weighted MRI and conventional angiography revealed a superior sagittal sinus (SSS) dural arteriovenous fistula (DAVF). After endovascular embolization and surgical interruption of all arteries feeding the DAVF, the dementia reversed. We should be aware of the possibility of DAVF as the cause of rapidly progressive dementia even if routine MRI reveals no or only minimal abnormality.

[Differentiation between a thoracic spinal intramedullary lipoma and an intramedulary hematoma on fast spin-echo MRI scans: a case report].

We describe differentiation of a spinal intramedullary lipoma from an intramedullary hematoma on magnetic resonance images (MRI) with fast spin-echo (FSE) sequences. A 60-year-old man with dysesthesia in the legs and gait disturbance, was suspected of having myelopathy at a middle thoracic lesion. MRI with FSE sequences revealed an intramedullary lesion at T7 to T8. On the basis of hyperintensity on both T1-and T2-weighted images and a perilesional hypointense rim on T2-weighted images we made a diagnosis of subacute hematoma and planned observation. However, computed tomography for associated vertebral degeneration revealed a hypodense area (-97 Hounsfield units) in the region corresponding to the lesion depicted by MRI. We revised our diagnosis to an intramedullary lipoma and debulked the lesion. The lipoma was surrounded by a thick whitish capsule. Histopathologically, the capsule contained mature fat tissue and abundant collagen. The initial diagnosis was mainly attributable to specific FSE characteristics, i. e., the depiction of fat tissue as hyperintense on both T1-and T2-weighted images. Conventional spin-echo MRI depicts fat tissue as hyperintense on T1-and as hypointense on T2-weighted images. Other factors contributing to our initial diagnosis were MRI findings suggestive of an intramedullary hematoma, i. e., the intrinsic location of the lesion and the perilesional hypointense rim on T2-weighted images ascribable to collagen present in the capsule. The accurate diagnosis of an intramedullary lipoma on FSE requires correct interpretation of the signal, which is different from the signal on conventional spin-echo MRI.

[Distribution of Clostridium tetani in topsoil from Sagamihara, central Japan].

Despite reports of Clostridium tetani being isolated from soil in Kanazawa, Okinawa, and Tokyo, Japan, little has been studied about C. tetani distribution in other regions. We studied C. tetani in topsoil samples collected from private gardens, public road shoulders, a university campus, mountains, and fields in Sagamihara. C. tetani occurred in 8 of 35 soil samples (22.9%) and tetanus toxin in 7 of the 8 C. tetani-positive samples (87.5%). Contamination was clearly higher in soils from mountains near Tsukui-gun (Kanagawa Prefecture), Minamitsuru-gun, and Uenohara and Koshu cities (Yamanashi Prefecture) than in other regions. These findings suggest that tetanus toxin-producing strains of C. tetani tend to inhabit the topsoil of western Sagaminaha region, as a geographical feature.

[Inactivation of Cryptosporidium parvum oocysts in copper tubing].

We studied whether the infectivity of Cryptosporidium parvum oocysts for suckling mice could be inactivated by copper tubing or by other types of tubing used to construct water distribution systems, including stainless steel, rigid polyvinyl chloride (PVC), PVC-lined steel, polyethylene (PE), cross-linked PE, and polybutene (PB), using glass tubing as the control. Oocysts were incubated in each tubings for 24 hours. The extent of inactivation of infectious oocysts by copper tubing was -1.303 log, which significantly inactivated of infectivity. In contrast, other types of tubing had no significant effect on some oocyst infectivity, although PB did show a maximum inactivation of -0.313 log. 25% of oocysts showed degeneration morphologically after passing through copper tubing, while 0.3% to 1.8% showed degeneration after passing through other tubing. Significant inactivation of infectious oocysts was not caused by water in which copper tubing had been let stand for 24 hours, although it had a cupric ion (Cu2+) concentration of 2.4 mg/L. The direct contact of oocysts with copper surface resulted in a decrease in the recovery percentage of oocysts and generation of hydrogen peroxide (0.5 mg/L) after 24 h of incubation. The percentage of degenerating oocysts was 29%. Such cryptosporidicidal effects of the copper surface on oocysts were completely inhibited by overlaying the surface with a Millipore filter before adding oocysts and incubating oocysts in the presence of catalase, an antioxidant enzyme. These findings suggest that copper tubing inactivates infectious C. parvum oocysts cytotoxically which may be due to oxygen radicals generated by the interaction between Cu2+ and hydrogen peroxide on the tubing surface.

[Antimicrobial ceramic for killing Legionella pneumophila in hot spring waters].

Killing of Legionella pneumophila by an antimicrobial ceramic was evaluated during culture in nine kinds of hot spring water at 40 degrees C. After 24 hours, the efficacy against L. pneumophila varied, depended on water quality. The strongest antibacterial effect was seen in chloride hot spring water from Wakayama and in deionized water. In four hot spring water samples (sulfur and hydrogen carbonate springs from Fukushima, simple thermals from Mie, and radioactive spring from Tottori), the decrease was < -2 log cfu after 48 hours. These results suggest that the antimicrobial ceramic is able to eradicate Legionella from hot spring waters.

[Physicochemical factors influencing distribution of Legionella species in Japanese hot springs].

We examined the relationship between the distribution of Legionella bacteria and various physicochemical characteristics of hot springs in Japan. Legionella bacteria were isolated from 52 (49.5%) out of 105 water samples, particularly from outdoor hot springs (67.3%). The bacterial count in the water samples positive for Legionella (86.5%) ranged from 10(1) to < 10(3) cfu/100 mL. L. pneumophila serogroup (SG) 4 (27.8%) was predominant in the water samples, followed by SG 5 (12.2%). The pulsefield gel electrophoresis (PFGE) patterns of chromosomal DNA for L. pneumophila SG 4 isolated from different parts of a hot spring resort were identical. Isolation of Legionella species from hot spring waters did not occur at pH 1.8-3.3, SO4(2-): > 780 mg/L, and H2SiO3: > 146 mg/L. The hot water-recirculating systems were applied to 18 out of 20 (90%) hot spring facilities which were found positive for Legionella. These results indicate that Legionella species are widespread in hot springs throughout Japan, except for waters with a low pH and non-recirculating waters, and that a single strain of L. pneumophila SG 4 is predominant in a particular hot spring resort.