Bilateral brachial pull-through technique for stenting in a patient with stenosis of the vertebral artery origin: technical case report.

Stenting for stenosis of the proximal vertebral artery (VA) is commonly performed via a femoral approach. However, iliofemoral occlusive disease such as arteriosclerosis obliterans sometimes prevents safe transfemoral access. In certain situations where both femoral access and ipsilateral brachial access are difficult because of a concomitant vascular diseases or particular anatomic setting, a contralateral brachial approach using the brachiobrachial pull-through technique may allow efficient and accurate stenting. A case of VA origin symptomatic stenosis successfully treated with stenting using the new pull-through technique from the contralateral brachial artery to the brachial artery on the affected side is described.

Ultrasound evaluation of the fibrosis stage in chronic liver disease by the simultaneous use of low and high frequency probes.

A liver biopsy is currently considered the definitive diagnostic modality for establishing the severity of hepatic fibrosis. We analysed the diagnostic sensitivity and accuracy of ultrasound (US) using both low frequency and high frequency probes as a repeatable, inexpensive, and reliable method to determine the fibrosis stage in chronic liver disease and then compared our results with the histological findings. A total of 103 patients with chronic liver disease (60 males and 43 females, average age 51 years old) who had undergone both a liver biopsy and US with 2-5 MHz frequency and 5-12 MHz frequency probes were prospectively evaluated in this study. An US scoring system using both the low frequency and high frequency probes was performed by evaluating the edge, surface and parenchymal texture of the liver. Each score was obtained by evaluating three parameters; the bluntness of the liver edge, the irregularity of the surface and the coarseness of the parenchymal texture were evaluated and then compared with the histological findings. The US scores of the liver edge (rs: 0.6668), liver surface (rs: 0.9007) and liver parenchymal texture (rs: 0.8853) correlated significantly with the fibrosis stage obtained based on the biopsy findings. The accumulated US scores of these three parameters, however, was found to be the most reliable indicator (rs: 0.9524). Patients with an accumulated score of 6.5 or more were all found to have fibrosis stage 4 in which the accuracy of our scoring system for correctly predicting cirrhosis was found to be 100% sensitive. When an accumulated US score of 3 was interpreted to indicate mild fibrosis (a fibrosis score of 0 or 1), all 42 patients with stage 0 or 1 fibrosis were found to have an accumulated US score of 3 or less (a probability of 100%) and 42 of 53 patients with a score of 3 or less were found to have stage 0 or 1 fibrosis (specificity of 79.2%). An ultrasound evaluation of the liver fibrosis stage based on the scoring system using both low and high frequency probes was found to be a reliable and effective alternative to the histological staging in chronic liver diseases.

Successful second allogeneic peripheral blood stem cell transplantation and donor lymphocyte infusion in patients with relapsed acute leukemia using the same donors as for the initial allogeneic bone marrow transplantation.

We report the successful treatment of two acute lympho- blastic leukemia (ALL) patients who relapsed following allogeneic bone marrow transplantation (allo-BMT) with allogeneic peripheral blood sem cell transplantation(allo-PBSCT) and donor lymphocyte infusion (DLI) from the same HLA-identical related donors as those used for the first allo-BMT. The patients relapsed on days 154 and 351 from the initial allo-BMT, respectively. Since conventional reinduction chemotherapy failed, allo-PBSCT was undertaken while the patients were still myelosuppressed immediately after reinduction chemotherapy. To induce and/or enhance GVL effects following allo-PBSCT, we performed rapid tapering of CsA and added DLI. After allo-PBSCT and DLI, the patients maintained their complete remission at 55 and 48 months post allo-PBSCT, respectively. From these findings, allo-PBSCT and DLI may be a useful treatment strategy for acute leukemia relapsing after allo-BMT.

[Efficacy and toxicity of lenograstim (glycosylated recombinant human granulocyte-colony stimulating factor) on peripheral blood stem cell harvest in healthy donors].

