Aggravation of Food Allergy by Skin Sensitization via Systemic Th2 Enhancement.

BACKGROUND: Recently, the relationship between antigen contact via skin (skin sensitization) and the development of food allergies has gained increasing attention. However, few studies have examined the effects of skin sensitization on healthy skin. OBJECTIVE: To examine the effect of sensitization in healthy skin on IgE and cytokine production during food allergy development. METHODS: The effect of skin sensitization on food allergy was evaluated using DO11.10 mice whose T cells express ovalbumin (OVA)-specific T-cell receptors. OVA was applied to the back skin of mice dehaired by various methods, and then food allergy was induced by providing them with an OVA-containing diet. OVA-specific IgE production in the sera and decreases in body temperature due to anaphylactic reaction were measured as indicators of food allergy. In addition, IL-4 production and proliferation of splenocytes were measured in mice with food allergy after skin sensitization. RESULTS: Skin sensitization in healthy skin increased IgE production and exacerbated anaphylactic symptoms induced by ingesting the antigen. Moreover, skin sensitization enhanced IL-4 production from splenocytes during the onset of food allergy. In contrast, oral tolerance was induced even after establishing skin sensitization. CONCLUSION: Skin sensitization temporarily exacerbated food allergy by enhancing systemic Th2 responses. These findings will help identify the mechanisms involved in food allergy and help develop treatments.

Clinicopathological and prognostic evaluations of anorectal cancer after fecal diversion for patients with Crohn's disease.

PURPOSE: Colorectum diversion with a proximal stoma is often the preferred surgical approach in patients with Crohn's disease-related anorectal lesions or refractory colitis. To date, few studies have assessed the incidence and prognosis of cancer in the diverted anorectal segments. This study aimed to evaluate the clinical characteristics and prognosis of anorectal cancer associated with Crohn's disease following fecal diversion. METHODS: This was a retrospective study based on medical records of patients diagnosed with Crohn's disease between 1999 and 2020. It was conducted at Yokohama Municipal Citizen's Hospital. Patients diagnosed with anorectal cancer following fecal diversion were identified, and their prognosis was the primary outcome measure. RESULTS: Among 1615 patients, 232 patients (14%) underwent colorectum diversion. Of those 232 patients, 11 were diagnosed with anorectal cancer following fecal diversion, ten were diagnosed with advanced cancer, 10 underwent abdominoperineal resection, and eight died. 1 could not undergo resection due to multiple lung metastasis and died. The overall five-year survival rate in patients diagnosed with anorectal cancer following fecal diversion was 20%. CONCLUSION: Crohn's disease-associated anorectal cancer following fecal diversion was challenging to diagnose early, and patients had a poor prognosis even after curative resection. Early abdominoperineal resection may be considered for patients with Crohn's disease who cannot benefit from cancer screening and surveillance due to difficulty accessing the anorectal stricture via endoscopy.

Gold nanoparticle-adjuvanted S protein induces a strong antigen-specific IgG response against severe acute respiratory syndrome-related coronavirus infection, but fails to induce protective antibodies and limit eosinophilic infiltration in lungs.

The spike (S) protein of coronavirus, which binds to cellular receptors and mediates membrane fusion for cell entry, is a candidate vaccine target for blocking coronavirus infection. However, some animal studies have suggested that inadequate immunization against severe acute respiratory syndrome coronavirus (SARS-CoV) induces a lung eosinophilic immunopathology upon infection. The present study evaluated two kinds of vaccine adjuvants for use with recombinant S protein: gold nanoparticles (AuNPs), which are expected to function as both an antigen carrier and an adjuvant in immunization; and Toll-like receptor (TLR) agonists, which have previously been shown to be an effective adjuvant in an ultraviolet-inactivated SARS-CoV vaccine. All the mice immunized with more than 0.5 µg S protein without adjuvant escaped from SARS after infection with mouse-adapted SARS-CoV; however, eosinophilic infiltrations were observed in the lungs of almost all the immunized mice. The AuNP-adjuvanted protein induced a strong IgG response but failed to improve vaccine efficacy or to reduce eosinophilic infiltration because of highly allergic inflammatory responses. Whereas similar virus titers were observed in the control animals and the animals immunized with S protein with or without AuNPs, Type 1 interferon and pro-inflammatory responses were moderate in the mice treated with S protein with and without AuNPs. On the other hand, the TLR agonist-adjuvanted vaccine induced highly protective antibodies without eosinophilic infiltrations, as well as Th1/17 cytokine responses. The findings of this study will support the development of vaccines against severe pneumonia-associated coronaviruses.

