Number Of CD34-positive Cells Research Articles

Kinetic analysis of superparamagnetic iron oxide nanoparticles in the liver of body-temperature-controlled mice using dynamic susceptibility contrast magnetic resonance imaging and an empirical mathematical model

The purpose of this study was to develop a method for analyzing the kinetic behavior of superparamagnetic iron oxide nanoparticles (SPIONs) in the murine liver under control of body temperature using dynamic susceptibility contrast magnetic resonance imaging (DSC-MRI) and an empirical mathematical model (EMM).First, we investigated the influence of body temperature on the kinetic behavior of SPIONs in the liver by controlling body temperature using our temperature-control system. Second, we investigated the kinetic behavior of SPIONs in the liver when mice were injected with various doses of GdCl3, while keeping the body temperature at 36°C. Finally, we investigated it when mice were injected with various doses of zymosan, while keeping the body temperature at 36°C. We also investigated the effect of these substances on the number of Kupffer cells by immunohistochemical analysis using the specific surface antigen of Kupffer cells (CD68).To quantify the kinetic behavior of SPIONs in the liver, we calculated the upper limit of the relative enhancement (A), the rates of early contrast uptake (α) and washout or late contrast uptake (β), the parameter related to the slope of early uptake (q), the area under the curve (AUC), the maximum change of transverse relaxation rate (ΔR2) (ΔR2max), the time to ΔR2max (Tmax), and ΔR2 at the last time point (ΔR2last) from the time courses of ΔR2 using the EMM.The β and Tmax values significantly decreased and increased, respectively, with decreasing body temperature, suggesting that the phagocytic activity of Kupffer cells is significantly affected by body temperature. The AUC, ΔR2max, and ΔR2last values decreased significantly with increasing dose of GdCl3, which was consistent with the change in the number of CD68-positive cells. They increased with increasing dose of zymosan, which was also consistent with the change in the number of CD68-positive cells. These results suggest that AUC, ΔR2max, and ΔR2last reflect the number of Kupffer cells.In conclusion, we presented a method for analyzing the kinetic behavior of SPIONs in the liver using DSC-MRI and EMM, and investigated the influence of body temperature, GdCl3, and zymosan using body-temperature-controlled mice. The present study suggests that control of body temperature is essential for investigating the kinetic behavior of SPIONs in the liver and that our method will be applicable and useful for quantifying the responses of Kupffer cells to various drugs under control of body temperature.

Anti-tumor effect of the extract from Qingyihuaji formula on pancreatic cancer by down-regulating Notch-4 and Jagged-1

ObjectiveTo investigate, in terms of Notch signaling pathway, the effect on pancreatic cancer of the extract of an anti-tumor prescription – Qingyihuaji formula (QYHJ) – from Traditional Chinese Medicine (TCM). MethodsNude mice were implanted subcutaneously with human pancreatic cancer cell line SW1990 and then randomly divided into four groups: Control, QYHJ extract, Gemcitabine, and Combination of QYHJ extract and gemcitabine. Treatments were given for 21 days and tumor growth was evaluated simultaneously. Then, expression of Notch receptors (Notch-1, Notch-2, Notch-3, and Notch-4) and their Jagged ligands (Jagged-1 and Jagged-2) in dissected tumor tissue were detected by real-time quantitative reverse transcription-polymerase chain reaction and Western blot. Finally, immunohistochemistry was performed to detect CD133, a marker of pancreatic cancer stem cells (CSCs), to evaluate the impact of QYHJ extract on pancreatic CSCs. ResultsQYHJ extract treatment effectively inhibited the tumor growth in nude mice. The expression of both Notch-4 and Jagged-1 were decreased significantly in QYHJ treatment groups (P < 0.05), while gemcitabine alone had no significant effect in down-regulating Jagged-1 (P > 0.05). No significant difference was observed in the expression of Notch-1, Notch-2, Notch-3, and Jagged-2 between three treatment groups and control group (P > 0.05). Moreover, immunohistochemical analysis showed that the number of CD133 positive cells was significantly reduced by QYHJ treatment (P < 0.05), and the combined treatment was more effective than gemcitabine alone (P < 0.05). ConclusionThe role of the extract in pancreatic cancer treatment was associated with down-regulation of Notch-4 and Jagged-1 in Notch signaling pathway. The extract could enhance the antitumor activity of gemcitabine and was more effective than gemcitabine in regulating Notch signaling pathway to some extent.

Influence of dietary supplementation with flaxseed and lactobacilli on the cells of local innate immunity response in the jejunal mucosa in piglets after weaning

