Long-term detection of seasonal influenza RNA in faeces and intestine.

Some cases of seasonal influenza virus (human influenza A virus (IAV)/human influenza B virus (IBV)) are associated with abdominal symptoms. Although virus RNA has been detected in faeces, intestinal infection has not been clearly demonstrated. We aimed to provide evidence that IAV/IBV infects the human intestine. This prospective observational study measured virus RNA in faecal and sputum samples from 22 patients infected with IAV/IBV (19 IAV positive and three IBV positive). Nineteen patients were included in the analysis and were assigned to faecal IAV-positive and -negative groups. Virus kinetics were examined in faecal samples from an IAV-infected patient (patient 1) and an IBV-infected patient (patient 2). Finally, intestinal tissue from an IAV-diagnosed patient who developed haemorrhagic colitis and underwent colonoscopy was examined for the presence of replicating IAV (patient 3). Virus RNA was detected in faecal samples from 8/22 IAV/IBV-infected patients (36.4%). Diarrhoea occurred significantly more often in the faecal IAV-positive group (p 0.002). In patients 1 and 2, virus RNA became undetectable in sputum on days 7 and 10 after infection, respectively, but was detected in faeces for a further 2 weeks. Virus mRNA and antigens were detected in intestinal tissues (mucosal epithelium of the sigmoid colon) from patient 3. These findings suggest that IAV/IBV infects within the intestinal tract; thus, the human intestine may be an additional target organ for IAV/IBV infection.

Folate intakes and folate biomarker profiles of pregnant Japanese women in the first trimester.

OBJECTIVE: To assess the status of dietary folate intake, serum and red blood cell (RBC) folate, and related nutritional biomarkers in healthy Japanese women in early pregnancy. DESIGN: A cross-sectional, observational study. SUBJECTS: Pregnant women in the first trimester, at 7-15 weeks gestation (n=70), who were not consuming any folate supplements or folate fortified foods. METHODS: Three-day dietary records were obtained from each subject to assess dietary folate intake. Blood samples were collected for measurement of biomarkers. Biomarkers and nutrient intake were analyzed in two groups defined by their serum folate concentrations: the low folate group (serum folate < 9 ng/ml) and the high folate group (serum folate > or = 9 ng/ml). RESULT: Mean serum and RBC folate concentrations in all subjects were 10.3 and 519 ng/ml, respectively. These levels were remarkably higher than the reported values from many other countries despite our subjects receiving no folic acids supplements. However, mean folate intake by our subjects from natural foods was 289 microg/day, which is thought to be low according to the Japanese dietary recommendation specified for pregnant women. The intake of spinach and fruits was significantly greater in the high folate group than in the low folate group. CONCLUSION: Folate intake was thought to be adequate to maintain a desirable level of serum folate concentration in Japanese pregnant women in the first trimester, although the intake of folate from natural food was not high enough to meet the recommended daily intake.

Immunohistochemical diagnosis of Cryptococcus neoformans var. gattii infection in chronic meningoencephalitis: the first case in Japan.

Cryptococcus neoformans (C. neoformans) var. gattii infection usually occurs in tropical and subtropical areas, and rarely in the northern hemisphere. We report the first Japanese with cryptococcal meningoencephalitis caused by C. neoformans var. gattii infection that occurred during a trip to Australia. This agent was identified in a cerebellar biopsy specimen by immunohistochemical technique with serotype-specific anti-sera. Because the meningitis caused by it did not respond well to conventional therapy, we used an aggressive therapeutic regimen to successfully treat the patient. Even in areas where C. neoformans var. gattii does not exist, this infection should be considered possible as a travel-related infection.

Effects of roxithromycin on adhesion molecules expressed on endothelial cells of the dermal microvasculature.

The objective of the study was to investigate the pharmacological action of roxithromycin, an oral macrolide antibiotic. The effects of roxithromycin on the cytokine-induced expression of endothelial leukocyte adhesion molecule (E-selectin) and intracellular adhesion molecule (ICAM)-1 on endothelial cells of the dermal microvasculature were investigated in vitro using flow cytometry. Roxithromycin at a concentration of 0.5 microgram/ml, which is lower than the therapeutic plasma concentration (ordinary daily dose, 150-300 mg), significantly inhibited the expression of E-selectin and ICAM-1 on endothelial cells of the dermal microvasculature induced by tumour necrosis factor-alpha. We conclude that roxithromycin may exert its anti-inflammatory action by inhibition of the in vivo expression of adhesion molecules on dermal microvascular endothelial cells.

