Blockade of the fractalkine-CX3CR1 axis ameliorates experimental colitis by dislodging venous crawling monocytes.

Chemokine systems modulate inflammatory and immune responses in inflammatory bowel disease (IBD). The colons of IBD patients show increased levels of fractalkine (FKN) and high numbers of FKN receptor-positive (CX3CR1+) cells; however, the FKN-CX3CR1 axis's role in intestinal inflammation, especially in intravascular leukocyte behaviors, still remains unclear. Here, we show that interruption of the FKN-CX3CR1 axis by anti-FKN monoclonal antibody (mAb) ameliorates murine colitis through regulation of intravascular monocyte behaviors in murine colitis models. FKN expression was detectable in vascular endothelium and CX3CR1+ macrophages accumulated in the mucosal lamina propria and submucosa of the inflamed colons. CD115+ monocytes tethered to the venous endothelium and expressed pro-inflammatory mediators. The anti-FKN mAb improved colitis symptoms, markedly reduced pro-inflammatory factors in the colon, maintained blood vessel integrity and reduced tethered monocytes in the inflamed veins. Intravital imaging revealed that CD115+Gr-1low/- monocytes crawled on the apical surfaces of venous endothelium, and anti-FKN mAb rapidly dislodged the crawling monocytes and inhibited their patrolling behavior. These findings suggest that the FKN-CX3CR1 axis triggers the patrolling behavior of crawling monocytes on the venous endothelium of inflamed colons, and accelerates the subsequent leukocyte activation and infiltration by locally producing inflammatory cytokines and chemokines. The mAb also ameliorated symptoms in another IBD model, T-cell-transferred colitis. Blocking the FKN-CX3CR1 axis with an anti-FKN mAb considerably inhibits the colitis-triggered inflammatory cascades, which may be an alternative strategy to treat IBD.

LIM homeobox 8 (Lhx8) is a key regulator of the cholinergic neuronal function via a tropomyosin receptor kinase A (TrkA)-mediated positive feedback loop.

Basal forebrain cholinergic neurons play an important role in cognitive functions such as learning and memory, and they are affected in several neurodegenerative diseases, including Alzheimer disease and Down syndrome. Despite their functional importance, the molecular mechanisms of functional maturation and maintenance of these cholinergic neurons after the differentiation stage have not been fully elucidated. This study demonstrates that the LIM homeobox 8 (Lhx8) transcription factor regulates cholinergic function in rat septal cholinergic neurons in primary cultures from E18.5 embryos and in the adult brain. Lhx8 expression modulated tropomyosin receptor kinase A (TrkA) expression in septal cholinergic neurons in vitro and in vivo, resulting in regulated acetylcholine release as an index of cholinergic function. In addition, Lhx8 expression and function were regulated by nerve growth factor (NGF), and the effect of NGF was potentiated by Lhx8-induced TrkA expression. Together, our findings suggest that positive feedback regulation between Lhx8, TrkA, and NGF is an important regulatory mechanism for cholinergic functions of the septum.

N-type calcium channel in the pathogenesis of experimental autoimmune encephalomyelitis.

One of the family of voltage-gated calcium channels (VGCC), the N-type Ca(2+) channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca(2+) channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca(2+) channel α(1B)-deficient (α(1B)(-/-)) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35-55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the α(1B)(-/-) mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from α(1B)(-/-) mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation, or cytokine production from splenocytes between the two genotypes. On the other hand, Western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from α(1B)(-/-) mice and was significantly inhibited by a selective N-type Ca(2+) channel antagonist, ω-conotoxin GVIA or a withdrawal of extracellular Ca(2+). These results suggest that the N-type Ca(2+) channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.

Tranilast, an anti-allergic drug, down-regulates the growth of cultured neurofibroma cells derived from neurofibromatosis type 1.