A histological study was designed to determine the influence of flaxseed and/or lactobacilli inclusion in the diet of piglets from 10 days before to 21 days after weaning. The selected inflammatory cell population incidence in the piglet jejunal mucosa was investigated. Significantly higher numbers of myeloperoxidase-positive (P<0.01) and CD163-positive (P<0.001) cells in the jejunal mucosa were recorded on the weaning day and for 7 days after (P<0.001 and P<0.01, respectively) in the flaxseed group compared with the basal diet. The number of intraepithelial lymphocytes was also significantly increased until 3 days after weaning (P<0.001). A prolonged significant increase in the myeloperoxidase-positive cells and intraepithelial lymphocyte numbers in the flaxseed+lactobacilli group was detected. In contrast, the number of CD163-positive cells in the flaxseed+lactobacilli group was significantly lower on the day of weaning (P<0.05) and 3 days after (P<0.01). The same effect was observed in the group with lactobacilli alone during the first 3 days after weaning (P<0.05 and P<0.01, respectively) and these findings indicate down-regulation of CD163 expression in the jejunal mucosa by lactobacilli. The presence of lactobacilli in the diet had a stimulatory effect on goblet cell quantity in the epithelium (P<0.001) and a distinct 50% reduction in the flaxseed group (P<0.01) compared with the basal diet was observed on the weaning day. A significant increase in myeloperoxidase-positive cell number in the jejunal mucosa in the flaxseed+lactobacilli group was the only significant difference (P<0.05 and P<0.01, respectively) found 21 days after weaning in comparison with all the other groups, indicating the pro-inflammatory effect of this feed additive combination. We conclude that dietary supplementation with flaxseed and lactobacilli on the cells of local innate immunity response in the jejunal mucosa in piglets after weaning might be linked with significant anti-inflammatory effects in the jejunal mucosa.

Psoriatic Arthritis Complicating Systemic Sclerosis: Possible Involvement of M2 Macrophages.

Dear Editor: Psoriatic arthritis (PsA) is a seronegative inflammatory arthritis that occurs in the presence of psoriasis. The synovial macrophages are a major source of proinflammatory cytokines1. The subset of macrophages that expresses alternatively activated macrophages (M2 macrophages) is usually greater in number in PsA synovitis than in rheumatoid arthritis synovitis2. Systemic sclerosis (SSc) is a rheumatological disorder characterized by excessive fibrosis and microvascular damage of the skin and various internal organs. The presence of macrophage infiltrates and increased numbers of M2 macrophages in fibrotic areas have been reported3. A 44-year-old Japanese man developed scaly erythema that had been present on the trunk and lower and upper extremities for 40 years. A primary care doctor had diagnosed his condition as psoriasis. His previous treatments included topical corticosteroid therapy, topical psoralen ultraviolet-A therapy, or oral cyclosporin. Sacroiliitis was also associated with the skin symptoms. For the previous 4 years, the scaly erythema had gradually improved but was then complicated by sclerodactylia. On examination, a high anti-nuclear antibody (ANA) titer with homogeneous, speckled, and nucleolar patterns (×640) was present. Detailed analysis of the ANA revealed that there was a significant increase in anti-topoisomerase I antibody (152 U/ml). Rheumatoid factor was negative. From these results, the presumptive diagnosis was SSc complicated by PsA, and the patient was admitted to our hospital. Psoriatic plaque was present on the elbows of the patient (Fig. 1A). The modified Rodnan total skin thickness score value of 22 was used to grade the degree of skin sclerosis (Fig. 1B). The levels of T helper 2 cytokines, such as interleukin (IL)-4 (17.0 pg/ml) and transforming growth factor (TGF)-β1 (15.1 ng/ml), were elevated. The serum Krebs von den Lungen-6 (1,182 U/ml) and surfactant protein D (226 ng/ml) levels were elevated, and high-resolution computed tomography revealed the presence of a bilateral consolidation of both lower lobes with peripheral ground glass opacity. Skin biopsies from the elbow and the forearm revealed the presence of markedly elongated rete ridges with parakeratosis (Fig. 1C), and sclerotic change that ascended from the lower dermis (Fig. 1D). We also performed immunostaining for CD68 (Fig. 1E, F) and CD163 (Fig. 1G, H) in lesional skin biopsy samples. Subequal infiltrated dermal cells expressed CD68, which suggested that macrophages infiltrated both types of samples of lesional skin. Furthermore, the numbers of CD163-positive cells were increased in lesional skin from SSc. This result suggests that M2 macrophages were increased in lesional skin from SSc compared with lesional skin from PsA (Fig. 1E~H). Fig. 1 Clinical and histopathological manifestations in the patient. (A) Psoriatic skin lesion affecting the elbow. (B) Widespread skin thickening, progressing from the fingers to the trunk. (C) The skin biopsy specimen from the elbow has characteristics consistent ... Polarized macrophages have been broadly classified into two groups: M1 and M2 macrophages. Tissue macrophages can exist in different activation states. They can be proinflammatory macrophages (M1; activated by interferon-γ or lipopolysaccharide) or anti-inflammatory macrophages (M2; activated by TGF-β or IL-4)4. They participate in tissue remodeling, immune modulation, parasite clearance, and tumor progression5. In this patient, M2 macrophages may be one of the key regulating cell types involved in the clinical manifestations. However, further investigations are needed for elucidating the involvement of M2 macrophages in skin disorders.

Histological evaluation of cell behavior in soft tissue around a 4-META/MMA-TBB resin cement applied to the bone tissue in rats

ObjectivesThe aim of this study was to evaluate the behavior of the soft tissue in rats around a 4-META/MMA-TBB resin and other types of bone cement. Methods4-META/MMA-TBB resin (SBV) and PMMA bone cement (SIMP) were applied to wounds made in the left femurs of 24 male Sprague-Dawley rats. Rats were sacrificed at 3 or 7 days after the operation; then, histological evaluation and immunohistochemical evaluation using a CD68 antibody were performed. ResultsHistologically, at 3 days after the operation, inflammation against operative stress was observed in all groups. At 7 days, marked formation of granulation tissue was recognized in the SIMP group compared with the SBV and control groups. Immunohistochemically, at 7 days, a number of CD68-positive cells were recognized in the SIMP group compared with the SBV and control groups. ConclusionThese results suggest that SBV has a superior biocompatibility to SIMP in the soft tissue around the rat femur.