Anti-GM1 antibody facilitates leakage in an in vitro blood-nerve barrier model.

Using an in vitro blood-nerve barrier (BNB) model, the authors tested the effect of various monoclonal antiganglioside antibodies on BNB function. Only anti-GM1 antibody significantly facilitated BNB leakage in a concentration-dependent, complement-independent manner. This study provided evidence that anti-GM1 antibody, frequently detected in sera from patients with inflammatory neuropathies, may participate BNB dysfunction and contribute to development of neuropathy.

Glycosphingolipid antibodies and blood-nerve barrier in autoimmune demyelinative neuropathy.

OBJECTIVE: To evaluate morphologic changes in small endoneurial vessels in patients with autoimmune demyelinative neuropathy and antiglycosphyngolipid antibodies. BACKGROUND: Although a humoral immune cause has been postulated for various demyelinating neuropathies, the mechanism by which large molecules like immunoglobulins can traverse the blood-nerve barrier (BNB) to enter the endoneurium is not understood. METHODS: We examined histologic and ultrastructural changes in small endoneurial vessels in sural nerve biopsy specimens from autoimmune demyelinative neuropathy patients, with or without anti-glycosphingolipid (GSL) antibodies. Density of small endoneurial vessels, mean endothelial area, mean luminal area, and the percentage of endothelial cell junctions (that make up the BNB) that showed evidence of disruption were evaluated. RESULTS: Only junctional disruption showed a significant difference: autoimmune demyelinative neuropathy patients with anti-GSL antibodies showed more BNB disruption than autoimmune demyelinative neuropathy patients without antibodies or control patients with nonautoimmune neuropathies. The most commonly observed endoneurial change in antibody-positive autoimmune demyelinative neuropathy patients was the finding of continuous, patent spaces lacking tight junctions between endothelial cells. CONCLUSIONS: Brain endothelial cells and endoneurial endothelial cells share various GSL antigens, including GM1 and GD1b gangliosides, with peripheral nerve tissues. Circulating anti-GSL antibodies could damage cell-to-cell attachments in endoneurial endothelium. This barrier disruption may permit the large molecules like immunoglobulins to enter the endoneurial space, contributing to development of autoimmune demyelinative neuropathy.

Effect of transcatheter arterial chemoembolization on kidney hemodynamics and function in patients with cirrhosis and hepatocellular carcinoma.

BACKGROUND/AIMS: Transcatheter arterial chemoembolization (TACE) may have deleterious effect on the kidney in patients with cirrhosis and hepatocellular carcinoma. The aim of the study was to test this hypothesis. METHODS: Twenty-four patients with cirrhosis and hepatocellular carcinomas were included. They consisted of 16 patients undergoing a single TACE and eight patients undergoing diagnostic angiography. Doppler ultrasonography was used to measure hepatic artery pulsatility index (HA-PI) and renal artery pulsatility index (RA-PI) before and 1 day and 10 days after the procedure. Similarly, kidney function was assessed by measuring creatinine clearance. In addition, plasma renin activity, noradrenaline, and endothelin-1 were also measured. RESULTS: In patients receiving diagnostic angiography, no significant changes in HA-PI were observed after the procedure. In contrast, HA-PI increased significantly 1 day after the procedure (19%, p<0.01) in patients undergoing TACE, although it returned to baseline value 10 days after the procedure. In patients undergoing diagnostic angiography, no significant changes in RA-PI were observed after the procedure. Similarly, no detectable changes in RA-PI were noted in patients undergoing TACE. A transient small reduction in creatinine clearance was noted after the procedure in patients undergoing diagnostic angiography (-12%, p<0.05) and in those undergoing TACE (-11%, p<0.05). However, the effect was similar in the two groups (two-way ANOVA, p=0.72). No significant changes in plasma renin activity, noradrenaline, and endothelin-1 were observed after either diagnostic angiography or TACE. CONCLUSIONS: These results suggest that TACE per se has no deleterious effect on the kidney hemodynamics and function in patients with cirrhosis and hepatocellular carcinoma.

Blood-nerve barrier in IgM paraproteinemic neuropathy: a clinicopathologic assessment.

We report the pathologic findings in a patient with sensorimotor neuropathy associated with Waldenström's macroglobulinemia, particularly in relation to blood-nerve barrier defects. The monoclonal IgM was of kappa type and possessed anti-HNK-1 activity. A sural nerve biopsy specimen revealed severe loss of myelinated and unmyelinated nerve fibers and gaps between adjacent endothelial cells of small endoneurial vessels. Postmortem findings 3 years later included severe loss of myelinated nerve fibers and diffuse infiltration by lymphoplasmacytic B cells throughout the peripheral nervous system, sparing the central nervous system. Findings in this case suggest an immune attack against endoneurial endothelial cells with permeation of IgM into peripheral nerve tissue.

