A Case of Congenital Leukemia With MYB-GATA1 Fusion Gene in a Female Patient.

We report a female newborn with acute myelogenous leukemia (AML) associated with a MYB-GATA1 fusion gene. Morphologic findings of myeloid lineage were obtained using light microscopy. Cytogenetic analysis of peripheral blood showed a complex karyotype: 46,X,-X,add(3)(q21),der(6)add(6)(q21)del(6)(q?), +mar1[5]/46,XX[15]. Targeted RNA sequencing revealed a MYB-GATA1 fusion gene. Reduced-dose AML-type chemotherapy resulted in remission and survival for >3 years without relapse. The present case demonstrated the feasibility of carrying out targeted RNA sequencing for identifying MYB-GATA1 and supports the notion that neonatal AML with MYB-GATA1 with reduced chemotherapy may show better prognosis than other highly toxic therapies.

Amazake made from sake cake and rice koji suppresses sebum content in differentiated hamster sebocytes and improves skin properties in humans.

Amazake is a traditional Japanese health drink. Here, we examined the effects of amazake on skin in cells and humans. Treatment with sake cake or rice koji suppressed intracellular lipid accumulation in differentiated hamster sebocytes, likely through the reduced expression of peroxisome proliferator-activated receptor-gamma (PPARγ) mRNA. In double-blind, placebo-controlled trial, seventeen Japanese women ingested either amazake or placebo for 4 weeks. Ingestion of the amazake decreased the sebum content compared to the placebo. The questionnaires showed improvements in "face color," "dark circles under the eyes," "glossy hair," and "waking up well", only in the amazake. In accordance with the questionnaires, additional analysis revealed the change in the L* values under the eyes was statistically increased in the amazake compared to the placebo. These results indicate that amazake may decrease sebum content in cells and humans and increase the L* values under the eyes, with some additional beneficial effects in humans.

Changes in physical function after hospitalization in patients with nursing and healthcare-associated pneumonia.

To clarify the functional changes after hospitalization due to pneumonia in elderly Japanese patients, we investigated the changes in physical functioning, nutritional routes, and diet that occurred after hospitalization in patients with nursing and healthcare-associated pneumonia (NHCAP). We analyzed 405 patients with NHCAP and compared findings with 448 patients with community-acquired pneumonia (CAP). Among the NHCAP patients, 140 (34%) patients showed a decline in activities of daily living function between baseline and discharge. After hospital discharge, 149 (37%) NHCAP patients did not return to the same residence location compared with where they were living prior to hospital admission. The frequency of this outcome was significantly higher in NHCAP patients than in CAP patients (p < 0.0001). After 6 months' follow-up, of the patients who transferred to different hospitals, 41 (73%) patients with CAP had returned to their own home, but only 16 (20%) patients with NHCAP could return home (p < 0.0001). Rates of alteration of nutritional route and type of diet from oral nutrition were significantly higher in NHCAP patients compared with CAP patients (22% vs 4%, p < 0.0001). Our results demonstrated that approximately one-third of hospitalized patients with NHCAP showed a decline in physical function. In addition, approximately one-fifth of NHCAP patients had changed their route of nutrition and type of diet. Our results indicated that physicians should attach greater importance to preventative measures against NHCAP rather than relying on antibiotic therapy post-infection in the management of pneumonia in elderly patients in order to extend their healthy life expectancy.

Transcriptional responses of human epidermal keratinocytes to cytokine interleukin-1.