[Diagnosis and treatment of six patients with Crohn's disease complicated by recto-perineal-urethral fistula].

Recto-perineal-urethral fistula caused by Crohn's disease is a rare, for which fundamental treatment has not been established yet. We analyzed the clinical characteristics, diagnosis, treatment, and prognosis of six male patients with anorectal lesions caused by Crohn's disease. Three patients were treated with abdominoperineal resection and the other three with rectal excision without anastomosis. Excluding one case, which presented worsening immediately after surgery, symptoms of urethral fistula improved without urethral stenosis, dysuria, or sexual dysfunction. Recto-perineal-urethral fistula is currently treated with various treatment modalities, including colostomy and drug therapy. Although the evaluation of these modalities is required, abdominoperineal resection or rectal excision without anastomosis may be a valuable treatment option because these approaches improve the urethral fistula while sparing the urethra without any significant complications.

Sex Differences and the Heritability of Scute Pattern Abnormalities in the Green Sea Turtle from the Ogasawara Archipelago, Japan.

Although scute pattern abnormalities in sea turtle species are considered to be strongly correlated with survival rate, there is little information available regarding these abnormalities and the primary cause for their development is unclear. For the conservation of sea turtle species, accumulating basic knowledge of scute pattern abnormalities is a fundamental step towards a better understanding of the causes of these abnormalities. In the present study, we counted vertebral and costal scutes from adults hunted for food (male and female) (n = 899), nesting females (n = 155), and hatchlings (n = 44,537) of the green sea turtle (Chelonia mydas) from the Ogasawara Archipelago, Japan. We found that the frequency of turtles with non-modal scute patterns was significantly greater in adult females than that in adult males (P = 0.02). Since females are produced by warmer incubation temperatures, and the period of sex determination coincides with the period of scute pattern determination, high incubation temperatures may be responsible for the induction of scute pattern abnormalities. Moreover, females with non-modal scute patterns produced a higher frequency of hatchlings with non-modal scute patterns than females with modal scute patterns (P < 0.01), indicating that scute abnormality may be heritable. For conservation of this species, our results suggest that decrease of the incubation temperature by cooling methods, such as provision of shade, may minimize the frequency of non-modal scute patterns, with consideration for the natural sex ratio.

Degradation of staphylococcal enterotoxin A by a Pseudomonas aeruginosa isolate from raw milk.

Recently, we found that staphylococcal enterotoxin A (SEA)-producing Staphylococcus aureus strains produced SEA in raw milk with microbial contaminants at high temperatures like 40 °C only. Moreover, the concentration of SEA produced in raw milk gradually decreased after the peak. The reason(s) for SEA degradation in raw milk was studied in this study. Degradation of SEA spiked in raw milk was observed at 40 °C, but not at 25 °C. A Pseudomonas aeruginosa isolate from raw milk degraded SEA spiked in broth at 40 °C. A sample partially purified with a chromatographic method from culture supernatant of the isolate degraded SEA. Two main proteolytic bands were observed in the sample by zymographic analysis with casein. These results suggested that the SEA in raw milk might be degraded by a protease(s) produced by the P. aeruginosa isolate. This finding might be the first report on SEA degradation by a proteolytic enzyme(s) derived from Pseudomonas bacteria to our knowledge.

New methods for isolation of keratolytic bacteria inducing intractable hoof wall cavity (Gidoh) in a horse; double screening procedures of the horn powder agar-translucency test and horn zymography.

To establish a new system to isolate keratolytic bacteria from the hoof wall cavity (Gidoh) of a racehorse, we invented the horn powder agar-translucency (HoPAT) test and horn zymography (HZ). Using routine bacteriological techniques and these methods, we isolated five strains of keratolytic soil bacteria, which were then identified by means of 16S ribosomal RNA (rRNA) gene sequencing analysis. The findings from the study on the horse suggested that Brevibacterium luteolum played the main role in the local fragility of the hoof, eventually forming a Gidoh in coordination with four other strains of keratolytic bacteria. The double screening procedures of the HoPAT test and HZ were useful and easy techniques for isolating the keratolytic bacteria from the horn lesions.

A case of Crohn's disease complicated with simultaneous double cancers of the small bowel.

A case of Crohn's disease complicated with simultaneous double cancers of the small bowel is reported. The patient is a 66-year-old man who had suffered from Crohn's disease for 20 years. He underwent surgery to identify the source of repeated episodes of intestinal obstruction. Two short segments of strictures and proximal dilatations were found in the distal ileum. Therefore, we performed an en bloc resection of the two stenotic sections instead of strictureplasty. Histological examination of the resected specimen revealed not only Crohn's disease but also a well-differentiated adenocarcinoma within each of the two strictures. One had invaded to the muscular layer and the other to the subserosal layer. In cases of Crohn's disease with longstanding stenosis, the probability of carcinoma should be considered.

