Proteomic analysis of CD44(+) and CD44(−) gastric cancer cells

CD44 is a cell surface protein and it is widely used as a cancer stem cell marker in various cancer types including gastric cancer. We conducted proteomic analysis in CD44(+) and CD44(?) gastric cancer cells to understand characteristics of CD44(+) and CD44(?) cells. In the present study, we sorted cells from the gastric cancer cell line MKN45 according to CD44 expression to separate out CD44(+) and CD44(?) cells. And we conducted RT-PCR to identify mRNA expression of cancer stem cell markers in CD44(+) and CD44(?) cells. Cancer stem cell markers showed upregulated expression in CD44(+) cells. Next, we performed two-dimensional electrophoresis analysis to determine the differential expression pattern of proteins in each group; control, CD44(+), and CD44(?) MKN45 cells. We found a total of 113 spots that varied in expression between CD44(+) and CD44(?) cells, and subjected 20 of those protein spots to MALDI-MS. We selected the three proteins (HSPA8; heat shock cognate 71 kDa protein isoform 1, ezrin, α-enolase) upregulated in CD44(+) cells than CD44(?) cells and one protein (prohibitin) showed increased expression in CD44(?) cells. We validated the protein expression levels of four selected proteins by Western blot. We suggest that our study could be a helpful background to study CD44(+) cancer stem-like cells and differences between CD44(+) and CD44(?) cells in gastric cancer.     

Cell kinetics,DNA integrity,differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells

Human adipose tissue has been described as a potential alternative reservoir for stem cells. Although studies have been performed in rabbits using autologous adipose-derived stem cells (ADSC), these cells have not been well characterized. The primary objectives of this study were to demonstrate the presence of adipose-derived stem cells isolated from rabbit inguinal fat pads and to characterize them through osteogenic and adipogenic in vitro differentiation and lipid fingerprinting analysis. The secondary objective was to evaluate cell behavior through growth kinetics, cell viability, and DNA integrity. Rabbit ADSCs were isolated to determine the in vitro growth kinetics and cell viability. DNA integrity was assessed by an alkaline Comet assay in passages 0 and 5. The osteogenic differentiation was evaluated by Von Kossa, and Alizarin Red S staining and adipogenic differentiation were assessed by Oil Red O staining. Lipid fingerprinting analyses of control, adipogenic, and osteogenic differentiated cells were performed by MALDI-TOF/MS. We demonstrate that rabbit ADSC have a constant growth rate at the early passages, with increased DNA fragmentation at or after passage 5. Rabbit ADSC viability was similar in passages 2 and 5 (90.7% and 86.6%, respectively), but there was a tendency to decreased cellular growth rate after passage 3. The ADSC were characterized by the expression of surface markers such as CD29 (67.4%) and CD44 (89.4%), using CD 45 (0.77%) as a negative control. ADSC from rabbits were successfully isolated form the inguinal region. These cells were capable to differentiate into osteogenic and adipogenic tissue when they were placed in inductive media. After each passage, there was a trend towards decreased cell growth. On the other hand, DNA fragmentation increased at each passage. ADSC had a different lipid profile when placed in control, adipogenic, or osteogenic media.     

Expression of cancer stem cell marker during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis

One of the theories regarding oral carcinogenesis is that the tumor growth is initiated from cancer stem cells (CSCs) that self-renew and give rise to differentiated tumor cells, like stem cells do in normal tissues. The most common methods of CSC identification are based on CSC marker expression in carcinogenesis. This study examined the expression of CD133 and CD44, the most commonly used CSC biomarkers in oral squamous cell sarcoma (SCC), with the goal of identifying molecular biomarkers whose expression is associated with the multistep oral carcinogenesis. The expression of CD133, CD44, proliferating cell nuclear antigen (PCNA), and Cytokeratin (CK) was examined by Western blot analysis and confirmed by immunohistochemistry in a 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis model. Also, the expression of aldehyde dehydrogenase 1 (ALDH1), OCT-4 and Nanog were investigated for alteration of cancer cell stemness by Western blot. Along with the progress of multistep carcinogenesis, there were slight increases of CD133 and CD44 expression in the dysplasia group compared with normal rats. However, CD133 protein level was significantly overexpressed in SCC. The expression of PCNA and CK were low in normal group, but sequentially increased in SCC. ALDH1, Nanog and OCT-4 expression were significantly increased according to SCC grade during carcinogenesis. The findings indicate that CD133 is useful in identifying oral CSCs, which suggests that CD133 may serve as a predictor to identify CSCs with a high risk of oral cancer development.     

