Macrophage depletion using clodronate liposomes reveals latent dysfunction of the hematopoietic microenvironment associated with persistently imbalanced M1/M2 macrophage polarization in a mouse model of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH), a hyperinflammatory syndrome, is caused by the incessant activation of lymphocytes and macrophages, resulting in damage to organs, including hematopoietic organs. Recently, we demonstrated that repeated lipopolysaccharide (LPS) treatment induces HLH-like features in senescence-accelerated (SAMP1/TA-1) mice but not in senescence-resistant control (SAMR1) mice. Hematopoietic failure in LPS-treated SAMP1/TA-1 mice was attributed to hematopoietic microenvironment dysfunction, concomitant with severely imbalanced M1 and M2 macrophage polarization. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment. Clodronate liposomes are useful tools for in vivo macrophage depletion. In this study, we depleted macrophages using clodronate liposomes to determine their role in the hematopoietic microenvironment in SAMP1/TA-1 and SAMR1 mice. Under clodronate liposome treatment, the response between SAMR1 and SAMP1/TA-1 mice differed as follows: (1) increase in the number of activated M1 and M2 macrophages derived from newly generated macrophages and M2-dominant and imbalanced M1 and M2 macrophage polarization in the BM and spleen; (2) severe anemia and thrombocytopenia; (3) high mortality rate; (4) decrease in erythroid progenitors and B cell progenitors in the BM; and (5) decrease in the mRNA expression of erythroid-positive regulators such as erythropoietin and increase in that of erythroid- and B lymphoid-negative regulators such as interferon-γ in the BM. Depletion of residual macrophages in SAMP1/TA-1 mice impaired hematopoietic homeostasis, particularly erythropoiesis and B lymphopoiesis, owing to functional impairment of the hematopoietic microenvironment accompanied by persistently imbalanced M1/M2 polarization. Thus, macrophages play a vital role in regulating the hematopoietic microenvironment to maintain homeostasis.

Imbalanced M1 and M2 Macrophage Polarization in Bone Marrow Provokes Impairment of the Hematopoietic Microenvironment in a Mouse Model of Hemophagocytic Lymphohistiocytosis.

Lipopolysaccharide (LPS) treatment induced hemophagocytic lymphohistiocytosis in senescence-accelerated mice (SAMP1/TA-1), but not in senescence-resistant control mice (SAMR1). SAMP1/TA-1 treated with LPS exhibited functional impairment of the hematopoietic microenvironment, which disrupted the dynamics of hematopoiesis. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment, which regulates hematopoiesis. Qualitative and quantitative changes in activated macrophages in LPS-treated SAMP1/TA-1 are thought to contribute to the functional deterioration of the hematopoietic microenvironment. Thus, we examined the polarization of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages, and the dynamics of macrophage production in the BM of SAMP1/TA-1 and SAMR1 after LPS treatment. After LPS treatment, the proportions of M1 and M2 macrophages and the numbers of macrophage progenitor (CFU-M) cells increased in both SAMP1/TA-1 and SAMR1. However, compared to the SAMR1, the increase in the M1 macrophage proportion was prolonged, and the increase in the M2 macrophage proportion was delayed. The increase in the number of CFU-M cells was prolonged in SAMP1/TA-1 after LPS treatment. In addition, the levels of transcripts encoding an M1 macrophage-inducing cytokine (interferon-γ) and macrophage colony-stimulating factor were markedly increased, and the increases in the levels of transcripts encoding M2 macrophage-inducing cytokines (interleukin (IL)-4, IL-10, and IL-13) were delayed in SAMP1/TA-1 when compared to SAMR1. Our results suggest that LPS treatment led to the severely imbalanced polarization of activated M1/M2 macrophages accompanied by a prolonged increase in macrophage production in the BM of SAMP1/TA-1, which led to the impairment of the hematopoietic microenvironment, and disrupted the dynamics of hematopoiesis.

Oral Coadministration of Zn-Insulin with d-Form Small Intestine-Permeable Cyclic Peptide Enhances Its Blood Glucose-Lowering Effect in Mice.

