Swelling of Doubly Magic

Interaction cross sections for ^{42-51}Ca on a carbon target at 280 MeV/nucleon have been measured for the first time. The neutron number dependence of derived root-mean-square matter radii shows a significant increase beyond the neutron magic number N=28. Furthermore, this enhancement of matter radii is much larger than that of the previously measured charge radii, indicating a novel growth in neutron skin thickness. A simple examination based on the Fermi-type distribution, and mean field calculations point out that this anomalous enhancement of the nuclear size beyond N=28 results from an enlargement of the core by a sudden increase in the surface diffuseness of the neutron density distribution, which implies the swelling of the bare ^{48}Ca core in Ca isotopes beyond N=28.

The influence of enteral obestatin administration to suckling rats on intestinal contractility.

This study investigated the effect of enteral administration of obestatin on the contractility of whole-thickness preparations of duodenum and middle jejunum, as well as on the morphology of the enteric nervous system (ENS). Suckling rats were assigned to 3 groups (n=12) treated with: C-saline solution; LO-obestatin (125nmol/kgb.wt); HO-obestatin (250nmol/kgb.wt). Saline solution or obestatin were administered twice daily, from the 14th to the 21st day of life. Sections were studied in an organ bath, for isometric recording in the presence of acetylocholine (ACh), atropine (ATR) and tetradotoxin (TTX). Thickness of intestinal muscularis layer, the number of interstitial cells of Cajal (ICC) were measured in the paraffin sections. The immunodetection of Muscarinic Acetylocholine Receptor 2 (M2 receptor) was performed in the intestinal segments. In both intestinal segments HO treatment decreased the amplitude of spontaneous contraction compared to that observed in the C group. In the middle jejunum, the LO treatment also decreased the amplitude. TTX and ATR had no effect on amplitude of spontaneous contraction in the jejunum of LO and HO-treated animals. Compared to the C group, duodenal sections from HO animals and middle jejunum sections from LO and HO groups displayed a lower amplitude in response to ACh and EFS evoked contraction. An increase in the thickness of the muscularis layer was observed in the duodenum of LO and HO groups whereas the number ICC did not change significantly after treatment with obestatin. Moreover, the enteral administration of obestatin did not effect significantly on the cytoplasmic expression of M2 receptor in the jejunum. Our study demonstrated that enteral administration of obestatin to suckling rats influences small intestine contractility in the segment specific manner.

Age-dependent effect of obestatin on intestinal contractility in Wistar rats.

Obestatin is a 23-amino acid peptide encoded by the ghrelin gene. We have investigated the effect of obestatin on intestinal contractility in rats ranging from the suckling period till adolescence. Duodenal and middle jejunum whole-thickness preparations from neonatal and adult rats were studied in an organ bath, for isometric recording under treatment with obestatin (1µmolL(-1)) in the presence of acetylocholine (ACh), atropine and tetradotoxin (TTX). Both the EFS and ACh-stimulated contractile response, as well as spontaneous contractile activity is age-dependent and specific for the segment of jejunum. Except for the middle jejunum of 7day old rats, treatment with obestatin caused a significant TTX-sensitive increase in the amplitude of EFS-stimulated off-contraction of both intestinal segments studied. Following injection of obestatin, the amplitude of spontaneous contraction in the duodenum increased in 7day old rats. In the middle jejunum, treatment with obestatin significantly increased both the amplitude and frequency of spontaneous contraction in rats till the 28th day of life, whereas in adult rats the observed effect of obestatin was the opposite (P<0.001 and P<0.0001, respectively). The effects of treatment with obestatin on stimulation with increasing doses of ACh were only observed in the preparations from suckling rats. ACh-stimulated contractility in the duodenum was decreased while in the middle jejunum the observed effect was opposite. These results indicate the importance of peripheral obestatin in the cholinergic control of intestinal contractility in both neonatal and adult rats.

Deletion of platelet-derived growth factor receptor-ß improves diabetic nephropathy in Ca²âº/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice.

