A novel cell-containing device for regenerative medicine: biodegradable nonwoven filters with peripheral blood cells promote wound healing.

The efficacy of skin regeneration devices consisting of nonwoven filters and peripheral blood cells was investigated for wound healing. We previously found that human peripheral blood cells enhanced their production of growth factors, such as transforming growth factor ß1 (TGF-ß1) and vascular endothelial growth factor, when they were captured on nonwoven filters. Cells on biodegradable filters were expected to serve as a local supply of growth factors and cell sources when they were placed in wounded skin. Nonwoven filters made of biodegradable polylactic acid (PLA) were cut out as 13-mm disks and placed into cell-capturing devices. Mouse peripheral blood was filtered, resulting in PLA filters with mouse peripheral blood cells (m-PBCs) at capture rates of 65.8 ± 5.2%. Then, the filters were attached to full-thickness surgical wounds in a diabetic db/db mouse skin for 14 days as a model of severe chronic wounds. The wound area treated with PLA nonwoven filters with m-PBCs (PLA/B+) was reduced to 8.5 ± 12.2% when compared with day 0, although the non-treated control wounds showed reduction only to 60.6 ± 27.8%. However, the PLA filters without m-PBCs increased the wound area to 162.9 ± 118.7%. By histopathological study, the PLA/B+ groups more effectively accelerated formation of epithelium. The m-PBCs captured on the PLA filters enhanced keratinocyte growth factor (FGF-7) and TGF-ß1 productions in vitro, which may be related to wound healing. This device is useful for regeneration of wounded skin and may be adaptable for another application.

Appropriate nonwoven filters effectively capture human peripheral blood cells and mesenchymal stem cells, which show enhanced production of growth factors.

Scaffolds, growth factors, and cells are three essential components in regenerative medicine. Nonwoven filters, which capture cells, provide a scaffold that localizes and concentrates cells near injured tissues. Further, the cells captured on the filters are expected to serve as a local supply of growth factors. In this study, we investigated the growth factors produced by cells captured on nonwoven filters. Nonwoven filters made of polyethylene terephthalate (PET), biodegradable polylactic acid (PLA), or chitin (1.2-22 µm fiber diameter) were cut out as 13 mm disks and placed into cell-capturing devices. Human mesenchymal stem cells derived from adipose tissues (h-ASCs) and peripheral blood cells (h-PBCs) were captured on the filter and cultured to evaluate growth factor production. The cell-capture rates strongly depended on the fiber diameter and the number of filter disks. Nonwoven filter disks were composed of PET or PLA fibers with fiber diameters of 1.2-1.8 µm captured over 70% of leukocytes or 90% of h-ASCs added. The production of vascular endothelial growth factor (VEGF), transforming growth factor ß1, and platelet-derived growth factor AB were significantly enhanced by the h-PBCs captured on PET or PLA filters. h-ASCs on PLA filters showed significantly enhanced production of VEGF. These enhancements varied with the combination of the nonwoven filter and cells. Because of the enhanced growth factor production, the proliferation of human fibroblasts increased in conditioned medium from h-PBCs on PET filters. This device consisting of nonwoven filters and cells should be investigated further for possible use in the regeneration of impaired tissues.

Inhibitory effects of low-dose aloe-emodin on the development of colorectal tumors in min mice.

Aloe-emodin (AE), a natural anthraquinone compound, has been reported to exhibit anticancer activity in various cancer cell lines and anti-inflammatory effects in murine macrophages. In the present study, we investigated the cancer chemopreventive effects of AE in an Apc-deficient Min mouse model. In the first experiment, male Min mice were fed a basal diet or diets containing 5 ppm AE and 10 ppm AE for 12 weeks. The dietary administration of 5 ppm AE significantly reduced the number of colorectal tumors. In a second experiment, we investigated the effects of AE on colitis-related colon carcinogenesis in Min mouse treated with dextran sodium sulfate (DSS). Female Min mice were administered 1% DSS in their drinking water for 7 days. AE was given to mice in their diet at a dose of 5 or 50 ppm for 5 weeks. Feeding with AE significantly reduced the number of colorectal tumors. When proliferation of cells in normal-appearing colonic mucosa was assessed by monoclonal anti-rat Ki-67 antibody (MIB-5) immunohistochemistry in experiments 1 and 2, the AE treatment significantly decreased the mean MIB-5-labeling index. These results suggest that the dietary administration of low-dose AE may have chemopreventive effects against development of colorectal tumors in Min mice, possibly in part by reducing cell proliferation in colorectal mucosa.

Peculiar tonsil-like structure near vagina of the laboratory shrew, Suncus murinus.

