Steatohepatitic hepatocellular carcinoma: imaging findings with clinicopathological correlation.

AIM: To evaluate the clinicopathological and computed tomography (CT) and magnetic resonance imaging (MRI) findings of steatohepatitic hepatocellular carcinoma (SH-HCC). MATERIALS AND METHODS: Clinicopathological and radiological features were evaluated in 20 patients with SH-HCC. The diagnosis of SH-HCC was made histologically if the tumour had four of the following five characteristics: steatosis (>5% tumour cells), ballooning, Mallory-Denk bodies, interstitial fibrosis, and inflammation. All patients underwent dynamic CT and MRI. CT and MRI images were reviewed for morphological features including tumour size, presence, and distribution of fat, and patterns and degree of contrast enhancement. RESULTS: Obesity, hypertension, and history of heavy alcohol intake were common clinical findings observed in 10 (50%), 13 (65%), and 11 (55%) of the 20 patients, respectively. Steatosis and steatohepatitis were pronounced in the background liver in 12 (60%) and 10 (50%) patients, respectively. SH-HCC was moderately differentiated in 18 patients (90%) and well differentiated in two (10%). Pathologically, steatohepatitic features were diffuse in 12 (60%) of the 20 tumours and focal in eight (40%). Tumour size and the percentage of intratumoural steatosis were not correlated (r=0.17, p=0.47). On CT, 16 (80%) patients showed arterial phase enhancement and delayed washout. On MRI, 16 (80%) of 20 tumours showed prominent fatty deposition (10 diffusely, six focally) with arterial phase enhancement. CONCLUSIONS: SH-HCC is likely to show prominent fatty deposits with arterial phase enhancement on CT and MRI. A hypervascular lesion with prominent fatty change should raise the diagnostic suspicion of SH-HCC.

Dynamics of the Vortex-Particle Complexes Bound to the Free Surface of Superfluid Helium.

We present an experimental and theoretical study of the 2D dynamics of electrically charged nanoparticles trapped under a free surface of superfluid helium in a static vertical electric field. We focus on the dynamics of particles driven by the interaction with quantized vortices terminating at the free surface. We identify two types of particle trajectories and the associated vortex structures: vertical linear vortices pinned at the bottom of the container and half-ring vortices traveling along the free surface of the liquid.

What causes alopecia areata?

The pathobiology of alopecia areata (AA), one of the most frequent autoimmune diseases and a major unsolved clinical problem, has intrigued dermatologists, hair biologists and immunologists for decades. Simultaneously, both affected patients and the physicians who take care of them are increasingly frustrated that there is still no fully satisfactory treatment. Much of this frustration results from the fact that the pathobiology of AA remains unclear, and no single AA pathogenesis concept can claim to be universally accepted. In fact, some investigators still harbour doubts whether this even is an autoimmune disease, and the relative importance of CD8(+) T cells, CD4(+) T cells and NKGD2(+) NK or NKT cells and the exact role of genetic factors in AA pathogenesis remain bones of contention. Also, is AA one disease, a spectrum of distinct disease entities or only a response pattern of normal hair follicles to immunologically mediated damage? During the past decade, substantial progress has been made in basic AA-related research, in the development of new models for translationally relevant AA research and in the identification of new therapeutic agents and targets for future AA management. This calls for a re-evaluation and public debate of currently prevalent AA pathobiology concepts. The present Controversies feature takes on this challenge, hoping to attract more skin biologists, immunologists and professional autoimmunity experts to this biologically fascinating and clinically important model disease.

Coudability hairs: a revisited sign of alopecia areata assessed by trichoscopy.

BACKGROUND: We have previously reported several trichoscopic (dermatoscopic) characteristics, such as black dots, 'exclamation-mark' hairs, broken hairs, yellow dots and clustered short vellus hairs as being useful clinical indicators for alopecia areata (AA). 'Coudability hairs', which are normal-looking hairs tapered at the proximal end, have been previously reported as another sign of AA. AIMS: To use trichoscopy to evaluate coudability hairs as a clinical indicator for the disease activity of AA and a substitute-marker for the hair-pull test. METHODS: Trichoscopic examinations of hair loss and perilesional areas on the scalps of 100 East Asian patients with AA were performed using a dermatoscope. Using Spearman's rank-order correlation coefficient by rank test, we examined the correlations of scores between coudability and AA disease activity, severity or duration and other trichoscopic features, and then evaluated the coudability score as a surrogate-marker for the hair-pull test. RESULTS: Coudability scores correlated positively with AA disease activity, hair-pull tests, short duration, black dots and exclamation-mark hairs, and correlated negatively with short vellus hairs. CONCLUSIONS: Coudability hairs, more closely perceived by trichoscopy, are useful-markers for disease activity in AA and provide a surrogate-marker for the hair-pull test.

