[Diagnosing radiation-induced liver injury in rabbit using 3.0 Tesla magnetic resonance diffusion-weighted imaging].

OBJECTIVE: To evaluate the clinical value of magnetic resonance (MR) diffusion-weighted imaging (DWI) for diagnosing radiation-induced liver injury (RILI) and detecting changes in hepatic pathology at different post-irradiation times. METHODS: Male New Zealand white rabbits received no irradiation (C0, control group; n = 10) or irradiation of 50 Gy/10F once every other day by virtual three dimensional conformal radiotherapy (3D-CRT) for one day (C1; n = 10), three days (C2; n = 10), two weeks (C3; n = 10), one month (C4; n = 10) or two months (C5; n = 10). One member of all groups were sacrificed for DWI examination and pathologic study on post-irradiation day 1, day 3, week 2, month 1 and month 2. The apparent diffusion coefficient (ADC) values were measured using a range of b values (50, 300, 600, 800 and 1000 s/mm2). RESULTS: Hematoxylin-eosin (H-E) staining showed that livers of rabbits in the C3, C4 and C5 groups had the characteristic features of veno-occlusive disease. DWI examination showed that the irradiated livers of rabbits in C2, C3, C4 and C5 groups had significantly lower ADC values than the livers of the non-irradiated rabbits at b values of 300, 600, 800 and 1000 s/mm2 (P less than 0.05). When the b value was 600 s/mm2, the best negative correlation between ADC values and pathological stage was seen for the irradiated livers (Spearman's rank, r = -0.459, P less than 0.01). The threshold ADC value to distinguish the normal group (C0) from an irradiated group (more than or equal toC1) was 1.955 * 10-3 mm2/s at 600 s/mm2 b value. When the b value was 1000 s/mm2, the threshold ADC value to predict an irradiated group with normal H-E staining (C1) from an irradiated group with abnormal H-E staining (more than or equal toC2) was 1.5250 * 10-3 mm2/s; the ADC threshold value was 1.5150 * 10-3 mm2/s to predict groups C0-2 and groups C3-5. CONCLUSION: DWI has high sensitivity for detecting RILI at three days after irradiation with proper b values. Use of the ADC value is feasible for estimating the evolutionary process of pathological features of RILI damage. DWI may represent an important clinical tool for detection of early pathological changes in RILI.

Papillary carcinoma of the duodenum combined with right renal carcinoma: a case report.

We report a case of papillary carcinoma of the duodenum combined with right renal carcinoma. A 58-year-old man underwent a physical examination that revealed intrahepatic and extrahepatic bile duct dilatation on B ultrasound. Intrahepatic bile duct dilatation could be seen on magnetic resonance imaging (MRI), but the head of the pancreas and distal bile duct showed no tumor signals, which led to a diagnosis of periampullary carcinoma and right renal carcinoma. Considering the trauma of pancreaticoduodenectomy combined with renal resection operation is greater, we carried out the laparoscopic right renal radical resection first, and then a pylorus-preserving pancreaticoduodenectomy was performed. However, postoperative intra-abdominal infections and bleeding occurred; our patient improved after vascular interventional microcoil embolization for the treatment of hemostasis. The second operation for celiac necrotic tissue elimination, jejunal fistulization and peritoneal lavage and drainage was performed 14 days latter. Our patient improved gradually and was discharged on the 58th postoperative day. There has been no tumor recurrence after a follow-up of 26 months.

Regulatory T cells increase in breast cancer and in stage IV breast cancer.