Peripheral blood stem cell harvest with lenograstim (glycosylated rhG-CSF) was performed 12 times from 10 normal donors. Five micrograms/kg of lenograstim was administered subcutaneously twice a day (10 micrograms/kg/day) for 4 to 6 days, and apheresis was performed on day 5 to 7 depending on the collected CD 34+ cell counts. We collected a sufficient number of CD 34+ cells in 9 mobilizations from 7 donors less than 50 years of age, with a total number of collected CD 34+ cells in each mobilization of 22.1 +/- 6.5 x 10(7). In contrast, we could not obtain a sufficient number of CD 34+ cells in 2 mobilizations from 3 donors above 50 years of age, with a total number of collected CD 34+ cells of 9.8 +/- 3.3 x 10(7). Although all donors had adverse events in response to lenograstim administration, all of them were grade 2 or less toxicity. These results indicate that peripheral blood stem cell mobilization and apheresis by lenograstim is safe and well tolerated, but the risk of poor mobilization may become higher in donors more than 50 years of age.

[Prevalence of cervical and cerebral atherosclerosis and silent brain infarction in patients with multivessel coronary artery disease].

OBJECTIVES: This clinical study investigated the prevalence of cervical and cerebral atherosclerosis and silent brain infarction in patients with coronary artery disease. METHODS: Cervical and cerebral magnetic resonance angiography(MRA) was performed in 133 patients (98 males, 35 females, mean age 65.3 years) with suspected coronary artery disease, who were divided into a zero- and one-vessel disease group(n = 71) and a two- and three-vessel disease group(n = 62) depending on the number of major coronary branches with 75% or more stenosis. The MRA lesion was defined as more than 50% stenosis. Magnetic resonance imaging(MRI) of the brain was performed within 1 week of MRA in 78 patients without symptomatic stroke and atrial fibrillation. Silent brain infarction on MRI was defined as a focal high intensity area on T2-weighted images larger than 3 mm. RESULTS: The prevalence of MRA lesions was significantly greater in the two- and three-vessel group than in the zero- and one-vessel group(53% vs 14%, p < 0.01). The prevalence of MRI lesion was significantly higher in the two- and three-vessel group than in the zero- and one-vessel group(77% vs 36%, p < 0.01). The size and number of the MRI lesions were also significantly greater in the two- and three-vessel group than in the zero- and one-vessel group(p < 0.01). Neither age nor percentage of male gender was different between the groups. Diabetes mellitus was the common risk factor for coronary artery disease, MRA lesion and MRI lesion. CONCLUSIONS: Cervical and cerebral atherosclerosis and silent brain infarction are frequently observed in patients with multivessel coronary artery disease.

Expression and postnatal changes of adrenergic receptor subtype mRNA in rat submandibular glands.

Adrenergic receptors (ARs) are involved in regulating saliva secretion and composition in salivary glands. Nine AR subtypes, including three alpha1-ARs (alpha1a-, alpha1b- and alpha1d-ARs), three alpha2-ARs (alpha2A-, alpha2B- and alpha2C-ARs) and three beta-ARs (beta1,beta2- and beta3-ARs), have been identified through molecular cloning. The five subtype genes, alpha1a-, alpha1b-, alpha2A-, beta1-, and beta2-ARs, were expressed in rat submandibular glands. In contrast, the other four subtype mRNAs, alpha1d-, alpha2B-, alpha2C- and beta3-ARs, were not detected by reverse transcription-polymerase chain reaction (RT-PCR). The steady-state mRNA expression for the five AR subtypes in rat submandibular glands was measured by quantitative competitive RT-PCR using synthetic DNA as internal standard at different stages of postnatal development. The relative rank order of AR subtype mRNA expression was alpha1a>beta2>beta1>alpha2A>alpha1b at all stages except that beta1- and alpha2A-subtypes were reversed at 2 weeks of age. The gene expression of alpha1a-AR subtype relative to total AR was low at 2 weeks of age and increased and reached a maximum at 6 weeks of age, whereas those patterns of alpha2A-, beta1- and beta2-AR subtypes were similar to each other and their gene expressions were high at 2 weeks of age and then decreased. On the other hand, the gene expression of alpha1b-AR subtype did not change over the different stages in relation to that of a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase, and to total AR. Although rat submandibular glands contain the five AR subtype mRNAs, distinct subtype-specific expression is evident.

[Extraaxial primary malignant lymphoma associated with calcified chronic subdural hematoma: a case report].