Population doubling level-dependent change of secreted glycosaminoglycan in equine bone marrow-derived mesenchymal stem cells.

In regenerative medicine using transplantation of mesenchymal stem cells (MSCs), the importance of regulating the quality of MSCs has been well recognized; however, there is little information concerning the relationship between the population doubling level (PDL) and the stemness of MSCs in equine medicine. In this study, we showed that the amount of glycosaminoglycan (GAG) secreted by bone marrow-derived MSCs (BMSCs) decreases with increase of PDL. Enzymatic digestion and two-dimensional electrophoresis revealed that a main component of GAG produced by BMSCs was hyaluronan with a small amount of chondroitin sulfate. Increase of PDL downregulated the expression of MSC CD markers, including CD44, CD73, CD90, CD105, and CD146, along with loss of differentiation capacity. Thus, the effect of hyaluronan supplement to the growth medium on both expression of CD markers and the tri-lineage potential of BMSCs was evaluated. Expression of CD73 and CD90 was preserved by continuous addition of hyaluronan to the growth medium, whereas mRNA levels corresponding to CD44, CD105 and CD146 were not preserved by supplementation of hyaluronan. BMSCs subcultured with hyaluronan-supplemented growth medium to PDL-12 showed osteogenic capacity, however adipogenic and chondrogenic activities at PDL-12 were not preserved by exogenous hyaluronan. These results suggest that downregulation of CD44, CD105 and CD146 might not affect the osteogenic capacity. Taken together, the results suggested that supplementation of hyaluronan to the growth medium might be effective at maintaining the osteogenic capacity of equine BMSCs.

[Recent advances in medical and surgical treatment of ulcerative colitis].

Recent advances in both medical and surgical treatment of ulcerative colitis have been remarkable. Changes in medical treatment are mainly good results of therapy with the anti-TNF-α antibody, tacrolimus, and those in surgical treatment are an expansion of the surgical indications to include patients with intractable disease, such as treatment refractoriness and chronic corticosteroid dependence, by a better postoperative clinical course after pouch surgery, improred selection of surgical procedures and the timing of surgery in elderly patients. To offer the optimal treatment for patients with ulcerative colitis, new medical therapies should be analyzed from the standpoint of the efficacy and limitations of effect. Long postoperative clinical course of surgical patients including colitic cancer, prevention of postoperative complications should be also analyzed.

Regulation of Tenomodulin Expression Via Wnt/ß-catenin Signaling in Equine Bone Marrow-derived Mesenchymal Stem Cells.

Tenomodulin has been recognized as a biomarker for tendon differentiation, and its gene expression is regulated by several transcription factors including Scleraxis and Mohawk. In this study, we found a novel regulatory mechanism of tenomodulin expression. Equine bone marrow-derived mesenchymal stem cells (BMSCs) in monolayer culture showed a low mRNA level of tenomodulin in comparison with the level in the tendon. When cultured in collagen gel containing a glycogen synthase kinase-3 (GSK-3) inhibitor (BIO), expression of tenomodulin in BMSCs increased up to the level in the tendon. Participation of GSK-3 in its gene expression was further demonstrated by a gene silencing experiment with small interference RNA corresponding to GSK-3, suggesting that Wnt/ß-catenin signaling mediated expression of tenomodulin. These results were confirmed by nuclear translocation of ß-catenin in BIO-treated BMSCs cultured in collagen gel. Under this culture condition, expression of tenomodulin-related transcription factors including Scleraxis and Mohawk was not affected, suggesting that Wnt/ß-catenin signaling was independent from these transcription factors. Additionally, BIO strongly enhanced expression of type XIV collagen in collagen-embedded BMSCs up to the level in the tendon, and other tendon-related extracellular matrix components such as decorin and fibromodulin were also upregulated. Taken together, these results indicated that activation of Wnt/ß-catenin signaling could induce differentiation of BMSCs into tenomodulin-expressing tendon cells in collagen gel.

Characterization of senescent mesenchymal stem/stromal cells derived from equine bone marrow and the effects of NANOG on the senescent phenotypes.