Neurofibromas are benign tumors that comprise primarily of Schwann cells and fibroblasts. Mast cells have been found scattered in the tumor tissue, and their role in promoting the proliferation of neurofibroma has been suggested. Tranilast (N-[3,4-dimethoxycinnamolyl]anthranilic acid) is an anti-allergic drug that inhibits release of the chemical mediators from mast cells and it used for the treatment of keloids and hypertrophic scars by its inhibition of growth-promoting transforming growth factor (TGF)-beta(1) from fibroblasts. We assumed that tranilast would suppress neurofibroma cell growth. In order to prove this hypothesis, we investigated the effectiveness of tranilast in inhibiting the tumor growth using a new cell culture system obtained from patients with neurofibromas. We called this culture system with the mixture of Schwann cells and fibroblasts "NF1 cells culture". Mast cells were differentiated from CD34(+) peripheral blood mononuclear cells of normal healthy subjects, and were co-cultured with NF1 cells. Three days after tranilast (10 approximately 100 microM) added to the culture dishes, we counted viable cell numbers and measured the concentrations of TGF-beta(1), stem cell factor (SCF) and tryptase, which exists in the histamine granule, in the culture medium. Tranilast significantly suppressed proliferation of the NF1 cells and lowered the levels of TGF-beta(1), SCF and tryptase. These results suggest that tranilast retards tumor proliferation through not only suppression of cell growth factor, but also the inhibition of a chemical mediator released from mast cells. Thus, tranilast can be a potent therapeutic agent to inhibit the growth of neurofibromas.

Donepezil, an acetylcholinesterase inhibitor, enhances adult hippocampal neurogenesis.

Donepezil hydrochloride is a potent and selective acetylcholinesterase inhibitor and has been treated for Alzheimer's disease, in which the cholinergic dysfunction is observed. Recently, the degeneration of medial septal cholinergic nuclei in adult rat suppressed the neurogenesis in hippocampal dentate gyrus (DG) was reported. Then, we determined whether donepezil which activated the brain cholinergic system could modulate hippocampal neurogenesis in normal rats. After the injection of 5'-bromo-2'-deoxyuridine (BrdU) to label dividing cells, we orally treated with donepezil (0.5 or 2mg/kg) once a day for 4 weeks. In the other group, we performed 4-week subcutaneous infusion of scopolamine (0.75 or 3mg/day), a muscarinic acetylcholine receptor blocker. The doses of donepezil and scopolamine we used in this study were reported to activate and inhibit cholinergic activity in rats, respectively. One day after the completion of drug treatment, the animals were sacrificed, and immunohistochemical analysis was performed. Donepezil increased, but scopolamine decreased, the number of BrdU-positive cells in the DG as compared with the vehicle-treated control. Neither drug had any effects on the percentage of BrdU-positive cells that were also positive for a neuronal marker NeuN, nor the number of proliferating cell nuclear antigen-positive cells in the DG. These results indicate that donepezil enhances and scopolamine suppresses the survival of newborn neurons in the DG without affecting the proliferation of neural progenitor cell and the neuronal differentiation. We also found that chronic treatment of donepezil enhanced, and scopolamine suppressed phosphorylation of cAMP response element binding protein (CREB), which was involved in cell survival, in the DG. These results suggest that donepezil activates the central cholinergic transmission and enhances the survival of newborn neurons in the DG via CREB signaling.

Cryopreserved cultured epithelial allografts for pediatric deep partial dermal burns: Early wound closure and suppression of scarring.

