Use of the genomic subtractive hybridization technique to develop a real-time PCR assay for quantitative detection of Prevotella spp. in oral biofilm samples

Genomic subtractive hybridization was used to design Prevotella nigrescens-specific primers and TaqMan probes. Based on this technique, a TaqMan real-time PCR assay was developed for quantifying four oral black-pigmented Prevotella species. The combination of real-time PCR and genomic subtractive hybridization is useful for preparing species-specific primer-probe sets for closely related species.     

Local and systemic consequences of acute,low-dose ultraviolet B radiation are mediated by different immune regulatory mechanisms

Acute, low-dose ultraviolet B (UVB) radiation alters the local skin site such that epicutaneous application of hapten fails to induce contact hypersensitivity (CH), but induces tolerance in UVB-susceptible mice. Although the inability of irradiated skin to support CH induction may be a strictly local effect, there may also be systemic immune consequences of UVB radiation delivered in this manner. To examine this matter, abdominal skin of C57BL/6 mice was exposed to acute, low-dose UVB radiation. Dinitrofluorobenzene was immediately painted directly on the irradiated site, or at a distant (unirradiated) site. In separate experiments, epicutaneous application of the hapten on a distant site was delayed for 1–3 days. The mice were tested for acquisition of CH, and for tolerance, i.e. the capacity to become sensitized when exposed subsequently to hapten via normal body wall skin. It was found that, immediately after completion of the UVB regimen, CH was inducible via unirradiated, but not via irradiated, skin. At 3 days post-UVB exposure, CH was no longer inducible even through unirradiated skin. Mice that first encountered hapten via UVB-exposed skin developed tolerance, as did mice that first encountered hapten via unirradiated skin of UVB-treated mice. Neutralizing anti-tumor necrosis factor-α TNF-α antibodies failed (a) to restore the ability of unirradiated skin to support induction of CH, and (b) to interfere with tolerance induction, whether hapten was first painted on irradiated or unirradiates skin. The data indicate that the acute, low-dose regimen of UVB radiation produces effects on the immune system that are manifest locally as well as systemically. By demonstrating that the disruption of CH induction following UVB radiation is TNF-α dependent, whereas locally and systemically induced tolerance is not, our findings encourage further search for other UVB-related modulators of systemic immunity and tolerance.     

Tissue regeneration using macrophage migration inhibitory factor-impregnated gelatin microbeads in cutaneous wounds

Migration inhibitory factor (MIF) responds to tissue damage and regulates inflammatory and immunological processes. To elucidate the function of MIF in cutaneous wound healing, we analyzed MIF knockout (KO) mice. After the excision of wounds from the dorsal skin of MIF KO and wild-type (WT) mice, healing was significantly delayed in MIF KO mice compared to WT mice. Lipopolysaccharide treatment significantly increased [(3)H]thymidine uptake in WT mouse fibroblasts compared to MIF KO mouse fibroblasts. Furthermore, there was a significant reduction in fibroblast and keratinocyte migration observed in MIF KO mice after 1-oleoyl-2-lysophosphatidic acid treatment. We subsequently examined whether MIF-impregnated gelatin slow-release microbeads could accelerate skin wound healing. Injection of more than 1.5 microg/500 microl of MIF-impregnated gelatin microbeads around a wound edge accelerated wound healing compared to a single MIF injection without the use of microbeads. MIF-impregnated gelatin microbeads also accelerated skin wound healing in C57BL/6 mice and diabetic db/db mice. Furthermore, incorporating MIF-impregnated gelatin microbeads into an artificial dermis implanted into MIF KO mice accelerated procollagen production and capillary formation. These findings suggest that MIF is crucial in accelerating cutaneous wound healing and that MIF-impregnated gelatin microbeads represent a promising treatment to facilitate skin wound healing.     

