MR imaging features for improved diagnosis of hepatocellular carcinoma in the non-cirrhotic liver: Multi-center evaluation.

PURPOSE: To determine MR-imaging features for the differentiation between hepatocellular carcinoma (HCC) and benign hepatocellular tumors in the non-cirrhotic liver. MATERIAL AND METHODS: 107 consecutive patients without liver cirrhosis (46 male; 45 ± 14 years) who underwent liver resection due to suspicion of HCC were included in this multi-center study. The following imaging features were assessed: lesion diameter and demarcation, satellite-lesions, central-scar, capsule, fat-content, hemorrhage, vein-infiltration and signal-intensity (SI) on native T1-, T2- and dynamic-enhanced T1-weighted images (center versus periphery). In addition, contrast-media (CM) uptake in the liver specific phase was analyzed in a sub-group of 42 patients. RESULTS: Significant differences between HCC (n=55) and benign lesions (n=52) were shown for native T1-, T2- and dynamic-enhanced T1-SI, fat-content, and satellite-lesions (all, P<.05). Independent predictors for HCC were T1-hypointensity (odds-ratio, 4.81), T2-hypo-/hyperintensity (5.07), lack of central tumor-enhancement (3.36), and satellite-lesions (5.78; all P<0.05). Sensitivity and specificity of HCC was 91% and 75% respectively for two out-of four independent predictors, whereas specificity reached 98% for all four predictors. Sub-analysis, showed significant differences in liver specific CM uptake between HCC (n=18) and benign lesions (n=24; P<0.001) and revealed lack of liver specific CM uptake (odds-ratio, 2.7) as additional independent feature for diagnosis of HCC. CONCLUSION: Independent MRI features indicating HCC are T1-hypointensity, T2-hypo- or hyperintensity, lack of central tumor-enhancement, presence of satellite-lesions and lack of liver specific CM-uptake. These features may have the potential to improve the diagnosis of HCC in the non-cirrhotic liver.

TIMES-SS--recent refinements resulting from an industrial skin sensitisation consortium.

The TImes MEtabolism Simulator platform for predicting Skin Sensitisation (TIMES-SS) is a hybrid expert system, first developed at Bourgas University using funding and data from a consortium of industry and regulators. TIMES-SS encodes structure-toxicity and structure-skin metabolism relationships through a number of transformations, some of which are underpinned by mechanistic 3D QSARs. The model estimates semi-quantitative skin sensitisation potency classes and has been developed with the aim of minimising animal testing, and also to be scientifically valid in accordance with the OECD principles for (Q)SAR validation. In 2007 an external validation exercise was undertaken to fully address these principles. In 2010, a new industry consortium was established to coordinate research efforts in three specific areas: refinement of abiotic reactions in the skin (namely autoxidation) in the skin, refinement of the manner in which chemical reactivity was captured in terms of structure-toxicity rules (inclusion of alert reliability parameters) and defining the domain based on the underlying experimental data (study of discrepancies between local lymph node assay Local Lymph Node Assay (LLNA) and Guinea Pig Maximisation Test (GPMT)). The present paper summarises the progress of these activities and explains how the insights derived have been translated into refinements, resulting in increased confidence and transparency in the robustness of the TIMES-SS predictions.

[Recurrent Baker's cyst]. / Rezidivierende symptomatische Bakerzyste.

A 50-year old female patient with unilateral knee pain demonstrated a recurrent ultrasound-proven popliteal cyst (Baker's cyst). Even though a proper differential diagnosis was done, the MRT of the knee showed in a secondary step a tibial fissure as the cause of the treatment-refractory knee pain and Baker's cyst. A fracture of the tibia is a rare cause for a symptomatic Baker's cysts. Mechanical, degenerative or inflamed diseases of the joint are more frequent associated with a Baker's cyst.

pH dependence of the partitioning of triphenyltin and tributyltin between phosphatidylcholine liposomes and water.