Abstract 14771: Iron Plays a Role in the Thrombus Formation of a Rat Model of Deep Vein Thrombosis

Introduction: Deep vein thrombosis (DVT) is a major cause of pulmonary thromboembolism. On the other hand, iron is an essential element for maintaining physiological homeostasis in the body, whereas excess iron leads to toxicity and free radical damage. Hence, iron accumulation is associated with the pathogenesis of several cardiovascular diseases. We have previously reported that dietary iron restriction (IR) prevents the development of hypertension and chronic kidney disease (CKD) in animal models of several cardiovascular diseases. In addition, we have shown that intracellular iron transport protein, transferrin receptor 1 (TfR1), is involved in the mechanism of several cardiovascular diseases. In this study, we hypothesized that iron and TfR1 play roles in the pathogenesis of DVT. We investigate the effects of dietary IR on the thrombus formation and resolution in a rat model of DVT. Methods: We performed the ligation of inferior vena cava to induce DVT in 8-week-old male Sprague-Dawley rats. After the ligation, rats were given either a normal diet (DVT group) or iron-restricted diet (DVT+IR group). The thrombus was harvested at 5days after the ligation. Results: At first, TfR1 expression was increased in the thrombus of a rat model of DVT, suggesting that iron and TfR1 play roles in the pathogenesis of DVT. Interestingly, dietary iron restriction reduced the thrombus size compared with the DVT group (180±17 mg/cm vs 135±13 mg/cm, DVT vs DVT+IR ; p &lt; 0.05). Intrathrombotic collagen content was diminished in the DVT+IR group compared with the DVT group. Intrathrombotic gene expression and activity of matrix metalloproteinase (MMP)-9 were increased in the DVT+IR group compared with the DVT group. In addition, the numbers of CD68 positive cells in the thrombus were fewer in the DVT+IR group than in the DVT group. Furthermore, intrathrombotic gene expression of urokinase-type plasminogen activator and tissue-type plasminogen activator was higher in the DVT+IR group than in the DVT group. Conclusions: These results suggest that iron plays a role in the pathogenesis of DVT.

CD34 and p53 immunohistochemical stains differentiate hypocellular myelodysplastic syndrome (hMDS) from aplastic anemia and a CD34 immunohistochemical stain provides useful survival information for hMDS.

BackgroundThe presence of significant dysplasia in bone marrow (BM) aspirates helps to distinguish between hypocellular myelodysplastic syndrome (hMDS) and aplastic anemia (AA). Occasionally, diluted BM aspirates make it difficult to recognize dysplastic changes and can also negatively affect the detection of cytogenetic abnormalities in hMDS. We evaluated the usefulness of CD34 and p53 immunoreactivity for discriminating between hMDS and AA and for estimating survival outcomes in hMDS patients.MethodsBM clot section (BMC) or BM biopsy (BMB) specimens were obtained from 64 hMDS/AA patients (33 with hMDS and 31 with AA) and seven controls. Immunohistochemical (IHC) staining for CD34 and p53 was performed by using the EnVision detection system (Dako, Denmark). We compared the results of IHC staining, BM findings, and chromosomal analyses, and determined overall survival outcomes.ResultsThe number of CD34- and p53-positive BM cells was higher among the patients with hMDS than among the patients with AA (P<0.001 and P=0.001, respectively). hMDS patients with increased CD34-positive cells had significantly poorer survival outcomes compared with those with normal number of CD34-positive cells (P=0.013).ConclusionsCD34 and p53 IHC stains of BMC or BMB provide useful information for differentiating between hMDS and AA. CD34 IHC staining of BMC or BMB also provides useful information for estimating survival outcomes in hMDS patients.

Clinicopathological analysis of CD8-positive lymphocytes in the tumor parenchyma and stroma of hepatocellular carcinoma.

Tumor-infiltrating lymphocytes may be a manifestation of antitumor immunity. In the present study, hepatocellular carcinoma (HCC) and pericancerous non-tumor liver tissues samples were obtained from 86 surgical patients who had not received preoperative treatment. The cellular expression levels of CD4 and CD8 were immunohistochemically examined in the two tissue groups using tissue microarrays, to evaluate their clinicopathological relevance. Immunohistochemically, CD4 and CD8 T cells were observed in the tumor parenchyma and tumor stroma, and the intensity of CD4 and CD8 immunoreactivity was homogeneous in all HCC samples examined. Morphometrically, the average numbers of CD4- and CD8-positive T cells were significantly increased in the tumor stroma, compared with those in the tumor parenchyma (tumor stroma versus tumor parenchyma: 22±3.6 versus 7.4±0.9 in CD4, 32.8±4.2 versus 16±2.5 in CD8; both P<0.01). Furthermore, the average numbers of CD8-positive T cells in the tumor parenchyma and stroma were significantly increased, compared with the average numbers of CD4-positive cells (P<0.05). In addition, in the tumor parenchyma and stroma, the average numbers of CD8 T cells were significantly higher in patients with tumor diameters ≤5 cm compared with those in patients with tumor diameters >5 cm (diameter ≤5 cm versus diameter >5 cm: 18.1±3.3 versus 12.2±3.8 in tumor parenchyma, 36.5±4.8 versus 21.9±8.9 in tumor stroma; both P<0.05). In addition, CD8 expression was significantly enhanced in patients with chronic hepatitis and cirrhosis, compared with paired tumor parenchymal tissues (P<0.01). Furthermore, a significant positive correlation was observed between CD4 and CD8 expression in the tumor parenchyma and stroma (both P<0.001). These observations suggest that tumor parenchyma- or stroma-infiltrating CD8 T cells may be involved in HCC tumor diameter control.