Production of adenocarcinomas of the small intestines of Wistar rats fed panfuran-S containing 3-di(hydroxymethyl)amino-6-(5-nitro-2-furylethenyl)-1,2,4-triazine.

Panfuran-S in the diet produced a high incidence of invasive and metastatic adenocarcinomas of the duodenum and the jejunum in outbred Wistar rats. Depressed growth and incidence of cancer production were dose-dependent. Adenocarcinomas of the duodenum were present in 2 of 18 (11%) rats that received 1,750 ppm 3-di(hydroxymethyl)amino-6-(5-nitro-2-furylethenyl)-1,2,4-triazine (DHNT) in the diet and in 11 of 19 (58%) rats that received 3,500 ppm DHNT. Adenocarcinomas of the jejunum were present in 10 of 18 (56%) and 16 of 19 (84%) rats that received 1,750 and 3,500 ppm DHNT, respectively, for 35-37 weeks. The only other tumors observed were squamous cell papillomas of the forestomach. Histologically, most of the adenocarcinomas appeared tubular or papillary and were similar to adenocarcinomas in the small intestine of man and also to human intestinal-type adenocarcinoma of the stomach. This system provided a useful experimental model for the study of the pathogenesis of carcinoma of the small intestine.

Conversion of a poorly differentiated human adenocarcinoma to ascites form with invasion and metastasis in nude mice.

Cells (1 X 10(7)/0.5 ml) from a Borrmann type III poorly differentiated adenocarcinoma of the human stomach were injected ip into nude mice. The injection resulted in ascites carcinoma with invasion (carcinomatous peritonitis) and liver metastasis. The inoculum was obtained from subcutaneous tumors at passage 9 in nude mice that had received serial transplants from the patient with Borrmann type III poorly differentiated adenocarcinoma of the stomach. Serial transfers of 1.5 X 10(6) dispersed cancer cells/0.5 ml into the peritoneal cavity of nude mice converted this adenocarcinoma to an ascites form. Hemorrhagic ascites accumulated within 3 weeks at the first passage and 4-6 weeks in serial passages. Carcinomas peritonitis occurred consistently and was observed in the diaphragm, mesenteries, omentum, and pancreas; metastases were seen in the liver and spleen. Subsequently, iv injection of ascites at passage 3 (6 X 10(5) cells/0.2 ml) into nude mice produced metastatic lesions in the lung and the heart. The histology of the invasive and metastatic lesions in the nude mice was similar to that of the original tumor in the patient with stomach carcinoma.

Induction of TR4 orphan receptor by retinoic acid in human HaCaT keratinocytes.

Human TR4 orphan receptor (TR4) can modulate the transcriptional activity of the reporter gene containing an AGGTCA direct repeat-hormone response element. Here we studied the potential role of TR4 in human HaCaT keratinocytes. Using a chloramphenicol acetyl-transferase reporter gene assay, it was shown that TR4 can suppress retinoic acid-induced transactivation by 47.3% in human HaCaT keratinocytes. Electrophoretic mobility shift assay indicated that this suppression may be due to TR4 binding with higher affinity to the retinoic acid response element than retinoid receptors. Western blot analysis further suggested that retinoic acid can increase the expression of TR4 protein in human HaCaT keratinocytes, indicating that TR4 acts as a negative feedback modulator for retinoic acid action. Interestingly, TR4 expression is increased in normal human keratinocytes when substituting a low calcium medium with a high calcium medium. Together, our data suggested, for the first time, that an orphan receptor, such as TR4, may play an important part in retinoid-mediated signaling pathways in human keratinocytes, providing a new insight into keratinocyte biology.

The gene structure and promoter analysis of mouse lymphocyte signal transduction molecule alpha 4 that is related to the yeast TAP42 involved in a rapamycin-sensitive pathway.

