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Purpose: Effective treatment of colorectal cancer could benefit from understanding molecular characteristics including mutation profiles of important genes. This study aimed to explore the molecular characteristics of colorectal cancer based on next generation sequencing. Methods: The mutational characteristics by targeted next generation sequencing in 172 colorectal tumor samples from Korean patients were evaluated to explore their associations with clinical features. Targeted sequencing of 375 genes was performed with an average target-depth of 800X. Results: TP53 and APC showed higher mutation frequencies from the left-sided tumors, while CTNNB1 were more frequent from the right-sided tumors. The tumor suppressor NOTCH1 and the DNA strand break repair gene PALB2 were more frequently mutated in early onset tumors. KRAS and PTEN mutations were more frequent from patients with advanced cancers by cancer antigen markers. TP53 and BRAF mutations were more frequent from patients of T3 and T4 stages, where their variant allele fractions were generally higher in T4 tumors, implying that advanced tumors have higher fraction of cancer cells with TP53 and BRAF mutations. Mutational profiles of these patients were also assessed with other clinical features. Comparison of mutational characteristics with the Caucasian subjects from independent data showed that the identified mutational characteristics are largely Korean-specific except for a few key colorectal cancer genes. Conclusion: Next generation sequencing-based targeted sequencing can provide valuable information on molecular characterization of colorectal cancer patients, and its clinically relevant information can provide benefits to better understand colorectal cancer.

Amyloid-β (Aβ) accumulation in the hippocampus is an essential event in the pathogenesis of Alzheimer’s disease. Insoluble Aβ is formed through the sequential proteolytic hydrolysis of the Aβ precursor protein, which is cleaved by proteolytic secretases. However, the pathophysiological mechanisms of Aβ accumulation remain elusive. Here, we report that rats fed high-phytate diets showed Aβ accumulation and increased apoptotic neuronal cell death in the hippocampus through the activation of the amyloidogenic pathway in the hippocampus. Immunoblotting and immunohistochemical analyses confirmed that the overexpression of BACE1 β-secretase, a critical enzyme for Aβ generation, exacerbated the hippocampal Aβ accumulation in rats fed high-phytate diets. Moreover, we identified that parathyroid hormone, a physiological hormone responding to the phytate-mediated dysregulation of calcium and phosphate homeostasis, plays an essential role in the transcriptional activation of the Aβ precursor protein and BACE1 through the vitamin D receptor and retinoid X receptor axis. Thus, our findings suggest that phytate-mediated dysregulation of calcium and phosphate is a substantial risk factor for elevated Aβ accumulation and apoptotic neuronal cell death in rats.

Phosphate overload contributes to mineral bone disorders that are associated with crystal nephropathies. Phytate, the major form of phosphorus in plant seeds, is known as an indigestible and of negligible nutritional value in humans. However, the mechanism and adverse effects of high-phytate intake on Ca2+and phosphate absorption and homeostasis are unknown. Here, we show that excessive intake of phytate along with a low-Ca2+diet fed to rats contributed to the development of crystal nephropathies, renal phosphate wasting, and bone loss through tubular dysfunction secondary to dysregulation of intestinal calcium and phosphate absorption. Moreover, Ca2+supplementation alleviated the detrimental effects of excess dietary phytate on bone and kidney through excretion of undigested Ca2+-phytate, which prevented a vicious cycle of intestinal phosphate overload and renal phosphate wasting while improving intestinal Ca2+bioavailability. Thus, we demonstrate that phytate is digestible without a high-Ca2+diet and is a risk factor for phosphate overloading and for the development of crystal nephropathies and bone disease.

Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is an inflammatory sinonasal disorder characterized by eosinophilic inflammation and T-helper 2 skewing. Eosinophil accumulation in sinonasal mucosa comprises a major feature of CRSwNP. The study aimed to investigate the effect of the flavone wogonin in nasal polyposis by assessing its ability to induce eosinophil apoptosis in vitro and attenuate eosinophilic CRSwNP in mice. Double immunofluorescence, immunohistochemistry, flow cytometry, and immunoblotting were performed to evaluate hypoxia-inducible factor (HIF)-1α, survivin, and apoptotic markers in the human eosinophilic EoL-1 cell line or sinonasal tissues from patients with CRS with or without NPs. In sinonasal specimens from patients with CRS, HIF-1α and survivin were up-regulated in eosinophils from patients with NPs compared with levels in patients without NPs. Under hypoxia, HIF-1α and survivin expression was up-regulated in EoL-1 cells. Wogonin down-regulated both HIF-1α and survivin in EoL-1 cells. In addition, overexpression of survivin protected EoL-1 cells against apoptosis in response to wogonin. Moreover, wogonin attenuated nasal polyp formation in a murine model. Our findings suggest that wogonin could induce caspase-3 activation by suppressing HIF-1α and survivin expression in EoL-1 cells. Further studies regarding novel therapeutic options for CRSwNP targeting eosinophil apoptosis are needed.

Jang et al. show that eosinophils in the small intestine can suppress Th17 cell differentiation through the secretion of the IL-1 receptor antagonist., Eosinophils play proinflammatory roles in helminth infections and allergic diseases. Under steady-state conditions, eosinophils are abundantly found in the small intestinal lamina propria, but their physiological function is largely unexplored. In this study, we found that small intestinal eosinophils down-regulate Th17 cells. Th17 cells in the small intestine were markedly increased in the ΔdblGATA-1 mice lacking eosinophils, and an inverse correlation was observed between the number of eosinophils and that of Th17 cells in the small intestine of wild-type mice. In addition, small intestinal eosinophils suppressed the in vitro differentiation of Th17 cells, as well as IL-17 production by small intestinal CD4+ T cells. Unlike other small intestinal immune cells or circulating eosinophils, we found that small intestinal eosinophils have a unique ability to constitutively secrete high levels of IL-1 receptor antagonist (IL-1Ra), a natural inhibitor of IL-1β. Moreover, small intestinal eosinophils isolated from IL-1Ra−deficient mice failed to suppress Th17 cells. Collectively, our results demonstrate that small intestinal eosinophils play a pivotal role in the maintenance of intestinal homeostasis by regulating Th17 cells via production of IL-1Ra.

Accumulating data have indicated a fundamental role of eosinophils in regulating adipose tissue homeostasis. Here, we performed whole-genome RNA sequencing of the small intestinal tract, which suggested the presence of impaired lipid metabolism in eosinophil-deficient ΔdblGATA mice. ΔdblGATA mice fed a high-fat diet (HFD) showed reduced body fat mass, impaired enlargement of adipocytes, decreased expression of adipogenic genes, and developed glucose intolerance. HFD induced accumulation of eosinophils in the perigonadal white adipose tissue. Concordantly, adipocyte-differentiated 3T3-L1 cells promoted the migration of eosinophils through the expression of CCL11 (eotaxin-1) and likely promoted their survival through the expression of interleukin (IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor. HFD-fed ΔdblGATA mice showed increased infiltration of macrophages, CD4+ T-cells, and B-cells, increased expression of interferon-γ, and decreased expression of IL-4 and IL-13 in white adipose tissue. Interferon-γ treatment significantly decreased lipid deposition in adipocyte-differentiated 3T3-L1 cells, while IL-4 treatment promoted lipid accumulation. Notably, HFD-fed ΔdblGATA mice showed increased lipid storage in the liver as compared with wild-type mice. We propose that obesity promotes the infiltration of eosinophils into adipose tissue that subsequently contribute to the metabolic homeostasis by promoting adipocyte maturation.

Although atopic dermatitis (AD) is characterized by cytokine production predominantly mediated by T helper (Th) 2 cells, AD pathogenesis also involves innate immune and Th1 cells. To optimize the cytokine milieu required for accurate reproduction of AD-related gene expression profile in vitro, we evaluated the expression pattern of CCL22, CCL17, IL5, IL13, IL33, IL25, TSLP, FLG, and LOR in human lesional AD skin and cytokine-stimulated HaCaT cells. An increase in Th2 mediators (IL5, IL13, CCL22, CCL17, IL25, IL33, and TSLP) and a decrease in genes related to cornified cell envelope (filaggrin and loricrin) were observed in human AD lesions. Innate (tumor necrosis factor-α) and/or Th1/Th2 adaptive cytokines (interferon-γ/IL-4) were required for inducing these inflammatory changes in HaCaT cells, implying that a complex network of innate, Th1, and Th2 cytokines drives AD-like changes. Therefore, stimulation with various combinations of cytokines, beyond Th2 polarization, is necessary when HaCaT cell line is used to study genetic changes implicated in AD pathogenesis.