The authors report the case of a 54-year-old male with extraaxial primary malignant lymphoma associated with calcified chronic subdural hematoma. He slowly developed progressive headache accompanied by a bulge in the left forehead. Skull radiogram showed a large biconvex calcification in the left frontoparietal region, with concave change in the overlying bone. Computed tomograms and magnetic resonance images revealed a left frontoparietal chronic subdural hematoma surrounded by a calcified rim, with marginal enhancement in the frontal portion extending upward to the subcutaneous tissue through the underlying bone. The lesion was suspected to be an infectious calcified hematoma. The patient underwent a craniotomy for the removal of the hematoma. It was observed that the tumor was located mainly in the epidural and subdural space. The extent of the tumor corresponded with the enhanced area of the lesion in the preoperative neuroimages. The histological diagnosis was malignant lymphoma of B cell origin. General examination, which included bone marrow study and Ga scintigraphy, failed to prove systemic lymphoma. Extraaxial primary malignant lymphoma is extremely rare, and this is the first report of a lymphoma associated with calcified chronic subdural hematoma. The authors review the literature and discuss the clinical features and the pathogenesis of the lesion.

Early onset of hemophagocytic syndrome following allogeneic bone marrow transplantation.

We report a 40-year-old man who presented with acute onset of hemophagocytic syndrome (HPS) after allogeneic bone marrow transplantation (alloBMT) for acute myelogenous leukemia. On day 8 after alloBMT, the patient suddenly manifested high-grade fever, transfusion-resistant severe anemia, and thrombocytopenia. Neither veno-occlusive disease nor thrombotic microangiopathy was documented. The level of ferritin in serum was elevated to 1192 ng/mL. A bone marrow aspiration test on day 16 showed a markedly increased number of activated macrophages showing massive hemophagocytosis. Serum levels of interferon-gamma, soluble interleukin-2 receptor, interleukin-6, tumor necrosis factor-alpha, and macrophage colony-stimulating factor (M-CSF) were elevated. From these findings, we determined his transfusion-resistant cytopenias to be attributable to HPS. No viruses (including cytomegalovirus, Epstein-Barr virus, human herpes-virus-6, parvovirus B19, and adenovirus B11) were detected in serum or urine by polymerase chain reaction amplification. We speculate that in addition to the administration of M-CSF, hypercytokinemia during the early phase post-alloBMT might have contributed to the onset of HPS in this patient. Methylprednisolone pulse therapy was very effective for the treatment of the HPS. This case reveals that HPS could develop after alloBMT, even when engraftment of hematopoietic cells is not confirmed.

Cytokines prevent dexamethasone-induced apoptosis via the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways in a new multiple myeloma cell line.

A new human myeloma cell line, OPM-6, was established from the peripheral blood of a patient with advanced IgG-kappa plasma cell leukemia. Cytogenetic and phenotypic analysis confirmed that the cells were derived from the patient's leukemic cells. Insulin-like growth factor-1 (IGF-1) acts as an autocrine growth factor in these cells. In addition, OPM-6 cells were particularly sensitive to dexamethasone (DEX), when endogenous IGF-1 was blocked. Under these conditions, >95% of the DEX-treated cells died within 36 h. Therefore, OPM-6 represents a potentially powerful tool for the analysis of the molecular mechanisms of DEX-induced apoptosis, because it is possible to easily analyze the direct effects of DEX using this system. Using this culture system of OPM-6, we demonstrated that the treatment with DEX plus a monoclonal antibody to the human IGF-1 receptor (alphaIGF-1R) leads to the down-regulation of the gene expression of Bcl-xL, an antiapoptotic gene, and the activation of CPP32 during this apoptotic process. IFN-alpha as well as IL-6 prevented DEX plus alphaIGF-1R-induced apoptosis, and this prevention was blocked by the mitogen-activated protein kinase kinase inhibitor, PD098059, or the phosphatidylinositol 3-kinase inhibitor, wortmannin. Therefore, both IL-6 and IFN-alpha blocked DEX plus alphaIGF-1R-induced apoptosis through activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.

Integrin-associated protein/CD47 regulates motile activity in human B-cell lines through CDC42.

