Focal hypohidrosis in lesional skin in a probable case of confluent and reticulated papillomatosis: A case report with insight into the pathomechanism of recurrence.

Confluent and reticulated papillomatosis (CRP) is a rare skin disorder that develops in young adults and presents as persistent brown papules and plaques predominantly affecting the intertriginous areas, however, its etiopathogenesis remains elusive. Herein, we report a probable case of CRP with lesional hypohidrosis as detected by sweat test and provide insight into the pathomechanism. A 23-year-old man presented with nine-months history of painful sensation on his trunk without any skin change. The result of sweat test was compatible with acquired idiopathic generalized anhidrosis. Topical heparinoid and physical exercise improved the symptoms. However, he started to notice asymptomatic brownish reticulated macules on the trunk. Intriguingly, focal hypohidrosis, as detected by sweat test was evident on the macules. In histology, the lesional skin demonstrated hyperkeratosis, acanthosis, basal melanosis, mild papillomatosis, and obstruction of the sweat duct in the upper dermis, which were not observed in the peri-lesional skin. Accumulation of the sweat in the luminal aspect of the secretory portion and dilation of the sweat duct in the deeper dermis was detected in the lesional skin, as highlighted by anti-dermcidin staining. Aquaporin 5 expression in the secretory portion was more confined to the cell membrane in the lesional skin. Both brownish macules and lesional hypohidrosis simultaneously improved in summer and exacerbated in winter. Literature review found nine reports on recurrent CRP, and obesity was thought to be a major comorbidity in recurrent CRP cases. Obesity is often associated with sweat dysregulation. This, together with the findings in our case, implied the possible contribution of focal sweating abnormality in the pathogenesis of reticulated skin lesion in our case.

Clinicopathological digital image analyses before and after thermal stimulation subdivide acquired idiopathic generalized anhidrosis into inflammatory and non-inflammatory type.

BACKGROUND: Acquired idiopathic generalized anhidrosis (AIGA) manifests varying degrees of syringotropic inflammation. OBJECTIVE: To better understand the basis of inflammation in AIGA. METHODS: Changes in the extent of cell infiltration around the sweat gland/duct and the difference in the expression level of immune privilege (IP)-related/sweat gland markers before and after thermal stimulation were assessed in AIGA. We also adopted a semi-quantitative digital image analysis of sweating as detected by the starch-iodine method. The changes in sweating before and after treatment was defined as the improvement index. RESULTS: Nine AIGA cases were analyzed. Two cases with minimal inflammation were defined as non-inflammatory type (non-inf)AIGA, while others with evident cell infiltration were defined as inflammatory type (inf)AIGA. MHC class I was significantly upregulated with downregulation of macrophage migration inhibitory factor and alpha-melanocyte-stimulating hormone exclusively in the sweat duct of infAIGA after thermal stimulation (p < 0.05). Furthermore, the extent of inflammation and the ductal dermcidin expression prior to thermal stimulation were inversely correlated (r = - 0.807), while that and the ductal claudin-1 expression after thermal stimulation was positively correlated (r = 0.875). The improvement index positively correlated with the degree of inflammation after thermal stimulation implying possible contribution of inflammation in AIGA pathophysiology. In addition, interferon-induced protein 10 and claudin-1 expression level in the sweat duct before thermal stimulation respectively correlated with the improvement index (r = 0.750, and 0.762). CONCLUSION: The pathophysiology of AIGA may be subcategorized into two groups: IP-collapse possibly play some roles in infAIGA, while ductal dysfunction may exist in non-infAIGA.

Human iPS Cell-Derived Cell Aggregates Exhibited Dermal Papilla Cell Properties in in vitro Three-Dimensional Assemblage Mimicking Hair Follicle Structures.