Triorganotin compounds are very toxic contaminants. The site of their basic mechanism of action of acute toxicity is the biomembrane. Liposome-water distribution ratios of triphenyltin and tributyltin were determined between pH 3 and pH 8 with the equilibrium dialysis method in the micromolar concentration range, which is the concentration range where acute toxicity is observed. In addition, biomembrane-water distribution ratios of tributyltin were determined with chromatophores of Rhodobacter sphaeroides that contain approximately 70% protein intercalated in the lipid bilayer. The liposome-water distribution of both compounds showed only weak pH dependence. For tributyltin, the apparent distribution ratio decreased from 4100 at low pH to 2000 at high pH, while this ratio decreased from 70 000 to 22 000 for TPT. The distribution ratio of the triorganotin cation exceeded that of the neutral hydroxo complex by a factor of 2. The distribution ratio of both the cation and the hydroxo complex of triphenyltin exceeded that of tributyltin by a factor of 10. It is postulated that the sorption of the cation is governed by complex formation with ligands in the phospholipids, presumably the phosphate group. The biomembrane-water distribution ratio of tributyltin was found to be lower than the liposome-water distribution ratio at high pH. The hydroxo complex appears to partition only to the lipid fraction of the biomembrane. Yet, at low pH the biomembrane-water distribution ratio exceeded the liposome-water distribution ratio, which is attributed to complex formation of the cationic species with ligands of the protein fraction.

Interaction of phenolic uncouplers in binary mixtures: concentration-additive and synergistic effects.

The uncoupling activities of 14 binary mixtures of substituted phenols and of 4 binary mixtures of phenols and anisols were investigated at different pH values. Experiments were performed with time-resolved spectroscopy on membrane vesicles (chromatophores) of the photosynthetic bacteria Rhodobacter sphaeroides. Phenols are known to destroy the electrochemical proton gradient in energy-transducing membranes by a protonophoric mechanism. Anisols do not have protonophoric activity but disturb membrane structure and functioning as a nonspecific baseline toxicant. It was postulated in the literature that, for certain substituted phenols, the formation of a dimer between the phenoxide and the neutral phenol may contribute significantly to the overall protonophoric activity. In 13 of 14 mixtures of substituted phenols but in none of the mixtures of phenols with anisols, such a dimer appears to be formed between two different mixture partners. An extended shuttle mechanism of uncoupling, which includes a term for the contribution of such a mixed dimer, provided a good description of all experimental data. Opposite speciation favors interaction and ortho substituents abate interaction, which adds evidence for the dimerformation via a hydrogen bond between the phenol-OH and the phenoxide. These findings are significant not only regarding the mechanism of protonophoric action but also for the risk assessment process of chemical mixtures in the environment. When assessing the effect of mixtures, concentration addition is regarded as a reference X concept to estimate effects of similarly acting compounds. The substituted phenols in this work act according to the same action mechanism of uncoupling. Nevertheless, the overall effect of four of the investigated mixtures, which exhibit stronger dimer formation as compared to the single compounds or for which the resulting dimer is intrinsically more active, exceeded the effect calculated according to concentration addition considerably. In future work, this synergistic effect observed in-vitro has to be validated in-vivo to deduce its implications for the risk assessment process.

Acute toxicity of (chloro-)catechols and (chloro-)catechol-copper combinations in Escherichia coli corresponds to their membrane toxicity in vitro.

(Chloro-)catechols are toxic for bacteria and higher organisms, but the mode of action is not yet clearly understood. We have compared the acute toxicity of different chlorinated catechols to Escherichia coli with membrane toxic effects, namely narcosis and uncoupling that we have determined in an in vitro assay. In vitro membrane toxicity was quantified by measuring the accelerated decay of the membrane potential of chromatophores isolated from Rhodobacter sphaeroides. Both acute and membrane toxicity increased with increasing degree of chlorination. Analysis of dose-response curves, pH dependence, and estimated membrane concentrations gave a consistent picture of the mechanisms of membrane toxicity: At pH 7, the higher-chlorinated catechols acted as uncouplers of oxidative and photophosphorylation, and the lower-chlorinated catechols and catechol acted as narcotics. In the case of 3,5-dichlorocatechol and 4-monochlorocatechol at pH 8.8, both mechanisms appeared to contribute to the overall toxicity. Copper exhibited a diverging effect on the toxicity of catechols and of (chloro-)catechols to E. coli. Whereas the presence of copper increased the toxicity of catechol and 4-monochlorocatechol, the toxicity of 3,5-dichlorocatechol, 3,4,5-trichlorocatechol, and tetrachlorocatechol decreased. Again, the results obtained with in vitro assays agreed with the acute toxicity observed in E. coli: The presence of copper accelerated decay of the membrane potential of catechol and 4-monochlorocatechol; however, the effect was reversed by copper in experiments with 3,5-dichlorocatechol, 3,4,5-trichlorocatechol, and tetrachlorocatechol. We have proposed a mechanistic model to explain the diverging effects of copper on the uncoupling activities of the different catechols.