BACKGROUND: In deep partial thickness dermal burns (DDB) where greater than 50% of the dermis is lost, severe pain, scarring and contractures occur. Therefore, skin grafting may be required. In children, scar contracture occurs because scarred skin does not stretch with growth creating the need for additional scar-releasing or skin-grafting surgeries. In order to resolve this problem, we used cryopreserved cultured epithelial allograft (cryopreserved allo-CEG), which can be grafted shortly after sustaining a wound. We reevaluated the promotion of early wound closure of burns and suppression of scarring by this treatment. METHODS: Cryopreserved allo-CEGs were used to treat 50 cases of pediatric DDB from 1992 to 2000. These cases were reviewed with regard to the time until epithelialization, take percentage, and pain level. Also, in order to examine why cryopreserved allo-CEG promotes healing of burns and suppresses scarring, growth factors and cytokines in the cryopreserved allo-CEG were measured. Cryopreserved allo-CEG sheets were solubilized and concentrations of TGF-α, TGF-ß1, IL-1α, IL-1ß, PDGF-AA, VEGF, KGF, IL-6, b-FGF, as well as metalloprotease-1 (MMP-1) and HGF, which are noted to have scarring suppression effects, were measured before grafting. RESULTS: Grafting of cryopreserved allo-CEGs in 50 cases of childhood DDB resulted in early epithelialization (9.32 ± 3.63 days on the average) and an almost 100% take rate. Also, pain relief (pain reduction or elimination, reduced need for anesthetics) was seen in all cases. Although 15-23 years have now elapsed, adverse events have not been observed. Cryopreserved allo-CEG contains IL-1α, IL-1ß, PDGF-AA, TGF-α, TGF-ß1, VEGF, and IL-6 have wound healing effects. The concentration of IL-1α was higher than the concentrations of other components, and this was followed by TGF-α, TGF-ß1, b-FGF and VEGF. Although the concentration of MMP-1, which has a scarring suppression effect, was high, HGF was not detected. CONCLUSION: Cryopreserved allo-CEG contains growth factors that promote wound healing and factors that suppress scarring. Three effects, namely (1) early wound closure, (2) scarring suppression, and (3) pain relief were seen with grafts of cryopreserved allo-CEG in cases of childhood DDB. These observations show that cryopreserved allo-CEG is clinically useful and effective for the treatment of childhood DDB.

False aneurysm of the ascending aorta concomitant with chronic mediastinitis after tube graft replacement in octogenarian.

An 81-year-old man developed impending rupture of a false aneurysm of the ascending aorta concomitant with chronic mediastinitis lasting for 10 years after tube graft replacement. He had undergone various infection-related mediastinal surgical procedures. He was successfully treated by debridement of infected tissues, patch repair of the false aneurysm, and transposition of the right latissimus dorsi muscle flap. The postoperative course was uneventful except for seromas. A chest computed tomographic scan performed 5 and 24 months after surgery showed no signs of recurrent aneurysm formation. A conservative surgical treatment including muscle flap transposition was effective in octogenarian.

Effects of TGF-beta and TGF-beta-neutralizing antibody on normal skin fibroblasts and scar-derived fibroblasts.

Transforming growth factor-beta (TGF-beta) is known as an important cytokine for scar formation in wound healing. The purpose of the present study was to investigate the effects of TGF-beta and its neutralizing antibody on normal skin fibroblasts and scar-derived fibroblasts in culture. Endogenous TGF-beta levels were similar in all fibroblasts. Cell proliferation increased when TGF-beta 1 or beta 2 was added to the cultures, and the increase was higher and started at a lower level in the scar-derived fibroblasts (p < 0.05). The increase of fibroblasts was suppressed by the addition of TGF-beta-neutralizing antibody to the cultures, and the suppression rate was higher in the scar-derived cells (p < 0.05). Percentages of the cells in the growth phases of cell cycle decreased in the normal skin fibroblasts (p < 0.05) when TGF-beta-neutralizing antibody was added. Our findings showed that scar-derived fibroblasts and normal skin fibroblasts have a different sensitivity to TGF-beta. Further study is needed on the effect of the neutralizing antibody to the cell counts and cell cycle of scar-derived fibroblasts.

Minimally invasive functional reconstruction after extended oropharyngeal resection including soft palate and base of tongue using a pectoralis major myocutaneous flap.