Cell migration requires a dynamic interaction between the cell, its substrate, and the cytoskeleton-associated motile apparatus. Integrin-associated protein (IAP)/CD47 is a 50-kd cell surface protein that is physically associated with beta3 integrins and that modulates the functions of beta3 integrins in various cells. However, in B-lymphocytes that express beta1 integrins but few beta3 integrins, the roles of IAP/CD47 remain to be determined. Cross-linking of IAP/CD47 by the immobilized anti-IAP/CD47 monoclonal antibody (mAb) B6H12, but not 2D3, produced signals to promote polarization with lamellipodia, a characteristic morphology during leukocyte migration, in pre-B and mature B-cell lines (BALL, Nalm6, ONHL-1, Daudi), but not in myeloma cell lines (RPMI8226, OPM-2). In the presence of the immobilized fibronectin (FN), soluble B6H12 could increase the rate of the polarization and activate migratory activity of BALL cells to FN in a transwell filter assay. Furthermore, the dominant-negative form of CDC42 completely blocked B6H12-induced morphologic and functional changes without inhibiting phorbol 12-myristate 13-acetate-induced spreading on FN in BALL cells, whereas the dominant-negative form of Rac1 inhibited all these changes. These findings demonstrate that in B-lymphocytes, IAP/CD47 may transduce the signals to activate the migratory activity, in which CDC42 may be specifically involved, and that IAP/CD47 shows synergistic effect with alpha4beta1 on B-cell migration. These findings would provide new insight into the role of IAP/CD47 on B-cell function.

Kupffer cell-derived prostaglandin E(2) is involved in alcohol-induced fat accumulation in rat liver.

Destruction of Kupffer cells with gadolinium chloride (GdCl(3)) and intestinal sterilization with antibiotics diminished ethanol-induced steatosis in the enteral ethanol feeding model. However, mechanisms of ethanol-induced fatty liver remain unclear. Accordingly, the role of Kupffer cells in ethanol-induced fat accumulation was studied. Rats were given ethanol (5 g/kg body wt) intragastrically, and tissue triglycerides were measured enzymatically. Kupffer cells were isolated 0-24 h after ethanol, and PGE(2) production was measured by ELISA, whereas inducible cyclooxygenase (COX-2) mRNA was detected by RT-PCR. As expected, ethanol increased liver triglycerides about threefold. This increase was blunted by antibiotics, GdCl(3), the dihydropyridine-type Ca(2+) channel blocker nimodipine, and the COX inhibitor indomethacin. Ethanol also increased PGE(2) production by Kupffer cells about threefold. This increase was also blunted significantly by antibiotics, nimodipine, and indomethacin. Furthermore, tissue triglycerides were increased about threefold by PGE(2) treatment in vivo as well as by a PGE(2) EP(2)/EP(4) receptor agonist, whereas an EP(1)/EP(3) agonist had no effect. Moreover, permeable cAMP analogs also increased triglyceride content in the liver significantly. We conclude that PGE(2) derived from Kupffer cells, which are activated by ethanol, interacts with prostanoid receptors on hepatocytes to increase cAMP, which causes triglyceride accumulation in the liver. This mechanism is one of many involved in fatty liver caused by ethanol.

Impact of burn injury on hepatic TGF-beta1 expression and plasma TGF-beta1 levels.

BACKGROUND: The liver plays a critical regulatory role in the acute inflammatory response to injury, although the mechanisms of this regulation are not well understood. transforming growth factor-beta1 (TGF-beta1) is induced after burn injury and may contribute to an inhibitory or fatal effect on hepatocytes. We investigated the association over time between plasma concentration of TGF-beta1, expression of TGF-beta1 m-RNA in liver tissue, and histologic analysis of liver apoptosis after burn injury. METHODS: Male BALB/c mice were anesthetized and randomized to receive 0% (sham), moderate (approximately 25%) (M), or large (approximately 50%) (L) body surface area full-thickness contact burn, followed by resuscitation and analgesia. Animals were killed over a time course from 15 minutes to 24 hours after burn injury, and liver tissue and peripheral blood were collected. Plasma levels of TGF-beta1 (nanograms per milliliter) were measured by enzyme-linked immunosorbent assay. TGF-beta1 m-RNA was extracted from liver and measured by reverse transcription-polymerase chain reaction. Histology of liver apoptosis was examined after fixation and staining with TdT-mediated dUTP nick-end labeling (TUNEL) method. RESULTS: The plasma concentration of TGF-beta in burn group L was significantly increased at 4 hours after burn when compared with sham and M burn groups. This rise in plasma TGF-beta1 was preceded by an increase in hepatic TGF-beta1 m-RNA expression at 30 minutes, 1, 2, and 4 hours after burn in the L group. Histologic analysis found greater hepatocyte death in the L group than in the M group at 8 hours after burn. CONCLUSION: The levels of induced TGF-beta1 and TGF-beta1 m-RNA after L burn injury are higher and peak earlier than after M burn injury. Elevated TGF-beta1 may be associated with cell death in hepatocytes. The TGF-beta1 rise may be associated with hepatocyte injury and systemic response to massive burn.