Properties of stem cells isolated from subcutaneous and subepicardial adipose tissues

Stem cells (SCs) vary in morphological, immunophenotypic, proliferative, and differentiation characteristics depending on their tissue source. Comparative analysis of their biological properties is essential for making an optimal SC choice for regenerative therapy. Using immunocytochemistry, flow cytometry, histochemistry, and RT-PCR, we have investigated SCs obtained from human subepicardial (SEC-AT) and subcutaneous (SC-AT) adipose tissues and cultured under similar conditions without any differentiation-promoting factors. The cultures were similar in having a high proportion of proliferating cells positive for nuclear antigen (PCNA). In both cultures, immunophenotyping has revealed high expression of mesenchymal stem-cell surface markers CD29, CD44, CD73, and CD105; low expression of CD31, CD34, and CD45; and variability in CD117, CD146, and CD309 expression. The only difference in the CD marker profile was the significantly lower expression of CD90 in the culture of SCs from SC-AT than from SEC-AT. Histochemical analysis showed a lack of Oil Red O-positive cells in both cultures and an about ten times higher number of alkaline phosphatase-positive cells among SCs from SC-AT. In both cultures, immunocytochemistry detected low expression of the slow myosin heavy chain marker MAB1628 and smooth muscle actin marker α-hSMA. Expression of the gap junction protein connexin-43 was markedly higher in cells from SC-AT cultures. Only the cells of these cultures expressed the epithelial cell marker cytokeratin-19. GATA4 mRNA expression detected with RT-PCR was identified in SEC-AT rather than in SC-AT cells. Our results suggest that SC-AT is enriched compared to SEC-AT with epithelial cell and osteogenic progenitors. In turn, SEC-AT possesses cardiomyogenic SCs and can be considered an alternative source for cell cardiotherapy.     

Effects of Bone Marrow Mesenchymal Stem Cells on Hematopoietic Recovery and Acute Graft-Versus-Host Disease in Murine Allogeneic Umbilical Cord Blood Transplantation Model

To investigate the effect of bone marrow mesenchymal stem cells (MSC) on hematopoietic recovery and acute graft-versus-host disease (GVHD) in a murine allogeneic umbilical cord blood transplantation (allo-UCBT) model. MSCs were obtained from C57/BL mouse bone marrow. The MSC phenotypes were identified by flow cytometry (FCM), and their ability to differentiate into osteoblasts and adipocytes was tested. Once murine allo-UCBT and aGVHD models were established, mice were divided into five groups: (1) total body irradiation (TBI) group, each mouse receiving 0.3 ml sterile saline infusion after TBI and used as control; (2) UCB group, receiving 2 × 10⁶ umbilical cord blood mononuclear cells (UCB-MNC) after TBI; (3) UCB+MSC group, receiving 2 × 10⁶ UCB-MNC and 2 × 10⁷ MSC after TBI; (4) UCB+SC group, receiving 2 × 10⁶ UCB-MNC and 2 × 10⁶ spleen cells after TBI; and (5) UCB+SC+MSC group, receiving 2 × 10⁶ UCB-MNC, 2 × 10⁷ MSC and 2 × 10⁶ spleen cells after TBI. To evaluate the engraftment of HSC, the white blood cells, red blood cells, and platelets counts were tested at different time points after transplantation, and the ratio of chimerism was identified by FCM. The acute GVHD clinical scores, recipient mice survival, and the histopathological analyses were used to evaluate the effect of MSC on acute GVHD. MSCs were successfully obtained in vitro and FCM analysis showed that these cells are highly positive for CD90.2, CD44, and negative for CD34, CD45, and they are capable to differentiate into osteoblasts and adipocytes after being induced. Compared to UCB group, the UCB+MSC mice had shorter duration of myelosuppression and higher percentage of donor-derived cells which was up to 22.87 ± 4.3 % and the white blood cell (WBC), red blood cell (RBC), and platelet counts started to increase by day 6 after transplantation. Moreover, the average survival time for UCB+MSC mice was 25.0 ± 10.55 days, while for the UCB group it was 15.5 ± 12.50 days. The UCB+SC mice showed fatigue, loss of appetite, weight loss, arched back, and hair ruffling on day 13 post transplantation. Approximately 50 % of mice showed skin ulcers, had diarrhea and other manifestations of acute GVHD, and all mice were died within 20 days. Histopathological analysis confirmed grade II–IV GVHD manifestation. In addition to transient weight loss, some UCB+SC+MSC mice developed arched back, hair ruffling, diarrhea and other manifestations of acute GVHD. The clinical scores in UCB+SC+MSC mice with acute GVHD (grade I–II or without GVHD) were lower than UCB+SC group (P < 0.05). Bone marrow MSCs can promote hematopoietic recovery and decrease the incidence of acute GVHD in murine allo-UCBT model.     