Expression of the mutant thyroid hormone receptor PV in the pituitary of transgenic mice leads to weight reduction.

Resistance to thyroid hormone (RTH) is a genetic disease caused by mutations of the thyroid hormone receptor beta gene (TRbeta). One of the symptoms in some affected individuals is growth retardation. To understand the molecular basis of growth retardation in these patients with RTH, a transgenic mouse was prepared in which the expression of the TRbeta1 mutant PV was targeted to the pituitary using the promoter of the glycoprotein hormone alpha-subunit. The PV mutant was originally identified in a patient with severe growth impairment. The PV mutation is a C-insertion at codon 448 of the TRbeta gene and leads to a frame-shift of the carboxyl-terminal 14 amino acids of TRbeta1, resulting in total loss of triiodothyronine (T3) binding and transcriptional activation. PV was selectively expressed in the pituitary of the transgenic mouse and not in other tissues examined. The transgenic mice showed a significant impairment in weight gain. However, no changes in the serum level of thyroid-stimulating hormone were seen, and no elevation of thyroid hormones was detected in the transgenic mice. The circulating levels of growth hormone and insulin-like growth factor I were not affected in the transgenic mice, suggesting that the growth impairment in RTH is complex and is mediated by pathways that are yet to be elucidated.

Enteric lesions in SCID mice infected with "Helicobacter typhlonicus," a novel urease-negative Helicobacter species.

BACKGROUND AND PURPOSE: Several rodent helicobacters have been associated with chronic active hepatitis or inflammatory bowel disease. Severe combined immunodeficient (SCID) mice appear to be inherently susceptible to disease attributable to these emerging pathogens. With the advent of polymerase chain reaction (PCR) analysis, it has become clear that several as yet unidentified Helicobacter species may also colonize rodents, but their capacity to cause disease is unknown. METHODS: A Helicobacter species isolated from feces of a BALB/c mouse and provisionally named "H. typhlonicus" was used to inoculate helicobacter-free 4-week-old SCID mice (n = 11 males and 11 females). At various weeks after inoculation, mice were sacrificed and liver and intestinal specimens were collected for histologic examination and PCR analyses. RESULTS: The C.B-17 scid/scid mice inoculated with "H. typhlonicus" developed moderate to severe proliferative typhlocolitis, similar to that seen in SCID mice infected with H. hepaticus or H. bilis. However, in contrast to mice infected with H. hepaticus or H. bilis, lesions of chronic active hepatitis were not detected in mice inoculated with "H. typhlonicus." A similar disease syndrome developed in SCID mice cohabitated with B6D2F1 mice naturally infected with a novel Helicobacter species that was genetically identical to "H. typhlonicus." CONCLUSION: "Helicobacter typhlonicus" joins a growing list of helicobacters that are capable of inducing enteric disease in immunodeficient mice.

NK and CTL recognition of a single chain H-2Dd molecule: distinct sites of H-2Dd interact with NK and TCR.

We generated transgenic mice expressing a single-chain beta2-microglobulin (beta2m)-H-2Dd. The cell-surface beta2m-H-2Dd molecule was expressed on a beta2m-deficient background and reacted with appropriate mAbs. It was of the expected m.w. and directed the normal development of CD8+ T cells in the thymus of a broad TCR repertoire. It also presented both exogenously provided and endogenous peptide Ags to effector CD8+ T cells. In tests of NK cell education and function, it failed to reveal any interaction with NK cells, suggesting that the site of the interaction of NK receptors with H-2Dd was disrupted. Thus, the sites of TCR and NK receptor interaction with H-2Dd are distinct, an observation consistent with independent modes of TCR and NK receptor evolution and function.

Transgenic mice demonstrate AP-1 (activator protein-1) transactivation is required for tumor promotion.