Oral delivery of insulin remains a challenge owing to its poor permeability across the small intestine and enzymatic digestion in the gastrointestinal tract. In a previous study, we identified a small intestine-permeable cyclic peptide, C-DNPGNET-C (C-C disulfide bond, cyclic DNP peptide), which facilitated the permeation of macromolecules. Here, we showed that intraintestinal and oral coadministration of insulin with the cyclic DNP derivative significantly reduced blood glucose levels by increasing the portal plasma insulin concentration following permeation across the small intestine of mice. We also found that protecting the cyclic DNP derivative from enzymatic digestion in the small intestine of mice using d-amino acids and by the cyclization of DNP peptide was essential to enhance cyclic DNP derivative-induced insulin absorption across the small intestine. Furthermore, intraintestinal and oral coadministration of insulin hexamer stabilized by zinc ions (Zn-insulin) with cyclic D-DNP derivative was more effective in facilitating insulin absorption and inducing hypoglycemic effects in mice than the coadministration of insulin with the cyclic D-DNP derivative. Moreover, Zn-insulin was more resistant to degradation in the small intestine of mice compared to insulin. Intraintestinal and oral coadministration of Zn-insulin with cyclic DNP derivative also reduced blood glucose levels in a streptozotocin-induced diabetes mellitus mouse model. A single intraintestinal administration of the cyclic D-DNP derivative did not induce any cytotoxicity, either locally in the small intestine or systemically. In summary, we demonstrated that coadministration of Zn-insulin with cyclic D-DNP derivative could enhance oral insulin absorption across the small intestine in mice.

Dynamics of hematopoiesis is disrupted by impaired hematopoietic microenvironment in a mouse model of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening systemic hyperinflammatory disorder. We found recently that repeated lipopolysaccharide (LPS) treatment induces HLH-like features in senescence-accelerated mice (SAMP1/TA-1) but not in senescence-resistant control mice (SAMR1). In this study, we analyzed the dynamics of hematopoiesis in this mouse model of HLH. When treated repeatedly with LPS, the numbers of myeloid progenitor cells (CFU-GM) and B-lymphoid progenitor cells (CFU-preB) in the bone marrow (BM) rapidly decreased after each treatment in both strains. The number of CFU-GM in SAMP1/TA-1 and SAMR1, and of CFU-preB in SAMR1, returned to pretreatment levels by 7 days after each treatment. However, the recovery in the number of CFU-preB in SAMP1/TA-1 was limited. In both strains, the BM expression of genes encoding positive regulators of myelopoiesis (granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and interleukin (IL)-6), and negative regulators of B lymphopoiesis (tumor necrosis factor (TNF)-α) was increased. The expression of genes encoding positive regulators of B lymphopoiesis (stromal-cell derived factor (SDF)-1, IL-7, and stem cell factor (SCF)) was persistently decreased in SAMP1/TA-1 but not in SAMR1. Expression of the gene encoding p16INK4a and the proportion of ß-galactosidase-positive cells were increased in cultured stromal cells obtained from LPS-treated SAMP1/TA-1 but not in those from LPS-treated SAMR1. LPS treatment induced qualitative changes in stromal cells, which comprise the microenvironment supporting appropriate hematopoiesis, in SAMP1/TA-1; these stromal cell changes are inferred to disrupt the dynamics of hematopoiesis. Thus, hematopoietic tissue is one of the organs that suffer life-threatening damage in HLH.