Ecto-Mesenchymal Stem Cells from Facial Process: Potential for Muscle Regeneration

Ecto-mesenchymal stem cells (EMSCs) originate from the cranial neural crest and participate in the formation of tooth, salivary, and muscle in early development stage. The transplantation of EMSCs, a potential source of myoblast stem cell, might improve muscle regeneration. The purpose of this study was to explore whether EMSCs have the potential to differentiate and display a myogenic phenotype in vitro the in vitro. Here, we characterized the EMSCs isolated from the facial process, and p75 + EMSCs were collected by a FACS calibur flow cytometer. In vitro, p75 + EMSCs induced by DMSO can accumulate and fuse into multinucleated myotubes and further differentiate into the skeletal muscle cells in form of cell sheet. Functional myoblast phenotypes of p75 + EMSCs were found in vivo model of muscle injury. The remarkable ability of stem cells to regenerate skeletal muscle indicated their potential role in the cell therapy and tissue engineering of the skeletal muscle.     

A comparative study of PKH67, DiI,and BrdU labeling techniques for tracing rat mesenchymal stem cells

Mesenchymal stem cells (MSCs) have generated a great deal of promise as a potential source of cells for cell-based therapies. Various labeling techniques have been developed to trace MSC survival, migration, and behavior in vitro or in vivo. In the present study, we labeled MSCs derived from rat bone marrow (rMSCs) with florescent membrane dyes PKH67 and DiI, and with nuclear labeling using 5 μM BrdU and 10 μM BrdU. The cells were then cultured for 6 d or passaged (1–3 passages). The viability of rMSCs, efficacy of fluorescent expression, and transfer of the dyes were assessed. Intense fluorescence in rMSCs was found immediately after membrane labeling (99.3?±?1.6% PKH67+ and 98.4?±?1.7% DiI+) or after 2 d when tracing of nuclei was applied (91.2?±?4.6% 10 μM BrdU+ and 77.6?±?4.6% 5 μM BrdU+), which remained high for 6 d. Viability of labeled cells was 91?±?3.8% PKH67+, 90?±?1.5% DiI+, 91?±?0.8% 5 μM BrdU+, and 76.9?±?0.9% 10 μM BrdU+. The number of labeled rMSCs gradually decreased during the passages, with almost no BrdU+ nuclei left at final passage 3. Direct cocultures of labeled rMSCs (PKH67+ or DiI+) with unlabeled rMSCs revealed almost no dye transfer from donor to unlabeled recipient cells. Our results confirm that labeling of rMSCs with PKH67 or DiI represents a non-toxic, highly stable, and efficient method suitable for steady tracing of cells, while BrdU tracing is more appropriate for temporary labeling due to decreasing signal over time.     