Interleukin-1 is a proinflammatory and immunomodulatory cytokine that plays a crucial role in inflammatory diseases of the skin, including bacterial infections, bullous diseases, UV damage, and especially psoriasis. To characterize the molecular effects of IL-1 in epidermis, we defined the transcriptional changes in human epidermal keratinocytes 1, 4, 24, and 48 h after treatment with IL-1alpha. IL-1 significantly regulated 388 genes, including genes associated with proteolysis, adhesion, signal transduction, proliferation, and epidermal differentiation. IL-1 induces many genes that have antimicrobial function. Secreted cytokines, chemokines, growth factors, and their receptors are the prominent targets of IL-1 regulation, including IL-8, IL-19, elafin, C3, and S100A proteins, which implicate IL-1 in the pathogenesis of inflammatory diseases. IL-1 induced not only proliferation-associated genes but also differentiation marker genes such as transglutaminase-1 and involucrin, which suggests that IL-1 plays an important role in the aberrant proliferation and differentiation seen in psoriasis. Correlation of IL-1 regulated genes with the TNFalpha and IFNgamma regulated ones showed more similarities between IL-1 and TNFalpha than IL-1 and IFNgamma, whereas Oncostatin-M (OsM) affected a largely unrelated set of genes. IL-1 regulates many genes previously shown to be specifically over-expressed in psoriasis. In summary, IL-1 regulates a characteristic set of genes that define its specific contribution to inflammation and aberrant differentiation in skin diseases.

Serum anti-Fcgamma receptor autoantibodies in patients with alopecia areata.

Although anti-Fcgamma receptor antibodies (Abs) are detected in various autoimmune diseases, there have been no studies about the anti-Fcgamma receptor Abs in alopecia areata (AA). To detect the anti-Fcgamma receptor Abs in patients with AA and their clinical correlations, Serum samples from 72 patients with AA and 23 normal controls were examined by enzyme-linked immunosorbent assay assessing anti-Fcgamma receptor Ab levels. Anti-Fcgamma receptor I Abs were significantly frequently detected in patients with AA compared with normal controls. Furthermore, the detection of anti-Fcgamma receptor I Abs significantly inversely correlated with the disease duration. These results suggest that anti-Fcgamma receptor I Ab and Fcgamma receptor I play an important role in the regulation of AA, are useful for a marker of the disease prognosis and are worth intense research for the reasonable and specific therapy of AA.

Transcriptional profiling of epidermal differentiation.

In epidermal differentiation basal keratinocytes detach from the basement membrane, stop proliferating, and express a new set of structural proteins and enzymes, which results in an impermeable protein/lipid barrier that protects us. To define the transcriptional changes essential for this process, we purified large quantities of basal and suprabasal cells from human epidermis, using the expression of beta4 integrin as the discriminating factor. The expected expression differences in cytoskeletal, cell cycle, and adhesion genes confirmed the effective separation of the cell populations. Using DNA microarray chips, we comprehensively identify the differences in genes expressed in basal and differentiating layers of the epidermis, including the ECM components produced by the basal cells, the proteases in both the basal and suprabasal cells, and the lipid and steroid metabolism enzymes in suprabasal cells responsible for the permeability barrier. We identified the signaling pathways specific for the two populations and found two previously unknown paracrine and one juxtacrine signaling pathway operating between the basal and suprabasal cells. Furthermore, using specific expression signatures, we identified a new set of late differentiation markers and mapped their chromosomal loci, as well as a new set of melanocyte-specific markers. The data represent a quantum jump in understanding the mechanisms of epidermal differentiation.

Activation of Akt by mechanical stretching in human epidermal keratinocytes.

Mechanical stretching represents an important part of the signaling in skin. We have previously demonstrated that mechanical stretching induced proliferative phenotypes in human keratinocytes, as shown in increased 5-bromo-2'-deoxyuridine (BrdU) incorporation, ERK1/2 activation, and keratin K6 induction. Here we have further investigated the antiapoptotic signals in human keratinocytes provoked by mechanical stretching in vitro. Keratinocytes were plated on flexible silicone supports to transmit mechanical stretching to keratinocytes, involving continuous stretching by +20%. Stretching of these cells for 15-30 min caused the phosphorylation and activation of Akt. Inhibition of mitogen and extracellular signal-regulated kinase (MEK1/2) with U0126 and phosphoinositide 3-OH kinase (PI 3-K) with Wortmannin attenuated Akt activation. The epidermal growth factor (EGF) receptor kinase inhibitor, AG1478, and calcium channel inhibitor, gadolinium (Gd3+), also inhibited Akt activation. In summary, our results demonstrate the activation of the Akt signaling pathway by mechanical stretching, generating not only proliferative but also antiapoptotic signals in human keratinocytes.