Transforming growth factor-beta1 and splenocyte apoptotic cell death after burn injuries.

Transforming growth factor (TGF)-beta1 is a multifunctional cytokine that mediates apoptotic cell death in human lymphocytes in vitro. To better understand the mechanism through which TGF-beta1 exerts its apoptotic effect, we investigated the role of TGF-beta1 in the relationship between burn injury and cell death of splenocytes in a mouse model of either 0%, 25%, or 40% full-thickness burns. Mice were killed and spleens were harvested at 15 and 30 minutes and at 1, 2, 4, 8, 12, and 24 hours after the burn. The spleens were divided and used for both histologic analyses with H-E stain and TUNEL stain and for total messenger RNA isolation and reverse transcriptase-polymerase chain reaction amplification. Amplified polymerase chain reaction products were analyzed for signal strength by electrophoresis. TGF-beta1 RNA expression was highest at 2 hours after the burn injuries in the 40% full-thickness burns and at 4 hours after the burn injuries in the 25% full-thickness burns. The relative increase in TGF-beta1 RNA was 3 times greater with the larger burn than with the smaller burn. In histologic analysis, splenocyte apoptotic cell death was observed at 4 to 24 hours after the burn in the 40% full-thickness burns but at only 4 to 12 hours in the 25% full-thickness burns. TGF-beta1 RNA peak expression was observed at different times after the burn in 25% and 40% full-thickness burns. Histologic analysis showed apoptotic cell death in proportion with respective messenger RNA expressions. This suggests that TGF-beta1 may be associated with apoptosis of splenocytes in vivo and that the effect of TGF-beta1 after a burn injury may be important in the immune system.

Gene expression and cytokine and enzyme activation in the liver after a burn injury.

The liver plays a critical role in the inflammatory response to injury; however, the mechanisms by which the liver is affected and how it influences the rest of the immune system are not well understood. Partial hepatectomy is a direct injury to the liver, whereas a burn is an indirect injury to liver, but both injuries appear to produce damage to the liver. In this study, we used a mouse model of 25% total body surface area and 40% total body surface area full-thickness burns to investigate the mechanism of liver damage and response to burn injury by measuring levels of c-Jun messenger (m)RNA, NFkappaB nuclear protein, interleukin-6, transaminases, and liver tissue histology over time. c-Jun and NFkappaB are 2 transcription factors that are induced by partial hepatectomy and related to hepatocyte injury and growth. In both groups of mice with burns, expression of c-Jun mRNA and NFkappaB nuclear protein was activated within 30 minutes after the burn injury, followed by increased levels of interleukin-6 and, finally, elevated enzyme levels. Liver injuries were similar in both groups despite the magnitude of the burns. We believe that these gene products are initiated in the hepatocyte injury after a burn and that they precede other inflammatory responses such as cytokine release, plasma transaminase levels, and histologic changes.

[Congenital arteriovenous malformation of the scalp with a drainage to the transverse sinus: a case report].