Eosinophils are multifunctional leukocytes that reside in the gastrointestinal (GI) lamina propria, where their basal function remains largely unexplored. In this study, by examining mice with a selective deficiency of systemic eosinophils (by lineage ablation) or GI eosinophils (eotaxin-1/2 double deficient or CC chemokine receptor 3 deficient), we show that eosinophils support immunoglobulin A (IgA) class switching, maintain intestinal mucus secretions, affect intestinal microbial composition, and promote the development of Peyer's patches. Eosinophil-deficient mice showed reduced expression of mediators of secretory IgA production, including intestinal interleukin 1β (IL-1β), inducible nitric oxide synthase, lymphotoxin (LT) α, and LT-β, and reduced levels of retinoic acid-related orphan receptor gamma t-positive (ROR-γt(+)) innate lymphoid cells (ILCs), while maintaining normal levels of APRIL (a proliferation-inducing ligand), BAFF (B cell-activating factor of the tumor necrosis factor family), and TGF-β (transforming growth factor β). GI eosinophils expressed a relatively high level of IL-1β, and IL-1β-deficient mice manifested the altered gene expression profiles observed in eosinophil-deficient mice and decreased levels of IgA(+) cells and ROR-γt(+) ILCs. On the basis of these collective data, we propose that eosinophils are required for homeostatic intestinal immune responses including IgA production and that their affect is mediated via IL-1β in the small intestine.

Eosinophils are proinflammatory granulocytes that are involved in the pathogenesis of various inflammatory reactions. However, their roles in psoriasis remain largely unknown. In this study, by examining the inflammatory features of the eosinophilic cell line EoL-1 and an imiquimod-induced murine model of psoriasis, we show that eosinophils provide inflammatory signals that accelerate the pathogenesis of psoriasis. EoL-1 cells constitutively expressed TLR7, which mediates acute and rapidly developing psoriatic inflammation. The activation of TLR7 on EoL-1 cells using R837 resulted in the secretion of inflammatory mediators that support the migration, activation, and survival of neutrophils. The underlying pathologic role of eosinophils in psoriatic inflammation was further substantiated by markedly decreased psoriasiform inflammation in imiquimod-treated ΔdblGATA mice, which have a systemic eosinophil deficiency. While imiquimod-treated wild-type mice showed a significant increase in the eosinophils in their skin, neutrophils remarkably outnumbered the eosinophils in the skin, lymph nodes, and spleen in wild-type mice after imiquimod application. In addition, lesional skin samples from psoriasis patients also showed up-regulated eosinophil cytotoxic granules, accompanied by marked neutrophil infiltration. Based on these collective data, we propose that eosinophils accelerate psoriatic inflammation by supporting inflammatory microenvironments to favor the activation and infiltration of neutrophils.

The antibody levels against the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1c) were measured in 276 patients with P. vivax malaria (patient group), 320 malaria-naïve healthy individuals (control group 1), and 70 malaria-naïve individuals with various disorders (control group 2) using the immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the direct sandwich ELISA. To evaluate the antibody response during relapse, 5 relapsed patients were tested using the IgM capture ELISA. The IgM antibodies were negative in 99.7% of control group 1 and in 100% of control group 2; they were positive in 90.6% of the patient group. The total antibody levels were positive in 88.4% of the patient group with the direct sandwich ELISA. The sera from the second malaria episode, i.e., relapsed patients, were 100% positive on the IgM capture ELISA. The results of this study suggest that the IgM capture ELISA may be a useful diagnostic method for P. vivax malaria for both primary infection and relapse.