Senescence-accelerated mice (SAMP1/TA-1) treated repeatedly with lipopolysaccharide develop a condition that resembles hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis is a life-threatening systemic hyperinflammatory disorder with primary and secondary forms. Primary hemophagocytic lymphohistiocytosis is associated with inherited defects in various genes that affect the immunological cytolytic pathway. Secondary hemophagocytic lymphohistiocytosis is not inherited, but complicates various medical conditions including infections, autoinflammatory/autoimmune diseases, and malignancies. When senescence-accelerated mice (SAMP1/TA-1) with latent deterioration of immunological function and senescence-resistant control mice (SAMR1) were treated repeatedly with lipopolysaccharide, SAMP1/TA-1 mice displayed the clinicopathological features of hemophagocytic lymphohistiocytosis such as hepatosplenomegaly, pancytopenia, hypofibrinogenemia, hyperferritinemia, and hemophagocytosis. SAMR1 mice showed no features of hemophagocytic lymphohistiocytosis. Lipopolysaccharide induced upregulation of proinflammatory cytokines such as interleukin-1ß, interleukin-6, tumor necrosis factor-α, and interferon-γ, and interferon-γ-inducible chemokines such as c-x-c motif chemokine ligands 9 and 10 in the liver and spleen in both SAMP1/TA-1 and SAMR1 mice. However, upregulation of proinflammatory cytokines and interferon-γ-inducible chemokines in the liver persisted for longer in SAMP1/TA-1 mice than in SAMR1 mice. In addition, the magnitude of upregulation of interferon-γ in the liver and spleen after lipopolysaccharide treatment was greater in SAMP1/TA-1 mice than in SAMR1 mice. Furthermore, lipopolysaccharide treatment led to a prolonged increase in the proportion of peritoneal M1 macrophages and simultaneously to a decrease in the proportion of M2 macrophages in SAMP1/TA-1 mice compared with SAMR1 mice. Lipopolysaccharide appeared to induce a hyperinflammatory reaction and prolonged inflammation in SAMP1/TA-1 mice, resulting in features of secondary hemophagocytic lymphohistiocytosis. Thus, SAMP1/TA-1 mice represent a useful mouse model to investigate the pathogenesis of bacterial infection-associated secondary hemophagocytic lymphohistiocytosis.

Organic anion transporters, OAT1 and OAT3, are crucial biopterin transporters involved in bodily distribution of tetrahydrobiopterin and exclusion of its excess.

Tetrahydrobiopterin (BH4) is a common coenzyme of phenylalanine-, tyrosine-, and tryptophan hydroxylases, alkylglycerol monooxygenase, and NO synthases (NOS). Synthetic BH4 is used medicinally for BH4-responsive phenylketonuria and inherited BH4 deficiency. BH4 supplementation has also drawn attention as a therapy for various NOS-related cardio-vascular diseases, but its use has met with limited success in decreasing BH2, the oxidized form of BH4. An increase in the BH2/BH4 ratio leads to NOS dysfunction. Previous studies revealed that BH4 supplementation caused a rapid urinary loss of BH4 accompanied by an increase in the blood BH2/BH4 ratio and an involvement of probenecid-sensitive but unknown transporters was strongly suggested in these processes. Here we show that OAT1 and OAT3 enabled cells to take up BP (BH4 and/or BH2) in a probenecid-sensitive manner using rat kidney slices and transporter-expressing cell systems, LLC-PK1 cells and Xenopus oocytes. Both OAT1 and OAT3 preferred BH2 and sepiapterin as their substrate roughly 5- to 10-fold more than BH4. Administration of probenecid acutely reduced the urinary exclusion of endogenous BP accompanied by a rise in blood BP in vivo. These results indicated that OAT1 and OAT3 played crucial roles: (1) in determining baseline levels of blood BP by excluding endogenous BP through the urine, (2) in the rapid distribution to organs of exogenous BH4 and the exclusion to urine of a BH4 excess, particularly when BH4 was administered, and (3) in scavenging blood BH2 by cellular uptake as the gateway to the salvage pathway of BH4, which reduces BH2 back to BH4.

Pulmonary Injury from Waterproofing Spray During a Hike.

A 48-year-old man developed general fatigue, dyspnea, and fever at an altitude of 1562 m from the morning of the first day of a 3-day hike. Despite pharyngeal discomfort and mild general fatigue, he felt that the symptoms were not sufficient to abandon his plan. He usually required 1.5 hours to reach Tokusawa (6.4 km from the starting point at an altitude of 1500 m), but this time he required 2.5 hours and slept briefly upon arrival at Tokusawa due to extreme fatigue and respiratory discomfort. His symptoms became aggravated, so he presented at a mountain clinic with oxygen saturation at 80% and body temperature of 37.6ºC. He was diagnosed with hypoxemia due to pneumonia and/or other disease(s) and was evacuated to a hospital where a chest computed tomography scan revealed ground glass opacity and infiltrative shadows. He was treated for pneumonia, but another doctor discovered during follow-up that the patient had sprayed 300 mL of a waterproofing aerosol on mountain equipment in a nonventilated, enclosed area of his home on the night before starting out on the hike. Therefore, waterproofing spray was considered to have caused pulmonary damage. Self-reporting or appropriate questionnaires are the only means of identifying this type of injury. The differential diagnosis of pulmonary problems in an outdoor setting should include toxic aerosol exposure from waterproofing spray.