Arteriovenous malformation (AVM) of the scalp is uncommon, and a subtype which has connection with the intracranial dural sinus is extremely rare. Only 3 cases have been reported. We present a case of congenital AVM of the scalp which was connected with the intracranial venous sinus. A 27-year-old woman had been noted as having a pulsatile soft mass in the midline of the occipital region since her birth. She visited our hospital because of pain and enlargement of the mass. The patient had had no history of trauma. Physical examination revealed a pulsatile scalp mass in the midline of the occipital region, measuring 3.5 x 3.5 x 1 cm. A loud bruit was ausculated. Tenderness was noted. The skin over the mass was slightly reddish. No focal neurological deficits were noted. Plain skull films demonstrated a round defect in the midline of the occipital bone. Magnetic resonance imaging (MRI) demonstrated a subcutaneous mass with low signal intensity and an infratentorial mass with flow void. 3D CT angiograms demonstrated a subcutaneous vascular mass with a single large vein draining into the right transverse sinus. External carotid angiograms revealed a vascular lesion within the scalp with supply from the branches of the bilateral occipital arteries and the meningeal arteries. The nidus penetrated the skull and connected to a dilated varix, which had a draining vein shunting into the right transverse sinus. After embolization of the right meningeal feeding arteries, surgery was performed. The vascular lesion penetrated the skull and the dura. The infratentorial mass was in the epiarachnoid space and was fed by a small pial artery. The mass was excised completely after interruption of the pial artery and the draining vein. Postoperative course was uneventful. Histologically, the subcutaneous mass and infratentorial vascular mass were shown to be AVM and varix, respectively.

Physiologically based pharmacokinetics of KNI-272, a tripeptide HIV-1 protease inhibitor.

The effects of dose on the pharmacokinetic characteristics of KNI-272 were evaluated in rats after intravenous (iv) administration. The plasma kinetics of KNI-272 were dose-independent within a dose range of 1.0 to 10.0 mg/kg. However, when the dose was increased to 50.0 mg/kg, the area under the plasma concentration-time curve (AUC)/dose significantly increased and the total plasma clearance (Cl(tot)) significantly decreased, possibly due to saturation of hepatic metabolism. On the other hand, the terminal elimination half-life (t(1/2,lambda(z))) was independent of dose. Using biochemical and physiological parameters obtained from in vitro and in vivo studies, we developed a physiologically based pharmacokinetic (PBPK) model for KNI-272 in rats in which concentration-dependent nonlinear hepatic metabolism (Michaelis-Menten type metabolism) was considered. Using this PBPK model, plasma KNI-272 concentration-time profiles were simulated. From these profiles it was demonstrated that the terminal elimination phase was proportional to the dose at lower doses. However, as the dose increased to 50.0 mg/kg, the simulated plasma concentrations at the terminal elimination phase increased more than the increase of dose in the same way as the observed data. Accordingly, the dose-dependent plasma kinetics observed after a 50.0 mg/kg dose was considered to be attributable in part to concentration-dependent hepatic metabolism in rats.

Differential expression of human lysyl hydroxylase genes, lysine hydroxylation, and cross-linking of type I collagen during osteoblastic differentiation in vitro.

The pattern of lysyl hydroxylation in the nontriple helical domains of collagen is critical in determining the cross-linking pathways that are tissue specific. We hypothesized that the tissue specificity of type I collagen cross-linking is, in part, due to the differential expression of lysyl hydroxylase genes (Procollagen-lysine,2-oxyglutarate,5-dioxygenase 1, 2, and 3 [PLOD1, PLOD2, and PLOD3]). In this study, we have examined the expression patterns of these three genes during the course of in vitro differentiation of human osteoprogenitor cells (bone marrow stromal cells [BMSCs]) and normal skin fibroblasts (NSFs). In addition, using the medium and cell layer/matrix fractions in these cultures, lysine hydroxylation of type I collagen alpha chains and collagen cross-linking chemistries have been characterized. High levels of PLOD1 and PLOD3 genes were expressed in both BMSCs and NSFs, and the expression levels did not change in the course of differentiation. In contrast to the PLOD1 and PLOD3 genes, both cell types showed low PLOD2 gene expression in undifferentiated and early differentiated conditions. However, fully differentiated BMSCs, but not NSFs, exhibited a significantly elevated level (6-fold increase) of PLOD2 mRNA. This increase coincided with the onset of matrix mineralization and with the increase in lysyl hydroxylation in the nontriple helical domains of alpha chains of type I collagen molecule. Furthermore, the collagen cross-links that are derived from the nontriple helical hydroxylysine-aldehyde were found only in fully differentiated BMSC cultures. The data suggests that PLOD2 expression is associated with lysine hydroxylation in the nontriple helical domains of collagen and, thus, could be partially responsible for the tissue-specific collagen cross-linking pattern.

Development of a new, simple rat model of early alcohol-induced liver injury based on sensitization of Kupffer cells.