Spinophilin expression determines cellular growth, cancer stemness and 5-flourouracil resistance in colorectal cancer.

The putative tumor suppressor gene spinophilin has been involved in cancer progression in several types of cancer. In this study, we explored the prognostic value of spinophilin expression in 162 colon adenocarcinoma patients. In addition, we generated stably expressing spinophilin-directed shRNA CRC cell lines and studied the influence of spinophilin expression on cellular phenotypes and molecular interactions. We independently confirmed that low spinophilin expression levels are associated with poor prognosis in CRC patients (p = 0.038). A reduction of spinophilin levels in p53 wild-type HCT116 and p53-mutated Caco-2 cells led to increased cellular growth rates and anchorage-independent growth (p<0.05). At molecular level, reduced spinophilin levels increased the expression of the transcription factor E2F-1. In addition, we observed an increased formation of tumor spheres, increased number of CD133 positive cells and an increased resistance to 5-flourouracil (p<0.05). Finally, treatment with the de-methylating agent 5-aza-dC increased spinophilin expression in CRC cells (p<0.05), corroborated by a correlation of spinophilin expression and extent of methylated CpG sites in the gene promoter region (p<0.001). In conclusion, gain of aggressive biological properties of CRC cells including cellular growth, cancer stem cell features and 5-flourouracil resistance partly explains the role of spinophilin in CRC.

Thymosin-β4 prevents cardiac rupture and improves cardiac function in mice with myocardial infarction.

Thymosin-β4 (Tβ4) promotes cell survival, angiogenesis, and tissue regeneration and reduces inflammation. Cardiac rupture after myocardial infarction (MI) is mainly the consequence of excessive regional inflammation, whereas cardiac dysfunction after MI results from a massive cardiomyocyte loss and cardiac fibrosis. It is possible that Tβ4 reduces the incidence of cardiac rupture post-MI via anti-inflammatory actions and that it decreases adverse cardiac remodeling and improves cardiac function by promoting cardiac cell survival and cardiac repair. C57BL/6 mice were subjected to MI and treated with either vehicle or Tβ4 (1.6 mg·kg(-1)·day(-1) ip via osmotic minipump) for 7 days or 5 wk. Mice were assessed for 1) cardiac remodeling and function by echocardiography; 2) inflammatory cell infiltration, capillary density, myocyte apoptosis, and interstitial collagen fraction histopathologically; 3) gelatinolytic activity by in situ zymography; and 4) expression of ICAM-1 and p53 by immunoblot analysis. Tβ4 reduced cardiac rupture that was associated with a decrease in the numbers of infiltrating inflammatory cells and apoptotic myocytes, a decrease in gelatinolytic activity and ICAM-1 and p53 expression, and an increase in the numbers of CD31-positive cells. Five-week treatment with Tβ4 ameliorated left ventricular dilation, improved cardiac function, markedly reduced interstitial collagen fraction, and increased capillary density. In a murine model of acute MI, Tβ4 not only decreased mortality rate as a result of cardiac rupture but also significantly improved cardiac function after MI. Thus, the use of Tβ4 could be explored as an alternative therapy in preventing cardiac rupture and restoring cardiac function in patients with MI.

Macrophages and bisphosphonate-related osteonecrosis of the jaw (BRONJ): evidence of local immunosuppression of macrophages in contrast to other infectious jaw diseases.

Bisphosphonates (BIP) are well established in bone diseases. A serious side effect is the bisphosphonate-related osteonecrosis of the jaw (BRONJ). Among different aetiology factors, local suppression of immune functions is gaining interest. The aim of this study was to analyze the function of macrophages in BRONJ in contrast to patients with osteoradionecrosis (ORN) and secondary chronic osteomyelitis (OM) of the jaws. Samples were also taken from patients with bisphosphonate medication (BP) without signs of infection, radiation therapy (RA), and osteoporosis (OP) as controls. One hundred five patients with surgery to the jaw were included in this study: 33 patients with BRONJ, 17 with ORN, 11 with secondary chronic OM, 8 with RA, 25 with BP medication and 11 with OP. Samples were histologically analysed and monocytes/macrophages stained using CD14 and CD68. The number of positively marked cells was counted per view (pv), and the CD68/CD14 ratio was calculated. Statistically, the Naïve-Bayes and decision-tree classifier were used. The number of CD14 positive cells was 10.3 cells/pv in the BRONJ-group in as compared to 5 in the ORN- and 3.8 in the OM-group respectively. The number of CD68 positive cells was 11.4/pv (BRONJ-group) as compared to 14/pv (ORN-group) and 12.7/pv (OM-group). With 0.89, the BRONJ-group showed a statistically different CD68/CD14 ratio than ORN-group with 3.39 and OM-group with 3.03. Our results indicate a different expression of CD14 and CD68 markers of monocytes/macrophages in BRONJ as compared to other jaw infections. This could be a sign of macrophage immunosuppression by BPs. In contrast, patients receiving BP medication without BRONJ showed no differences to other controls. This is the first study that clinically indicates a compromised macrophage function at BRONJ sites in contrast to ORN or secondary OM sites. The BRONJ itself could be forwarded by this effect.