Nerve alterations showing autophagy in 2 patients with lichen aureus

Lichen aureus is a rare, chronic, persistent purpuric dermatosis clinically characterized by striking yellow‐ to bronze‐colored lesions. Histologically, lichen aureus differs from other pigmented purpuric dermatoses in containing dense, band‐like infiltrates closely associated with the epidermis. This report describes 2 patients with lichen aureus, a 20‐year‐old woman with a lesion on her right arm and a 51‐year‐old man with a lesion on the right side of his groin. Skin biopsy specimens revealed almost identical findings in both patients, including dense band‐like infiltrates containing lymphocytes, histiocytes with hemosiderin deposits scattered extravasated red blood cells and nerve alterations at the dermo‐epidermal interface. The nerves within the lesions were filled with granules, which stained positive with antibody to microtubule‐associated protein 1A/1B‐light chain 3, suggesting autophagy within the nerves. These altered nerves were present only in areas of band‐like dermal lymphocytic infiltration. Electron microscopy of the lesions showed the accumulation of autophagosomes in Schwann cells.     

Hepatocyte growth factor gene transfer with naked plasmid DNA ameliorates dimethylnitrosamine-induced liver fibrosis in rats

Aim: Hepatocyte growth factor (HGF) has various biological properties, including antifibrogenic activity. In the present study, we tested the efficacy of HGF gene therapy using naked plasmid DNA in dimethylnitrosamine (DMN)-induced liver fibrosis in a rat model. Methods: Naked plasmid DNA encoding human HGF was injected once, together with a hypertonic solution, into the hepatic artery after DMN treatment on three consecutive days per week for 3 weeks. Naked plasmid DNA encoding beta-galactosidase was injected similarly in the DMN-treated control rats. DMN treatment was continued once weekly after gene transfer for additional 3 weeks. Results: The human HGF protein expression was detected in livers transfected with human HGF naked plasmid DNA, gradually decreasing by day 21. The expression of the endogenous rat HGF protein was also upregulated after human HGF gene transfer. Phosphorylation of c-Met, a HGF receptor, was detected only in livers transfected with human HGF plasmid DNA. Fibrosis was attenuated significantly in livers transfected with the human HGF plasmid. Attenuation wasaccompanied by decreased expression of alpha-smooth muscle actin. Increased portal vein pressure after treatment with DMN was suppressed significantly by HGF gene transfer. The upregulated hepatic protein expression of transforming growth factor-beta (TGF-beta) in response to DMN was markedly attenuated by HGF gene transfer accompanied by the increased protein expression for matrix metalloproteinases (MMP)-3 and -13. Conclusion: The hepatic arterial injection of human naked plasmid HGF DNA was effective in suppressing liver fibrosis induced in rats by DMN. The mechanisms by which HGF expression attenuated liver fibrosis may include the suppression of hepatic TGF-beta expression and the induction of MMP expression.     

Detection of coronary stenosis and myocardial viability using a single intravenous bolus injection of BR14

OBJECTIVES: The aim of the study was to determine whether coronary stenosis can be detected and myocardial viability assessed after myocardial infarction from a single venous bolus injection of BR14, a new ultrasound contrast agent. BACKGROUND: BR14 is an ultrasound contrast agent that, like (201)Tl, demonstrates redistribution. Whether this principle can be used to determine myocardial viability is not known. METHODS: Non-critical (n = 6) or flow-limiting (n = 4) stenoses were placed on coronary arteries of 10 open-chest dogs, which then underwent 2 h of coronary occlusion followed by reperfusion through the stenosis. Hyperemia was induced to create flow mismatch in the dogs with non-critical stenosis. Hyperemia was not induced in dogs with reduced resting coronary blood flow. All dogs were given 2 ml of BR14 as a bolus injection and serial images were obtained. Myocardial blood flow (MBF) was measured using radiolabeled microspheres. At the end of the experiment, tissue staining was performed to determine infarct size and topography. RESULTS: Initial images demonstrated flow mismatch between the normal bed and that subtended by the stenosis (during hyperemia in dogs without critical stenosis and during rest in those with reduced resting MBF). The perfusion defect size correlated well with radiolabeled microsphere-derived hypoperfused zone (r = 0.89). Regions within the hypoperfused zone that had not undergone necrosis showed redistribution, whereas the necrotic regions showed a persistent defect, the size of which correlated well with infarct size (r = 0.80). CONCLUSIONS: Because of its ability to redistribute, BR14 can define regions of relative hypoperfusion and also discriminate between infarcted and viable tissue within the hypoperfused zone after a single venous injection. This property lends itself to assessing myocardial perfusion during exercise stress.     