Activator protein-1 (AP-1) is a transcription factor that consists of either a Jun-Jun homodimer or a Jun-Fos heterodimer. Transactivation of AP-1 is required for tumor promoter-induced transformation in mouse epidermal JB6 cells and for progression in mouse and human keratinocytes. Until now, the question of whether AP-1 transactivation is required for carcinogenesis in vivo has remained unanswered, as has the issue of functionally significant target genes. To address these issues we have generated a transgenic mouse in which transactivation mutant c-jun (TAM67), under the control of the human keratin-14 promoter, is expressed specifically in the basal cells of the epidermis where tumor induction is initiated. The keratin-14-TAM67 transgene was expressed in the epidermis, tongue, and cervix, with no apparent abnormalities in any tissue or organ. TAM67 expression blocked 12-O-tetradecanoylphorbol 13-acetate (TPA, phorbol 12-tetradecanoate 13-acetate) induction of the AP-1-regulated luciferase in AP-1 luciferase/TAM67 mice, but did not inhibit induction of candidate AP-1 target genes, collagenase-1 or stromelysin-3. More interestingly, TAM67 expression did not inhibit TPA-induced hyperproliferation. In two-stage skin carcinogenesis experiments, the transgenic animals showed a dramatic inhibition of papilloma induction. We conclude that transactivation of a subset of AP-1-dependent genes is required for tumor promotion and may be targeted for cancer prevention.

A dominant-negative mutant of c-Jun inhibits cell cycle progression during the transition of CD4(-)CD8(-) to CD4(+)CD8(+) thymocytes.

While Jun/Fos-containing transcription factors are known to be necessary for many TCR-mediated events in mature T cells, relatively little is known about their roles in thymocyte development. We have generated transgenic mice that express a trans-dominant-negative mutant of c-Jun (TAM-67) specifically in thymocytes. Expression of TAM-67 inhibited the up-regulation of AP-1-responsive genes such as c-jun and IL-2 in stimulated thymocytes from transgenic mice. In addition, altered thymocyte development in TAM-67-expressing mice was revealed by a decrease in thymic cellularity ( approximately 50%) which could be accounted for primarily by a reduction in the number of CD4(+)CD8(+) thymocytes, a large percentage of which retained CD25. The decrease in the number of CD4(+)CD8(+) thymocytes did not appear to be due to an enhanced rate of apoptosis but rather to a decrease in the number of CD4(-)CD8(-)CD25(-) cells in the S + G(2)/M stages of the cell cycle. These results indicate that Jun/Fos-containing transcription factors promote the proliferative burst that accompanies the transition from the CD4(-)CD8(-) to the CD4(+)CD8(+) stage of thymocyte development.

[Chronic hepatitis B and C in HIV-infected patients]. / Chronische Hepatitis B und C bei HIV-infizierten Patienten.

BACKGROUND AND AIM: This retrospective study examined the prevalence of co-infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) and the frequency of chronic hepatitis in HIV-infected patients with respect to both the different risk groups and the serological results. PATIENTS AND METHODS: All Zurich participants of the Swiss HIV Cohort Study were evaluated who had available results of hepatitis B and C serology and ALT. RESULTS: Of the total 279 patients, 52% belonged to the intravenous drug user, 34% to the homosexual, and 11% to the heterosexual risk category. Serologically, previously acquired infection with HBV alone could be demonstrated in 92 (33%), HCV alone in 9 (3%), and both HBV and HCV in 130 (47%) patients. Only 3% of patients with sexually acquired HIV infection had anti-HCV antibodies, whereas co-infection with HBV and HCV was present in 87% of intravenous drug users. Among the 222 patients with previous HBV contact, 25 (11%) had positive HBsAg and 91 (41%) had "anti-HBc alone", both assumed to represent active HBV infection. 66 (24%) of 279 patients had chronic hepatitis with ALT elevation lasting > or = 6 months. Chronic hepatitis was present in 46% of those with active HBV and HCV co-infection, in 36% of those with HCV infection alone and in 18% of those with active HBV infection alone (P < 0.001). Of the 66 cases of chronic hepatitis, 58 were associated with HCV infection, and only 2 cases had no serological signs of active HBV or HCV infection. CONCLUSION: In patients with sexually acquired HIV infection, HBV had frequently been co-transmitted. In contrast, almost all of those infected by means of intravenous drug use had a co-infection with both HBV and HCV. The latter seems to play the strongest role in the development of chronic hepatitis with persistent ALT elevation. A chronic ALT elevation was almost always associated with serologically active HBV or HCV infection.