C9,t11-Conjugated linoleic acid ameliorates steatosis by modulating mitochondrial uncoupling and Nrf2 pathway

Oxidative stress, hepatic steatosis, and mitochondrial dysfunction are key pathophysiological features of nonalcoholic fatty liver disease. A conjugated linoleic acid (CLA) mixture of cis9,trans11 (9,11-CLA) and trans10,cis12 (10,12-CLA) isomers enhanced the antioxidant/detoxifying mechanism via the activation of nuclear factor E2-related factor-2 (Nrf2) and improved mitochondrial function, but less is known about the actions of specific isomers. The differential ability of individual CLA isomers to modulate these pathways was explored in Wistar rats fed for 4 weeks with a lard-based high-fat diet (L) or with control diet (CD), and, within each dietary treatment, two subgroups were daily administered with 9,11-CLA or 10,12-CLA (30 mg/day). The 9,11-CLA, but not 10,12-CLA, supplementation to CD rats improves the GSH/GSSG ratio in the liver, mitochondrial functions, and Nrf2 activity. Histological examination reveals a reduction of steatosis in L-fed rats supplemented with both CLA isomers, but 9,11-CLA downregulated plasma concentrations of proinflammatory markers, mitochondrial dysfunction, and oxidative stress markers in liver more efficiently than in 10,12-CLA treatment. The present study demonstrates the higher protective effect of 9,11-CLA against diet-induced pro-oxidant and proinflammatory signs and suggests that these effects are determined, at least in part, by its ability to activate the Nrf2 pathway and to improve the mitochondrial functioning and biogenesis.

Neuron-derived Neurotrophic Factor Functions as a Novel Modulator That Enhances Endothelial Cell Function and Revascularization Processes

Strategies to stimulate revascularization are valuable for cardiovascular diseases. Here we identify neuron-derived neurotrophic factor (NDNF)/epidermacan as a secreted molecule that is up-regulated in endothelial cells in ischemic limbs of mice. NDNF was secreted from cultured human endothelial cells, and its secretion was stimulated by hypoxia. NDNF promoted endothelial cell network formation and survival in vitro through activation of Akt/endothelial NOS (eNOS) signaling involving integrin αvβ3. Conversely, siRNA-mediated knockdown of NDNF in endothelial cells led to reduction of cellular responses and basal Akt signaling. Intramuscular overexpression of NDNF led to enhanced blood flow recovery and capillary density in ischemic limbs of mice, which was accompanied by enhanced phosphorylation of Akt and eNOS. The stimulatory actions of NDNF on perfusion recovery in ischemic muscles of mice were abolished by eNOS deficiency or NOS inhibition. Furthermore, siRNA-mediated reduction of NDNF in muscles of mice resulted in reduction of perfusion recovery and phosphorylation of Akt and eNOS in response to ischemia. Our data indicate that NDNF acts as an endogenous modulator that promotes endothelial cell function and ischemia-induced revascularization through eNOS-dependent mechanisms. Thus, NDNF can represent a therapeutic target for the manipulation of ischemic vascular disorders.

Granulocyte colony-stimulating factor fails to enhance leptomeningeal collateral growth in spontaneously hypertensive rats

The promotion of collateral artery growth is an attractive approach for the treatment of chronic brain hypoperfusion due to occlusive artery disease. We previously reported that hypertension impaired the collateral artery growth of leptomeningeal anastomoses after brain hypoperfusion. Granulocyte colony-stimulating factor (G-CSF) enhances arteriogenesis in a mouse model via a mechanism involving monocyte/macrophage mobilization. However, the arteriogenic effect of G-CSF in hypertension remains unknown. In the present study, we tested whether G-CSF affected collateral artery growth in both normotensive and hypertensive model rat. Left common carotid artery (CCA) occlusion was performed to induce hypoperfusion in the brains of Wistar rats and spontaneously hypertensive rats (SHR). G-CSF was administered subcutaneously for 5 consecutive days. The superficial angioarchitecture of the leptomeningeal anastomoses and the circle of Willis after CCA occlusion and G-CSF treatment were visualized by latex perfusion. Circulating blood monocytes and CD68-positive cells, which represented the macrophages on the dorsal surface of the brain, were counted. G-CSF enhanced leptomeningeal collateral growth in Wistar rats, but not in SHR. G-CSF increased circulating blood monocytes in both Wistar rats and SHR. The number of CD68-positive cells on the dorsal surface of the brain was increased by G-CSF in Wistar rats, but not in SHR. The increase in macrophage accumulation correlated with the observed arteriogenic effects. In conclusion, G-CSF promotes collateral artery growth in the normotensive model rat, but not in the hypertensive model rat.