The Rho kinase inhibitor Y-27632 facilitates the differentiation of bone marrow mesenchymal stem cells

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell-based regenerative therapies, but technical challenges have limited cell yield and thus the success of these potential treatments. The Rho GTPases and downstream Rho kinases (Rho coiled-coil kinases or ROCKs) are central regulators of cytoskeletal dynamics during the cell cycle and thus help determine the balance between stem cells self-renewal, lineage commitment, and apoptosis. Here, we examined if suppression of ROCK signaling enhances the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) differentiation into neurons and neuroglial cells. BMSCs were cultured in epidermal growth factor (EGF, 10 µg/l) and basic fibroblastic growth factor (bFGF, 10 µg/l) in the presence or absence of the Rho kinase inhibitor Y-27632 (10 µM). The expression levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence and Western blotting. The average number of NSE-positive cells increased from 83.20 ± 8.677 (positive ratio 0.2140 ± 0.0119) to 109.20 ± 8.430 (positive ratio 0.3193 ± 0.0161) per visual field in the presence of Y-27632, while GFAP-positive cell number increased from 96.30 ± 8.486 (positive ratio 0.18 ± 0.0152) to 107.50 ± 8.683 (positive ratio 0.27 ± 0.0115) (P < 0.05 for both). Both NSE and GFAP protein expression levels were enhanced significantly by Y-27632 treatment (NSE: 0.74 ± 0.05 vs. 1.03 ± 0.06; GFAP: 0.64 ± 0.08 vs. 0.97 ± 0.05, both P < 0.01) as indicated by Western blots. The Rho kinase inhibitor Y-27632 concomitant with EGF and bFGF stimulation promotes BMSC differentiation into neural cells. Control of Rho kinase activity may enhance the efficiency of stem cell-based treatments for neurodegenerative diseases.     

Rapid selection of XO embryonic stem cells using Y chromosome-linked GFP transgenic mice

We developed a transgenic mouse line with Y chromosome-linked green fluorescent protein expressing transgenes (Y-GFP) by the conventional microinjection into the pronucleus of C57BL/6J fertilized oocytes. Embryonic stem (ES) cells derived from Y-GFP mice enabled not only sexing but also the identification of 39, XO karyotype by the lack of Y chromosome. Actually, when fluorescence activated cell sorting (FACS) was applied to Y-GFP ES cells, non-fluorescent ES cells were conveniently collected and showed the lack of Y chromosome by PCR genotyping and Southern blot analysis. FACS analysis revealed Y chromosome loss occurred at 2.9 % of 40, XY ES cells after five passages. These Y-GFP ES cells are potentially applicable to reduce the time, cost and effort needed to generate the gene-targeted mice by the production of male and female mice derived from the same ES cell clone.     

Effect of Intestinal Microbiota Alteration on Hepatic Damage in Rats with Acute Rejection After Liver Transplantation

The previous studies all focus on the effect of probiotics and antibiotics on infection after liver transplantation. Here, we focus on the effect of gut microbiota alteration caused by probiotics and antibiotics on hepatic damage after allograft liver transplantation. Brown-Norway rats received saline, probiotics, or antibiotics via daily gavage for 3 weeks. Orthotopic liver transplantation (OLT) was carried out after 1 week of gavage. Alteration of the intestinal microbiota, liver function and histopathology, serum and liver cytokines, and T cells in peripheral blood and Peyer’s patch were evaluated. Distinct segregation of fecal bacterial diversity was observed in the probiotic group and antibiotic group when compared with the allograft group. As for diversity of intestinal mucosal microbiota and pathology of intestine at 2 weeks after OLT, antibiotics and probiotics had a significant effect on ileum and colon. The population of Lactobacillus and Bifidobacterium in the probiotic group was significantly greater than the antibiotic group and the allograft group. The liver injury was significantly reduced in the antibiotic group and the probiotic group compared with the allograft group. The CD4/CD8 and Treg cells in Peyer’s patch were decreased in the antibiotic group. The intestinal Treg cell and serum and liver TGF-β were increased markedly while CD4/CD8 ratio was significantly decreased in the probiotic group. It suggested that probiotics mediate their beneficial effects through increase of Treg cells and TGF-β and deduction of CD4/CD8 in rats with acute rejection (AR) after OLT.     