Quantitative Analysis of Formaldehyde-induced Fluorescence in Paraffin-embedded Specimens of Malignant Melanomas and Other Melanocytic Lesions.

Inter-observer agreement is problematic in the histopathological diagnosis of melanoma and melanocytic naevi, even among expert pathologists. Formaldehyde-induced fluorescence (FIF) has been used for histochemical demonstration of catecholamines, 5-hydroxytryptamine and their immediate precursors. FIF can detect melanogenic activity and may be useful in differentiating malignant melanoma from other melanocytic lesions. The fluorescence of various types of melanocytic lesions has been previously studied quantitatively in formalin-fixed and paraffin-embedded sections. This study compared 2 sets of excitation and emission bands: 450-490 nm excitation/510-560 nm absorption filters (filter unit A) and 480 nm excitation/510< nm absorption filters (filter unit B). Higher FIF was observed with filter unit A than with filter unit B. FIF intensity of central regions was found to be higher than that of the peripheral regions. Mean FIF was significantly higher in malignant melanomas than in naevi. Fluorescence imaging with filter unit A gave better diagnostic performance. In conclusion, quantitative measurement of FIF is a useful marker of malignant potential.

Differential Regulation of Lympho-Myelopoiesis by Stromal Cells in the Early and Late Phases in BALB/c Mice Repeatedly Exposed to Lipopolysaccharide.

Chronic lipopolysaccharide (LPS) exposure to mice reduces the lymphoid compartment and skews the hematopoietic cell compartment toward myeloid-cells, which is considered to be a direct effect of LPS on hematopoietic stem cells. However, the effect of chronic LPS exposure on stromal-cells, which compose the hematopoietic microenvironment, has not been elucidated. Here, we investigated early- and late-phase effects of repeated LPS exposure on stromal-cells. During the early phase, when mice were treated with 5 or 25 µg LPS three times at weekly intervals, the numbers of myeloid-progenitor (colony forming unit-granulocyte macrophage (CFU-GM)) cells and B lymphoid-progenitor (CFU-preB) cells in the bone-marrow (BM) rapidly decreased after each treatment. The number of CFU-GM cells recovered from the initial decrease and then increased to levels higher than pretreatment levels, whereas the number of CFU-preB cells remained lower than pretreatment levels. In the BM, expression of genes for positive-regulators of myelopoiesis including granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and interleukin (IL)-6 and negative-regulators of B lymphopoiesis including tumor necrosis factor (TNF)-α was up-regulated, whereas expression of positive-regulators of B lymphopoiesis including stromal cell-derived factor (SDF)-1, IL-7, and stem cell factor (SCF) was down-regulated. During the late phase, the number of CFU-preB cells remained lower than pretreatment levels 70 d after the first treatments with 5 and 25 µg LPS, whereas the number of CFU-GM cells returned to pretreatment levels. IL-7 gene expression in the BM remained down-regulated, whereas gene-expression levels of SDF-1 and SCF were restored. Thus, chronic LPS exposure may impair stromal-cell function, resulting in prolonged suppression of B lymphopoiesis, which may appear to be senescence similar to the hematological phenotype.

Kinetics of hematopoietic stem cells and supportive activities of stromal cells in a three-dimensional bone marrow culture system.

In the bone marrow, hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment. Previously, we established a 3D bone marrow culture system. In this study, we analyzed the kinetics of hematopoietic cells, and more than 50% of hematopoietic progenitor cells, including CFU-Mix, CFU-GM and BFU-E in 3D culture were in a resting (non-S) phase. Furthermore, we examined the hematopoietic supportive ability of stromal cells by measuring the expression of various mRNAs relevant to hematopoietic regulation. Over the 4 weeks of culture, the stromal cells in the 3D culture are not needlessly activated and "quietly" regulate hematopoietic cell proliferation and differentiation during the culture, resulting in the presence of resting hematopoietic stem cells in the 3D culture for a long time. Thus, the 3D culture system may be a new tool for investigating hematopoietic stem cell-stromal cell interactions in vitro.