In equine regenerative medicine using bone marrow-derived mesenchymal stem/stromal cells (BM-MSC), the importance of the quality management of BM-MSC has been widely recognized. However, there is little information concerning the relationship between cellular senescence and the stemness in equine BM-MSC. In this study, we showed that stemness markers (NANOG, OCT4, SOX2 and telomerase reverse transcriptase) and colony forming unit-fibroblast apparently decreased accompanied with incidence of senescence-associated ß-galactosidase-positive cells by repeated passage. Additionally, we suggested that down-regulation of cell proliferation in senescent BM-MSC was related to increased expression of cyclin-dependent kinase inhibitor 2B (CDKN2B). On the other hand, forced expression of NANOG into senescent BM-MSC brought upregulation of several stemness markers and downregulation of CKDN2B accompanied with restoration of proliferation potential and osteogenic ability. These results suggested that expression of NANOG was important for the maintenance of the stemness in equine BM-MSC.

[Long-term outcomes of ileal pouch-anal canal anastomosis in children with ulcerative colitis].

The present study investigated the long-term outcomes of restorative proctocolectomy in pediatric patients with ulcerative colitis (UC). We report a series of 25 patients who underwent total proctocolectomy with ileal pouch-anal canal anastomosis (IACA). Surgery was performed for medically intractable colitis and severe colitis in 14 and 11 patients, respectively. Early and late complications were observed in 6 (24%) and 14 (56%) patients, respectively. The long-term quality of life outcomes were satisfactory, including both bowel function and social function. Growth retardation was observed in 6 patients. Five patients exhibited catch-up growth and 3 patients overcame growth retardation. Patients with growth retardation tended to have a younger onset and longer duration of UC, as well as a longer duration and higher total dose of steroid use. In conclusion, the long-term outcomes of pediatric patients with UC undergoing IACA are satisfactory. Furthermore, to minimize the risk of growth retardation, surgery should be performed without delay.

Superoxide dismutase 6 is required during metamorphosis for the development of properly movable legs in Tribolium castaneum.

The body form of holometabolous insects dramatically transforms from larval to adult stages during metamorphosis that occurs in the pupal stage. The larval disorganization and then new adult tissues are built up at this time. In motoneuron, larval neuronal cells degenerate, and new adult neurons are remodeled. Finally, adult neurons reconnect to new adult muscles. However, the factors that control metamorphosis have not yet been fully elucidated. Here, we show that an antioxidant enzyme, Tribolium castaneum superoxide dismutase 6 (TcSOD6), is secreted into the haemolymph and is required for proper movable legs during metamorphosis. TcSOD6 has a unique domain architecture and is mainly expressed in the pupal stage. The depletion of TcSOD6 expression in the pupa inhibits normal axon development and results in adults that display dysfunctional leg motions, suggesting that SOD6 expression is required for the development of properly movable legs. Therefore, we speculate that TcSOD6 might participate in some of the processes for larval neurons to be remodelled to new adult functions in the legs during metamorphosis, providing new insight into the evolution of SOD functions.

Injury induces a change in the functional characteristics of cells recovered from equine tendon.

Injury initiates a repair process characterized by influx of fibroblasts and the rapid formation of fibrous scar tissue and subsequent tissue contraction. The response to injury and behavior of the different tendon fibroblast populations, however, has been poorly characterized. We hypothesized that the fibroblasts recovered from tendon with acute injury would exhibit different cell properties relating to adhesion, migration and tensegrity. To test this hypothesis we evaluated the ability of fibroblasts recovered from normal and injured equine superficial digital flexor tendons (SDFTs). The injured tendon-derived cells showed greater contraction of the collagen gel but poorer adhesion to pepsin-digested collagen, and migration over extracellular matrix proteins compared to normal SDFT-derived fibroblasts. Thus, the cells present within the tendon after injury display different behavior related to wound healing.

Two cases of esophageal ulcer after surgical treatment for ulcerative colitis.

The incidence of postoperative esophageal ulcers has been rarely reported associated with severe ulcerative colitis (UC). We report two cases of esophageal ulcers accompanied by acute necrotizing esophagitis after undergoing surgery for severe UC. Both patients, 47- and 53-year-old, were diagnosed with severe UC and underwent subtotal colectomy with sigmoid mucous fistula and ileostomy. In both cases, they had epigastralgia or digestive track bleeding and upper gastrointestinal endoscopy revealed an esophageal ulcer with acute necrotizing esophagitis accompanied by a black degeneration of mucosa after surgery. Conservative treatments improved the lesions. Esophageal stricture requiring endoscopic dilatation occurred in both cases. An acute UC requiring surgery seems to warrant caution in the merger of esophageal ulcer and acute necrotizing esophagitis.

Investigating the effects of nest shading on the green turtle (Chelonia mydas) hatchling phenotype in the Ogasawara islands using a field-based split clutch experiment.