Neutrophils and eosinophils, 2 prominent granulocytes, are commonly derived from myelocytic progenitors through successive stages in the bone marrow. Our previous genome-wide transcriptomic data unexpectedly showed that genes encoding a multitude of neutrophil primary granule proteins (NPGPs) were markedly downregulated during the end period of eosinophilic terminal differentiation when cord blood (CB) cluster of differentiation (CD) 34+ cells were induced to differentiate toward the eosinophil lineage during a 24-day culture period. Accordingly, this study aimed to examine whether NPGP genes were expressed on the way to eosinophil terminal differentiation stage and to compare their expression kinetics with that of genes encoding eosinophil-specific granule proteins (ESGPs). Transcripts of all NPGP genes examined, including proteinase 3, myeloperoxidase, cathepsin G (CTSG), and neutrophil elastase, reached a peak at day 12 and sharply declined thereafter, while transcript of ESGP genes including major basic protein 1 (MBP1) attained maximum expression at days 18 or 24. Growth factor independent 1 (GFI1) and CCAAT/enhancer-binding protein α (C/EBPA), transactivators for the NPGP genes, were expressed immediately before the NPGP genes, whereas expression of C/EBPA, GATA1, and GATA2 kinetically paralleled that of eosinophil granule protein genes. The expression kinetics of NPGPs and ESGPs were duplicated upon differentiation of the eosinophilic leukemia cell line (EoL-1) immature eosinophilic cells. Importantly, confocal image analysis showed that CTSG was strongly coexpressed with MBP1 in differentiating CB eosinophils at days 12 and 18 and became barely detectable at day 24 and beyond. Our results suggest for the first time the presence of an immature stage where eosinophils coexpress NPGPs and ESGPs before final maturation.

Brown adipose generates heat via oxidation of fatty acids by a mitochondrial uncoupling protein 1 (UCP1)-dependent process. In addition, a subpopulation of cells within subcutaneous white adipose tissue, known as beige adipocytes, also plays a role in thermogenesis. The biogenesis of beige adipocytes is induced by thermogenic signals, such as chronic cold exposure. Recently, it has been reported that eosinophils, type 2 cytokines of IL-4/13, and alternatively activated macrophages control the thermogenic cycle of beige adipocytes. Alternatively, activated macrophages induce UCP1 beige adipocytes through secretion of catecholamines. These results define the role of type 2 immune responses in the regulation of energy homeostasis.

Eosinophils are multifunctional leukocytes implicated in protection against helminth infections. Although eosinophils comprise between 1~5% of peripheral blood leukocytes, they primarily reside in the gastrointestinal tract under homeostatic conditions, and rapidly proliferate upon parasitic infection. Intestinal infection with Trichinella spiralis (T. spiralis) induces eosinophilia when the parasite enters the larval stages and larvae finally migrate to the skeletal muscle. Eosinophils are known to mediate parasite death through antibody-dependent cellular cytotoxicity. In this study, we aimed to address the functional significance of eosinophils in the intestinal phase of T. spiralis infection by analysis of immune responses in the Peyer’s patch (PP) of infected BALB/c and eosinophil-ablated ΔdblGATA mice. Trafficking of eosinophils to the PP was significantly increased, with upregulation of interleukin-5 at 14 days post infection. Eosinophil deficiency led to a significant augmentation of serum immunoglobulin (Ig) M and IgG1 antibody levels. In accordance with this, IgG1 + B cells in the PP were substantially increased in ΔdblGATA mice compared to that in BALB/c mice. Transforming growth factor-β expression in the PP of infected ΔdblGATA mice was significantly decreased compared to that in BALB/c mice, whereas the number of T. spiralis larvae in the diaphragm was increased. Taken together, these findings indicate that eosinophils contribute to the regulation of Th2 immune responses, and protect the host from T. spiralis attempting to establish larvae in the skeletal muscle.