The mouse alpha 4 phosphoprotein encoding a component associated with the B cell antigen receptor (BCR)-mediated signal transduction is suggested to be involved in a unique rapamycin-sensitive pathway. We studied the structure and the molecular mechanism of the expression of alpha 4 gene by isolating two phage clones, named #10 and #23, covering entire exons of the mouse alpha 4 gene. The alpha 4 gene is located within about 25 kb and composed of six exons. To analyze the regulation of alpha 4 gene expression, we determined the nucleotide sequence toward 2 kb upstream of the translation start site of the alpha 4 gene. The 5'-flanking region does not contain a typical TATA box or the initiation consensus sequence, but it contains a CCAAT box, E-boxes, and several DNA binding motifs such as c-Myc, c-Myb, and c-Ets. Transcription of the alpha 4 gene starts at four different sites, determined by primer extension analysis, that were surrounded by Y-rich sequences. We further characterized the functional promoter of the alpha 4 gene at the region between -263 and the transcription start site of alpha 4 gene by luciferase assay system and suggested that the 5' upstream region of alpha 4 gene contains the silencer element of MT repetitive sequence.

Alpha4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases.

The catalytic activity of the C subunit of serine/threonine phosphatase 2A is regulated by the association with A (PR65) and B subunits. It has been reported that the alpha4 protein, a yeast homolog of the Tap42 protein, binds the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases such as protein phosphatase 4 and protein phosphatase 6. In the present study, we showed that alpha4 binds these three phosphatases and the association of alpha4 reduces the activities of these phosphatases in vitro. In contrast, PR65 binds to the C subunit of serine/threonine phosphatase 2A but not to protein phosphatase 4 and protein phosphatase 6. These results suggest that the alpha4 protein is a common regulator of the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases.

Establishment of a murine pre-B cell clone dependent on interleukin-7 and stem cell factor.

To identify cytokines required for proliferation of murine pre-B cells, we established a pre-B cell clone MH11 (B220+ MB-1+ sIgM-) on a stromal cell line ST2 from day 13 fetal liver. The growth of MH11 is dependent on ST2. Another stromal cell line PA6, non-secretor of IL-7, could not support MH11 unless IL-7 was added. We investigated the effect of cytokines on proliferation of MH11 with or without stromal cells. IL-7 had a stimulatory effect on proliferation of MH11, but IL-7 alone could not support MH11 growth without ST2. Recombinant stem cell factor (rSCF) also had a positive effect on MH11. rSCF and rIL-7, when added together, could maintain the growth of MH11 in the absence of stromal cells. Moreover, the growth of MH11 on ST2 was inhibited almost completely by anti-c-kit monoclonal antibody (mAb). These results demonstrate that direct SCF/c-kit interaction is involved in the stimulation of pre-B cells.

Involvement of a rapamycin-sensitive pathway in CD40-mediated activation of murine B cells in vitro.

Activation of resting B cells requires an initial triggering of the B cell antigen receptor (BCR) and secondary stimuli through various cytokine receptors and B cell activation molecules including CD40. We found that activation of B cells through CD40 is selectively inhibited by an immunosuppressant drug, rapamycin. This effect of rapamycin on anti-CD40-mediated activation of B cells was observed using three different in vitro assays. Rapamycin suppressed the anti-CD40-induced proliferation of splenic B cells, suppressed differentiation to surface IgMhigh/IgDlow B cells, and inhibited an anti-CD40-mediated prevention of apoptosis induced by BCR cross-linkage of WEHI-231 cells. We next examined several known CD40 signal transduction pathways to identify the target of rapamycin in stimulated B cells. Rapamycin did not inhibit the activation of c-Jun N-terminal kinases (JNKs) induced by anti-CD40 stimulation nor the activation of immediate nuclear transcription factors of NF-kappaB. Therefore, rapamycin affects a novel element of the CD40 signal transduction pathway which influences the proliferation, differentiation, and prevention of apoptosis of B cells.

Anti-CD40 monoclonal antibody induces the proliferation of murine B cells as a B-cell mitogen through a distinct pathway from receptors for antigens or lipopolysaccharide.