Epithelioid sarcoma associated with lung adenocarcinoma.

Epithelioid sarcoma is an uncommon soft tissue malignancy which is often misdiagnosed as necrobiotic granuloma or chronic inflammation. Many patients have local recurrence and distant metastasis because of its infiltrating growth. We report a 49-year-old Japanese man who had an ulcer with surrounding erythema on the left forearm after he sustained a bruise on this spot. His mother and father had died of rectal and bladder carcinoma, respectively, and he had also had a seminoma previously. Therefore, there was familial accumulation of malignancies. A skin biopsy revealed polygonal, plump, or spindle-shaped epithelioid cells and lymphocytes infiltrating through the dermis. Immunohistochemical studies showed positive staining for cytokeratins, epithelial membrane antigen, CD34 and vimentin and negative staining for desmin, CD68, HHF-35, 1A4 and p53. These clinical and histological features were diagnostic of epithelioid sarcoma. CT scans detected nodules in the right lung. A lung biopsy revealed that he had well-differentiated adenocarcinoma which expressed p53. Mutations in the TP53 gene were not searched for. We selected conservative treatment because the patient did not want surgical wide resection or amputation. Therefore, radiation, thermotherapy, application of 0.5% doxorubicin hydrochloride ointment and right lower lobectomy with lymph node dissection were performed. The size of the ulcer and the tumor invasion along the fasciae of muscles were decreased, however, metastasis of the epithelioid sarcoma was detected in the lymph node of the left axilla four years after the diagnosis.

Characterization and localization of side population cells in mouse skin.

Recently, the detection of side population (SP) cells, which have the ability to strongly efflux Hoechst 33342 fluorescence dye, has attracted attention as a method of stem cell isolation. We identified SP cells from mouse skin using the same method as from bone marrow. This population almost completely disappeared after treatment with the calcium channel blocker verapamil. SP cells were mainly localized in the epidermis, with a few in the dermis. The ratio of SP cells decreased as the mouse became older. Surface marker analysis revealed that the sorted SP cells expressed alpha6-integrin, beta1-integrin, Sca-1, keratin 14, and keratin 19, which are proliferating and progenitor cell markers, at levels higher than in non-SP cells, while they expressed E-cadherin, CD34, and CD71 at lower levels. The expression of breast cancer resistance protein 1 (BCRP1), which participates in dye efflux, was expressed at high levels at both the protein and mRNA level in sorted SP cells. Immunohistochemical analysis showed that BCRP1 was expressed in the basal layers and hair bulge regions of mouse skin. BCRP1 mRNA was found in basal layers and hair follicles of newborn skin by in situ hybridization. These results indicate that the localization of BCRP1-positive cells is compatible with that of keratinocyte stem cells. Based on the close relationship between BCRP1 and the SP cell phenotype, we conclude that keratinocyte stem cells are closely related to the SP- or BCRP1-positive cells.

Mechanical stretching in vitro regulates signal transduction pathways and cellular proliferation in human epidermal keratinocytes.