Genetic variation in low-dose UV-induced suppression of contact hypersensitivity and in the skin photocarcinogenesis response.

Two of the major cutaneous consequences of ultraviolet (UV) radiation exposure are immunosuppression and the development of skin cancer. This study examined whether these effects are genetically determined. Suppression of contact hypersensitivity by local, low-dose UV radiation was examined in what have been termed "UV-susceptible" and "UV-resistant" strains of mice. C3H/HeJ mice ("UV resistant") were resistant to the adverse effects of low-dose UV radiation when normal doses of hapten were applied to UV-irradiated skin; however, they were sensitive when the amount of hapten used for sensitization was reduced. A similar effect was observed in BALB/c mice ("UV resistant") and when the hapten was dimethylbenz(a)anthracene, thus indicating that the genetic variation was not strain or hapten specific. Despite the fact that some strains were sensitive and some were resistant to low-dose UV radiation when high doses of hapten were employed, all strains initially sensitized to hapten through UV-irradiated skin were found to be unresponsive when rechallenged on normal skin, no matter what the initial sensitizing dose of hapten was. To determine whether other biologic effects of UV also exhibited genetic variation, C3H/HeN and C3H/HeJ mice were compared for susceptibility to UVB-induced skin cancer formation. C3H/HeJ mice developed significantly more tumors than C3H/HeN mice when subjected to a single dose of UV radiation followed by repeated exposure to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. These studies provide strong evidence that genetic factors influence individual susceptibility to the biologic effects of UV radiation.

Loss of the tumor suppressor p53 gene at the liver cirrhosis stage in Japanese patients with hepatocellular carcinoma.

The incidence of p53 gene aberrations is reported to be about 20-50% in hepatocellular carcinomas (HCCs). In most cases, HCC is clinically preceded by liver cirrhosis, but the genetic changes in cirrhosis are not known well. Therefore, we studied the loss of heterozygosity (LOH) of the p53 gene in cirrhotic and neoplastic foci in the livers of patients with HCC. To assess the relationship between the LOH status of the p53 gene in the liver cirrhosis stage and that in HCC, we analyzed the samples microdissected from paraffin-embedded tissues using the polymerase-chain-reaction-based assay. We studied 18 patients with HCC. Fourteen of the 18 cases showed constitutional heterozygosity for the microsatellite markers. In 8 (57%) of the 14 informative cases, LOH was detected in primary HCCs. Among these 8 doubly informative (informative and LOH positive in primary HCC) cases, 5 cases (63%) showed LOH in liver cirrhosis lesions. The pattern of p53 allelic loss in the cirrhotic foci was identical with that in the corresponding tumor. The remaining 6 cases without LOH of the p53 gene in HCC showed on p53 loss in any cirrhotic foci. LOH of the p53 gene may occur before the development of HCC.

Inflammatory pseudotumor of the liver--MR imaging findings.

Using retrospective studies, we have investigated the possibility of obtaining characteristic findings of inflammatory pseudotumor of the liver by magnetic resonance (MR) imaging. We examined 8 patients (involving 8 masses) who had been histologically diagnosed as having an inflammatory pseudotumor in the liver. The histological studies were performed on an excised specimen of 1 mass, and on aspiration needle biopsy specimens and the clinical courses of the other 7 masses. T1 weighted images (T1WI) and T2 weighted images (T2WI) were obtained on MR imaging. MR imagings were analyzed for visualized patterns, patterns of internal structure and patterns of contrast enhancement of dynamic MR imaging. The 8 masses were visualized as hypointense on T1WI and hyperintense on T2WI by MR imaging. Dynamic MR imaging revealed that 1 mass was markedly enhanced peripherally while another mass was homogeneously enhanced, and that enhancement was most marked immediately after injection of contrast medium and then gradually disappeared. Vessels were observed in 4 masses (the portal vein in 2 masses, the hepatic vein in 1 mass, and portal and hepatic veins in 1 mass), and these vessels were clearly visualized on T1WI. The MR imaging findings from the early stage of an inflammatory pseudotumor showed a pattern similar to that of hepatic tumors with rich blood flow. The portal vein or hepatic vein was found in the tumor in half the patients, suggesting that this characteristic was useful for diagnosis of an inflammatory pseudotumor in the liver.