Excision of large oropharyngeal carcinomas that affect the base of the tongue and the soft palate severely impairs swallowing and articulation. In the present study we describe a minimally invasive technique that effectively restores swallowing and articulation by the insertion of a pectoralis major myocutaneous flap with a bilobular skin island. One lobe of the skin island is used to reconstruct the base of the tongue and the other to reconstruct the oropharynx. The soft palate is reconstructed by folding the tip of the lobe that is used to reconstruct the oropharynx in half along the long axis to fill the rhinopharynx. We have done this procedure for 13 patients with oropharyngeal carcinoma. Six months postoperatively all 13 were able to swallow without aspiration. Nine of the 13 patients were able to hold a normal conversation, but the remaining four had severe rhinolalia aperta. However, this condition was easily corrected by secondary reconstruction using a pharyngeal flap and a palatal mucoperiosteal flap (n = 3) or by the use of a small speech aid (n = 1).

V-Y fasciocutaneous flap of the medial thigh including the long saphenous vein for reconstruction of intrapelvic dead space.

Some patients develop an intrapelvic infection and fistula caused by the presence of intrapelvic dead space after the resection of rectal cancer, and the treatment is sometimes quite difficult. We have developed a new surgical technique for the treatment and prevention of such fistulas that uses a fasciocutaneous flap from the medial thigh. A V-shaped fasciocutaneous flap with a pedicle on the anterior side of the thigh is designed on the medial thigh and gluteal region. After raising the fasciocutaneous flap that contains the long saphenous vein, the gluteal section including a thick layer of fatty tissue is de-epithelialised, and the flap is rotated and advanced towards the dead space to fill it. Four patients were operated on using our technique. One was a secondary reconstruction: the patient had developed a small fistula after reconstructive surgery, but it healed with conservative treatment. As a result, all four patients achieved satisfactory outcomes. The advantages of our technique include: no change in the position of the body is required for reconstruction; operations are simple; sufficient volume of tissue is obtained from the thick fatty tissues of the gluteal region; and the fasciocutaneous flap contains the long saphenous vein and has good venous circulation. We consider this technique useful for the reconstruction of intrapelvic dead space.

Investigation of the dominant hydration structures among the ionic species in aqueous solution: novel quantum mechanics/molecular mechanics simulations combined with the theory of energy representation.

In the present work, we have performed quantum chemical calculations to determine preferable species among the ionic complexes that are present in ambient water due to the autodissociation of water molecule. First, we have formulated the relative population of the hydrated complexes with respect to the bare ion (H(3)O(+) or OH(-)) in terms of the solvation free energies of the relevant molecules. The solvation free energies for various ionic species (H(3)O(+), H(5)O(2) (+), H(7)O(3) (+), H(9)O(4) (+) or OH(-), H(3)O(2) (-), H(5)O(3) (-), H(7)O(4) (-), H(9)O(5) (-)), categorized as proton or hydroxide ion in solution, have been computed by employing the QM/MM-ER method recently developed by combining the quantum mechanical/molecular mechanical (QM/MM) approach with the theory of energy representation (ER). Then, the computed solvation free energies have been used to evaluate the ratio of the populations of the ionic complexes to that of the bare ion (H(3)O(+) or OH(-)). Our results suggest that the Zundel form, i.e., H(5)O(2) (+), is the most preferable in the solution among the cationic species listed above though the Eigen form (H(9)O(4) (+)) is very close to the Zundel complex in the free energy, while the anionic fragment from water molecules mostly takes the form of OH(-). It has also been found that the loss of the translational entropy of water molecules associated with the formation of the complex plays a role in determining the preferable size of the cluster.

New continuous negative-pressure and irrigation treatment for infected wounds and intractable ulcers.