Epidermal keratinocytes are continuously exposed to mechanical forces. The human skin surface can be thickened and enlarged by various stresses such as tissue expander or abrasive pressure. To investigate the mechanism of epidermal hyperproliferation by mechanical stress, keratinocytes were plated on flexible silicone dishes, which were continuously stretched by +20%. Stretching of cells for 24 h caused upregulation of 5-bromo-2'-deoxyuridine (BrdU)-positive cells to 200%-220% and activation of extracellular signal-regulated kinases (ERK)1/2. Inhibition of mitogen and ERK with U0126 and phosphoinositide 3-OH kinase attenuated BrdU incorporation and ERK1/2 activation. The EGF receptor kinase inhibitor and the calcium channel inhibitor also inhibited BrdU incorporation and the activation of ERK1/2. Twenty-four hours of stretching stimulated reporter activity driven by activator protein 1 (AP-1), induction of K6, and suppression of K10, which were inhibited by U0126. Our results indicate that mechanical stretching induces proliferative signals on human keratinocytes via induction of calcium influx, phosphorylation of epidermal growth factor receptor (EGFR), and ERK1/2. These mechanisms may contribute to the hyperproliferative nature of the epidermis, which is mechanically stretched by various stimuli.

Both IL-4 and IL-13 inhibit the TNF-alpha and IFN-gamma enhanced MDC production in a human keratinocyte cell line, HaCaT cells.

BACKGROUND: Macrophage-derived chemokine (MDC) is a Th2 type chemokine and its receptor CC chemokine receptor 4 (CCR4) is preferentially expressed on Th2 cells. Recent reports demonstrated that MDC is expressed not only by macrophages, dendritic cells and lymphocytes, but also by cultured human keratinocytes (KCs). However, the regulation of MDC production in KCs by various cytokines has not been well documented. OBJECTIVE: In this study, we investigated how Th1/Th2 cytokines regulate MDC production in a human KC cell line, HaCaT cells. METHODS: HaCaT cells were cultured with or without various cytokines for 24 h and RT-PCR was performed using these cells to evaluate MDC mRNA levels. ELISA was carried out using supernatant of HaCaT cells to calculate secreted MDC protein levels. RESULTS: MDC mRNA was weakly expressed in HaCaT cells, and upon stimulation with TNF-alpha or IFN-gamma, MDC expression was strongly upregulated. The supernatant MDC levels when stimulated with TNF-alpha or IFN-gamma were significantly higher than those without stimulation, and were synergistically increased when stimulated with a combination of TNF-alpha and IFN-gamma. Both interleukin-4 (IL-4) and IL-13 inhibited TNF-alpha and IFN-gamma enhanced MDC production in HaCaT cells in a dose-dependent manner. CONCLUSION: Th2-type cytokines IL-4 and IL-13 downregulate the production of MDC, a Th2 type chemokine, by KCs. This may partially contribute to maintaining Th1/Th2 balance in inflammatory skin diseases like atopic dermatitis.

Interleukin 15 induces the signals of epidermal proliferation through ERK and PI 3-kinase in a human epidermal keratinocyte cell line, HaCaT.

Interleukin 15 (IL-15) is a potent stimulator of proliferation and an inhibitor of apoptosis in lymphocytes. We attempted to elucidate the mechanism of IL-15 function in HaCaT keratinocytes. We found that 5-bromo-2(')-deoxyuridine incorporation increased in a dose-dependent manner with IL-15. This was blocked by MEK inhibitor U0126 or PI 3-K inhibitor LY294002. ERK1/2 and Akt phosphorylation by IL-15 were detected in a dose- and time-dependent manner. U0126 and LY294002 abolished ERK1/2 and Akt phosphorylation, respectively. DNA fragmentation and Annexin V binding accompanied by UVB-induced apoptosis were reduced by 30-50% with IL-15. Taken together, IL-15 induced cellular proliferation and had an anti-apoptotic effect on keratinocytes, in which ERK1/2 and Akt phosphorylation played crucial roles. The signal transduction pathways of IL-15 in keratinocytes were partially elucidated; they share a substantial part with growth signals induced by EGF. These results suggest a therapeutic approach to inflammatory skin diseases by controlling these signals.