Immunophenotypic Characterization of CD45RO+ and CD45RA+ T Cell Subsets in Peripheral Blood of Peripheral T Cell Lymphoma Patients

To study the distribution profile of CD45RO⁺ and CD45RA⁺ T cells in the peripheral blood of peripheral T cell lymphoma (PTCL) patients and its clinical significance. 27 patients with PTCL were enrolled in this study, together with 30 healthy individuals as the control group. Flow cytometry analysis was employed to examinate the differences in the distribution of CD45RO⁺ and CD45RA⁺ T cells in peripheral blood between two groups. In PTCL patient’s lymphnode tissues, the T cell population displayed diverse antigenic expression, with CD4⁺ T cells as the major subset. No B cell-related antigen was expressed. The percentage of CD4⁺/CD8⁺ and CD4⁺CD45RO⁺ T cells in patients’ peripheral blood were significantly lower than that in the control samples, while the percentage of CD4⁺CD45RA⁺, CD8⁺CD45RA⁺, and CD8⁺CD45RO⁺ T cells in patients’ peripheral blood were significantly higher than that in the control samples. The percentage of CD4⁺/CD8⁺, CD4⁺CD45RO⁺ cells in stage I/II PTCL patients’ peripheral blood were significantly higher than that in the samples from patients with stage III/IV PTCL. The percentage of CD4⁺CD45RA⁺, CD8⁺CD45RA⁺, and CD8⁺CD45RO⁺ T cells were notably lower than that in the samples from III/IV period PTCL patients. Both CD45RO⁺ and CD45RA⁺ T cells play important roles in the process of PTCL. The immunophenotypic profile from this study will help to develop the differential diagnosis and treatment of PTCL patients in the future, and improve the accuracy rate of diagnosis and to ameliorate the prognosis.     

Nanosecond pulsed electric fields modulate the expression of Fas/CD95 death receptor pathway regulators in U937 and Jurkat Cells

In this publication, we demonstrate that exposure of Jurkat and U937 cells to nanosecond pulsed electrical fields (nsPEF) can modulate the extrinsic-mediated apoptotic pathway via the Fas/CD95 death receptor. An inherent difference in survival between these two cell lines in response to 10 ns exposures has been previously reported (Jurkat being more sensitive to nsPEF than U937), but the reason for this sensitivity difference remains unknown. We found that exposure of each cell line to 100, 10 ns pulses at 50 kV/cm caused a marked increase in expression of cFLIP (extrinsic apoptosis inhibitor) in U937 and FasL (extrinsic apoptosis activator) in Jurkat, respectively. Measurement of basal expression levels revealed an inherent difference between U937 cells, having a higher expression of cFLIP, and Jurkat cells, having a higher expression of FasL. From these data, we hypothesize that the sensitivity difference between the cells to nsPEF exposure may be directly related to expression of extrinsic apoptotic regulators. To validate this hypothesis, we used siRNA to knockdown cFLAR (coding for cFLIP protein) expression in U937, and FasL expression in Jurkat and challenged them to 100, 10 ns pulses at 150 kV/cm, a typical lethal dose. We observed that U937 survival was reduced nearly 60 % in the knockdown population while Jurkat survival improved ~40 %. These findings support the hypothesis that cell survival following 10 ns pulse exposures depends on extrinsic apoptotic regulators. Interestingly, pretreatment of U937 with a 100-pulse, 50 kV/cm exposure (to amplify cFLAR expression) significantly reduced the lethality of a 150 kV/cm, 100-pulse exposure applied 24 h later. From these data, we conclude that the observed survival differences between cells, exposed to 10 ns pulsed electric fields, is due to inherent cell biochemistry rather than the biophysics of the exposure itself. Understanding cell sensitivity to nsPEF may provide researchers/clinicians with a predicable way to control or avoid unintended cell death during nsPEF exposure.     