Pharmacokinetics and pharmacodynamics of FSK0808 and Gran after single intravenous drip administration or single subcutaneous administration: comparative study in healthy Japanese adult male subjects

FSK0808 is a recombinant human granulocyte colony-stimulating factor developed by Fuji Pharma Co., Ltd and Mochida Pharmaceutical Co., Ltd. as a biosimilar product of Gran®. We verified the pharmacokinetic/pharmacodynamic equivalence of FSK0808 and commercially available Gran® by a randomized crossover study of single intravenous dose (200 µg/m2) and single subcutaneous dose (400 µg/m2) in healthy Japanese adult male subjects. According to the bioequivalence guidelines, the area under the blood concentration – time curve by 48 hours after administration (AUC0–48) in a single intravenous drip (IVD) study, and AUC0–48 and maximum blood concentration (Cmax) in a single subcutaneous (SC) dose study were used as primary endpoints, and the pharmacodynamic parameters including absolute neutrophil count (ANC) or number of CD34 positive cells (CD34+ cells) as secondary endpoints. The safety was evaluated based on the characteristics and incidence of adverse reactions. As a result, the 90% confidence interval (CI) of the difference in mean value for AUC0–48 among drugs ranged from log(0.8) to log(1.25), in the IVD study, and those for Cmax and AUC0–48 were within the range of log(0.8)–log(1.25) in the SC study. Those for secondary endpoints were all within the range of log(0.8)–log(1.25). Thus, the pharmacokinetics/pharmacodynamics of both drugs were considered equivalent for all routes of administration, and the profiles of adverse reactions were also very similar.

Immunolocalization of CD34 Positive Progenitor Cells in Diabetic and Non Diabetic Periodontitis Patients - A Comparative Study.

Little research has been documented to determine the CD34 positive cells in healthy periodontium, chronic periodontitis and in chronic periodontitis with diabetes mellitus individuals. The aim of the present study was to evaluate and compare the CD34 positive progenitor cells of the gingiva in patients with healthy periodontium, chronic periodontitis and chronic periodontitis with Diabetes Mellitus. A total number of 75 patients were divided into 3 groups which included Group I (healthy periodontium), Group II (chronic periodontitis) and Group III (chronic periodontitis with diabetes mellitus). Periodontal examination included Plaque index, Gingival index, Gingival bleeding index, Probing pocket depth and Clinical attachment levels. Gingival biopsies were collected from each participant and they were fixed in formalin embedded in paraffin which was later subjected to immunohistochemical procedure with anti-CD34 (a stemness marker). T-Test and Regression analysis (R-square) was used to analyse the data. The mean number of CD34 positive cells were higher in group III (5.71±1.97) compared to Group chronic periodontitis group I (4.98± 2.08) and II (4.48± 1.24). Although CD34 is a non specific stemness marker, results suggest that there is a significant difference in the number of CD34 positive progenitor cells between Group II and Group III but no significant difference was observed between Group I, II and Group I, III.

Tissue Factor and Tissue Factor Pathway Inhibitor in Chronically Inflamed Gallbladder Mucosa

We characterised a tissue factor (TF) and tissue factor pathway inhibitor (TFPI) expression in relation to severity of inflammatory infiltration of the gallbladder mucosa in a chronic cholecystitis. We prospectively studied the gallbladder specimens obtained from 54 patients who had undergone cholecystectomy due to chronic calculous cholecystitis and 16 calculosis-free gallbladder specimens obtained from patients who underwent cholecystectomy due to the polyp/polyps as well as in cases of gallbladder injury. To assess TF and TFPI immunoreactivity by immunohistochemistry, the monoclonal anti-human TF and TFPI antibodies were used. The inflammatory infiltration of the gallbladder mucosa was reflected by the number of CD3 and CD68 positive cells. The expression of TF and TFPI differed significantly between the cholecystitis and the control group. Most capillary endothelial cells of the cholecystitis group presented weak expression for TFPI. The mean number of CD3 positive lymphocytes in the cholecystitis group was 18.6 ± 12.2, but the mean number of CD68 positive cells was 29.7 ± 13.9. In the control sections, it was 3.1 ± 1.9 and 8.8 ± 3.9, respectively (P < 0.001). The results of the current study suggest that the tissue procoagulant state found may be engaged in the etiopathogenesis of the cholecystitis.

Here comes the cord.