Lipopolysaccharide reciprocally alters the stromal cell-regulated positive and negative balance between myelopoiesis and B lymphopoiesis in C57BL/6 mice.

Hematopoiesis in the bone marrow (BM) and spleen is controlled by stromal cells. Inflammation promotes myelopoiesis and simultaneously suppresses B lymphopoiesis. However, the role of the reciprocal regulation of myelopoiesis and B lymphopoiesis by stromal cells during inflammation is not fully understood. We investigated inflammation-induced alteration of hematopoietic regulation in lipopolysaccharide (LPS)-treated mice. C57BL/6 female mice were intravenously injected with a single, 5-µg dose of LPS, which induced a rapid decrease in the number of granulocyte-macrophage progenitors (colony-forming unit granulocyte-macrophage; CFU-GM) and B cell progenitors (CFU-preB) in BM. The CFU-GM count rapidly recovered, whereas the recovery of CFU-preB was delayed. LPS induced a marked increase in the number of CFU-GM but not in the number of CFU-preB in spleen. After LPS treatment, gene expression levels of positive regulators of myelopoiesis such as granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in BM and spleen were markedly upregulated whereas levels of positive regulators for B lymphopoiesis such as stromal cell-derived factor (SDF)-1, stem cell factor (SCF), and IL-7 remained unchanged. Meanwhile, the negative regulator of B lymphopoiesis tumor necrosis factor (TNF)-α was markedly up-regulated. The number of CFU-GM in S-phase in BM increased after LPS treatment, whereas the number of CFU-preB in S-phase decreased. These results suggest that LPS-activated stromal cells induce positive-dominant regulation of myelopoiesis and negative-dominant regulation of B lymphopoiesis, which facilitates emergency myelopoiesis during inflammation by suppressing B lymphopoiesis, thereby contributing to the host defense against infection.

Rapid clearance of supplemented tetrahydrobiopterin is driven by high-capacity transporters in the kidney.

Tetrahydrobiopterin (BH(4)) is an essential cofactor of aromatic amino acid hydroxylases and NO synthase. Supplementation of BH(4) potentially targets cardiovascular dysfunction as well as inherited BH(4) deficiencies and BH(4)-responsive phenylketonuria. However, the high cost/effect ratio of the recommended daily dose of BH(4) supplementation acts against further popularization of this therapy. The aim of this study was to attenuate urinary excretion with the intention of improving efficacy of BH(4) supplementation. The rapid excretion of BH(4) in the urine was confirmed to be the major route of supplemented BH(4) loss. In addition to glomerular filtration into the urine, a dominant rapid exclusion by renal secretion was observed in rats (T((1/2))=16 min) when the plasma BH(4) was higher than about 1 nmol/mL (more than 10 times higher than normal), due to BH(4) supplementation. The rapidity of the process was slowed by prior administration of cyclosporin A, a representative anti-excretory drug, and the excretion decelerated to a moderate rate (T((1/2))=53 min). By the combined administration of BH(4) plus cyclosporin A, the blood BH(4) levels were dramatically elevated. It was hypothesized that the drug interfered with kidney excretion of BH(4) rather than by attenuating organ tissue distribution by inhibiting biopterin uptake from the plasma. Consistent with this hypothesis, biopterin levels after BH(4) administration were elevated in major organs in the presence of anti-excretory drugs without notable change in their BH(4) fraction which was consistently 95% or higher regardless of combined administration with the drugs. Targeting these putative transporters would be a promising approach for improving the efficiency of BH(4) supplementation therapy.

Combination of serum 5-S-cysteinyldopa, melanoma inhibitory activity and IL-8 improves the diagnostic accuracy of malignant melanoma compared with individual markers.