Objective To investigate the effect of extracorporeal shock wave therapy (ESWT) on myofascial pain syndrome (MPS). Method Thirty patients with MPS in trapezius muscle were randomly divided into two groups, ESWT group (n=15), and trigger point injections (TPI)+transcutaneous electrical nerve stimulation (TENS) group (n=15). For a total of 3 weeks, ESWT was undertaken with 1,500 pulse each time at one week interval totaling 4,500 pulse, TPI for once a week totaling three times and TENS for five times a week totaling three weeks. Results The changes in pain threshold (lb/cm2) showed the values of 6.86±1.35 before first therapy, 11.43±0.27 after first therapy, and 12.57±0.72 after third therapy, while TPI+TENS group showed the values of 6.20±1.92 before first therapy, 8.80±0.48 after first therapy, and 9.60±2.19 after third therapy, and the changes between the groups were significantly different (p=0.045). The changes in visual analog scale were estimated to be 6.86±0.90 before first therapy, 2.86±0.90 after first therapy, and 1.86±0.69 after third therapy in case of ESWT group, whereas the figures were estimated to be 7.20±1.30 before first therapy, 4.60±0.55 after first therapy, and 2.80±0.84 after third therapy in case of TPI+TENS group, and the changes between the groups were significantly different (p=0.010). The changes in McGill pain questionnaire (p=0.816) and pain rating scale (p=0.644) between the groups were not significantly different. The changes in neck ROM were also not significantly different between the groups (p>0.05). Conclusion The ESWT in patients with MPS in trapezius muscle are as effective as TPI and TENS for the purpose of pain relief and improving cervical range of motion.

Objective To understand the injury pattern of contact burns from therapeutic physical modalities. Method A retrospective study was done in 864 patients with contact burns who discharged from our hospital from January 2005 to December 2008. The following parameters were compared between patients with contact burns from therapeutic modalities and from other causes: general characteristics, burn extent, cause of burn injury, place of occurrence, burn injury site, treatment methods, prevalence of underlying disease, and length of hospital stay were compared between patients with contact burns. Results Of the 864 subjects, 94 patients were injured from therapeutic modalities. A hot pack (n=51) was the most common type of therapeutic modality causing contact burn followed by moxibustion (n=21), electric heating pad (n=16), and radiant heat (n=4). The lower leg (n=31) was the most common injury site followed by the foot & ankle (n=24), buttock & coccyx (n=9), knee (n=8), trunk (n=8), back (n=6), shoulder (n=4), and arm (n=4). Diabetes mellitus was associated with contact burns from therapeutic modalities; the odds ratio was 3.99. Injuries took place most commonly at home (n=56), followed by the hospital (n=33), and in other places (n=5). Conclusion A hot pack was the most common cause of contact burns from therapeutic modalities, and the lower leg was the most common injury site. Injuries took place most commonly at home. The patients with contact burns from therapeutic modalities showed high correlation to presence of diabetes mellitus. These results would be helpful for the prevention of contact burns due to therapeutic modalities.

Eosinophils are abundant in the lamina propria of the small intestine, but they rarely show degranulation in situ under steady-state conditions. In this study, using two novel mAbs, we found that intestinal eosinophils constitutively expressed a high level of an inhibitory receptor signal regulatory protein α (SIRPα)/CD172a and a low, but significant, level of a tetraspanin CD63, whose upregulation is closely associated with degranulation. Cross-linking SIRPα/CD172a on the surface of wild-type eosinophils significantly inhibited the release of eosinophil peroxidase induced by the calcium ionophore A23187, whereas this cross-linking effect was not observed in eosinophils isolated from mice expressing a mutated SIRPα/CD172a that lacks most of its cytoplasmic domain (SIRPα Cyto−/−). The SIRPα Cyto−/− eosinophils showed reduced viability, increased CD63 expression, and increased eosinophil peroxidase release with or without A23187 stimulation in vitro. In addition, SIRPα Cyto−/− mice showed increased frequencies of Annexin V-binding eosinophils and free MBP+CD63+ extracellular granules, as well as increased tissue remodeling in the small intestine under steady-state conditions. Mice deficient in CD47, which is a ligand for SIRPα/CD172a, recapitulated these phenomena. Moreover, during Th2-biased inflammation, increased eosinophil cell death and degranulation were obvious in a number of tissues, including the small intestine, in the SIRPα Cyto−/− mice compared with wild-type mice. Collectively, our results indicated that SIRPα/CD172a regulates eosinophil homeostasis, probably by interacting with CD47, with substantial effects on eosinophil survival. Thus, SIRPα/CD172a is a potential therapeutic target for eosinophil-associated diseases.