Granulocyte Colony-Stimulating Factor–Producing Cholangiocellular Carcinoma

A 61-year-old female was admitted to our hospital with epigastric pain and fever. The laboratory data showed severe inflammatory reactions. Computed tomography revealed an irregular tumor in the left hepatic lobe and swelling of lymph nodes. ¹⁸F-fluorodeoxy-glucose positron emission tomography (FDG-PET) showed high uptake by the tumor, with diffuse uptake in the spine. Based on the elevated leukocyte count and FDG-PET findings, the patient was diagnosed with a granulocyte colony-stimulating factor (G-CSF)-producing tumor (G-CSF, 213 pg/mL). We performed left trisegmentectomy of the liver, bile duct resection, and lymph node dissection. Histologically, the tumor was a poorly differentiated adenocarcinoma with some lymph nodes metastasis. Immunohistochemical staining of the tumor cells was positive for G-CSF. Therefore, the tumor was diagnosed as G-CSF–producing cholangiocellular carcinoma. The inflammatory reactions and serum G-CSF level transiently improved immediately after surgery. However, 1 month later, the leukocyte count and serum G-CSF level increased again, and recurrence was observed in the remnant liver. The patient died 3 months after the operation. G-CSF–producing cholangiocellular carcinoma is rare. This tumor progresses rapidly, and surgical treatment for advanced condition should be carefully selected.Key words: Granulocyte colony-stimulating factor, Cholangiocellular carcinoma, FDG-PET, Immunohistochemistry, LeukocytosisGranulocyte colony-stimulating factor (G-CSF)-producing tumors were first reported in 1977.¹ G-CSF-producing cholangiocellular carcinomas (CCCs) are rare, with only 5 other reported cases. We herein report a surgical case of G-CSF–producing CCC with early recurrence and include bibliographic comments.     

Clinical outcome of up-front surgery in patients with asymptomatic, incurable synchronous peritoneal carcinomatosis

Purposes

The purpose of this study was to show the clinical features of up-front surgery of the primary tumor in asymptomatic patients with incurable colorectal PC.

Methods

Forty-six patients that were diagnosed between 1998 and 2007 with asymptomatic colorectal PC who could not be successfully cured by surgery were assessed retrospectively.

Results

A univariate analysis revealed the presence of liver metastases, without the use of oxaliplatin (OX)/irinotecan (IRI) and without a primary tumor resection to be poor prognostic factors for survival (p = 0.044, p = 0.030, p < 0.001, respectively). According to a multivariate analysis, no use of OX/IRI and no primary tumor resection, were found to be independent poor prognostic factors for survival (HR 2.57; p = 0.047, HR 6.62; p = 0.003, respectively). The median survival time of patients treated with and without OX/IRI was 18 and 7 months, respectively. The median survival time of patients with and without primary tumor resection was 10 and 2 months, respectively. The number of patients needing surgical intervention for intestinal obstruction after surgery significantly increased in patients treated with OX/IRI (p = 0.001).

Conclusion

Improvement of survival may be related to widespread use of modern systemic chemotherapy and primary tumor resection. However, an increased number of patients that needed surgery for an intestinal obstruction were observed, even after up-front surgery, when patients were treated with modern systemic chemotherapy.     

Solitary mediastinal metastasis of hepatocellular carcinoma treated by video-assisted thoracic surgery: report of a case

Solitary mediastinal metastasis of hepatocellular carcinoma (HCC) is extremely rare. We report a case of solitary mediastinal metastasis arising from HCC treated by video-assisted thoracic surgery (VATS). A 75-year-old man underwent hepatic resection and transcatheter arterial embolization for HCC. The level of protein induced by vitamin K antagonist was subsequently found to be elevated. Computed tomography showed a 2-cm mediastinal tumor. ¹⁸F-fluorodeoxy-glucose positron emission tomography revealed slight uptake by the tumor. VATS was performed. The histopathological diagnosis was metastatic HCC of the lymph nodes. The postoperative course was uneventful, with a postoperative hospital stay of 6 days. There has been no recurrence at 4 months after surgery. VATS is a minimally invasive and useful treatment for solitary mediastinal metastasis of HCC.