AIMS/HYPOTHESIS: The activation of platelet-derived growth factor receptor-ß (PDGFR-ß) signalling is increased in the glomeruli and tubules of diabetic animals. In this study, we examined the role of PDGFR-ß signalling during the development of diabetic nephropathy. METHODS: We recently generated pancreatic beta cell-specific Ca(2+)/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice (CaMKIIα mice), which show very high plasma glucose levels up to 55.5 mmol/l and exhibit the features of diabetic nephropathy. These mice were crossed with conditional knockout mice in which Pdgfr-ß (also known as Pdgfrb) was deleted postnatally. The effect of the deletion of the Pdgfr-ß gene on diabetic nephropathy in CaMKIIα mice was evaluated at 10 and 16 weeks of age. RESULTS: The plasma glucose concentrations and HbA(1c) levels were elevated in the CaMKIIα mice from 4 weeks of age. Variables indicative of diabetic nephropathy, such as an increased urinary albumin/creatinine ratio, kidney weight/body weight ratio and mesangial area/glomerular area ratio, were observed at 16 weeks of age. The postnatal deletion of the Pdgfr-ß gene significantly decreased the urinary albumin/creatinine ratio and mesangial area/glomerular area ratio without affecting the plasma glucose concentration. Furthermore, the increased oxidative stress in the kidneys of the CaMKIIα mice as shown by the increased urinary 8-hydroxydeoxyguanosine (8-OHdG) excretion and the increased expression of NAD(P)H oxidase 4 (NOX4), glutathione peroxidase 1 (GPX1) and manganese superoxide dismutase (MnSOD) was decreased by Pdgfr-ß gene deletion. CONCLUSIONS/INTERPRETATION: The activation of PDGFR-ß signalling contributes to the progress of diabetic nephropathy, with an increase in oxidative stress and mesangial expansion in CaMKIIα mice.

Biological effects of probiotics: what impact does Lactobacillus casei shirota have on us?

Probiotics have been defined as live bacteria beneficial to the host when administered in adequate amounts. To evaluate the effect of probiotics on the prevention of carcinogenesis, Lactobacillus casei Shirota (LcS) was given to the patients who had undergone the resection of superficial bladder cancer, and administration of LcS significantly reduced the recurrence rate of bladder cancer. When LcS was given to the patients whose colonic polyps were surgically removed, the recurrence of colorectal cancer with moderate or severe atypia was suppressed. To assess the putative actions of LcS on innate immune responses, we examined the effect of LcS on natural killer (NK) cell activity in humans. Daily ingestion of fermented milk containing LcS restored NK cell activity in healthy subjects with low NK cell activity as well as human T lymphotropic virus (HTLV)-1-associated myelopathy patients. When peripheral blood mononuclear cells from healthy humans were cultured in the presence of heat-killed LcS, NK cell activity was augmented, which were partly mediated by monocyte-derived interleukin (IL)-12. These findings suggest that LcS may help the reinforcement of our defense system against cancer by modulating innate immune functions.

Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells.

Hematopoietic cells are important targets for genetic modification with retroviral vectors. Attempts at human gene therapy of stem cells have achieved limited success partly because of low gene transfer efficiency. Chymotryptic fragments of the extracellular matrix molecule fibronectin used during infection have been shown to increase transduction of human hematopoietic progenitor cells. Here, we demonstrate that this enhanced gene transfer into mammalian target cells is due to direct binding of retroviral particles to sequences within the fibronectin molecule. Transduction of mammalian cells, including murine long-term repopulating hematopoietic cells, is greatly enhanced when cells are adherent to chimeric fragments containing these retroviral binding sequences. In addition, colocalization of retrovirus and target cells on fibronectin peptides allows targeted transduction of specific cell types by exploiting unique ligand/receptor interactions.

Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy.

The development of stem-cell gene therapy is hindered by the absence of repopulation assays for primitive human hematopoietic cells. Current methods of gene transfer rely on in vitro colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, as well as inference from other mammalian species. We have identified a novel human hematopoietic cell, the SCID-repopulating cell (SRC), a cell more primitive than most LTC-ICs and CFCs. The SRC, exclusively present in the CD4+CD8- fraction, is capable of multilineage repopulation of the bone marrow of nonobese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). SRCs were rarely transduced with retroviruses, distinguishing them from most CFCs and LTC-ICs. This observation is consistent with the low level of gene marking seen in human gene therapy trials. An SRC assay may aid in the characterization of hematopoiesis, as well as the improvement of transduction methods.

Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells.

Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.

Experimental systemic amyloidosis induced by immunization with syngeneic organ extracts in mice.