Current approaches for human hair follicle (HF) regeneration mostly adopt cell-autonomous tissue reassembly in a permissive murine intracorporeal environment. This, together with the limitation in human-derived trichogenic starting materials, potentially hinders the bioengineering of human HF structures, especially for the drug discovery and treatment of hair loss disorders. In this study, we attempted to reproduce the anatomical relationship between an epithelial main body and the dermal papilla (DP) within HF in vitro by three-dimensionally assembling columnarly molded human keratinocytes (KCs) and the aggregates of DP cells and evaluated how HF characteristics were reproduced in the constructs. The replaceability of human-induced pluripotent stem cell (hiPSC)-derived DP substitutes was assessed using the aforementioned reconstruction assay. Human DP cell aggregates were embedded into Matrigel as a cluster. Subsequently, highly condensed human KCs were cylindrically injected onto DP spheroids. After 2-week culture, the structures visually mimicking HFs were obtained. KC-DP constructs partially reproduced HF microanatomy and demonstrated differential keratin (KRT) expression pattern in HFs: KRT14 in the outermost part and KRT13, KRT17, and KRT40, respectively, in the inner portion of the main body. KC-DP constructs tended to upregulate HF-related genes, KRT25, KRT33A, KRT82, WNT5A, and LEF1. Next, DP substitutes were prepared by exposing hiPSC-derived mesenchymal cells to retinoic acid and subsequently to WNT, BMP, and FGF signal activators, followed by cell aggregation. The resultant hiPSC-derived DP substitutes (iDPs) were combined with KCs in the invented assay. KC-iDP constructs morphologically resemble KC-DP constructs and analogously mimicked KRT expression pattern in HF. iDP in the constructs expressed DP-related markers, such as vimentin and versican. Intriguingly, KC-iDP constructs more intensely expressed KRT33A, KRT82, and LEF1, which were stepwisely upregulated by the addition of WNT ligand and the mixture of WNT, SHH, and EDA signaling activators, supporting the idea that iDP exhibited biological properties analogous to DP cell aggregates in the constructs in vitro. These preliminary findings suggested the possibility of regenerating DP equivalents with in vitro hair-inductive capacity using hiPSC-derived cell composites, which potentially reduce the necessity of human tissue-derived trichogenic cell subset and eventually allow xeno-free bioengineering of human HFs.

Development of pemphigus foliaceus in a mucous membrane pemphigoid patient: An insight into possible mechanism eliciting a distinct autoimmune bullous disease in a rare anti-basement membrane autoimmunity.

Mucous membrane pemphigoid (MMP) rarely coexists with another autoimmune bullous disease (AIBD). Herein, we report an extremely rare MMP case who sequentially developed pemphigus foliaceus (PF). A 72-year-old man had been treated by azathioprine monotherapy for anti-BP180 MMP for 1.5 years. Clinical findings suggestive of PF, represented by scaly erythema and erosions, started to appear approximately 1 month after the episode of diarrhea. Serological and immunohistochemical examinations confirmed the diagnosis of PF. The mucocutaneous lesions were controlled by oral azathioprine and topical corticosteroids. To our knowledge, this is a previously unreported case of PF coexistent with MMP. A literature review of MMP cases associated with AIBD elucidated that 16 out of 18 cases simultaneously developed MMP and AIBD, while only two cases were diagnosed sequentially by the changes in clinical symptoms similar to our case. The titer of anti-desmoglein 1 antibodies lineally correlated with the changes in the severity of scaly erythema. Mild but noticeable exacerbation of mucosal erosion prior to the gradual increase in anti-BP180-NC16a antibodies was also noted. Unlike in other cases where MMP/AIBD coexisted, sequential development of autoantibodies in our case cannot be explained by the epitope-spreading theory as autoantigens are micro-anatomically isolated from one other. The preceding viral infection and/or continuous moderate inflammation due to azathioprine monotherapy for MMP might have contributed to the development of PF in our case.

Development of a scoring system to predict outcomes of i.v. corticosteroid pulse therapy in rapidly progressive alopecia areata adopting digital image analysis of hair recovery.

Alopecia areata (AA) is a common autoimmune disease manifesting varying degrees of hair loss. Rapidly progressive AA (RP-AA) is a severe subtype of AA and often resistant to skin-directed treatments. i.v. corticosteroid pulse therapy has been applied for RP-AA; however, the treatment outcome can only become evaluable several months after the intervention, discomposing the patients. In this study, we attempted to develop a scoring system to predict treatment outcomes based on statistical correlations between newly identified predictors and the recovery rates calculated by digital image analysis. Thirty RP-AA patients (15 men and 15 women) who underwent pulse therapy and demonstrated total hair loss during the clinical course were included. The percentages of hair regrowth (%HR) at 6 months after the treatment were quantitatively calculated by image analysis software. The correlation between %HR and clinicopathological and immunological variables were statistically assessed. The analysis identified four confirmatory contributors including female sex (P = 0.015), absence of previous AA history (P = 0.02), lower peripheral blood eosinophil count (P = 0.02) and mild to moderate cell infiltration around the hair bulb (P = 0.034), together with a potential contributor, namely absence of atopic dermatitis in their medical history (P = 0.08). The scoring system was developed by double counting confirmatory variables and single counting a potential variable. Importantly, the scores obtained by this system demonstrated significant correlation with %HR (r = 0.61, P < 0.001). The usefulness of this scoring system was further validated by assessing additional 20 cases of RP-AA. When combined with a recently published algorithm for early detection of self-healing subset, the current scoring system may help strategize the therapeutic approach for RP-AA.