After finding tonsil-like structures near the entrance of vagina of a laboratory shrew (Suncus murinus), which we subsequently designated as vaginal tonsils, we performed detailed immunohistochemical and developmental studies. The location of T and B cells in the vaginal tonsils differed from that in the palatine tonsils or that in the lymphoid nodes of other animals. The boundary between the germinal center region and the region encompassing follicular interfollicular tissue was not clear. B cells were widely distributed and very dense in the parenchyma, but they were scattered in the epithelial area (B cells were present in around 90% of the vaginal tonsil tissue). In contrast, T cells were scattered in the parenchyma and in the epithelial area (T cells were present in around 10% of the vaginal tonsil tissue). B cells were more prominent than T cells throughout the development of these structures and the epithelium was invaded by many immigrating cells. The size of the vaginal tonsils changed during postnatal development. Vaginal tonsils are structurally similar to other tonsils, and they may function to protect the vagina from infection.

Inhibition of ENNG-induced pyloric stomach and small intestinal carcinogenesis in mice by high temperature- and pressure-treated garlic.

High temperature- and pressure-treated garlic (HTPG) has been shown to have enhanced antioxidative activity and polyphenol contents. Previously, we reported that HTPG inhibited 1,2-dimethylhydrazine-induced mucin depleted foci (premalignant lesions) and O6-methylguanine DNA adduct formation in the rat colorectum. In the present study, we investigated the modifying effects of HTPG on N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)- induced pyloric stomach and small intestinal carcinogenesis in mice. Male C57BL/6 mice were given ENNG (100 mg/l) in drinking water for the first 4 weeks, then a basal diet or diet containing 2% or 5% HTPG for 30 weeks. The incidence and multiplicity of pyloric stomach and small intestinal (duodenal and jejunal) tumors in the 2% HTPG group (but not in the 5% HTPG group) were significantly lower than those in the control group. Cell proliferation of normal-appearing duodenal mucosa was assessed by MIB-5 immunohistochemistry and shown to be significantly lower with 2% HTPG (but again not 5% HTPG) than in controls. These results in dicate that HTPG, at 2% in the diet, inhibited ENNG-induced pyloric stomach and small intestinal (especially duodenal) tumorigenesis in mice, associated with suppression of cell proliferation.

The distribution of T lymphocytes, B lymphocytes, and dendritic-like cells of the anal tonsil in the laboratory shrew, Suncus murinus.

We investigated the distribution of T lymphocytes, B lymphocytes, and S-100 protein-immunoreactive dendritic-like in the anal tonsil of the laboratory shrew, Suncus murinus. In adult animals, T lymphocytes were located mainly at the periphery of the anal tonsil, especially around small blood vessels. B lymphocytes were located in the central and subepithelial region of the anal tonsil, which includes primary lymphoid follicles, and in which there are small numbers of scattered T lymphocytes. B and T lymphocytes were distributed over 72.7 and 27.3% of the tonsillar area, respectively. However, their areas of distribution were not clearly distinguished. The areas containing B lymphocytes were enriched in S-100 protein antibody-immunoreactive cells, which exhibited a dendritic shape. These S-100-positive cells appeared to be identical to the follicular dendritic cells (FDC) seen in the follicles of lymphoid organs. These results suggest that the anal tonsils constitute one of the gut-associated lymphoid tissues (GALT), and that a function of the anal tonsil includes the capture of intruding antigens that would generate protective antibody responses.

Effect of osteopontin on diarrhea duration and innate immunity in suckling mice infected with a murine rotavirus.

We studied the role of osteopontin (OPN) in host responses against rotavirus (RV) infection. OPN knockout (OPN-KO) suckling mice were more susceptible to RV (strain EW) infection and showed prolonged diarrhea duration compared to wild-type (WT) suckling mice. OPN in the small intestine of WT mice was expressed after 48 h post-infection. On day 2 postinfection, mRNA levels of interleukin-1beta, tumor necrosis factor-alpha, and interleukin-15 in OPN-KO mice were lower than in WT mice, although mRNA expression of Th-1- and Th-2-related cytokines in the small intestine were nearly the same between OPN-KO and WT mice. These results suggested that OPN is involved in innate responses against RV infection.

Histochemical study of the definitive erythropoietic foci in the chicken yolk sac.

It is well known that avian yolk sac is involved in both primitive and definitive erythropoiesis during embryonic development. Definitive erythropoiesis is first detected at about 4-5 days incubation and its maximum activity is reached between day 10 and 15 of incubation, ending between days 18 and 20 of incubation. We confirmed the definitive erythropoietic foci in the chicken yolk sac throughout the 5th to 19th day of incubation by histochemical light and electron microscopy. The definitive erythropoietic foci were observed in the yolk sac endodermal layer from day 5 until day 19, just before hatching. Ultrastructurally, definitive erythropoietic foci were observed extravascularly in the yolk sac endodermal cell layer in direct contact with the vitellolysis zone. These findings provide a basis for clarifying definitive erythropoiesis in vertebrates.