Systemic amyloidosis was induced consistently in mice by intramuscular injection of syngeneic organ (liver and kidney) extracts mixed with CFA six times at weekly intervals. Syngeneic organ extract with CFA also induced amyloidosis of a lesser degree. All three strains of mice (C57BL/6, C3H/He, and BALB/c) injected with a syngeneic liver extract mixed with CFA developed systemic amyloidosis; the most prominent amyloid deposition occurred in C57BL/6 (B6) mice, followed by C3H/He and BALB/c. The amyloid substance deposited in these animals was identified as mouse amyloid A protein (AA). Furthermore, an organ specificity of the immunogen in inducing amyloidosis was suggested with liver and kidney extracts. Primed spleen cells of the immunized B6 mice were fractionated by a nylon-wool column and injected to normal recipient mice via the tail vein. Organs of the recipient mice developed systemic amyloidosis 8 wk after the transfer, and the most prominent histological changes occurred in the recipient mice given nylon-wool column adherent spleen cells. Using anti-Thy-1,2; Ly-1; Ly-2, antibody and complement, it was suggested that T cells, especially Ly-1,2,3+ T cell populations in the primed nylon-wool adherent cells, play an important role in the induction of systemic amyloidosis. It was shown further that the amyloidosis-inducing substance in liver extract was composed of unstable proteins or protein-bound substance.

Immunosuppressive effect of prolactin-induced protein: a new insight into its local and systemic role in chronic allergic contact dermatitis.

BACKGROUND: Prolactin-induced protein (PIP) has been shown to bind to CD4 and is speculated to block CD4-HLA-DR interaction. However, the immunomodulatory effect of PIP on chronic allergic contact dermatitis (ACD) remains to be elucidated. OBJECTIVES: To define the role of PIP during the immunoresponse. METHODS: Using a low-dose oxazolone-induced mouse chronic ACD model, expression of PIP was examined immunohistologically. Furthermore, effects of continued exposure to a peptide mimicking the major binding site of PIP (amino acids 106-132) for CD4 was examined in a mouse chronic ACD model. RESULTS: We clarified that keratinocytes, dermal infiltrating cells and spleen infiltrating mononuclear cells are positively stained with anti-PIP antibody. The PIP peptide significantly downregulated oxazolone-induced mouse ACD compared with controls. We also found that inflammation of the control ear, to which the PIP peptide had not been applied, was also suppressed in a synchronized manner in the late phase of ACD. CONCLUSIONS: These findings suggest that PIP might have a local and systemic immunosuppressive effect in mouse chronic ACD.

Centrally administered neuromedin S inhibits feeding behavior and gastroduodenal motility in mice.

Neuromedin S (NMS) was recently identified as an endogenous ligand for the FM-4/TGR-1 receptor in the rat hypothalamus. No previous studies have examined the effect of NMS on gut motility. We examined the effects of intracerebroventricular administration of NMS on food intake in food-deprived and free-feeding mice, and on gastroduodenal motility by using a manometric method, and gastric emptying in mice. We found that NMS decreased food intake and the gastric emptying rate. It also disrupted the motor activity in the antrum and duodenum of conscious food-deprived mice. These results suggest that NMS influences gut motility as well as feeding behavior.

Influence of obestatin on the histological development of the small intestine in piglets during the first week of postnatal life.

Obestatin is a gastrointestinal peptide having wide-ranging effects on cell proliferation; however, its mechanism of action remains poorly understood. Thus, the aim of the study was to elucidate the effect of exogenous obestatin on the postnatal structural development of the small intestine. Seven-day-old piglets with an average BW of 1.56 ± 0.23 kg were divided into four groups (n = 10) that received intragastrically obestatin (2, 10 or 15 µg/kg BW) or vehicle. After a 6-day experimental period, morphological analysis of gastrointestinal tract and small intestine wall (mitosis and apoptosis indexes, histomorphometry of mucosa and muscularis layers) was performed. The study revealed a seemingly incoherent pattern of the histological structure of the small intestine among the experimental groups, suggesting that the effect of obestatin is both intestinal segment specific and dose dependent. Histomorphometric analysis of the small intestine showed that higher doses of obestatin seem to promote the structural development of the duodenum while simultaneously hindering the maturation of more distal parts of the intestine. Intragastric administration of obestatin increased the crypt mitotic index in all segments of the small intestine with the strongest pro-mitotic activity following the administration of obestatin at a dose of 10 and 15 µg/kg BW. The significant differences in the number of apoptotic cells in the intestinal villi among the groups were observed only in proximal jejunum and ileum. In conclusion, it seems that obestatin shows a broad-spectrum of activity in the gastrointestinal tract of newborn piglets, being able to accelerate its structural development. However, the varied effect depending on the intestinal segment or the concentration of exogenous obestatin causes that further research is needed to clarify the exact mechanism of this phenomenon.

Rapid alphabeta TCR-mediated responses in gammadelta T cells transduced with cancer-specific TCR genes.