Immunoregulation Effects of Bone Marrow-Derived Mesenchymal Stem Cells in Xenogeneic Acellular Nerve Grafts Transplant

This study evaluated whether bone marrow-derived mesenchymal stem cells (BM-MSCs) combined with xenogeneic acellular nerve grafts (xANGs) would reduce the inflammation reaction of xANGs transplantation. BM-MSCs were extracted, separated, purified, and cultured from the bone marrow of rats. Then BM-MSCs were seeded into 5 mm xANGs as experimental group, while xANGs group was chosen as control. Subcutaneous implantation and nerve grafts transplantation were done in this study. Walking-track tests, electrophysiological tests, H&E staining, and immunostaining of CD4, CD8, and CD68 of subcutaneous implantations, cytokine concentrations of IL-2, IL-10, IFN-γ and TNF-α in lymphocytes supernatants and serum of the two groups were evaluated. Walking-track tests and electrophysiological tests suggested the group of BM-MSCs with xANGs obtained better results than xANGs group (P < 0.05). H&E staining and immunostaining of CD4, CD8, and CD68 of subcutaneous implantations showed there were less inflammatory cells in the group of BM-MSCs when compared with the xANGs group. The cytokine concentrations of IL-2, IFN-γ, and TNF-α in BM-MSCs group were lower than xANGs group in lymphocytes supernatants and serum (P < 0.05). However, IL-10 concentrations in BM-MSCs group were higher than xANGs group (P < 0.05). xANG with BM-MSCs showed better nerve repair function when compared with xANG group. Furthermore, xANG with BM-MSCs showed less inflammatory reaction which might indicate the reason of its better nerve regeneration.     

Involvement of Upregulated SYF2 in Schwann Cell Differentiation and Migration After Sciatic Nerve Crush

SYF2 is a putative homolog of human p29 in Saccharomyces cerevisiae. It seems to be involved in pre-mRNA splicing and cell cycle progression. Disruption of SYF2 leads to reduced α-tubulin expression and delayed nerve system development in zebrafish. Due to the potential of SYF2 in modulating microtubule dynamics in nervous system, we investigated the spatiotemporal expression of SYF2 in a rat sciatic nerve crush (SNC) model. We found that SNC resulted in a significant upregulation of SYF2 from 3 days to 1 week and subsequently returned to the normal level at 4 weeks. At its peak expression, SYF2 distributed predominantly in Schwann cells. In addition, upregulation of SYF2 was approximately in parallel with Oct-6, and numerous Schwann cells expressing SYF2 were Oct-6 positive. In vitro, we observed enhanced expression of SYF2 during the process of cyclic adenosine monophosphate (cAMP)-induced Schwann cell differentiation. SYF2-specific siRNA-transfected Schwann cells did not show significant morphological change in the process of Schwann cell differentiation. Also, we found shorter and disorganized microtubule structure and a decreased migration in SYF2-specific siRNA-transfected Schwann cells. Together, these findings indicated that the upregulation of SYF2 was associated with Schwann cell differentiation and migration following sciatic nerve crush.     

Granulocyte Colony Stimulating Factor Reduces Brain Injury in a Cardiopulmonary Bypass-Circulatory Arrest Model of Ischemia in a Newborn Piglet

Ischemic brain injury continues to be of major concern in patients undergoing cardiopulmonary bypass (CPB) surgery for congenital heart disease. Striatum and hippocampus are particularly vulnerable to injury during these processes. Our hypothesis is that the neuronal injury resulting from CPB and the associated circulatory arrest can be at least partly ameliorated by pre-treatment with granulocyte colony stimulating factor (G-CSF). Fourteen male newborn piglets were assigned to three groups: deep hypothermic circulatory arrest (DHCA), DHCA with G-CSF, and sham-operated. The first two groups were placed on CPB, cooled to 18 °C, subjected to 60 min of DHCA, re-warmed and recovered for 8–9 h. At the end of experiment, the brains were perfused, fixed and cut into 10 µm transverse sections. Apoptotic cells were visualized by in situ DNA fragmentation assay (TUNEL), with the density of injured cells expressed as a mean number ± SD per mm². The number of injured cells in the striatum and CA1 and CA3 regions of the hippocampus increased significantly following DHCA. In the striatum, the increase was from 0.46 ± 0.37 to 3.67 ± 1.57 (p = 0.002); in the CA1, from 0.11 ± 0.19 to 5.16 ± 1.57 (p = 0.001), and in the CA3, from 0.28 ± 0.25 to 2.98 ± 1.82 (p = 0.040). Injection of G-CSF prior to bypass significantly reduced the number of injured cells in the striatum and CA1 region, by 51 and 37 %, respectively. In the CA3 region, injured cell density did not differ between the G-CSF and control group. In a model of hypoxic brain insult associated with CPB, G-CSF significantly reduces neuronal injury in brain regions important for cognitive functions, suggesting it can significantly improve neurological outcomes from procedures requiring DHCA.     