Objective To assess the effectiveness of modified dynamic metacarpophalangeal joint flexion orthoses for treatment of post-burn hand contractures. Method We enrolled 42 hand burn patients with limited range of motion at the metacarpophalangeal joints in this study. The patients were randomly assigned into either a control or an orthotic group. Both groups received the standard rehabilitation therapy focused on hand therapy; 21 subjects in the orthotic group wore a splint for 3 hours per day for 8 weeks. Hand function was measured by active range of motion, grip strength and other assessment tools. All parameters were estimated using the Mann-Whitney U test at the beginning and the end of the treatment after 8 weeks. Results The 21 subjects that had an orthotic intervention showed significant improvement in the range of motion at 2nd, 3rd, 4th and 5th metacarpophalangeal joints (p0.05). There was a significant difference in the hand function scales between the 2 groups (p

Graft-versus-host disease (GvHD) remains a major complication after allogeneic hematopoietic cell transplantation (HCT). Recent literature demonstrates a potential benefit of human mesenchymal stromal cells (MSC) for the treatment of refractory GvHD; however, the optimal dose remains uncertain. We set out to develop an animal model that can be used to study the effect of MSC on GvHD.A GvHD mouse model was established by transplanting C3H/he donor bone marrow (BM) cells and spleen cells into lethally irradiated BALB/c recipient mice. MSC were obtained from C3H/he mice and the C3H/10T1/2 murine MSC line.The mRNA expression of Foxp3 in regional lymph nodes (LN) localized with T cells was markedly increased by the addition of C3H10T1/2 cells in a real-time polymerase chain reaction (PCR). Using a mixed lymphocyte reaction, we determined the optimal splenocyte proliferation inhibition dose (MSC:splenocyte ratios 1:2 and 1:1). Three different C3H10T1/2 cell doses (low, 0.5 x 10(6), intermediate, 1 x 10(6), and high, 2 x 10(6)) with a consistent splenocyte dose (1 x 10(6)) were evaluated for their therapeutic potential in an in vivo GvHD model. The clinical and histologic GvHD score and Kaplan-Meier survival rate were improved after MSC transplantation, and these results demonstrated a dose-dependent inhibition.We conclude that MSC inhibit GvHD in a dose-dependent manner in this mouse model and this model can be used to study the effects of MSC on GvHD.

Background Mesenchymal stromal cells (MSC) comprise one of the BM stromal cells that are known to support hematopoiesis. It has also been suggested recently that MSC display immunosuppressive capacities through inhibiting the differentiation of monocyte-derived DC. DC travel to the lymph nodes (LN) to present Ag to T cells, and CCL21 is the chemokine that plays an important role in DC migration into the T-cell area of LN. We addressed the effect of MSC on this chemotactic activity of DC, one of the typical characteristics upon maturation. Methods BM cells were isolated and then cultured for generation of myeloid DC in the presence of GM-CSF and/or lipopolysaccharide with or without MSC. MSC were identified by flow cytometry of the immunologic markers and by performing colony-forming unit fibroblast assay. Migration of DC was observed with a newly developed time-lapse video microscopic technique. Results MSC co-culture inhibited the initial differentiation of DC, as well as their maturation. The matured DC actively migrated directionally in response to CCL21, a powerful DC-attracting chemokine, whereas the MSC co-cultured DC did not. Discussion Collectively, the findings of these experiments raise the possibility that MSC suppress the migratory function of DC and so they may serve immunoregulatory activities through the modulation of the Ag-presenting function of DC.

Purification of enough numbers of circulating eosinophils is difficult because eosinophils account for less than 5% peripheral blood leukocytes. Human eosinophilic leukemia EoL-1 cells have been considered an in vitro source of eosinophils as they can differentiate into mature eosinophil-like cells when incubated with dibutyryl cAMP (dbcAMP) or butyric acid. In this study, the viability and phenotypic maturation of EoL-1 cells stimulated by either dbcAMP or butyric acid were comparatively analyzed. After treatment with 100 µM dbcAMP or 0.5 µM butyric acid, EoL-1 cells showed morphological signs of differentiation, although the number of nonviable EoL-1 cells was significantly increased following butyric acid treatment. Stimulation of EoL-1 cells with 0.5 µM butyric acid more effectively induced the expression of mature eosinophil markers than stimulation with dbcAMP. These results suggest that treatment of EoL-1 cells with 0.5 µM butyric acid for limited duration could be an effective strategy for inducing their differentiation. Considering that expression of CCR3 was not sufficient in EoL-1 cells stimulated with 0.5 µM butyric acid, treatment of the chemically stimulated EoL-1 cells with cytokines, which primarily support eosinophil maturation, would help to obtain differentiated EoL-1 cells with greater functional maturity.