BACKGROUND: Continuous irrigation and the vacuum-assisted closure system are effective methods for the treatment of infected wounds and intractable ulcers. The objective of this study was to simultaneously use both of the above methods as a new approach for obtaining more satisfactory, accelerated wound healing. METHODS: After debridement of the wound, indwelling irrigation and aspiration tubes are placed in the wounds that have been sutured closed. With open wounds, a sponge with the same shape as the wound is placed directly onto the wound surface, and after the two tubes are inserted in the sponge, the wound is covered with film dressing to make the wound completely airtight. A bottle of physiologic saline solution is then attached to the irrigation tube, and a continuous aspirator (Mera Sacume) is attached to the aspiration tube. The bottle of physiologic saline solution is placed at the same height as the wound, and with a pressure gradient between the two of 0, continuous aspiration is applied. RESULTS: All nine cases treated as closed air cavity wounds with this method healed after 2 to 3 weeks. In eight cases of open wound, recurrence of infection was observed in only one case. CONCLUSIONS: The two treatments of continuous irrigation and negative pressure were observed to have an additive and synergistic effect for earlier wound healing. Furthermore, the present method can dramatically reduce the number of dressing changes required, patient pain, psychological stress, and treatment cost.

A surgical treatment of severe late posttraumatic enophthalmos using sliced costal cartilage chip grafts.

The efficacy of sliced costal cartilage chip grafts for the treatment of late posttraumatic enophthalmos was investigated. Surgery was conducted based on the method reported by Matsuo et al. in 1989. After making an incision in the lower eyelid, dissecting the subperiosteum of the medial orbital wall, orbital floor and lateral orbital wall was performed to the posterior of the orbit, and then costal cartilage chips were gradually grafted in a step-like configuration to the subperiosteum from a location posterior to the equatorial plane of the eyeball. At this time, as well as to the area of concave depression in the orbital bone caused by the fracture, grafts were made to the subperiosteum of the non-deformed medial and lateral orbital wall, to move all of the orbital tissue, including the eyeball, forward. This was performed for five cases of severe late posttraumatic enophthalmos. Among the five cases, there were four cases of severe orbital fracture and one case for which malignant orbital tumor extirpation and radiation therapy had been performed. Following surgery, although mild enophthalmos remained in three of the five cases, esthetically satisfactory results were obtained for all cases. Costal cartilage chip grafts were shown to be an effective method for the treatment of late posttraumatic enophthalmos.

Refinements in the elevation of reconstructed auricles in microtia.

BACKGROUND: In the treatment of microtia, the search has been for surgical techniques that prevent postoperative complications and realize sufficient and stable projection of the constructed ear. METHODS: Cartilage was fixed with absorbable synthetic thread instead of wire because wire has a high risk of exposure. A subcutaneous pedicle was added to the concha to prevent skin necrosis. Dead space and hematoma creation were prevented with vacuum aspiration, bolster fixation, and microdrainage with small tubes. A triangular skin flap connecting to the ear lobe was used to prevent shrinkage on the posteroinferior portion of the concha. Projection of the inferior portion of the auricle was supported with a hydroxyapatite-tricalcium phosphate ceramic. RESULTS: Our technique was applied to 42 patients, and none of them experienced slip of the fixed cartilage, auricular deformation, skin necrosis, or infections. Shrinkage of the inferior portion of the auricle was minimal, and good projection was obtained. CONCLUSIONS: The authors' technique prevents complications and realizes good shape and projection of the auricle in total reconstruction of the auricle. Hydroxyapatite-tricalcium phosphate ceramic is a useful material that complements the cartilage shortage.

Reconstruction of the form and function of lateral malleolus and ankle joint.

Soft-tissue reconstruction alone cannot obtain normal ankle function in patients with large defects in the area of the lateral malleolus. The authors report a functional reconstructive method for the lateral malleolus, utilized in a male patient whose osteosarcoma in the fibula was resected with surrounding soft tissue. In order to reconstruct the lateral malleolus, the remaining half of the fibula at the knee was removed, and the fibular head was fixed with the tibia at the ankle joint. Ligaments were reconstructed with tendon grafts. Skin and soft-tissue defects were reconstructed with a combined composite flap comprised of a latissimus dorsi myocutaneous flap and a serratus anterior muscle flap. Dead space around the bone graft was filled with the serratus anterior muscle flap that was divided into two portions. The surface was covered with the latissimus dorsi myocutaneous flap. The patient regained almost normal function of the ankle joint. This technique would be a useful functional reconstructive method for patients with large defects in the area of the lateral malleolus.