To study the activation and differentiation of murine B cells, we prepared a hybridoma secreting monoclonal antibody, LB429, which can directly induce the proliferation of murine B cells in vitro. LB429 recognizes a B cell specific surface molecule of 45 kDa. It recognizes an epitope of murine CD40 produced as a soluble fusion protein with glutathione S-transferase. LB429 stains COS-7 transfectant with murine CD40 cDNA and mature B-cell lines but does not stain pre-B cell lines. Two color staining demonstrated that the epitope recognized with LB429 appears on the surface of B220+ cells of spleen and bone marrow. LB429 can induce a strong proliferation of murine B cells from spleen in the absence of initial triggering with anti-IgM antibody or with anti-IgM antibody + IL-4. LB429 induced the cell size enlargement and the cell cycle transition of resting B cells as well as lipopolysaccharide (LPS). LB429 and LPS stimulate B cells synergistically in vitro by accumulating 44.7% of cells in S/G2/M phases of cell cycle. However, stimulation of spleen B cells with LB429 resulted in the increase of sIgM high+ sIgD(high)+ B cells, in contrast LPS showed the proliferation of both sIgM(high)+ sIgD(high)+ B cells and sIgM(low)+ sIgD(high)+ B cells. These results suggested that LB429 and LPS cause the proliferation of B cells through different stimulatory pathways. This anti-mouse CD40 antibody (LB429) is a very useful reagent to study the activation and differentiation of B cells in vitro.

Production of pancreatic acinar cell carcinoma by combined administration of 4-hydroxyaminoquinoline 1-oxide and azaserine in partial pancreatectomized rats.

The effect of azaserine on the pancreatic tumorigenesis of 4-hydroxyaminoquinoline 1-oxide (4-HAQO) after partial pancreatectomy in rats was studied. Pancreatic acinar cell carcinomas were produced in 7 out of 10 rats (70%), which received 7 mg/kg body wt. 4-HAQO 3 days after partial pancreatectomy, followed by 10 weekly injections of 30 mg/kg body wt. azaserine. Partial pancreatectomy enhanced the carcinogenesis of 4-HAQO, which was further promoted by azaserine.

Lack of androgen receptor transcriptional activity in human keratinocytes.

Since detection of androgen receptor (AR) expression in keratinocytes by immunostaining is controversial, we investigated whether keratinocytes can act as androgen target cells using transient transfection assays. Chloramphenicol acetyltransferase (CAT) assays for the endogenous AR transcriptional activity in HaCaT keratinocytes indicated that DHT (10(-9)-10(-8) M) can induce less than 1.5-fold of mouse mammary tumor virus CAT, which is quite low, compared with 38-fold induction by 10(-7) M 1,25-dihydroxyvitamin D(3) of P450cc24-CAT. Furthermore, this low DHT-mediated induction could not be enhanced by the AR co-activators, ARA70 or ARA55. Western blotting analysis indicated that HaCaT and normal keratinocytes do not express AR protein. Transfection of exogenous AR into HaCaT keratinocytes, however, could install AR transcriptional activity, suggesting that HaCaT keratinocytes have all the necessary accessory factors for AR transcription activity. In conclusion, keratinocytes are unlikely to be target cells for androgen.

Potential anti-androgenic activity of roxithromycin in skin.

Since acne formation is a multistep process accelerated by androgens, we examined whether a new anti-acne antibiotic roxithromycin (RXM) may act as anti-androgen using transient transfection assays in human skin fibroblasts. The result showed no significant effect of 0.5, 1 and 5 microg/ml RXM on 10(-9) M R1881-induced androgen receptor (AR) transcriptional activity. While the cotransfection of exogenous ARA55, a novel AR coactivator, increased AR transactivation up to 2.59-fold, this increase was attenuated by 5 microg/ml RXM to 64.7%. Semiquantitative RT-PCR results showed that 0.1 mM H(2)O(2) treatment increased ARA55 mRNA expression level, indicating that reactive oxygen species increase the expression of ARA55 in skin. These results suggest that RXM may serve as anti-androgen only in the hypersensitive state to androgen, but not in the physiological state, through modulating end-organ hypersensitive condition to androgen possibly involving the pathway from reactive oxygen species to ARA55.

Synchronization of normal human keratinocyte in culture: its application to the analysis of 1,25-dihydroxyvitamin D3 effects on cell cycle.

In this report, we applied the isoleucine-deprivation method to normal human keratinocytes in cultivation with serum-free MCDB153 medium. The growing cells were synchronized by transfer to MCDB153 medium prepared without isoleucine, and the degree of synchronization was analyzed by the cell cycle distribution and phosphorylation of retinoblastoma protein (pRB), one of the tumor suppressor gene products. 1,25-dihidroxyvitamin D3 (1,25(OH)2D3), a biologically active form of vitamin D3, is supposed to induce the differentiation and growth inhibition of human keratinocytes caused by cell cycle arrest. We examined its effect on cell cycle kinetics, especially progression from G1/G0 to S-phase, by using this synchronized system. We showed that 10(-6) M 1,25(OH)2D3 inhibited the progression from G1/G0 to S-phase strikingly in synchronized normal human keratinocytes.