Expression levels of VEGF and Her-2, levels of T-regulatory (Treg) cells, levels of CD3+ cells, and ratios of Th (CD4+ T cells)/Tr (Treg) cells were compared between stage I, II, III, and IV breast cancer patients (n = 120) prior to chemotherapy and healthy women (n = 30). Cells from peripheral blood were counted by flow cytometry, Her-2 and VEGF expression was detected by pathological examination, and Her-2 was detected by FISH. Breast cancer patients had more Treg cells and a lower ratio of Th/Tr cells than the healthy women. Stage IV breast cancer patients had more Treg cells and a lower ratio of Th/Tr cells than stage I, II, or III breast cancer patients. Patients positive for VEGF had a lower ratio of Th/Tr cells compared with patients negative for VEGF, and those positive for both VEGF and Her-2 also had a lower ratio of Th/Tr cells compared with patients not positive for both VEGF and Her-2. The decreased Th/Tr cells ratio indicates impaired immune function, suggesting that the stage IV breast cancer and the Her-2/VEGF-positive breast cancer patients have lower immune function.

Two PKC consensus sites on human acid-sensing ion channel 1b differentially regulate its function.

Human acid-sensing ion channel 1b (hASIC1b) is a H(+)-gated amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker psalmotoxin-1 decrease the migration and proliferation of glioma cells. PKC also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1b's COOH terminus has been shown to interact with protein interacting with C kinase (PICK)1, which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine, to prevent phosphorylation, and to glutamic acid or aspartic acid, to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating the modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild type (WT), whereas S40E, S499A, and S499D currents were decreased. The PKC activators PMA and phorbol 12,13-dibutyrate inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A but had no effect on S499A or S40A/S499A. PKC activators or the inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information that may help in designing potentially beneficial therapies in their treatment.

[Histopathologic analysis of 2 micron continuous wave laser for the partial cystectomy of bladder urothelial carcinoma].

OBJECTIVE: To observe the histopathologic characteristics of 2 micron continuous wave laser transurethral partial cystectomy for the treatment of bladder tumor. METHODS: A total of 54 patients with 65 bladder tumors underwent 2 micron laser via transurethral by caudal or surface anesthesia from October 2007 to December 2008. It included 41 male and 13 female cases, and the age ranged from 27 to 81 years old with a mean of (66.2 +/- 12.4) years old. The operation evaporated and exsected the wall of urinary bladder, including tumor, submucosa and all muscular layers. Specimens were sent for pathology examination. The histomorphologic changes of raw surfaces were observed 1 week, 1 month, 3 months, 6 months and 1 year postoperation by cystoscopic and pathologic examinations. RESULTS: All the patients tolerated in the operation. Clinical stages of the tumor: T1 for 42 cases, T2 for 12 cases. All cases were followed-up for 1 to 14 months, with a mean of 8.5 months. Tumor recurrences were found in 2 cases, no one had recurrence in situ. The tumor, submucosa and all muscular layers can be resected completely by 2 micron continuous wave laser transurethral partial cystectomy. Pathologic staging can be judged correctly. The umbilication raw surface were infiltrated by fibrous connective tissue and chronic inflammatory cells 1 week postoperation. The umbilication changed shallow and transitional epithelial cells began to cover it 1 month postoperation. The umbilication disappeared and transitional epithelial cells cover the raw surface 3 months postoperation. There was no difference between the raw surface and normal bladder mucosa. CONCLUSIONS: 2 micron continuous wave laser for the treatment of bladder tumor can get the same clinical result as partial cystectomy. The pathologic staging can be judged correctly by the specimens.

[Morphologic diagnosis and clinical significance of prostatic atypical small acinar proliferation suspicious but not diagnostic of cancer].