Eosinophils are multifunctional leukocytes that reside in several tissues, most abundantly in the small intestinal lamina propria under the steady state. To date, the phenotypic and functional characteristics of small intestinal eosinophils have remained poorly understood. In this study, we found that proliferation of ovalbumin (OVA)-specific CD4 + T cells isolated from the mesenteric lymph nodes of eosinophil-deficient ΔdblGATA mice were decreased relative to wild-type mice after oral immunization with OVA and cholera toxin (CT), the typical mucosal adjuvant that induces CD4 + T cell-dependent responses. ΔdblGATA mice showed reduced mucosal secretion of OVA-specific IgA and IgG1 while maintaining a systemic level of anti-OVA IgG1 upon oral immunization with OVA and CT. These findings suggest that eosinophils might have a role in the modulation of T cell-mediated immune responses including mucosal antibody responses in the gastrointestinal tract following oral immunization.

Objective To investigate the effect of extracorporeal shock wave therapy (ESWT) on painful stump neuroma. Methods Thirty patients with stump neuroma at the distal end of an amputation site were assigned randomly to the ESWT group (n=15) and the transcutaneous electrical nerve stimulation (TENS)+desensitization+pharmacological treatment group (n=15). For 3 weeks, the ESWT group received a weekly session involving 1,500 pulses at 0.10 mJ/mm2, while the control group was treated 10 times each, 40 minutes per day with TENS and desensitization treatment, and daily medication for 3 weeks. ESWT stimulation was given by focusing on the area at the neuroma site clearly identified by ultrasound. Results The changes in the McGill pain questionnaire were 38.8±9.0 prior to treatment and 11.8±3.1 following the treatment. The corresponding values for the control group were 37.2±7.7 and 28.5±10.3. The changes between groups were significantly different (p=0.035). The change in visual analog scale prior to and after treatment was 7.0±1.5 and 2.8±0.8 in the ESWT group, respectively, and 7.2±1.4 and 5.8±2.0 in the control group. These changes between the groups were also significantly different (p=0.010). The outcome in the pain rating scale also showed significant differences between groups (p0.05). Conclusion The study findings imply that ESWT for stump neuroma is superior to conventional therapy.

The traditional light microscopy has limitations for precise growth assays of malaria parasites in culture or for assessment of new compounds for antimalarial activity; the speed and high reproducibility of flow cytometry can overcome these limitations. A flow cytometric method using PicoGreen, a DNA-binding fluorochrome, was developed with optimal precision suitable for performing growth assays of low-parasitemia field isolates. In addition, intra- and inter-person reproducibility of the flow cytometric and the microscopic method were compared in order to quantitatively demonstrate the improved precision. RNase treatment contributed to the precision of the flow cytometric measurements by enhancing the signal-to-noise ratios. Coefficients of variation of the method were smaller than 10% for 0.1% or higher parasitemia samples. The intra- and inter-person coefficients of variation of the flow cytometric method were three to six times smaller than those of the microscopic method. The flow cytometric method developed in this study yielded substantially more precise results than the microscopic method, allowing determination of parasitemia levels of 0.1% or higher, with coefficients of variation smaller than 10%. Thus, the PicoGreen method could be a reliable high sensitivity assay for analysis of low parasitemia samples and might be applied to a high throughput system testing antimalarial drug activity.