Effect of E6060 [4-{5-[7-fluoro-4-(trifluoromethyl)benzo[b]furan-2-yl]-1H-2-pyrrolyl}benzoic acid], a novel subtype-selective retinoid, on lupus-like nephritis in female (NZBxNZW)F1 mice.

Disease amelioration by retinoids in various nephritic models has been reported from either immunological or pathophysiologic viewpoints. It has also been reported that retinoids exert immunosuppressive effects in a retinoic acid receptor (RAR)-alpha-dependent manner. In particular, synthetic retinoid agonists with selectivity to RAR-alpha have been reported to have a remarkable disease-ameliorating effect in some immune disease models via their potent immunosuppressive activities; however, there has been no report in which the effect of RAR-alpha-selective agonists in the nephritic models was examined. In this report, we investigated the effect of a newly synthesized RAR-alpha-selective retinoid agonist, E6060 [4-{5-[7-fluoro-4-(trifluoromethyl)benzo[b]furan-2-yl]-1H-2-pyrrolyl}benzoic acid], on the disease progression in a murine lupus nephritis model. Female (NZBxNZW)F1 mice were prophylactically treated with E6060 from 5 months of age, and their nephritic (proteinuria, blood urea nitrogen) and immunological parameters (serum anti-DNA autoantibodies and total serum immunoglobulins) were monitored with age up to 10 months old. E6060 at 0.03 and 0.1 mg/kg (once daily, p.o.) significantly improved survival rate and prevented the development of proteinuria in (NZBxNZW)F1 mice. Anti-DNA autoantibodies and total serum IgG were also significantly reduced in the E6060-treated mice. Among IgG isotypes, IgG2a was substantially reduced by E6060 treatment, indicating reduced T helper 1 responses in E6060-treated mice. In accordance with this possibility, elevation of serum interleukin-12 (p40) in old female (NZBxNZW)F1 mice was significantly inhibited by E6060 treatment. Our data suggest that the RAR-alpha-selective retinoid E6060 is a promising candidate of new remedy for lupus nephritis in systemic lupus erythematosus patients.

A library construction of 2,5-disubstituted pyrrole compounds by using solid/solution-phase syntheses.

Using solid- and solution-phase synthesis, a library of 2,5-disubstituted pyrrole compounds was constructed. This is the first report that Stetter reaction was applied to the solid-phase synthesis for C-C bond formation. Some of 2,5-disubstituted pyrrole compounds showed the inhibitory activity of LPS-induced mouse B-lymphocyte proliferation.

Prevention of acute and chronic allograft rejection by a novel retinoic acid receptor-alpha-selective agonist.

To investigate the involvement of retinoic acid receptor (RAR)-alpha in allograft rejection, we investigated the effect of a novel selective agonist to the receptor, ER-38925, in a mouse cardiac allograft model. Prophylactic treatment with ER-38925 inhibited the acute rejection of the mouse cardiac allograft (BALB/c --> C3H/HeN) at 0.3 and 3 mg/kg, and its effect was enhanced in combination with tacrolimus. In this model, ER-38925 remarkably inhibited cytotoxic T lymphocyte induction and alloantigen-stimulated production of cytokines, i.e. IL-2, IL-12 and IFN-gamma. In the chronic rejection model, combined treatment with tacrolimus and ER-38925 reduced the grade and incidence of arteriosclerosis in the cardiac allografts significantly more potently than tacrolimus monotherapy. ER-38925 inhibited the proliferation of rat aortic smooth muscle cells stimulated in vitro, presumably through the induction of a cyclin-dependent kinase inhibitor, p27(kip-1). Those results provide a rationale for using RAR-alpha agonists as immunosuppressants in human organ transplantation.