Split tolerance to the MHC class I molecule H-2Dd in animals transgenic for its soluble analog.

To determine whether the function of MHC molecules in tolerance and education is related to cell surface expression, we have produced two strains of transgenic mice in the C57Bl/6 background that express soluble analogs of the H-2D(d) class I protein. The transgenes were stably integrated and genetically transmitted in a Mendelian fashion. Messenger RNA for the hybrid genes was detected in all tissues analyzed in a class I-like pattern of expression, with the highest levels in lymphoid tissues. All mice bearing the transgenes expressed relatively high levels (0.1 mg/ml) of the encoded protein in their serum as assessed by Western blotting and enzyme-linked immunosorbent assay (ELISA). Gel filtration chromatography showed that the soluble H-2D(d) protein exists as a heterodimer with beta2-microglobulin and as higher order multimers in serum. Lymphoid cells from the transgenic mice showed no cell surface expression of the soluble class I protein in indirect immunofluorescence assays. Splenocytes from two independently derived transgenic lines generated primary cytotoxic and proliferative responses directed against membrane H-2D(d) antigens. Mice of both strains rejected tail skin from donors that differed from the B6 background at the H-2D(d) locus only, but with delayed kinetics compared to nontransgenic littermate controls. Mice expressing the transgenic protein on immunization did not produce antibodies that recognized soluble H-2D(d) in ELISA, whereas B6 mice generated strong antibody responses to challenge with splenocytes bearing cell surface H-2D(d). Thus, transgenic mice expressing soluble H-2D(d) were partially tolerant to stimulation by membrane-bound H-2D(d). As with the activation of T-cells, the induction and maintenance of immunologic tolerance apparently displayed different requirements depending upon the T-cell subpopulation involved.

Increased interleukin 4 and immunoglobulin E production in transgenic mice overexpressing NK1 T cells.

Natural Killer (NK)1.1+ (NK1) T cells are a specialized subset of alpha/beta T cells that coexpress surface receptors that are normally associated with the NK cell lineage of the innate immune system. On recognition of the conserved, major histocompatibility complex class I-like CD1 molecule, these cells are able to release explosive bursts of interleukin 4 (IL-4), a cytokine that promotes the T helper type 2 (Th2) effector class of an immune response. A unique feature of their T cell receptor (TCR) repertoire is the expression of an invariant TCR alpha chain, V alpha 14-J alpha 281, and of a restricted but polyclonal set of V beta gene families, V beta 8, V beta 7, and V beta 2. Here, we show that transgenic expression of this TCR alpha chain during thymic development is sufficient information to bias the differentiation of mainstream thymocytes towards the NK1 developmental pathway. It markedly increases the frequency of cells with the NK1 pattern of T cell differentiation and also has drastic consequences for the selection of the V beta repertoire. Transgenic CD4 cells exhibited a 10-100-fold increase in IL-4 production on mitogen stimulation in vitro and in vivo, and baseline levels of the Th2-controlled serum immunoglobulin isotypes, IgE and IgG1, were also selectively elevated in vivo.

A targeted glucocorticoid receptor antisense transgene increases thymocyte apoptosis and alters thymocyte development.

The exquisite sensitivity of thymocytes to steroid-induced apoptosis, the steroidogenic potential of thymic epithelial cells, and the ability of steroid synthesis inhibitors to enhance antigen-specific deletion of thymocytes in fetal thymic organ cultures suggest a role for glucocorticoids in thymocyte development. To address this further, transgenic mice that express antisense transcripts to the glucocorticoid receptor (GR) specifically in immature thymocytes were generated. The consequent hyporesponsiveness of thymocytes to glucocorticoids was accompanied by a reduction in thymic size, primarily owing to a decrease in the number of CD4+CD8+ cells. While an enhanced susceptibility to T cell receptor (TCR)-mediated apoptosis appeared to be partially responsible for this reduction, thymocyte loss could also be detected before thymocytes progressed to the CD4+CD8+ TCR alpha beta-expressing stage. These results suggest that glucocorticoids are necessary for survival and maturation of thymocytes, and are consistent with a role for steroids in both the transition from CD4-CD8- to CD4+CD8+ cells and the survival of CD4+CD8+ cells stimulated via the TCR.