Early diagnosis of malignant melanoma is critical for effective treatment and reduced patient mortality. However, current clinical and histological variables show limited accuracy in diagnosis. Serum or urine level of 5-S-cysteinyldopa (5-S-CD) is a commonly used melanoma biomarker in Japan owing to its increased sensitivity compared with other melanoma markers. However, its use as a diagnostic marker has shown some limitations. Therefore, here we examined the combination of 5-S-CD with melanoma inhibitory activity, which showed sensitivity in detecting melanoma comparable with that of 5-S-CD, and interleukin-8, a cytokine linked with melanoma progression, in a cohort of Japanese patients with melanoma. Our results revealed that the triple combination of 5-S-CD, melanoma inhibitory activity, and interleukin-8 showed high diagnostic accuracy in detecting melanoma compared with each of the individual factors. Importantly, the triple marker showed specificity and utility in detecting early-stage melanoma. Our results suggest the utility of the triple marker as a diagnostic biomarker for melanoma patients.

Mast cell development and biostresses: different stromal responses in bone marrow and spleen after treatment of myeloablater, 5-fluorouracil, and inflammatory stressor, lipopolysaccharide.

Mast-cell-development in the bone-marrow (BM) and the spleen is restrictedly controlled by stromal-cells which produce positive-regulators such as stem cell factor (SCF), and negative-regulators such as transforming growth factor-ß (TGF-ß). How the balance between positive- and negative-regulation is achieved or maintained by stromal-cells is not well understood. We intravenously injected 5-fluorouracil (5-FU) and lipopolysaccharide (LPS) into C3H/HeN mice to disrupt mast-cell-development in order to reveal mechanisms of mast-cell-regulation. 5-FU treatment induces a rapid decrease in the number of mast-cell-progenitor (colony-forming unit (CFU)-mast) cells in the BM and spleen, followed by rapid recovery of CFU-mast numbers. Expression of the SCF gene is one-fiftieth the level of that of TGF-ß during the steady-state in BM and spleen. After 5-FU treatment, SCF mRNA levels in the BM markedly increased, approaching TGF-ß mRNA levels, whereas SCF levels in the spleen showed limited oscillations whose increases paralleled those in TGF-ß levels. In contrast, LPS treatment induces a rapid decrease in CFU-mast number in the BM and a rapid increase in of CFU-mast number in the spleen. After LPS treatment, SCF mRNA levels in the BM markedly decreased, whereas SCF levels in the spleen remained unchanged. These results suggest that regulation of mast-cell-development is dominated by negative-signals in the BM and spleen during the steady-state, and, under biostress-conditions such as 5-FU and LPS treatment, the balance between positive- and negative-regulation can be changed in the BM but not in the spleen. The difference in the regulation of mast-cell-development in the BM versus the spleen probably reflects the different roles of tissue-specific stromal-cells.

Superior mesenteric artery syndrome: risk factor for duodenal involvement in Henoch-Schönlein purpura.

BACKGROUND: The anatomical location of the third portion of the duodenum is between the superior mesenteric artery (SMA) and the abdominal aorta (AA). When the aorto-mesenteric angle (AMA) is small and the aorto-mesenteric distance (AMD) is short, the duodenum becomes tightly compressed between these two blood vessels. Severe compression can obstruct the duodenum, resulting in vomiting and abdominal pain. This clinical condition is termed superior mesenteric artery syndrome (SMA syndrome). The duodenum is frequently affected in Henoch-Schönlein purpura (HSP). The aim of the present study was to verify that duodenal anatomy mimicking SMA syndrome is a risk factor for duodenal involvement and abdominal pain in HSP. METHODS: A prospective case-control study involving 12 HSP patients compared with 48 age- and sex-matched children (controls) was conducted. Bowel abnormalities were defined as thickened wall >3 mm and paralytic ileus detected on ultrasound. AMA, AMD and obesity index (OI) were compared between the two groups on Mann-Whitney testing. The correlation between duodenal bowel abnormalities and SMA syndrome was examined using Fisher's exact test. RESULTS: AMA, AMD and OI were significantly smaller in the HSP patients (P < 0.001, 0.003 and 0.026, respectively). All HSP patients, but only 10 controls, met the ultrasound diagnostic criteria for SMA syndrome (100% vs 20.8%, P < 0.001). CONCLUSIONS: Duodenal compression by SMA against the AA, which mimics SMA syndrome, may lead to duodenal bowel abnormalities with abdominal pain in thin children with HSP.