The continuous intragastric in vivo enteral feeding model in the rat developed by Tsukamoto and French has been very useful; however, it requires surgical expertise. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hours later. Accordingly, these experiments were designed to determine if a new, simple animal model of ethanol hepatotoxicity could be developed based on Kupffer cell sensitization. Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hours intragastrically. Livers were stained with hematoxylin-eosin to assess steatosis, inflammation, and necrosis, and tissue triglycerides, serum transaminases, and plasma endotoxin were measured. Kupffer cells were isolated 0 to 24 hours after one intragastric dose of ethanol daily, and intracellular Ca2+ ([Ca2+]i) was measured using fura-2, while tumor necrosis factor alpha (TNF-alpha) was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western and Northern analysis. Ethanol caused steatosis, necrosis, and inflammation in only a few weeks, and after 8 weeks, serum aspartate transaminase (AST) levels were doubled. Values were similar to levels achieved in the enteral feeding model. Triglycerides were also increased significantly by ethanol as expected, and endotoxin levels were increased to 70 to 80 pg/mL. This latter increase was prevented (<20 pg/mL) by antibiotics implicating endotoxin. In isolated Kupffer cells from untreated control rats, [Ca2+]i increased to 82 +/- 7 nmol/L after addition of lipopolysaccharide (LPS) (100 ng/mL), and levels were elevated about twofold by ethanol given 24 hours earlier (174 +/- 15 nmol/L). In addition, TNF-alpha production by Kupffer cells was increased fourfold in cells isolated from rats treated with ethanol 24 hours earlier. Sterilization of the gut with antibiotics blocked all effects of ethanol on [Ca2+]i and TNF-alpha release completely. Moreover, 4 weeks after ethanol, CD14 in Kupffer cells was elevated about twofold. A new, simple chronic model of ethanol hepatotoxicity has been developed here based on sensitization of Kupffer cells to endotoxin.

Stromal cell CD9 regulates differentiation of hematopoietic stem/progenitor cells.

CD9 belongs to the transmembrane 4 superfamily, and has been shown to influence cell proliferation, motility, and adhesion. We show here that ligation of CD9 modifies proliferation and/or differentiation of hematopoietic stem/progenitors. Pluripotent EML-C1 hematopoietic cells were cocultured with MS-5 stromal cells in the presence of KMC8.8, an anti-CD9 antibody. Numbers of recovered EML-C1 cells were slightly reduced and the antibody caused the hematopoietic cells to migrate beneath the adherent stromal cell layer. Of particular interest, EML-C1 cells recovered from CD9-ligated cultures had undifferentiated properties. Separate pretreatment of the two cell types with antibody showed that stromal-cell CD9 mediated these responses. Spontaneous expression of erythroid marker was completely blocked and there was a shift towards undifferentiated clonogenic progenitors. Immunoprecipitation studies showed that stromal-cell CD9 associates with the beta1 subunit of integrin, as well as a novel 100 kD protein. Antibody cross-linking of cell surface CD9 increased the amount of 100 kD protein that was subsequently coprecipitated with CD9. These observations show that stromal-cell CD9 influences physical interactions with hematopoietic cells and may be one factor that determines the degree of stem cell differentiation.

Postnatal changes of gene expression for tissue inhibitors of metalloproteinase-1 and -2 and cystatins S and C, in rat submandibular gland demonstrated by quantitative reverse transcription-polymerase chain reaction.

The rat submandibular gland is not fully developed at birth and definitive differentiation takes place postnatally. The steady-state mRNA expression for the four proteinase inhibitor molecules, tissue inhibitors of metalloproteinase (TIMP)-1 and -2, and cystatins S and C, and for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), in rat submandibular glands was measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) at different stages of postnatal development. The gene-expression patterns of TIMP-1 and -2 relative to G3PDH were similar to each other. The TIMP-2 and cystatin C genes were more highly expressed than those of TIMP-1 and cystatin S at all stages. Moreover, the gene expressions of TIMP-1 and -2, and of cystatins S and C, were predominant between 1 and 7, and 7 and 12 weeks of age, respectively, and coincided developmentally with the regression of terminal tubule cells and the differentiation of granular convoluted tubule cells, respectively. Quantitative competitive RT-PCR allowed accurate measurement of small changes in the steady-state concentrations of these proteinase-inhibitor mRNA molecules.