Digital immunohistological dissection of immune privilege collapse in syringotropic autoimmune diseases: Implication for the pathogenesis.

BACKGROUND: Syringotropic cell infiltration is a histological hallmark of some autoimmune diseases. However, its underlying mechanism remains unclear. OBJECTIVES: To assess the immune privilege (IP) of the human sweat gland (SwG) in homeostasis and in syringotropic autoimmune diseases. METHODS: We combined quantitative digital image microdissection with immunohistochemisty to analyze IP molecule expression in SwG of normal and diseased skin. The human skin organ culture model was used to examine the influence of proinflammatory conditions on IP in SwG. RESULTS: In the normal subjects (n = 10), major histocompatibility complex (MHC) class І expression was significantly reduced in SwGs compared to the epidermis. In contrast, IP-guardians, macrophage migration inhibitory factor (MIF) and alpha-melanocyte stimulating hormone (α-MSH) were upregulated in SwGs. MHC class І was upregulated in whole SwGs in lupus erythematosus (LE; n = 7) and scleroderma/morphea (Scl; n = 9), whereas differential expression was noted only in the secretory portion in Sjögren's syndrome (SjS) (n = 4). MIF expression level inversely correlated with that of MHC class I in all samples tested, and downregulation of α-MSH was detected in LE SwGs alone. The severity of inflammatory changes and MIF and ⍺-MSH expression were inversely correlated in LE. CD200 expression was decreased exclusively in atrophic stage of Scl. In a human skin organ culture model, intratissue injection of interferon-gamma up-regulated MHC class I and downregulated MIF and α-MSH. CONCLUSIONS: These findings indicate that SwGs enjoy IP. Dysregulated IP molecule expression may lead to SwG IP collapse and contribute to distinct inflammatory cell distribution in syringotropic autoimmune disorders.

Altered FGF expression profile in human scalp-derived fibroblasts upon WNT activation: implication of their role to provide folliculogenetic microenvironment.

BACKGROUND: Hair follicle (HF) formation and growth are sustained by epithelial-mesenchymal interaction via growth factors and cytokines. Pivotal roles of FGFs on HF regeneration and neogenesis have been reported mainly in rodent models. FGF expression is regulated by upstream pathways, represented by canonical WNT signaling; however, how FGFs influence on human folliculogenesis remains elusive. The aim of this study is to assess if human scalp-derived fibroblasts (sFBs) are able to modulate their FGF expression profile in response to WNT activation and to evaluate the influence of WNT-activated or suppressed FGFs on folliculogenesis. METHODS: Dermal papilla cells (DPCs), dermal sheath cells (DSCs), and sFBs were isolated from the human scalp and cultured independently. The gene expression profile of FGFs in DPCs, DSCs, and sFBs and the influence of WNT activator, CHIR99021, on FGF expression pattern in sFBs were evaluated by reverse transcription polymerase chain reaction, which were confirmed at protein level by western blotting analysis. The changes in the expression of DPC or keratinocyte (KC) biomarkers under the presence of FGF7 or 9 were examined in both single and co-culture assay of DPCs and/or KCs. The influence of FGF 7 and FGF 9 on hair morphogenesis and growth was analyzed in vivo using mouse chamber assay. RESULTS: In single culture, sFBs were distinguished from DPCs and DSCs by relatively high expression of FGF5 and FGF 18, potential inducers of hair cycle retardation or catagen phase. In WNT-activated state, sFBs downregulated FGF7 while upregulating FGF9, a positive regulator of HF morphogenesis, FGF16 and FGF 20 belonging to the same FGF subfamily. In addition, CHIR99021, a WNT activator, dose-dependently modulated FGF7 and 9 expression to be folliculogenic. Altered expressions of FGF7 and FGF9 by CHIR99021 were confirmed at protein level. Supplementation of FGF9 to cultured DPCs resulted in upregulation of representative DP biomarkers and this tendency was sustained, when DPCs were co-cultured with KCs. In mouse chamber assay, FGF9 increased both the number and the diameter of newly formed HFs, while FGF7 decreased HF diameter. CONCLUSION: The results implied that sFBs support HF formation by modulating regional FGF expression profile responding to WNT activation.