CD39/NTPDase-1 expression and activity in human umbilical vein endothelial cells are differentially regulated by leaf extracts from Rubus caesius and Rubus idaeus

Many experimental studies have demonstrated the favorable biological activities of plants belonging to the genus Rubus, but little is known of the role of Rubus leaf extracts in the modulation of the surface membrane expression and activity of endothelial apyrase. The aim of this study was to assess the influence of 1–15 μg/ml Rubus extracts on CD39 expression and enzymatic activity, and on the activation (ICAM-1 expression) and viability of human umbilical vein endothelial cells (HUVEC). The polyphenolic contents and antioxidative capacities of extracts from dewberry (R. caesius L.) and raspberry (R. idaeus L.) leaves were also investigated. The techniques applied were flow cytometry (endothelial surface membrane expression of ICAM-1 and CD39), malachite green assay (CD39 activity), HPLC-DAD (quantitative analysis of polyphenolic extract), ABTS, DPPH and FRAP spectrometric assays (antioxidant capacity), and the MTT test (cell viability). Significantly increased CD39 expressions and significantly decreased ATPDase activities were found in the cells treated with 15 μg/ml of either extract compared to the results for the controls. Neither of the extracts affected cell proliferation, but both significantly augmented endothelial cell ICAM-1 expression. The overall antioxidant capacities of the examined extracts remained relatively high and corresponded well to the determined total polyphenol contents. Overall, the results indicate that under in vitro conditions dewberry and raspberry leaf extracts have unfavorable impact on endothelial cells.     

Evaluation of Angiogenesis in Non-small Cell Lung Carcinoma by CD34 Immunohistochemistry

The objective of the study was to investigate angiogenesis in non-small lung cancer by measuring the expression of CD34. Immunohistochemistry was used to detect CD34 at the endothelial cell surface in 81 surgically resected non-small cell lung cancer specimens. CD34 immunohistochemistry had high specificity and sensitivity with minimal background, which enabled efficient identification of CD34-positive staining. Statistical analysis showed that increased microvessel density (MVD) was closely correlated to tumor progression as reflected by the clinical stage (P < 0.05). However, MVD was not significantly associated with lymph node metastasis and other clinical and pathological features (P > 0.05). In conclusion, microvascular density may play an important role in the development and progression of lung cancer.     

Inhibition of intestinal polyp growth by oral ingestion of bovine lactoferrin and immune cells in the large intestine

Studies using animal models have demonstrated that ingestion of bovine lactoferrin (bLF) inhibits carcinogenesis in the colon and other organs of experimental animals. As a result of these studies, a blinded, randomized, controlled clinical trial was conducted in the National Cancer Center Hospital, Tokyo, Japan to determine whether ingestion of bLF had an effect on the growth of colorectal polyps in humans. Patients with colorectal polyps ≤5 mm diameter and likely to be adenomas ingested 0, 1.5, or 3.0 g bLF daily for 1 year. Ingestion of 3.0 g bLF suppressed the growth of colorectal polyps and increased the level of serum human lactoferrin in trial participants 63 years old or younger. The purpose of the present study was to investigate correlations between immune parameters and changes in polyp size. Trial participants with regressing polyps had increased NK cell activity, increased serum hLF levels (indicating increased neutrophil activity), and increased numbers of CD4+ cells in the polyps. These findings are consistent with a correlation between higher immune activity and suppression of colorectal polyps. In addition, participants with regressing polyps had lower numbers of PMNs and increased numbers of S100A8+ cells in the polyps, consistent with a correlation between lower inflammatory potential in the colon and suppression of colorectal polyps. Trial participants ingesting bLF had increased serum hLF levels, a possible increase in systemic NK cell activity, and increased numbers of CD4+ and CD161+ cells in the polyps. Taken together, our findings suggest that bLF suppressed colorectal polyps by enhancing immune responsiveness.