While methicillin-resistant Staphylococcus aureus (MRSA) isolated from urinary tract infection (UTI) has recently increased in elderly adult urology patients, it has been only rarely reported in infants. Therefore, in this study to understand whether MRSA may be involved in UTI of infants who run fever without other apparent causes, we identified and counted S. aureus and S. epidermidis in suprapubic urine from 750 febrile infants via microbiological methods, and confirmed the counts via multiplex PCR. And we also detected four virulence genes, mecA, PVL, bbp and icaA genes for S. aureus and S. epidermidis via multiplex PCR in the same specimens. S. aureus (28 cases) counts were as follows: ＞10⁴ CFU/㎖ (3/28), 10²~10³ CFU/㎖ (1/28) and ＜10²~10³ CFU/㎖ (24/28). S. epidermidis (26 cases) counts were as follows: ＞10⁴ CFU/㎖ (2/26), 10²~10³ CFU/㎖ (4/26) and 10²~10³ CFU/㎖ (20/26). S. aureus virulence genes were detected in 26 cases as mecA (16/26, 59.3%), PVL (17/26, 63.0%), bbp (7/26, 26.9%) and icaA (20/26, 76.9%). S. epidermidis virulence genes were detected in 22 cases as mecA (17/22, 81.0%), PVL (15/22, 71.4%), bbp (3/22, 13.6%) and icaA (13/22, 50.1%). Therefore, mecA, PVL and icaA genes of MRSA and MRSE were detected with high positivity in urines from infants with fever. The results demonstrate that community-acquired MRSA or MRSE may be responsible for UTI incidence in febrile infants.

This study was designed to investigate the role of the hepatocyte growth factor (HGF) with regards to differentiation of somatic stem cells originating from the human umbilical cord blood (UCB) into hepatic lineage cells in vitro culture system. Methods: Mononuclear cells from UCB were cultured with and without HGF based on the fibroblast growth factor (FGF)-1, FGF-2, and stem cell factor. The cultured cells were confirmed by immunofluorescent staining analysis with albumin (ALB), cytokeratin-19 (CK-19), and proliferating cell nuclear antigen (PCNA) MoAb. ALB and CK-18 mRNA were also evaluated by reverse transcription-polymerase chain reaction. In order to observe changes in proliferating capacity with respect to the cultured period, CFSE with affinity to proliferating cells were tagged and later underwent flow cytometry. Results: In the HGF-treated group, cultured cells had a large oval shaped appearance with adherent, but easily detachable characteristics. In the HGF-non treated group, these cells were spindle-shaped with strong adherent characteristics. Expressions of ALB and CK-19 were evident in HGF-treated group compared to non-expression of those in to HGF-non treated group. Dual immunostaining analysis of the ALB producing cells showed presence of PCNA in their nuclei, and ALB and CK-18 mRNA were detected on the 21st day of cultured cells in the HGF-treated group. Conclusion: Our findings suggest that HGF has a pivotal role in differentiating somatic stem cells of human UCB into hepatic lineage cells in vitro.

Chemotaxis is one of the cardinal functions of leukocytes, which enables them to be recruited efficiently to the right place at the right time. Analyzing chemotactic activities is important not only for the study on leukocyte migration but also for many other applications including development of new drugs interfering with the chemotactic process. However, there are many technical limitations in the conventional in vitro chemotaxis assays. Here we applied a new optical assay to investigate chemotactic activities induced in differentiated HL-60 cells. Methods: HL-60 cells were stimulated with 0.8% dimethylformamide (DMF) for 4 days. The cells were analyzed for morphology, flow cytometry as well as chemotactic activities by a time-lapse videomicroscopic assay using a chemotactic microchamber bearing a fibronectin-coated cover slip and an etched silicon chip. Results: Videomicroscopic observation of the real cellular motions in a stable concentration gradient of chemokines demonstrated that HL-60 cells showed chemotaxis to inflammatory chemokines (CCL3, CCL5 and CXCL8) and also a homeostatic chemokine (CXCL12) after DFM-induced differentiation to granulocytic cells. The cells moved randomly at a speed of 6.99±1.24μm/min (n=100) in the absence of chemokine. Chemokine stimulation induced directional migration of differentiated HL-60 cells, while they still wandered very much and significantly increased the moving speeds. Conclusion: The locomotive patterns of DMF-stimulated HL-60 cells can be analyzed in detail throughout the course of chemotaxis by the use of a time-lapse videomicroscopic assay. DMF-stimulated HL-60 cells may provide a convenient in vitro model for chemotactic studies of neutrophils.