Presence of erythrocytes in the villous trophoblast cell layer of normal first trimester and term human placentae.

Light and electron microscopic examination of first-trimester and term human placental tissues were performed to identify erythrocytes containing hemoglobin in the villous trophoblast cell layer. Erythrocytes were not identified in chorionic villous epithelium at week 7 of gestation. These cells first appeared in the villous cytotrophoblast at week 8, and continued to be present in the villous cytotrophoblast until week 9, as shown by benzidine staining. At week 12 gestation, a cluster of erythrocytes was present in a villous syncytial sprout. At 40 and 41 weeks gestation, erythrocytes were located in the villous cytotrophoblast cell layer. Electron microscopic observations focused on the cytoplasm of villous cytotrophoblast at week 8, the syncytial sprout at week 12 and the cytotrophoblast cell layer at term, confirmed the presence of erythrocytes at an extravascular location, as observed by light microscopy.

Osteopontin participates in Th1-mediated host resistance against nonlethal malaria parasite Plasmodium chabaudi chabaudi infection in mice.

Osteopontin (OPN) knockout mice (OPN-KO mice) died of Plasmodium chabaudi chabaudi infection, although wild-type (WT) mice had self-limiting infections. OPN was detected in the WT mice at 2 days postinfection. OPN-KO mice produced significantly smaller amounts of interleukin-12 and gamma interferon than WT mice produced. These results suggested that OPN is involved in Th1-mediated immunity against malaria infection.

Effects of aloe arborescens ingestion on azoxymethane-induced intestinal carcinogenesis and hematological and biochemical parameters of male F344 rats.

We examined the modifying effect of freeze-dried whole-leaf Aloe arborescens Miller var. natalensis Berger (Kidachi aloe in Japan; designated as 'ALOE') on azoxymethane (AOM)-induced intestinal carcinogenesis in rats. Male F344 rats (4 weeks old) were fed basal diet or experimental diet containing 0.2% or 1% ALOE for 28 weeks. Starting two weeks later, the animals received subcutaneous injections of AOM once weekly for 10 weeks. The incidence of colorectal adenocarcinomas in the 0.2% (but not 1%) ALOE group showed a strong tendency for decrease (p = 0.056) from the control group. Further, the adenocarcinoma incidence in the entire intestine (small and large intestines) in the 0.2% ALOE group was significantly (p = 0.024) decreased compared to the control value. However, there were no significant differences in tumor multiplicities of colorectal or entire intestines among the 3 groups. In addition, we also studied the safety of long-term ingestion of ALOE as a health food or natural thickening stabilizer. Rats were fed the basal diet or 1% ALOE diet for 35 weeks without AOM treatment. Feeding with 1% ALOE did not affect most hematological and serum biochemical parameters in the rats. These results indicate that a low level of ALOE ingestion might have a mild suppressive effect on intestinal tumor growth without harmful side effects.

Composite tumor of mucinous cystadenoma and somatostatinoma of the kidney.

Approximately 30 cases of carcinoid tumor of the kidney have been reported in the English literature, including three cases found as components of teratomas. Renal composite tumors associated with somatostatinoma have not been described. A 53-year-old female presented with an incidentally found right renal cystic lesion. Computed tomography demonstrated a cystic lesion associated with a solid nodule in the right kidney and postcontrast dynamic MRI revealed enhancement of the solid nodule. The patient underwent radical nephrectomy for the kidney lesion and is now well without recurrence 21 months after the operation. From the histopathological findings we diagnosed the cystic lesion as a composite tumor composed of mucinous cystadenoma and carcinoid tumor. Immunohistochemistry demonstrated the majority of cells of in carcinoid portion to be positive for antisomatostatin staining. The present case is the first documented composite tumor of mucinous cystadenoma and somatostatinoma of the kidney.

Appearance of erythrocyte-like globules in the mouse visceral yolk sac endodermal cells on embryonic day 12, with special reference to blood islands.

The mouse visceral yolk sac (VYS) is widely known to play an important role as erythropoietic tissue during embryonic periods. Mouse VYS from embryonic days 9 to 12 was examined by light microscopy, electron microscopy and histochemical analysis with benzidine to detect the presence of hemoglobin with special reference to the development of VYS, the disappearance of the blood islands in VYS, and the appearance of a novel structure in the form of erythrocyte-like globules in VYS endodermal cells. The villous appearance of VYS became complicated by the development of VYS endodermal cells. The blood islands positive for the benzidine reaction were light microscopically detected on embryonic days 9, 10, and 11. They disappeared on embryonic day 12, however. Erythrocyte-like globules positive for the benzidine reaction were not observed in VYS endodermal cells on embryonic days 9, 10, and 11, but then appeared on embryonic day 12, by light and electron microscopy. Erythrocyte-like globules in VYS endodermal cells, which appear after the disappearance of blood islands in VYS, may participate in erythropoiesis during embryonic development.