UVB radiation suppresses the TNF-alpha-induced expression of E-selectin and ICAM-1 on cultured human umbilical vein endothelial cells.

Endothelial cells, which are involved in the development of inflammatory and immune responses, can express various kinds of cell adhesion molecules (CAM) including E-selectin and intercellular adhesion molecules-I (ICAM-I). These cell adhesion molecules and their ligands on leukocytes play an essential role in the control of extravasation of inflammatory cells. Ultraviolet B (UVB) radiation can reach the upper dermis and modulate CAM expressions on vascular endothelial cells (EC). We examined the direct effect of UVB on E-selectin and ICAM-I expression on cultured human umbilical vein endothelial cells (HUVEC) and also examined its effect on these cells induced by tumour necrosis factor-alpha (TNF-alpha), which is a potent CAM-inducer and is released by UVB radiation on the skin. Various doses of UVB were exposed to human umbilical vein endothelial cells (HUVEC) and these expressions were examined by flow cytometric analysis using FACScan; 5, 10 and 25 mJ/cm2 UVB induced neither E-selectin nor ICAM-I expression. Irradiation of HUVEC with UVB 30 min after treatment with TNF-alpha inhibited these expressions. Although the inhibition of E-selectin was observed until 12 h in a dose-dependent manner, ICAM-I expression was almost completely inhibited, even at 5 mJ/cm2 UVB. UVB irradiation before TNF-alpha stimulation showed similar effects to those obtained post-irradiation. This study has demonstrated that UVB can directly down-regulate EC functions, and the results may have implications in action mechanisms of UVB therapy.

Expression and localization of Lewis(x) glycolipids and GD1a ganglioside in human glioma cells.

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 micrograms of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis(x) (fucosylneolactonorpentaosyl ceramide; Le(x)), difucosylneolactonorhexaosyl ceramide (dimeric Le(x)), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 micrograms of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le(x) and the complex type of sialyl-Le(x) derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le(x) glycolipids and sialyl-Le(x) were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le(x) glycolipid was located on the tip of fine cellular processes. The unique localization of the Le(x) glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.

Effect of nerve growth factor and forskolin on glycosyltransferase activities and expression of a globo-series glycosphingolipid in PC12D pheochromocytoma cells.

The glycosphingolipid (GSL) composition of cells changes dramatically during cellular differentiation. Nerve growth factor (NGF) or forskolin (FRK) are known to induce cellular differentiation including process formation in PC12 pheochromocytoma cells. In this respect, we present the NGF/FRK-dependent regulation of glycosyltransferase activities and the corresponding GSL expression in PC12D cells. After treatment of PC12D cells with NGF or FRK, the cell processes, including varicoses and growth cones, became strongly immunoreactive with an antibody against a unique globo-series neutral GSL, Gal alpha 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer (GalGb3), and the activity of GalGb3-synthase increased significantly. Other glycosyltransferase activities, including GM1 containing blood group B determinant (BGM1)-, GM3-, GD1a-, and GM2-synthases, also increased significantly upon NGF treatment, but the immunoreactivity against BGM1 did not show any appreciable change. For the parent PC12 cells, NGF/FRK treatment significantly increased the percentage of anti-GalGb3 positive cells and induced some immunoreactive cell processes. Because the parent PC12 cells do not express appreciable amounts of GalGb3, and because PC12D cells are considered to be more differentiated than the parent PC12 cells, the expression of GalGb3 and the increase of GalGb3-synthase activity may be closely related to the cellular differentiation process in this cell line.

Accumulation of isogloboside and ganglio-N-tetraosyl ceramide having blood group B determinant in the hepatomas of female LEC rats.

We have studied the neutral glycolipid composition of spontaneous hepatomas in LEC female rats. Neutral lipid fractions were isolated and purified by column chromatographies on DEAE-Toyopearl 650(M) and Iatrobeads. The neutral glycolipid fraction contained 3.2 to 4.4 micrograms lipid-bound glucose (Glc) per mg protein, and consisted of isogloboside (iso-Gb4, 50.8% of total neutral glycolipids) and IV3Gal, IV2Fuc, GgOse4Cer (asialo-BGM1, 13.5%) as the major neutral glycolipids and Gb3 and iso-Gb3 (9.2%), GlcCer (7.2%), LacCer (6.1%) as the other species. The structure of iso-Gb4 was elucidated by gas-liquid chromatography (GLC), permethylation study, liquid secondary ion (LSI) mass spectrometry, and nuclear Overhauser enhancement spectroscopy (NOESY) and that for asialo-BGM1 by GLC, LSI mass spectrometry, and high-performance thin-layer chromatography (HPTLC)-overlay method using anti-asialo-BGM1 antibody. Isogloboside and asialo-BGM1 which are found in negligible amounts in normal liver tissues may represent excellent markers for studying tumor metastasis and cellular adhesion.