Leakage of sweat into the dermo-epidermal junction as a possible trigger for lichen planus lesion development.

No previous studies have convincingly linked sweating disturbance with the subsequent development of lichen planus (LP). Therefore, we investigated whether sweating disturbance could be specifically detected in LP lesions and how it could trigger lesion development. We utilized the impression mold technique (IMT), which allows accurate quantification of individual sweat glands/ducts actively delivering sweat in a well-defined location, to evaluate sweating disturbance in LP lesions. Psoriasis vulgaris (PsV) lesions were included as controls. Leakage of sweat and subsequent induction of chemokine expression were immunohistochemically identified. Both baseline and thermal stimulus-induced sweating responses were markedly impaired in LP lesions, as well as in PsV lesions. A marked difference, however, was found in normal-appearing perilesional skin; "cold spots", which were defined as a 1 mm2 area with no sweat droplets, were specifically and abundantly detected in perilesional LP skin, but not perilesional PsV skin. Leakage of sweat as evidenced by the immunohistochemical detection of dermcidin was specifically observed around the acrosyringium of these "cold spots" in LP, but not PsV, lesions and associated with CXCL10 induction on neighboring keratinocytes and syringotropic migration of CXCR3+ T cells. Leakage of sweat into the dermo-epidermal junction would serve not only to decrease sweat delivery to the skin surface but also to induce T-cell recruitment to the inflammatory site. Therapies for LP may be directed not only at ameliorating inflammatory responses but also at preventing the leakage of sweat into the dermo-epidermal junction.

Monocytes are involved in the balance between regulatory T cells and Th17 cells in severe drug eruptions.

BACKGROUND: Drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms (DiHS/DRESS) is a distinct phenotype of severe drug eruptions characterized by sequential reactivations of herpesviruses. Although a progressive loss of suppressive function in regulatory T cells (Tregs) occurred during the course of DiHS/DRESS, but not in Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), no previous studies investigated the mechanism. Given the recent finding that Treg development could be differentially regulated by CD16+ patrolling monocytes (pMOs) and CD14+ classical monocytes (cMOs), we can hypothesize that a differential fine-tuned interaction between Tregs and monocytes is the driving force behind the possible shift from Tregs to Th17 cells over a prolonged period of time in DiHS/DRESS. OBJECTIVE: To investigate whether the shift from Treg to Th17 could specifically occur during the course of DiHS/DRESS and to elucidate which subsets of monocytes could be involved in the shift. METHODS: We performed a prospective longitudinal study on the frequencies of Tregs, Th17 cells and monocyte subsets after onset of DiHS/DRESS and SJS/TEN, and long after their clinical resolutions. We next examined whether pMOs and cMOs could have a strong impact on the Th17/Treg differentiation and which cytokines could be crucial for the interaction between Th17/Tregs and MO subsets, by in vitro cocultures. RESULTS: Selective depletion of pMOs occurring at the acute stage of DiHS/DRESS was associated with the relative increase in the frequencies of cMOs producing IL-10 and it did drive Treg expansions. After clinical resolution, pMOs producing IL-6 were alternatively recruited and contributed to the eventual shift from a Treg to Th17 responses. CONCLUSIONS AND CLINICAL RELEVANCE: The gradual shift from Treg to Th17 cell development observed during the clinical course of DiHS/DRESS is mediated by the predominance of cMOs at the acute stage and alternatively recruited pMOs at the resolution stage, respectively.

Chronological clinicopathological characterization of rapidly progressive alopecia areata resistant to multiple i.v. corticosteroid pulse therapies: An implication for improving the efficacy.