Age-related exacerbation of hematopoietic organ damage induced by systemic hyper-inflammation in senescence-accelerated mice.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening systemic hyper-inflammatory disorder. The mortality of HLH is higher in the elderly than in young adults. Senescence-accelerated mice (SAMP1/TA-1) exhibit characteristic accelerated aging after 30 weeks of age, and HLH-like features, including hematopoietic organ damage, are seen after lipopolysaccharide (LPS) treatment. Thus, SAMP1/TA-1 is a useful model of hematological pathophysiology in the elderly with HLH. In this study, dosing of SAMP1/TA-1 mice with LPS revealed that the suppression of myelopoiesis and B-lymphopoiesis was more severe in aged mice than in young mice. The bone marrow (BM) expression of genes encoding positive regulators of myelopoiesis (G-CSF, GM-CSF, and IL-6) and of those encoding negative regulators of B cell lymphopoiesis (TNF-α) increased in both groups, while the expression of genes encoding positive-regulators of B cell lymphopoiesis (IL-7, SDF-1, and SCF) decreased. The expression of the GM-CSF-encoding transcript was lower in aged mice than in young animals. The production of GM-CSF by cultured stromal cells after LPS treatment was also lower in aged mice than in young mice. The accumulation of the TNF-α-encoding transcript and the depletion of the IL-7-encoding transcript were prolonged in aged mice compared to young animals. LPS dosing led to a prolonged increase in the proportion of BM M1 macrophages in aged mice compared to young animals. The expression of the gene encoding p16INK4a and the proportion of ß-galactosidase- and phosphorylated ribosomal protein S6-positive cells were increased in cultured stromal cells from aged mice compared to those from young animals, while the proportion of Ki67-positive cells was decreased in stromal cells from aged mice. Thus, age-related deterioration of stromal cells probably causes the suppression of hematopoiesis in aged mice. This age-related latent organ dysfunction may be exacerbated in elderly people with HLH, resulting in poor prognosis.

Long-term remission of nephrotic syndrome with etanercept for concomitant juvenile idiopathic arthritis.

Etanercept is a tumor necrosis factor (TNF)-alpha inhibitor that has been applied beneficially for juvenile idiopathic arthritis (JIA). We experienced long-term remission of nephrotic syndrome (NS) in a boy treated with etanercept, which was initially used for concomitant JIA. He developed NS at age 3 years 7 months and had mostly been treated with cyclosporine because of steroid dependency and frequent relapses. Cyclosporine was halted at 10 years 7 months because of nephrotoxicity, and he was subsequently treated with mizoribine. However, he had three relapses in the first year and developed JIA at 11 years 7 months. He was treated with sulfasalazine, methotrexate, and prednisolone, but his arthritis persisted. Etanercept was started at 12 years 3 months. Thereafter, his arthritis went into complete remission. Surprisingly, he has remained relapse-free for both NS and JIA for more than 3 years with etanercept and mizoribine. It is difficult to know whether the NS remission after initiating etanercept was coincidental. However, there are many reports of increased TNF-alpha or soluble TNF-alpha receptor in NS relapse. To date, there are two reports of the efficacy of TNF-alpha inhibitors against NS. It is possible that TNF-alpha inhibitors may have potential as therapeutic agents for NS.

Kinetics of leukemic cells in 3D culture with stromal cells and with arginine deprivation stress.

Previously, we established a three-dimensional (3D) bone marrow culture system that maintains normal hematopoiesis, including prolongation of hematopoietic stem cell proliferation and differentiation. To analyze the role of bone marrow stromal cells that compose the microenvironment, the growth of a leukemic cell line (K562) in the 3D condition and with arginine deprivation stress was compared with two-dimensional stromal cell monolayers (2D) and suspension cultures without stromal cells (stroma (-)). Arginine is essential for the proliferation and differentiation of erythrocytes. The proliferation and differentiation of K562 cells cultured in the 3D system were stabilized compared with cells in 2D or stroma (-). Furthermore, the number of K562 cells in the G0/G1 phase in 3D was increased significantly compared with cells grown in 2D or stroma (-). Interestingly, the mRNA expression of various hematopoietic growth factors of stromal cells in 3D was not different from 2D, even though supportive activity on K562 cell growth was observed in the arginine deprivation condition. Thus, the hematopoietic microenvironment involves multi-dimensional and complex systems including biochemical and physiochemical factors that regulate quiescence, proliferation, activation, and differentiation of normal hematopoietic cells and cloned leukemic cells. Our 3D culture system may be a valuable new tool for investigating leukemic cell-stromal cell interactions in vitro.