It is true that, when Langerhans cells migrate from the skin to the lymph node, they are transported via lymph vessels.

BACKGROUND: Generally, Langerhans cells deliver antigen information from the skin to the draining lymph nodes via lymph vessels. METHODS: By immunohistopathology, we investigated the delivery route of Langerhans cells in human skin using CD1a and S-100 protein antibodies. RESULTS: We noted CD1a- and S-100-positive Langerhans cells in the lymph vessels of the dermis. These were shaped like dendritic cells and presented with some lymphocytes, melanophages, melanin granules and lymph in the same vessels. CONCLUSION: These observations support the concept that Langerhans cells deliver antigen peptides to regional lymph nodes via afferent lymph vessels.

A light and electron microscopic study of the mouse visceral yolk sac endodermal cells in the middle and late embryonic periods, showing the possibility of definitive erythropoiesis.

Hematological studies have revealed the importance of the visceral yolk sac (VYS) in the primitive erythropoiesis of mouse embryos at an early stage before day 12. We examined the possibility of the occurrence of extra-embryonic erythropoiesis at a stage later than embryonic day 12 by light and electron microscopic analyses. Surprisingly, a novel structure in the form of erythrocyte-like globules was observed in the VYS endodermal cells. They were consistently present in the VYS endodermal cells from embryonic day 12 until day 18 (birth is day 19), by immunocytochemical and enzyme histochemical analyses. They were immuno-positive for mouse erythrocyte antibody and also positive for the benzidine reaction showing the presence of hemoglobin. The erythrocyte-like globules were shown to be the erythrocytes present in the cytoplasm. These results indicated that erythropoiesis in the VYS endodermal cells continues from the early embryonic stage, as primitive erythropoiesis, until the late stage.

Inhibitory Effects of Heated Garlic on N-Ethyl-N'-nitro-N-nitrosoguanidine-induced Carcinogenesis in the Duodenum and Jejunum of C57BL/6 Mice.

We examined the modifying effects of heated garlic (Allium sativum L.) on N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)-induced duodenal and jejunal carcinogenesis in mice. Heated garlic powder used in this study was prepared as follows: unpeeled garlic bulbs were blanched in boiling water for 6 min, and then peeled, the cloves being crushed, homogenized, and finally freeze-dried. The garlic powder had almost undetectable alliinase activity and was rich in alliin (the main sulfur compound of heated garlic; 22.1 &mgr;/g dry weight). Male C57BL/6 mice were given ENNG (100 &mgr;/l) in drinking water for the first 4 weeks, and then basal diet (Group 1), or 10% (Group 2), 3% (Group 3) or 1% (Group 4) heated garlic in the diet for 30 weeks. At the termination of the experiment, the incidences of duodenal tumors in Groups 1-3 were significantly lower than those in Group 1, and the multiplicities in Group 2 were significantly lower than those in Group 1. Additionally, the incidences and/or multiplicities of the jejunal tumors in Groups 2 and 4 were also significantly lower than those in Group 1. In this study, we also examined changes in erythrocyte polyamine levels. Values for Group 1 were significantly greater than those in the control group, and this elevation in Group 1 were significantly inhibited by dietary heated garlic (10% in the diet; Group 2). These results indicated that the post-initiation-stage feeding of heated garlic, especially at 10% in the diet, inhibits ENNG-induced duodenal and jejunal carcinogenesis in mice.

Inhibition of N-ethyl-N'-nitro-N-nitrosoguanidine-induced Duodenal Tumorigenesis in Mice by Whole-leaf Aloe arborescens Miller var. natalensis Berger.

We examined the modifying effects of freeze-dried whole-leaf Aloe arborescens Miller var. natalensis Berger (designated as 'ALOE') on N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)-induced duodenal tumorigenesis in C57BL/6 mice. Experiment 1: Male mice were given ENNG in drinking water for the first 4 weeks, and then 10% ALOE in basal diet for 16 weeks. Experiment 2: Female mice were given ENNG for 5 weeks, and then 5%, 1% or 0.2% ALOE in the diet were given for 15 weeks. In Experiment 1, the tumor incidence and tumor multiplicity (tumors per mouse) of the duodenum in the ENNG + 10% ALOE group were significantly decreased compared with that in the ENNG alone group. Erythrocyte polyamine levels in the ENNG + 10% ALOE group were also significantly decreased. In Experiment 2, the incidence of duodenal tumors in the ENNG + 5% ALOE group were significantly decreased compared with that in the ENNG alone group. These results indicated that ALOE, especially at 10% in the diet, inhibits ENNG-induced duodenal tumorigenesis in mice.