IL-4, but not IL-13, modulates TARC (thymus and activation-regulated chemokine)/CCL17 and IP-10 (interferon-induced protein of 10kDA)/CXCL10 release by TNF-alpha and IFN-gamma in HaCaT cell line.

It is known that both interleukin-4 (IL-4) and IL-13 are produced by Th2-type cells and share similar biological functions with each other. However, recently accumulated evidences have revealed that IL-4 may be involved in the Th1-type response. Both thymus and activation-regulated chemokine (TARC/CCL17), a ligand for CC chemokine receptor 4 that is mainly expressed on Th2-type cells, and interferon-induced protein of 10kDa (IP-10/CXCL10), a ligand for CXC chemokine receptor 3 that is mainly expressed on Th1-type cells, are produced by keratinocytes after the stimulation with the primary cytokines such as tumor necrotic factor-alpha (TNF-alpha) and/or interferon-gamma (IFN-gamma). In this study, we investigated the regulation of TARC or IP-10 production from HaCaT cells, an immortalized human keratinocyte cell line, after stimulation with TNF-alpha, IFN-gamma, IL-4 and/or IL-13. Without stimulation, HaCaT cells did not produce TARC. When both TNF-alpha and IFN-gamma were added, they increased synergistically (P<0.003). In addition, when HaCaT cells were stimulated with IL-4, but not IL-13, in combination with TNF-alpha and IFN-gamma, the supernatant TARC levels significantly decreased compared to those with both TNF-alpha and IFN-gamma (P<0.009). This inhibition was completely abolished with the addition of neutralizing anti-IL-4 antibody. The supernatant IP-10 levels also increased synergistically by stimulation with TNF-alpha and IFN-gamma for 24h (P<0.001). When IL-4, but not IL-13, was added to the medium and the cells were co-cultured with these cytokines, the IP-10 levels significantly increased compared to those with both TNF-alpha and IFN-gamma (P<0.04). Furthermore, the effects of IL-4 on TARC and IP-10 production in these cells were detected in a dose-dependent manner. These data strongly suggest that IL-4 may act not only as a mediator of Th1-type response but also as a down-regulator of Th2-type response in terms of the regulation of chemokine production by HaCaT cells.

Interferon-gamma, a strong suppressor of cell proliferation, induces upregulation of keratin K6, one of the inflammatory- and proliferation-associated keratins.

Keratin K6 is known as an inflammatory and hyperproliferative keratin, and is induced by an inflammatory and hyperproliferative agent. In this study, we demonstrated that interferon-gamma, an antiproliferative agent, also induces keratin K6. We used normal human ex vivo skin, normal human cultured keratinocytes, HaCaT keratinocytes, and DJM cells to examine the induction of K6 by interferon-gamma, by immunohistochemical staining, Western blot analysis, promoter chloramphenicol acetyl transferase assay, and reverse transcriptase polymerase chain reaction of mRNA. We succeeded in demonstrating the induction of keratin K6 by interferon-gamma in ex vivo human skin and HaCaT keratinocytes at the protein and message level, and in cultured normal human keratinocytes at the promoter level. The inhibition of the signal transducing activator of transcription 1 pathway by a dominant-negative transfer gene caused the inhibition of K6 induction by interferon-gamma, and the blocking of nuclear factor kappaB using antisense oligonucleotides also inhibited the K6 induction. We also blocked the released interleukin-1alpha from keratinocytes after stimulation with interferon-gamma by neutralizing antibodies, which showed a decrease in the K6 induction. Our results suggest that a small amount of interleukin-1alpha, which cannot induce K6 by itself, is secreted upon stimulation by interferon-gamma, and that the induction of K6 occurs through the synergistic effect of the interferon-gamma/signal transducing activator of transcription 1 and interleukin-1alpha/nuclear factor kappaB pathways. This is the first report to describe K6 induction in epidermal keratinocytes by interferon-gamma and indicate a probable signal transduction pathway, and demonstrates that K6 is a possible partner of K17 in the inflammatory process.