Interferon-ß sensitizes human malignant melanoma cells to temozolomide-induced apoptosis and autophagy.

Malignant melanoma is a highly aggressive skin cancer that is highly resistant to chemotherapy. Adjuvant therapy is administered to patients with melanoma that possess no microscopic metastases or have a high risk of developing microscopic metastases. Methylating agents, including dacarbazine (DTIC) and temozolomide (TMZ), pegylated interferon (IFN)­α2b and interleukin­2 have been approved for adjuvant immuno­chemotherapy; however, unsatisfactory results have been reported following the administration of methylating agents. IFN­ß has been considered to be a signaling molecule with an important therapeutic potential in cancer. The aim of the present study was to elucidate whether antitumor effects could be augmented by the combination of TMZ and IFN­ß in malignant melanoma. We evaluated the efficacy of TMZ and IFN­ß by comparing O6­methylguanine­DNA transferase (MGMT)­proficient and ­deficient cells, as MGMT has been reported to be associated with the resistance to methylating agents. Cell viability was determined by counting living cells with a Coulter counter, and apoptosis was analyzed by dual staining with Annexin V Alexa Fluor® 488 and propidium iodide. The expression of proteins involved in the cell cycle, apoptosis and autophagy was evaluated by western blot analysis. The combined treatment with TMZ and IFN­ß suppressed cell proliferation and induced cell cycle arrest. We also demonstrated that a combination of TMZ and IFN­ß enhanced apoptosis and autophagy more efficiently compared with TMZ treatment alone. These findings suggest that antitumor activity may be potentiated by IFN­ß in combination with TMZ.

Predominant regeneration of B-cell lineage, instead of myeloid lineage, of the bone marrow after 1 Gy whole-body irradiation in mice: role of differential cytokine expression between B-cell stimulation by IL10, Flt3 ligand and IL7 and myeloid suppression by GM-CSF and SCF.

Irradiation of mice at doses of 1-1.5 Gy induced a predominant regeneration of the B-cell lineage but suppressed the regeneration of the myeloid lineage. The mechanisms underlying such reciprocal regulation of regeneration and the relationship between the two lineages remain unclear. Because the predominant regeneration of the B-cell lineage observed is considered to depend on the stromal cell function, and because the impairment of such stromal function may nullify such reciprocal responses, mouse models of senescent stromal cell impairment (SCI) and the less senescent stage of SCI (non-SCI) were compared to elucidate the mechanisms underlying the reciprocal regulation of both lineages after radiation exposure. In non-SCI mice irradiated with 1 Gy, the numbers of B-lymphocyte progenitor (CFU-preB) and granulocyte-macrophage progenitor (CFU-GM) cells in the bone marrow decreased rapidly during the first 24 h. Then the number of CFU-preB cells in the bone marrow promptly recovered from the nadir and exceeded the pretreatment level, whereas that of CFU-GM cells remained lower than the pretreatment level. The expression of genes encoding positive regulators of the B-lymphoid lineage [interleukin (IL)10, Flt3 ligand and IL7] was up-regulated; in contrast, expression of the positive regulators of the myeloid lineage [granulocyte macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF)] was down-regulated. In SCI mice irradiated with 1 Gy, the oscillatory changes in the numbers of femoral CFU-preB and CFU-GM cells and in the expression levels of cytokine genes were less marked than those in the non-SCI mice. These results thus imply that the reciprocal regeneration depends on the up-regulation of IL10, Flt3 ligand and IL7 expression and the down-regulation of GM-CSF and SCF expression in the bone marrow, possibly depending on the hematopoietic microenvironment.