OBJECTIVE: To study the morphologic features and clinical significance of atypical small acinar proliferation (ASAP) suspicious but not diagnostic of cancer in prostatic biopsies. METHODS: The slides of 11 cases of prostatic needle biopsies collected during a two-year period with the diagnosis of ASAP were reviewed. Immunohistochemical study for 34betaE12, p63 and P504S was performed on the archival paraffin sections. RESULTS: All the 11 ASAP cases were characterized by the presence of a few compacted small acini in the prostatic stroma. Six cases had acini of less than three in number. The acini were round or slightly irregular in shape. The nuclei were enlarged, round or irregular, arranged in single layer and focally separated by broad interval. The nucleoli were usually prominent. Cytoplasm was amphophilic or pale and the lumen border was often well-defined. Basophilic mucus was also seen in some of the lumen. Immunohistochemical study for 34betaE12 and p63 was negative, while that for P504S was positive. In 4 of the 11 cases, the acini were more than three in number, round or slightly irregular, but without cytologic atypia. The nuclei were slightly enlarged with small or inconspicuous nucleoli. Immunohistochemical study for 34betaE12 and p63 was negative or at most focally positive. P504S staining was either negative or weakly positive. Second repeat biopsy was carried out in all cases, and 4 of them (36%) showed definite adenocarcinomatous changes. The positive cases were those with fewer acini but definite cytologic atypia in the initial biopsy. CONCLUSIONS: ASAP is a morphologic interpretation closely associated with prostatic adenocarcinoma. The histologic features are suspicious of but not diagnostic of cancer, due to insufficient criteria in terms of acinar number, cytologic or architectural abnormalities. The positive rate in subsequent repeat biopsy is higher than that for cases with usual nodular hyperplasia.

[Surgical treatment of primary pulmonary cryptococcosis].

OBJECTIVE: To assess the clinical feature, diagnosis and treatment of primary pulmonary cryptococcosis. METHODS: From 1996 to 2004, 11 patients with primary pulmonary cryptococcosis were surgical treated and confirmed by histologic study. At the same period, 2715 patients with pulmonary abnormalities received surgery. Their clinical data were retrospectively reviewed. RESULTS: Sixty-four percent (7/11) of the patients were symptomatic at the time of diagnosis. All 11 cases were misdiagnosed as lung cancer or inflammatory or tuberculosis by X-ray and CT scan before surgery. Three cases received fluorine-18 fluorodeoxyglucose-positron emission tomography (FDG-PET) scan and their primary pulmonary lesions showed FDG avid. All 11 patients were treated by antibiotics and antituberculosis therapy but no responses appeared. Primary pulmonary cryptococcosis was diagnosed by ultrasound-guided fine needle aspiration biopsy in only 2 cases, but antifungal therapy was not effective. All 11 patients underwent thoracotomy and their pulmonary cryptococcosis were resected. Only 1 patient with multiple nodules received antifungal therapy postoperatively. No recurrence was found in any patients. CONCLUSIONS: Primary pulmonary cryptococcosis is non-specific and can be confused with lung cancer, tuberculosis, etc. The pulmonary abnormalities should be resected unless the diagnosis is established. Antifungal therapy is not necessary in patients whose abnormality has been resected thoroughly.

Molecular cloning and characterization of human acid sensing ion channel (ASIC)2 gene promoter.

Acid sensing ion channel (ASIC)2 belongs to the amiloride-sensitive Na(+)-channel/ degenerin family. Our previous studies suggested that differential regulation of ASIC2 expression occurs between high-grade glial-derived tumor cells and normal astrocytes. To investigate the mechanisms involved in the regulation of ASIC2 gene expression, the human ASIC2 promoter region (-1551 to +117) was cloned and characterized. The ASIC2 promoter lacked a canonical TATA box, but contained one putative CCAAT box. Nucleotide sequencing of the promoter revealed the presence of a number of transcription factor-binding sites and a 404 bp CpG island upstream the transcription start site. Nested deletion mutants and transfection results showed that the construct between -133 and +117 base pairs conferred basal transcription specific activity. Mutation of Sp1 and CP2 sites in this region resulted in a 70 and 95% decrease in promoter activity, respectively. Gel shift assays demonstrated the existence of specific protein binding to the SP1 and CP2 elements. There was no mutation in the CpG island in six glioma cell lines, but methylation-specific PCR showed methylation in some of glioma cell lines and tumor tissues, and treatment with the methylation inhibitor 5-Aza-2'-deoxycytidine could partially restore ASIC2 expression in cell lines, suggesting that epigenetic mechanisms may contribute to dysregulated ASIC2 expression.