The Ogasawara Islands are an important rookery for the green turtle (Chelonia mydas) in the North Pacific. Green turtles possess temperature-dependent sex determination, and warmer incubation temperatures produce more females than males. Therefore, conservation practices such as nest shading may be required for this population to mitigate the effect of global warming on their sex ratio. To consider the application of such conservation practices in the Ogasawara population, it is fundamental to understand how artificially modified nest environments will affect green turtle hatchling phenotypes that influence their fitness. Here, we investigated the effects of nest shading on green turtle hatchling phenotypes in the Ogasawara population by using a split clutch experiment equally separating the clutch, relocating each half-clutch into an outdoor hatchery area either with or without shading, and observing the subsequent hatchling phenotype. Our results showed that the shading treatment produced hatchlings with a better self-righting response and a larger carapace size. Additionally, the shading treatment mostly reduced the production of hatchlings with a nonmodal scute pattern and produced hatchlings with a smaller unabsorbed yolk sac, which may be associated with their residual yolk mass. These results suggest that conservation practices such as shading could alter not only the sex ratio but also the hatchling phenotype that influences their fitness. Hence, our results suggest that applications of such conservation strategies must be carefully considered.

Threshold dose of piperonyl butoxide that induces reactive oxygen species-mediated hepatocarcinogenesis in rats.

To determine the threshold dose of piperonyl butoxide (PBO) that induces hepatocellular tumor-promoting effects, reactive oxygen species (ROS) generation, and drug-metabolizing enzymes that protect against ROS generation, partial hepatectomized rats were fed diets containing 0, 0.015, 0.03, 0.06, 0.125, 0.25, or 0.5% PBO after an i.p. injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Histopathologically, Glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in a dose-dependent manner in rats given 0.25% PBO or higher. The formation of microsomal ROS in the liver was significantly increased in 0.25 and 0.5% PBO. Real-time RT-PCR showed that the expression of the CYP1A1, UDPGTr-2, and Mrp3 genes was significantly upregulated in rats given 0.03% PBO or higher. These results suggest that 0.25% is the threshold dose of PBO that induces ROS-mediated hepatocarcinogenesis in rats, although the CYP1A1 gene that is related to ROS generation and the UDPGTr-2 and Mrp3 genes that are involved in protection against ROS were induced in the livers of rats even at a PBO dose of 0.03%.

Superoxide dismutase down-regulation and the oxidative stress is required to initiate pupation in Bombyx mori.

Perhaps, oxidative stress progresses pupation in some Lepidopteran insects; however, the reasons for this remain obscure. In our previous study, we clarified Bombyx mori SOD1 (BmSOD1) and B. mori SOD2 (BmSOD2) proteins respond in common to ultraviolet irradiation (UV) oxidative stress and metamorphosis. This result strongly suggested pupation initiates by oxidative stress and might mediate by down-regulation of expression of BmSOD1 and BmSOD2 proteins. Thus, we examined about these relationships in B. mori in this study. In the microarray data reanalysis, we found the Notch signaling pathways as the common pathways in pupation and UV oxidative stress in B. mori. Also, we showed a molting hormone, 20-hydroxyecdysone, leads not only generation of superoxide but also downregulation of the expression of BmSOD proteins during pupation in B. mori. Our findings can contribute to a deeper understanding of how biological defense systems work against environmental oxidative stress.

Mechanical strain increases expression of type XII collagen in murine osteoblastic MC3T3-E1 cells.

In adult mouse, the mRNA corresponding to the alpha1 chain of type XII collagen (alpha 1(XII)) is predominantly detected in the bone. Additionally, murine osteoblastic cells, MC3T3-E1, increased the mRNA level of alpha 1(XII) response to the mechanical strain in the stretch culture system. Cyclic stretch stress resulted in a threefold increase in mRNA level of alpha 1(XII) as compared to the control experiment in MC3T3-E1. Transient transfection assays employing a reporter construct, together with site-directed mutagenesis studies, suggested that the AP-1 binding site in the first exon of mouse alpha 1(XII) gene is important for stretch stress-mediated upregulation of alpha 1(XII) expression. Electrophoretic mobility shift assay and associated antibody supershift experiments showed that stretch stress promotes the binding of c-Jun and JunD. Further chromatin immunoprecipitation experiments confirmed the participation of these transcription factors in the region. Also, the exogenous induction of the dominant negative form of c-Jun canceled the effect of stretch stress on the stimulation of the alpha 1(XII) gene. Here, we reported a potential responsive element to the stretch stress in mouse alpha 1(XII) gene. These data will provide new information on the mechanical strain-mediated transcriptional control of alpha 1(XII)-mediated fibrillogenesis in the bone.