Intravenous corticosteroid pulse therapy (pulse therapy) has been reported to be effective for rapidly progressive alopecia areata (RP-AA). Mostly, a single 3-day administration of corticosteroid (methylprednisolone 500 mg/day) has been performed in Japan; however, to what extent additional administrations improve the outcome has not been fully elucidated. To assess the advantage of repeating the pulse therapy to RP-AA cases refractory to the initial intervention, retrospective clinicopathological analysis was performed. Detailed chronological analysis was conducted in eight cases (one man and seven women; average age, 38.3 ± 10.4 years) demonstrating total scalp hair loss 3 months after the first pulse therapy and treated with additional rounds of the pulse therapy. All cases manifested total hair loss, scalp edema, itch or pain on the scalp after the initial intervention. Histopathological analyses of affected lesions prior to additional pulse therapies revealed persisting dense perifollicular lymphocytic inflammation in all cases. Interestingly, such inflammatory change tended to be severer when compared with previously reported pulse therapy good responders. Extra pulse therapy resulted in partial regrowth of terminal hairs in three out of eight cases, but all of them experienced relapse in the long run. The literature review also suggested limited efficacy of repeating pulse therapy to severe AA cases. These findings suggested that the efficacy of currently conducted repetitive pulse therapy is limited in RP-AA cases with extensive perifollicular inflammation and resistant to the initial pulse therapy. Modulation of the dose and the interval of intervention, in combination with alternative approaches, may be required to achieve a successful outcome.

A novel method to assess the potential role of sweating abnormalities in the pathogenesis of atopic dermatitis.

Although atopic dry skin is believed to be caused by defects in skin genes important for maintaining skin barrier function, the role of sweat in atopic dermatitis (AD) has been apparently underestimated. Given the great capacity of sweat to maintain and increase skin hydration, defective sweating responses may be a logical place to look for changes that predispose individuals to the disease. We investigated how disease process and sweating defects progress from early asymptomatic stages to the onset of clinically apparent disease by employing the impression mould technique, which allows an accurate quantification of individual sweat gland/duct activity in relation to skin surface topography. Insensible and sensible sweating responses under baseline conditions and after thermal stimulus, respectively, were measured in various stages of AD patients and healthy controls. In controls, under baseline conditions, sweat ducts/glands at the dermal folds secreted basal levels of sweat (insensible sweating), thereby maintaining skin hydration. Not only such insensible sweating but also sensible sweating markedly decreased even in the earliest asymptomatic stage and the decrease was followed by compensatory hyperhidrosis at the ridge: leakage of sweat into the dermis could represent the initial event resulting in the decreased sweating and inflammation. The defects eventually progressed involving all of the ducts/glands to develop systemic dry skin. AD skin is characterized by varying degrees of functional impairment of sweat ducts/glands depending on the stage, and this defect would be among the reasons for the inability of AD patients to maintain skin hydration.

Immunohistochemical dissection of cystic panfolliculoma focusing on the expression of multiple hair follicle lineage markers with an insight into the pathogenesis.

Panfolliculoma (PF) is a rare benign tumor with signs of differentiation toward all components of the hair follicle (HF). Cystic panfolliculoma (CPF) is a subset of PF with histological similarity to trichofolliculoma making the differential diagnosis difficult in some cases. Immunohistopathological investigations of PF have been reported; however, previous studies focused mostly on the expression profile of the outer root sheath markers. Herein, we report a case of CPF. A panel of antibodies was developed and used for the detection of the expression of cytokeratin (CK) 10, CK13, CK14, CK15, hair-hard keratin (AE13) and EpCAM within the lesion, supporting the differentiation of all epithelial lineages of the HF and the diagnosis of CPF. Immunohistopathological examinations clearly showed the spatial distribution pattern of each subset of tumor cells with distinct HF differentiation marker expression. Intriguingly, fibroblastic dermal cells were distributed preferentially near CK15-negative epithelium or CK13-positive HF-like structures, suggesting a role for epithelial-mesenchymal interactions (EMIs) in CPF pathogenesis. Further characterization of EMIs between the tumor and surrounding mesenchymal cells in CPF may provide an explanation for immature HF differentiation. These findings suggest that the more intense and coordinated EMI in the analogous tumorigenesis gives rise to mature HF structures, resulting in trichofolliculoma, which may explain their histological resemblance.

Importance of Water Content of the Stratum Corneum in Mouse Models for Contact Hypersensitivity.