Adoptive T-cell transfer of in vitro cultured T cells derived from cancer patients with naturally developed immune responses has met with some success as an immunotherapeutic approach, although only a limited number of patients showed spontaneous immune responses. To find alternative ways, such as cancer-specific T-cell receptor (TCR) gene transfer, in preparation for sufficient numbers of antigen-specific T cells is an important issue in the field of adoptive T-cell therapy. Given the inherent disadvantage of alphabeta TCR transfer to other alphabeta T cells, namely the possible formation of mixed TCR heterodimers with endogenous alpha or beta TCR, we employed gammadelta T cells as a target for retroviral transfer of cancer-specific TCR and examined whether gammadelta T cells were useful as an alternative population for TCR transfer. Although retroviral transduction to gammadelta T cells with TCR alphabeta genes alone, isolated from a MAGE-A4(143-151)-specific alphabeta CD8(+) cytotoxic T lymphocyte (CTL) clone, did not provide sufficient affinity to recognize major histocompatibility (MHC)-peptide complexes due to the lack of CD8 co-receptor, gammadelta T cells co-transduced with TCR alphabeta and CD8 alphabeta genes acquired cytotoxicity against tumor cells and produced cytokines in both alphabeta- and gammadelta-TCR-dependent manners. Furthermore, alphabeta TCR and CD8-transduced gammadelta T cells, stimulated either through alphabeta TCR or gammadelta TCR, rapidly responded to target cells compared with conventional alphabeta T cells, reminiscent of gammadelta T cells. We propose alphabeta TCR-transduced gammadelta T cells as an alternative strategy for adoptive T-cell transfer.

[Acute pulmonary thromboembolism in polysplenia syndrome].

We present a case of azygos continuation of the inferior vena cava, combined with polysplenia syndrome in a 72-year-old woman who presented with circulatory collapse due to acute pulmonary thromboembolism. Patients with polysplenia have multiple spleens, and their splenic function is usually normal, but this case was not. In this case, defective splenic function was associated with a high risk of fulminant bacterial infections, especially with encapsulated bacteria. The clinical features and prognosis of this entity are discussed.

Long-term phenotypic, functional and genetic stability of cancer-specific T-cell receptor (TCR) alphabeta genes transduced to CD8+ T cells.

In adoptive T-cell transfer as an intervention for malignant diseases, retroviral transfer of T-cell receptor (TCR) genes derived from CD8(+) cytotoxic T-lymphocyte (CTL) clones provides an opportunity to generate a large number of T cells with the same antigen specificity. We cloned the TCR-alphabeta genes from a human leukocyte antigen (HLA)-A(*)2402-restricted CTL clone specific for MAGE-A4(143-151). The TCR-alphabeta genes were transduced to 99.2% of non-TCR expressing SupT1, a human T-cell line, and to 12.7-32.6% of polyclonally activated CD8(+) T cells by retroviral transduction. As expected, TCR-alphabeta gene-modified CD8(+) T cells showed cytotoxic activity and interferon-gamma production in response to peptide-loaded T2-A(*)2402 and tumor cell lines expressing both MAGE-A4 and HLA-A(*)2402. A total of 24 clones were established from TCR-alphabeta gene-transduced peripheral blood mononuclear cells and all clones were functional on a transduced TCR-dependent manner. Four clones were kept in culture over 6 months for analyses in detail. The transduced TCR-alphabeta genes were stably maintained phenotypically, functionally and genetically. Our results indicate that TCR-transduced alphabeta T cells by retroviral transduction represent an efficient and promising strategy for adoptive T-cell transfer for long term.

VLA-5 is expressed by mouse and human long-term repopulating hematopoietic cells and mediates adhesion to extracellular matrix protein fibronectin.

Fibronectin (FN), an extracellular matrix protein, is involved in the adhesion and migration of hematopoietic cells and has been shown to enhance retroviral gene transfer into primitive hematopoietic cells by co-localization of target cells and retrovirus when used as a substrate in vitro. We have previously found that mouse hematopoietic stem cells could be transduced on a FN fragment that included the recognition sequence Arg-Gly-Asp (RGD), suggesting that stem cells may express the integrin very late antigen (VLA)-5. To address this, we investigated the binding of mouse and human hematopoietic cells to recombinant peptides that contained one or a combination of the three principle cell-binding domains of FN. These domains included the VLA-5- binding sequence RGD, the VLA-4-binding site CS1, and the high affinity heparin-binding domain. Here we show that mouse long-term in vivo repopulating stem cells, as well as primitive human NOD/SCID mouse repopulating cells, can bind extracellular matrix protein FN by using integrin VLA-5 in vitro. This binding is specific and can be inhibited by antibodies to VLA-5. In addition, preincubation of BM cells with peptide CH-296, which contains all three primary FN-binding domains, decreased the engraftment of cells in the bone marrow in vivo, while intravenous injection of the same peptide induced an increase of progenitor cells in the spleen. In summary, our data demonstrate that VLA-5 is expressed on primitive mouse and human hematopoietic cells and suggest that there may be significant cooperation between integrin receptors and proteoglycan molecules in the engraftment of bone marrow cells and hematopoietic cell adhesion in vivo.

Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates.

Recent reports suggest that cells in active cell cycle have an engraftment defect compared with quiescent cells. We used nonhuman primates to investigate this finding, which has direct implications for clinical transplantation and gene therapy applications. Transfer of rhesus CD34(+) cells to culture in stem cell factor (SCF) on the CH-296 fibronectin fragment (FN) after 4 days of culture in stimulatory cytokines maintained cell viability but decreased cycling. Using retroviral marking with two different gene transfer vectors, we compared the engraftment potential of cytokine-stimulated cells versus those transferred to nonstimulatory conditions (SCF on FN alone) before reinfusion. In vivo competitive repopulation studies showed that the level of marking originating from the cells continued in culture for 2 days with SCF on FN following a 4-day stimulatory transduction was significantly higher than the level of marking coming from cells transduced for 4 days and reinfused without the 2-day culture under nonstimulatory conditions. We observed stable in vivo overall gene marking levels of up to 29%. This approach may allow more efficient engraftment of transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.

Autoantibodies against CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) that impair glucose-induced insulin secretion in noninsulin- dependent diabetes patients.

Cyclic ADP-ribose (cADPR) has been shown to be a mediator for intracellular Ca2+ mobilization for insulin secretion by glucose in pancreatic beta cells, and CD38 shows both ADP-ribosyl cyclase to synthesize cADPR from NAD+ and cADPR hydrolase to hydrolyze cADPR to ADP-ribose. We show here that 13.8% of Japanese non-insulin-dependent diabetes (NIDDM) patients examined have autoantibodies against CD38 and that the sera containing anti-CD38 autoantibodies inhibit the ADP-ribosyl cyclase activity of CD38 (P

Development and prevention of advanced diabetic nephropathy in RAGE-overexpressing mice.

Vascular complications arising from multiple environmental and genetic factors are responsible for many of the disabilities and short life expectancy associated with diabetes mellitus. Here we provide the first direct in vivo evidence that interactions between advanced glycation end products (AGEs; nonenzymatically glycosylated protein derivatives formed during prolonged hyperglycemic exposure) and their receptor, RAGE, lead to diabetic vascular derangement. We created transgenic mice that overexpress human RAGE in vascular cells and crossbred them with another transgenic line that develops insulin-dependent diabetes shortly after birth. The resultant double transgenic mice exhibited increased hemoglobin A(1c) and serum AGE levels, as did the diabetic controls. The double transgenic mice demonstrated enlargement of the kidney, glomerular hypertrophy, increased albuminuria, mesangial expansion, advanced glomerulosclerosis, and increased serum creatinine compared with diabetic littermates lacking the RAGE transgene. To our knowledge, the development of this double transgenic mouse provides the first animal model that exhibits the renal changes seen in humans. Furthermore, the phenotypes of advanced diabetic nephropathy were prevented by administering an AGE inhibitor, (+/-)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide (OPB-9195), thus establishing the AGE-RAGE system as a promising target for overcoming this aspect of diabetic pathogenesis.

Obestatin stimulates the secretion of pancreatic juice enzymes through a vagal pathway in anaesthetized rats - preliminary results.

Obestatin is a 23 amino acid peptide derived from the preproghrelin precursor, and originally purified from the rat stomach mucosa. It was shown that obestatin may counteract the effects of its sister peptide, ghrelin, on food intake and gastrointestinal motility but the other roles in controlling the gastrointestinal function remain unknown. The aim of the present study was to determine the influence of exogenous obestatin on the secretion of pancreatic juice. In anesthetized male Wistar rats the external jugular vein was catheterized, and the common biliary-pancreatic duct was cannulated with polyethylene tubing for collection of pancreatic-biliary juice (P-BJ). Obestatin boluses (30, 100 and 300 nmol/kg b. wt.) were injected intravenously or intraduodenally every 30 min. Obestatin was also administered in vagotomized (subdiaphragmatic vagotomy) rats. In the examined rats, obestatin intravenous and intraduodenal boluses did not affect the P-BJ volume. On the other hand, obestatin boluses increased the protein output and trypsin activity. Vagotomy abolished the effects of exogenous obestatin administration. In conclusion, the present study demonstrates for the first time that exogenous obestatin may stimulate the secretion of pancreatic juice enzymes. The effect is dose-dependent and requires intact vagal supply.