A family of murine NK cell receptors specific for target cell MHC class I molecules.

The Ly-49A molecule is an NK cell receptor specific for MHC class I molecules on target cells. When Ly-49A engages H-2Dd, Ly-49A+ NK cells become globally incapable of killing their targets in vitro. This interaction also occurs in vivo. Ly-49A belongs to a family of highly related molecules, including Ly-49C (5E6 antigen) and LGL-1 that also determine NK cell specificity. In the NK gene complex, the Ly-49 family is genetically linked to genes encoding NKR-P1 and CD69 that are structurally related and capable of activating NK cells. Finally, Ly-49 may be related to human molecules that are selectively expressed on NK cells and influence NK cell specificity. These findings highlight the emerging significance of the Ly-49 family in NK cell activity.

Abnormal B-cell function in HTLV-I-tax transgenic mice.

Transgenic mice that carry the HTLV-I Tax gene develop an exocrinopathy with some similarities to Sjoegren's syndrome. Our experiments reveal that these mice have lymphadenopathy and splenomegaly composed primarily of B lymphocytes, as well as abnormal levels of secreted immunoglobulins. To gain insight into whether the lymphadenopathy manifested by these transgenic mice was the result of induction of cytokines by Tax, we utilized cell lines from these mice to study in vitro B-cell responses. Conditioned media (CM) derived from the cell lines caused B-cells to proliferate when a second signal, surface Ig cross-linking, was provided. The CM also caused a marked enhancement of IgM secretion by spleen cells or by purified B-cells treated with supplemental cytokines. The B-cell proliferative response and enhanced IgM secretion have not been attributed to a known cytokine. These results suggest that the CM from the cell lines contain a factor(s) involved in novel pathways of B-cell growth and differentiation that may participate in the pathologic development of autoimmune disease.

Host MHC class I molecules modulate in vivo expression of a NK cell receptor.

Target cell expression of certain MHC class I molecules correlates with resistance to lysis by NK cells. To explain this correlation, one hypothesis states that NK cells may possess two types of receptors; one may activate NK cells whereas another, specific for target cell MHC class I molecules, may inhibit natural killing by transducing negative signals. The cell surface molecule, Ly-49, is expressed on an NK cell subpopulation (15% to 20%) in spleens from C57BL/6 (H-2b) mice. Previously, we showed that lysis by Ly-49+ IL-2-activated NK cells was globally inhibited when targets expressed either H-2Dd or an H-2k-class I molecule, consistent with the hypothesis that Ly-49 is an inhibitory NK cell receptor that engages these MHC class I molecules. We now have determined the influence of specific host MHC class I molecules on Ly-49 expression. In two-color flow cytometric examination of splenic cells, Ly-49+ NK1.1+ cells were undetectable in MHC-congenic strains expressing Dd or Dk, in C57BL/6 mice transgenic for membrane-bound Dd, and in B10.D2dm1 mice. These data establish that Dd itself is sufficient for this effect and suggest that Ly-49 engages Dd-alpha 1/alpha 2 domains. Cross-linking of Ly-49 with membrane-bound Dd may be required because Ly-49+ NK1.1+ cells were readily detectable in C57BL/6 strains transgenic for soluble forms of Dd. To examine whether this effect could be the result of down-regulation of Ly-49 expression or negative selection of Ly-49+ cells, we determined Ly-49 expression on highly purified, freshly isolated NK cell populations (> 90% NK1.1+ CD3- cells). These experiments demonstrated that Ly-49+ cells were present in normal numbers but that Ly-49 expression was markedly decreased in congenic mice expressing H-2Dd or Dk, and in the strain transgenic for membrane-bound H-2Dd. Thus, expression of a putative MHC class I-specific NK cell receptor is modulated by its apparent interaction with alpha 1/alpha 2 domains of host MHC class I molecules.