Although a marked rise in the prevalence of allergic diseases over the past few decades may be related to environmental factors in industrialized countries, evidence for the protective effect of humidity on the barrier function of the skin is still awaited. We asked whether an increase in the water content of stratum corneum at the site of hapten application had a strong impact on the magnitude of contact hypersensitivity (CHS). The magnitude of CHS, induced by either lipid-soluble or water-soluble hapten, was inversely correlated with the water content of stratum corneum at the hapten application site in the elicitation phase. An increase in the water content induced by exposure to high humidity for 6 hours was sufficient to ameliorate the magnitude of CHS even in mice with the genetic defect in attenuating the CHS responses, such as flaky tail mice. The reduced CHS was associated with downregulation of IL-1α, IL-4, and IFN-γ mRNA expression. Epicutaneously applied hapten can penetrate more readily through the stratum corneum with lower water content than that with higher water content, even after tape-stripping. These findings indicate that increased levels of water in the stratum corneum serve to ameliorate the CHS beyond the genetic effects.

Epidermal Neuromedin U Attenuates IgE-Mediated Allergic Skin Inflammation.

Although keratinocyte-derived neuropeptide neuromedin U (NMU) mediates the proinflammatory effects of innate-type mast cell activation, no information is available on the physiological roles. Here, to investigate the effects of NMU on IgE-mediated allergic skin inflammation, we determined whether IgE-mediated inflammation associated with severe scratching was induced in Nmu-/- mice administered repeated hapten applications to the ear or footpad. Dry skin was induced by targeted deletion of Nmu. Mice administered repeated hapten application developed IgE-mediated allergic inflammation characterized by severe scratching and increased serum IgE levels only when the ear, and not the footpad, was subjected to scratching, indicating that depletion of NMU from the epidermis alone does not drive such allergic inflammation. Thus, the susceptibility of Nmu-/- mice to allergic inflammation depends primarily on scratching dry skin. Further, allergic skin inflammation mediated by FcεRI cross-linking in Nmu-/-mice was inhibited by prior injection of NMU. These results indicate that NMU plays an important physiological role as a negative regulator during the late stage of IgE-mediated allergic skin inflammation.

Pathological role of regulatory T cells in the initiation and maintenance of eczema herpeticum lesions.

It remains unknown why the occurrence of eczema herpeticum (EH) caused by an extensive disseminated cutaneous infection with HSV-1 or HSV-2 is associated with the exacerbation of atopic dermatitis lesions after withdrawal of treatment. Although regulatory T cells (Tregs) limit the magnitude of HSV-specific T cell responses in mice, their role in the induction and resolution of EH has not been defined. We initially investigated the frequencies, phenotype, and function of Tregs in the peripheral blood of atopic dermatitis with EH (ADEH) patients at onset and after clinical resolution, atopic dermatitis patients without EH, and healthy controls. Tregs with the skin-homing phenotype and the activated/induced phenotype were expanded at onset and contracted upon resolution. Treg-suppressive capacity was retained in ADEH patients and, the expanded Tregs suppressed IFN-γ production from HSV-1-specific CD8(+) and CD4(+) T cells. The increased frequency of CD14(dim)CD16(+) proinflammatory monocytes (pMOs) was also observed in the blood and EH skin lesions. Thus, pMOs detected in ADEH patients at onset were characterized by an increased ability to produce IL-10 and a decreased ability to produce proinflammatory cytokines, unlike their normal counterparts. Our coculture study using Tregs and pMOs showed that the pMOs can promote the expansion of inducible Tregs. Tregs were detected frequently in the vicinity of HSV-expressing and varicella zoster virus-expressing CD16(+) monocytes in the EH lesions. Expansions of functional Tregs, together with pMOs, initially required for ameliorating excessive inflammation occurring after withdrawal of topical corticosteroids could, in turn, contribute to the initiation and progression of HSV reactivation, resulting in the onset of EH.

Application of a heat- and steam-generating sheet increases peripheral blood flow and induces parasympathetic predominance.

To promote the practical application of a Japanese traditional medical treatment, such as hot compresses, we developed a plaster-type warming device consisting of a heat- and steam-generating sheet (HSG sheet). First, we tested its effects when applied to the anterior abdominal wall or lumbar region of women complaining of a tendency towards constipation. Application of the sheet to either region produced a feeling of comfort in the abdomen, as assessed by a survey of the subjects. The significant increases in the total hemoglobin observed in these regions suggested an increase in peripheral blood flow, and significant increases in the HF component on ECG and in the amplitude of gastric motility suggested parasympathetic predominance. We concluded that application of the HSG sheet improves the peripheral hemodynamics and autonomic regulation, induces a feeling of comfort in the abdomen, and provides a beneficial environment for the improvement of gastrointestinal movements.