Hematopoietic stem cell transplantation has been established as a lifesaving therapy for leukemias or other hematopoietic malignancies since more than a half century ago [1]. Human leukocyte antigen (HLA)-matched sibling is the preferred donor for the better clinical outcomes, whiles, even patients without suitable family donor could also receive the benefits from unrelated donor registries or cord blood (CB) banks. International collaboration has also been well-improved in this field. Nowadays, almost all patients who need allogeneic transplantation are able to find adequate donors. Advantages to use CB unit are the quick availability, lower risk of severe graft-versus-host disease (GVHD) even though HLA between recipients and CB grafts is mismatched in most cases, and no risk for CB donors medically and mentally. Since the first patient received the CB from his HLA-matched sister to treat Fanconi Anemia successfully in 1988 [2], many CB banks have been established and the numbers of transplants using unrelated CB units dramatically increased. There are approximately 621,000 CB units in the database of Bone Marrow Donors Worldwide (BMDW: http://www.bmdw.org/). Recently, more than 3,000 CB transplantation (CBT)s are carried out annually all over the world: one third in Europe, one third in North America and another one third in Asia (mostly in Japan). Pediatric patients were major users in early period, however, CBT are gradually adopted in adults in this decade. For example, we are performing more than 1,200 transplantations using single CB unit annually in Japan, and more than 80% are adult patients with hematology malignancies, and a half of the recipients are older than 50 years. The major reason of this increment in the world is the improved clinical outcomes. Recently, for example, Eapen and colleagues [3] compared results of 165 single CBT for adult patients, 888 peripheral blood stem cell transplant (PBSCT) patients, and 472 bone marrow transplant (BMT) patients. Transplant-related mortality was higher for the CBT patients, but chronic GVHD were lower. Leukemia-free survival was comparable among CBT, fully matched, and mismatched PBSCT/BMT patients [3]. These findings have contributed to increase the CBT activity among the international transplant societies and have gradually improved the reliability of this transplant strategy. There is still limitation in CBT such as delayed engraftment and poor immune cell reconstitution [4]. The quality assurance of CB unit is also key issue for the reliability of CBT among the patients, their families, and transplant physicians. Cell dose is the most significant parameters for engraftment capacity of CB unit and also for survival results after CBT. Especially, CD34-positive cell number indicates the hematopoietic stem or progenitor cell function. However, the diversity of technical difference between CB banks has been evoked and it is necessary that we adjust that standard. Efforts are being intensified by the Foundation for the Accreditation of Cellular Therapy and the Joint Accreditation Committee - International Society for Cellular Therapy and European Group for Blood and Marrow Transplantation (FACT-JACIE), the American Association of Blood Banks (AABB), and CB banks all over the world. Because the importance of cell dose is critical, researchers are focusing on CB expansion strategies and some of them are under laboratory and clinical assessment. Under the situation described as above, CBT is definitely contributing to improving the life prognosis of patients with hematological malignancies. On the other hand, the utilized percentage of donated CB units all over the world is 6.3% reported by World Marrow Donor Association (WMDA), however it was reported as 1.2% in Korea. In this issue of Blood Research, Choi et al. [5] has analyzed the reasons by the questionnaire survey using Audience Response System for 67 board-certified transplant physicians at the annual meeting of the Korean Society of Blood and Marrow Transplantation. According to ordinary expectations, Korean physicians are now not positive for choose CB units as stem cell source for transplantation, because of the fears for poor clinical results and for unsatisfied quality of CB units from domestic CB banks. However, some Korean transplant specialists and CB bank persons have continued to proceed with great efforts for achieving the satisfied clinical outcomes with CBT and for obtaining good enough quality of CB units. They have already known the education is also important to improve the reliabilities for the strategy. Please just remember the time when you started BMT or PBSCT, or even transplant with haplo just a couple of years ago. Nothing gains without going forward. Here comes the time of CBT.

Numbers and Percentages Of Peripheral Monocytes Is a Prognostic Marker For CNS Involvement In Diffuse Large B-Cell Lymphoma

BackgroundIn the rituximab era, there are several studies that have reported the risk factors for central nervous system (CNS) involvement in non-Hodgkin lymphoma, but the same factors emerge, such as high international prognostic index (IPI) score, >1 extranodal site, elevated lactate dehydrogenase (LDH) level, poor performance status (PS), advanced stage, bone marrow involvement. Macrophages are an important component of the tumor microenvironment and the immune response to malignancy. Recently, elevated peripheral blood monocyte counts have been shown to be an independent marker associated with poor prognosis in patients with both non-Hodgkin and Hodgkin lymphoma. Patients and methodsWe reviewed data from a total of 1238 lymphoma patients(1185 non-Hodgkin lymphoma, 53 Hodgkin lymphoma) at our institution between February 2005 and May 2013. Of these, 42 patients (3.4%) developed CNS complications during the clinical course. Thirty patients out of these 42 (71.4%) were diagnosed with diffuse large B-cell lymphoma (DLBCL). Therefore, we focused on DLBCL. In this study, we retrospectively analyzed data from a total of 557 DLBCL patients, 30 patients (5.4%) who developed CNS involvement and 527 patients with DLBCL but without CNS involvement. This study was approved by the Institutional Review Board of the Cancer Institute Hospital of the Japanese Foundation for Cancer Research. The clinical features of all 557 DLBCL patients, including 30 patients with CNS involvement, are summarized in Table 1. CNS involvement was defined by the presence of at least one histologically confirmed CNS involvement; neuroimaging findings compatible with CNS involvement with lymphoma, in conjunction with consistent clinical presentation; and the absence of other clinically feasible diagnosis or positive cerebrospinal fluid (CSF) (lymphoma cells detected by cytology). The absolute monocyte counts (AMC) and monocyte ratio were derived from pre-treatment complete blood counts.Table 1Characteristics of DLBCL patients with or without CNS involvementCharacteristicsDLBCL with CNS involvement n=30, n (%)DLBCL without CNS involvement n=527, n (%)PGender; male21 (70)295 (56.0)0.1840Age ≤60 years16 (53.3)172 (32.6)0.0277Stage III-IV23 (76.7)208 (39.5)<0.0001IPI score 3-519 (63.3)175 (33.2)0.0013PS 2-412(40.0)47 (8.9)<0.0001Elevated LDH level18 (60)231 (43.8)0.0916Elevated soluble IL-2R level28 (93.3)380 (72.1)0.0096Monocyte count ≥0.6 (×109/L)12 (40)117 (22.2)0.0420Monocyte ratio ≥8 (%)17 (56.7)192 (36.4)0.0325 Pathological studiesImmunohistochemical analysis was carried out using mAbs against CD68 at our institution. ResultsThe incidence of CNS involvement was 5.4%, 1.3% having CNS involvement at diagnosis with DLBCL. Intriguingly, absolute monocyte counts (AMC) ≥0.6 (×109/L) at diagnosis were significantly frequent in 30 DLBCL patients (p=0.0420) with CNS involvement, compared with in 527 DLBCL patients without CNS involvement. Furthermore, the monocyte ratio ≥8% in peripheral blood at diagnosis was significantly frequent in 30 DLBCL patients (p=0.0325) with CNS involvement, compared with in 527 DLBCL patients without CNS involvement. DLBCL patients with CNS involvement showed age ≤60 years, stage III-IV, IPI score ≥3, and PS ≥2, elevated soluble IL-2 receptor levels was significantly frequent, compared with in DLBCL patients without CNS involvement. Neither gender, elevated LDH level, white blood cell counts (WBC) differed significantly in the two groups. With regard to pathological immunohistochemistry, the numbers of CD68 positive cells in or around lymphoma samples did not differ in the 14 DLBCL patients with CNS involvement that we were able to analyze, compared with DLBCL patients without CNS involvement. CNS involvement free survival rate in DLBCL patients was significantly lower in AMC ≥0.6 (×109/L) and/or the monocyte ratio ≥8% (Log-rank test, P=0.0102) in peripheral blood at diagnosis, compared with in AMC less than 0.6 (×109/L) and the monocyte ratio less than 8%. ConclusionsThese results suggest that in DLBCL patients, AMC and monocyte ratios in peripheral blood at diagnosis are closely correlated with the risk of eventual CNS involvement. AMC and monocyte ratios in peripheral blood at diagnosis in DLBCL patients could be a useful prognostic marker for the risk of CNS involvement during the clinical course. Disclosures:Yokoyama:Chugai Pharmaceutical CO., LTD.: Consultancy. Nishimura:Chugai Pharmaceutical CO., LTD.: Consultancy.