Structure of Cytoplasmic Polyhedrosis Virus from Bombyx mori.

The structures of full and empty capsids of CPV were studied by negative staining and electron cryomicroscopy and computer reconstruction techniques. By comparing the structures and biochemical compositions, the CPV was identified as a single-layered capsid with its five structural proteins located on it. This single capsid is arranged according to T=1 icosahedral symmetry with 12 turret-like spikes at its icosahedral vertices. The empty and full CPV show identical capsid but differ inside. The dense and ordered genomic dsRNA is located inside the full CPV. The internal space of the empty CPV has almost no electron density except for 12 electron densities attributed the transcriptional enzyme complexes extending inward from the base part of CPV spikes.

Participation of the chaperone Hsc70 in the trafficking and functional expression of ASIC2 in glioma cells.

High-grade glioma cells express subunits of the ENaC/Deg superfamily, including members of ASIC subfamily. Our previous work has shown that glioma cells exhibit a basally active cation current, which is not present in low-grade tumor cells or normal astrocytes, and that can be blocked by amiloride. When ASIC2 is present within the channel complex in the plasma membrane, the channel is rendered non-functional because of inherent negative effectors that require ASIC2. We have previously shown that high-grade glioma cells functionally express this current because of the lack of ASIC2 in the plasma membrane. We now hypothesize that ASIC2 trafficking in glioma cells is regulated by a specific chaperone protein, namely Hsc70. Our results demonstrated that Hsc70 co-immunoprecipitates with ASIC2 and that it is overexpressed in glioma cells as compared with normal astrocytes. In contrast, there was no difference in the expression of calnexin, which also co-immunoprecipitates with ASIC2. In addition, glycerol and sodium 4-phenylbutyrate reduced the amount of Hsc70 expressed in glioma cells to levels found in normal astrocytes. Transfection of Hsc70 siRNA inhibited the constitutively activated amiloride-sensitive current, decreased migration, and increased ASIC2 surface expression in glioma cells. These results support an association between Hsc70 and ASIC2 that may underlie the increased retention of ASIC2 in the endoplasmic reticulum of glioma cells. The data also suggest that decreasing Hsc70 expression promotes reversion of a high-grade glioma cell to a more normal astrocytic phenotype.

Surface expression of ASIC2 inhibits the amiloride-sensitive current and migration of glioma cells.

Gliomas are primary brain tumors with a complex biology characterized by antigenic and genomic heterogeneity and a propensity for invasion into normal brain tissue. High grade glioma cells possess a voltage-independent, amiloride-inhibitable, inward Na+ current. This current does not exist in normal astrocytes or low grade tumor cells. Inhibition of this conductance decreases glioma growth and cell migration making it a potential therapeutic target. Our previous results have shown that the acid-sensing ion channels (ASICs), members of the epithelial Na+ channel (ENaC)/degenerin (DEG) family of ion channels are part of this current pathway. We hypothesized that one member of the ENaC/DEG family, ASIC2, is retained intracellularly and that it is the lack of functional expression of ASIC2 at the cell surface that results in hyperactivity of this conductance in high grade gliomas. In this study we show that the chemical chaperone, glycerol, and the transcriptional regulator, sodium 4-phenylbutyrate, inhibit the constitutively activated inward current and reduce cell growth and migration in glioblastoma multiforme. The results suggest that these compounds induce the movement of ASIC2 to the plasma membrane, and once there, the basally active inward current characteristic of glioma cells is abolished by inherent negative regulatory mechanisms. This in turn compromises the ability of the glioma cell to migrate and proliferate. These results support the hypothesis that the conductance pathway in high grade glioma cells is comprised of ENaC/DEG subunits and that abolishing this channel activity promotes a reversion of a high grade glioma cell to a phenotype resembling that of normal astrocytes.