Diagnosis of lymphoma in paraffin wax sections by nested PCR and immunohistochemistry.

AIMS: To investigate whether nested polymerase chain reaction (PCR) and immunohistochemistry can be used to diagnose malignant lymphoma. METHODS: Paraffin wax embedded tissue sections from 31 patients with malignant lymphoma were analysed by nested PCR and immunohistochemistry using standard protocols. RESULTS: Nested PCR amplification of 1 pg DNA confirmed monoclonality in B cell lymphoma; PCR amplification of 10 pg DNA confirmed monoclonality in T cell lymphoma. Twenty seven (87%) samples were diagnosed as malignant lymphoma by nested PCR, and 24 (77%) by immunohistochemistry. Seven samples were diagnosed as malignant lymphoma by nested PCR, but not by immunohistochemistry, whereas the use of both procedures gave a diagnosis of malignant lymphoma in all 31 samples. CONCLUSIONS: A combination of immunohistochemistry and nested PCR can be used to diagnose malignant lymphoma in routine paraffin wax embedded sections.

The first seven cases of chronic beryllium disease in ceramic factory workers in Japan.

In the present paper the first 7 cases in Japan of chronic beryllium disease found in workers employed in a ceramic factory utilizing beryllium have been described. Immunological examinations of these cases showed changes similar to those observed in sarcoidosis, that is, negative tuberculin test and increase in serum gamma globulin and immunoglobulins. The fact that a considerable number of workers in the same factories as the patients showed negative tuberculin reaction may suggest that there may be further cases of chronic beryllium disease among them that are still in a latent period.

Differential effects of 4-hydroxyaminoquinoline-1-oxide on pancreatic and liver DNA synthesis in rats.

The effect of 4-hydroxyaminoquinoline-1-oxide (4-HAQO) on DNA synthesis in the pancreas and liver, target and non-target organs for 4-HAQO carcinogenesis, respectively, were compared. Pancreatic and liver DNA synthesis were simultaneously induced in rats fed a protein deficient diet containing 0.5% DL-ethionine for 18 days, and DNA synthesis in both tissues was inhibited by hydroxyurea. A single i.v. injection of 4-HAQO at a dose of 7 mg/kg body weight also inhibited DNA synthesis in both tissues within 4 h. In the pancreas the inhibition was maximum at a dose of 7 mg/kg, and DNA synthesis was less than in the pancreas of rats fed a control grain diet. This inhibition continued for the subsequent 5 days which were tested. In the liver, the degree of inhibition was less than in pancreas but the value remained higher than in rats fed control diet. The inhibition of liver DNA synthesis at a dose of 7 mg/kg completely recovered within 1 day. These results suggest that the lesions of DNA induced by 4-HAQO and its repair might be different between the pancreas and the liver. A pancreatic chemical carcinogen, 4-HAQO, might thus have the same cytotoxic effect that liver carcinogens have toward the liver resulting in failure to respond to mitotic stimuli. This might be causally related to the organotropism of 4-HAQO toward the pancreas.

Dose-dependent suppression of toluene metabolism by isopropyl alcohol and methyl ethyl ketone after experimental exposure of rats.

In order to examine possible suppression of toluene metabolism due to coexposure to other solvents, female Wistar rats were exposed for 8 h to toluene alone (at 50 or 100 ppm), or in combination with either methyl ethyl ketone (at 50, 100, 200 or 400 ppm) or isopropyl alcohol (at 50, 100, 200, 400, 800 or 1600 ppm). Urine samples were collected for 24 h after initiation of each exposure, and subjected to analysis for two toluene metabolites, hippuric acid and o-cresol, both by HPLC. The excretion of hippuric acid, a major metabolite, was not modified when the concentrations of methyl ethyl ketone or isopropyl alcohol were low, i.e. 100 ppm or below, whereas it was reduced when methyl ethyl ketone or isopropyl alcohol concentrations were twice or more times higher than that of toluene. There were no changes in any cases in excretion of o-cresol, a minor metabolite. The observation after coexposure to methyl ethyl ketone or isopropyl alcohol at low concentration is in line with the negative interaction between toluene and methyl ethyl ketone as well as between toluene and isopropyl alcohol after occupational exposures at low concentrations. Metabolic interaction may take place when the exposure intensity is high, as observed in the present study and also after experimental exposure of volunteers to toluene and m-xylene, or occupational exposure to benzene and toluene.