Glycosphingolipid antigens in cultured bovine brain microvascular endothelial cells: sulfoglucuronosyl paragloboside as a target of monoclonal IgM in demyelinative neuropathy [corrected].

Since a number of anti-glycosphingolipid (GSL) antibody activities have been demonstrated in patients with various neurological disorders, the presence of common antigens between brain microvascular endothelial cells (BMECs) and the nervous tissues presents a potential mechanism for the penetration of macromolecules from the circulation to the nervous system parenchyma. We first investigated GSL composition of cultured bovine BMECs. Bovine BMECs express GM3(NeuAc) and GM3(NeuGc) as the major gangliosides, and GM1, GD1a, GD1b, GT1b, as well as sialyl paragloboside and sialyl lactosaminylparagloboside as the minor species. Sulfoglucuronosyl paragloboside was also found to be a component of the BMEC acidic GSL fraction, but its concentration was lower in older cultures. On the other hand, the amounts of neutral GSLs were extremely low, consisting primarily of glucosylceramide. In addition, we analyzed the effect of anti-SGPG IgM antibody obtained from a patient of demyelinative polyneuropathy with macroglobulinemia against cultured BMECs. Permeability studies utilizing cocultured BMEC monolayers and rat astrocytes revealed that the antibody facilitated the leakage of [carboxy-14C]-inulin and 125I-labeled human IgM through BMEC monolayers. A direct cytotoxicity of this antibody against BMECs was also shown by a leakage study using [51Cr]-incorporated BMECs. This cytotoxicity depended on the concentration of the IgM antibody, and was almost completely blocked by preincubation with the pure antigen, sulfoglucuronosyl paragloboside. Our present study strongly supports the concept that immunological insults against BMECs induce the destruction or malfunction of the blood-nerve barrier, resulting in the penetration of the immunoglobulin molecule to attach peripheral nerve parenchyma.

Paraneoplastic encephalo-myelo-ganglionitis: cellular binding sites of the antineuronal antibody.

The cellular binding sites of an antineuronal antibody were characterized in an autopsy case of the paraneoplastic encephalo-myelo-ganglionitis. A 61 year-old woman developed a subacute sensorimotor polyneuropathy and, later, multiple involvement of cranial nerves, disturbance of consciousness, and generalized seizure. An autopsy revealed a small cell lung carcinoma and neuropathological changes that included disseminated encephalitis, spinal anterior horn lesions, severe loss of dorsal root ganglion neurons, and secondary degeneration and loss of the nerve fibers in the spinal posterior column and peripheral nerves. The serum IgG from the patient contained antineuronal antibody(s) including an antibody to 35- to 37-kDa neuronal antigens called anti-Hu as demonstrated in Western blot. In immunohistochemical studies, the serum IgG immunostained neurons of the brains, spinal cords, and dorsal root ganglia of humans or rats. Confocal laser-scanning microscopy revealed binding of the patient's IgG in the neuronal nuclei and cytoplasm, but not in the nucleoli. In immunoelectron microscopic studies, immunolabelling with the IgG was found diffusely in the karyoplasm, excluding nucleoli, and in the cytoplasmic matrix between the cisternae of the reticulums, Golgi apparatus, and mitochondria. Encephalo-myeloganglionitis is a clinicopathological entity frequently associated with the presence of neoplasm and antineuronal antibody, however, the role of the antibody in the pathogenesis remains to be elucidated.

GM3 regulates protein kinase systems in cultured brain microvascular endothelial cells.

The barrier function of endothelial cells is known to be positively regulated by protein kinase A (PKA) and negatively regulated by protein kinase C (PKC). We found that exogenously administered GM3(NeuAc) promoted PKA activity in cultured brain microvascular endothelial cells (BMECs). Other glycolipids, including GM1, sulfoglucuronyl paragloboside, and GM3(NeuGc), did not have any effect on the PKA activity of BMECs. PC12 cells did not respond to exogenously applied GM3(NeuAc). GM3(NeuAc) also suppressed the PKC activity of BMECs. Thus, GM3(NeuAc) may function as a modulator of blood-brain barrier function via the two different kinase systems.