ENaC plays a role in regulated antibody secretion by hybridomas.

Hybridomas are fused immortal lymphocytes that typically secrete monoclonal antibodies to a known antigen. Hybridomas express two ionic conductances that have properties consistent with epithelial sodium channel (ENaC) and CFTR. Both ion channels are expressed by lymphocytes. Both of these channels are known to play a role in epithelial cell physiology. However, the physiological role of these channels in lymphocytes is unclear. We tested the hypothesis that ENaC plays a role in the process of regulated antibody secretion. We have been able to demonstrate that hybridomas can be provoked to acutely secrete monoclonal antibodies by a variety of agonists. Concurrently, we were able to show that these same agonists activate amiloride-sensitive sodium currents in whole cell clamped hybridomas. Inhibition of ENaC by amiloride inhibited the acute provoked antibody secretion, thereby linking ENaC to the process of acute antibody secretion. Interestingly, the concentration of amiloride necessary to completely inhibit the provoked secretion was approximately an order of magnitude higher than the concentration necessary to inhibit all of the transmembrane current. However, because amiloride is a weak base, the equilibrium concentration necessary to produce partial inhibition was precisely in accord with the K(i) for amiloride and ENaC, indicating that the inhibition was intracellular.

ATP masks stretch activation of epithelial sodium channels in A6 distal nephron cells.

The mechanosensitivity of the epithelial sodium channel (ENaC) is controversial. Using cell-attached patch-clamp techniques, we found that mechanical stretch stimulated ENaC in A6 distal nephron cells in only three of nine cell-attached patches. However, stretch consistently activated ENaC after apical ATP was scavenged with apical hexokinase plus glucose or after P(2) receptors in the patch were blocked. The mean open probability (P(o)) of ENaC was increased from 0.31 +/- 0.04 to 0.61 +/- 0.06 (P < 0.001; n = 9) when patch pipettes contained hexokinase and glucose, or from 0.24 +/- 0.05 to 0.55 +/- 0.11 (P < 0.01; n = 7) when patch pipettes contained suramin, respectively. A poorly hydrolyzable ATP analog, ATPgammaS, in the patch pipettes inhibited ENaC, reducing the P(o) from 0.41 +/- 0.06 to 0.19 +/- 0.05 (P < 0.01; n = 8). Pretreatment of A6 cells with the phospholipase C (PLC) inhibitor U-73122 abolished the effect of ATP on ENaC activity. These data together suggest that ATP, acting through a PLC-dependent purinergic pathway, masks stretch-induced ENaC activation.

Acidic ATP activates lymphocyte outwardly rectifying chloride channels via a novel pathway.

Using whole-cell patch-clamp techniques we found that ATP activated an outwardly rectifying current in Daudi human B lymphoma cells under acidic conditions. The substitution of Cl- for gluconate(-) shifted the reversal potential, while Cl- channel blockers, 4,4'-diisothiocyanostibene-2,2'-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC), blocked the current, indicating that ATP induces this current by activating the outwardly rectifying chloride channel (ORCC). The effect of ATP on ORCC was mimicked by ADP, but not by other P2 receptor agonists such as ATPgammaS (a poorly hydrolyzable analog of ATP), 2',3'-O-benzoyl-4-benzoyl-ATP (BzATP), and UTP. The ATP-induced ORCC current was completely blocked by 100 microM suramin (a P2 receptor antagonist), and was partially blocked by 100 microM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS), which is another P2 receptor antagonist. Neither inactivation of G proteins nor elimination of extracellular Ca2+ affected the ATP-induced current, indicating that G protein-coupled P2Y receptors and Ca(2+)-permeable P2X receptors are not involved. Based on the pharmacological profile and the fact that acidic conditions are required for ATP to activate the ORCC, we suggest that acidic ATP activates the lymphocyte ORCC via a novel pathway, which is not associated with any previously described purinergic receptors.