B Type Natriuretic Peptide (BNP) Less Than 200 Pg/Ml Represents Good Candidate For High Dose Melphalan and Predicts Longer Survival In Systemic Light Chain Amyloidosis

BackgroundHigh dose melphalan with autologous stem cell transplantation (ASCT) is a standard treatment for eligible patients with systemic light chain (AL) amyloidosis. Treatment-related mortality (TRM) of ASCT for AL amyloidosis was previously reported being as high as 40%; however, risk-adapted melphalan dosing reduced TRM to about 10% or less in experienced institutes. Several ways to determine the dose of melphalan have been proposed. They were focusing on organ failures especially cardiac dysfunction shown by ejection fraction (EF). EF represents contractile function of the heart; however, in AL amyloidosis, EF is known to be maintained until late stage whereas diastolic function is damaged earlier. We here present the outcomes of AL patients who received ASCT following risk-adapted melphalan with our criteria including B type natriuretic peptide (BNP) which is a practical marker for diastolic function of the heart and less affected by the renal function than NT-proBNP. Patients and MethodsA total of 12 patients with primary systemic AL amyloidosis treated with HD-Mel with ASCT at Sapporo Medical University Hospital between 2004 and 2012 were evaluated. Patients with age older than 65 years, poor performance status or severe organ dysfunction were determined not to eligible for ASCT. The dose of melphalan for conditioning regimen was modified due to patients' condition. A dose of 200 mg/m2 was administered to patients in performance status (ECOG) 0 or 1, number of organ involvement 2 or less, serum creatinine <1.5 mg/dl, EF >50%, and BNP <200 pg/ml; otherwise 140 mg/m2. Hematological response (HR) and organ response (OR) were evaluated according to the Consensus Opinion from the 10th International Symposium on Amyloid and Amyloidosis. The Kaplan-Meier method was used to estimate event-free survival (EFS) and overall survival (OS); both of them were measured from the day of ASCT. ResultsTwelve patients were included in this study, 4 were women. The median age at transplantation was 54 years (range, 32 - 65 years). Involvement of 1 organ was present in 1 patient (8.3%), 2 organs were involved in 8, and 3 or more organs in the remaining 3. Ten patients had a lambda monoclonal protein, and the median percentage of plasma cells in the bone marrow was 2.5% (range, 0.2% - 9.6%). No patients received treatment before HDM/ASCT, except 2 patients treated high-dose dexamethasone or 1 course of VAD regimen before referring to our hospital. Six patients received 200 mg/m2 of melphalan and remaining six received reduced dose based on the criteria in Patient and Methods. Notably, it was possible to screen patients with only the value of BNP; i.e., all the patients who received 140 mg/m2 of melphalan by the criteria had a high level (more than 200 pg/ml) of BNP. The median time from diagnosis of AL to ASCT was 95 days (range, 47 to 195 days). The median number of CD34-positive cells infused was 3.55 x106/kg, and all patients were engrafted. The hematologic CR was achieved in 5 patients and PR in 2. The organ response was observed in 2 patients who achieved hematologic CR. The median EFS of all patients was 21.1 months (range, 3.4 to 70.8 months), and median OS was 21.1 months (range, 3.4 to 112.5 months). EFS and OS were significantly longer for patients who received 200 mg/m2 of melphalan that are same with patients who had BNP less than 200 pg/ml, compared with a lower dose of melphalan and higher level of BNP (EFS: 15.0 months vs not reached, p=0.0166; OS: 15.0 months vs not reached, p=0.0166). No TRM was observed. ConclusionsAL patients with less than 200 pg/ml of BNP can be safely performed HD-Mel with ASCT and expected longer survival. Disclosures:No relevant conflicts of interest to declare.