O-RADS MRI score: analysis of misclassified cases in a prospective multicentric European cohort.

OBJECTIVE: To retrospectively review the causes of categorization errors using O-RADS-MRI score and to determine the presumptive causes of these misclassifications. METHODS: EURAD database was retrospectively queried to identify misclassified lesions. In this cohort, 1194 evaluable patients with 1502 pelvic masses (277 malignant / 1225 benign lesions) underwent standardized MRI to characterize adnexal masses with histology or 2 years' follow-up as a reference standard. An expert radiologist reviewed cases with two junior radiologists and lesions termed misclassified if malignant lesion was scored ≤ 3, a benign lesion was scored ≥ 4, the site of origin was incorrect, or a non-adnexal mass was incorrectly categorized as benign or malignant. RESULTS: There were 139 / 1502 (9.2%) misclassified masses in 116 women including 109 adnexal and 30 non-adnexal masses. False-negative cases corresponded to 16 borderline or invasive malignant adnexal masses rated score ≤ 3 (16 / 139, 11.5%). False-positive cases corresponded to 88 benign masses were rated score 4 (67 / 139, 48.2%) or 5 (18 / 139,12.9%) or considered suspicious non-adnexal lesions (3 / 139, 2.2%). Misclassifications were only due to origin error in 12 adnexal masses (8 benign, 4 malignant) (8.6%, 12 / 139) and 23 non-adnexal masses (18 benign, 5 malignant,16.5%, 23 / 139) perceived respectively as non-adnexal and adnexal masses. Interpretive error (n = 104), failure to recognize technical insufficient exams (n = 9), and perceptual errors (n = 4) were found. Most interpretive was due to misinterpretation of solid tissue or incorrect assignment of mass origin. Eighty-four out of 139 cases were correctly reclassified by the readers with strict adherence to the score rules. CONCLUSION: Most errors were due to misinterpretation of solid tissue or incorrect assignment of mass origin. KEY POINTS: • Prospective assignment of O-RADS-MRI score resulted in misclassification of 9.25% of sonographically indeterminate pelvic masses. • Most errors were interpretive (74.8%) due to misinterpretation of solid tissue as defined by the lexicon or incorrect assignment of mass origin. • Pelvic inflammatory disease is a common source of misclassification (8.9%) (12 / 139).

Comparison of breast density assessment between human eye and automated software on digital and synthetic mammography: Impact on breast cancer risk.

PURPOSE: To evaluate the agreement between automatic assessment software of breast density based on artificial intelligence (AI) and visual assessment by a senior and a junior radiologist, as well as the impact on the assessment of breast cancer risk (BCR) at 5 years. MATERIALS AND METHODS: We retrospectively included 311 consecutive women (mean age, 55.6±8.5 [SD]; range: 40-74 years) without a personal history of breast cancer who underwent routine mammography between January 1, 2019 and February 28, 2019. Mammographic breast density (MBD) was independently evaluated by a junior and a senior reader on digital mammography (DM) and synthetic mammography (SM) using BI-RADS (5th edition) and by an AI software. For each MBD, BCR at 5 years was estimated per woman by the AI software. Interobserver agreement for MBD between the two readers and the AI software were evaluated by quadratic κ coefficients. Reproducibility of BCR was assessed by intraclass correlation coefficient (ICC). RESULTS: Agreement for MBD assessment on DM and SM was almost perfect between senior and junior radiologists (κ=0.88 [95% CI: 0.84-0.92] and κ=0.86 [95% CI: 0.82-0.90], respectively) and substantial between the senior radiologist and AI (κ=0.79; 95% CI: 0.73-0.84). There was substantial agreement between DM and SM for the senior radiologist (κ=0.79; 95% CI: 0.74-0.84). BCR evaluation at 5 years was highly reproducible between the two radiologists on DM and SM (ICC=0.98 [95% CI: 0.97-0.98] for both), between BCR evaluation based on DM and SM evaluated by the senior (ICC=0.96; 95% CI: 0.95-0.97) or junior radiologist (ICC=0.97; 95% CI: 0.96-0.98) and between the senior radiologist and AI (ICC=0.96; 95% CI: 0.95-0.97). CONCLUSION: This preliminary study demonstrates a very good agreement for BCR evaluation based on the evaluation of MBD by a senior radiologist, junior radiologist and AI software.

Mouse testicular transcriptome after modulation of non-canonical oestrogen receptor activity.

The aims of this study were to shed light on the role of G-protein-coupled membrane oestrogen receptor (GPER) and oestrogen-related receptor (ERR) in mouse testis function at the gene expression level, as well as the involvement of GPER and ERR in cellular and molecular processes. Male mice were injected (50µg kg-1,s.c.) with the GPER antagonist G-15, the ERRα inverse agonist XCT790 or the ERRß/ERRγ agonist DY131. Next-generation sequencing (RNA-Seq) was used to evaluate gene expression. Bioinformatic analysis of read abundance revealed that 50, 86 and 171 transcripts were differentially expressed in the G-15-, XCT790- and DY131-treated groups respectively compared with the control group. Annotated genes and their protein products were categorised regarding their associated biological processes and molecular functions. In the XCT790-treated group, genes involved in immunological processes were upregulated. In the DY131-treated group, genes with increased expression were primarily engaged in protein modification (protein folding and small protein conjugation). In addition, the expression of genes recognised as oncogenes, such as BMI1 proto-oncogene, polycomb ring finger (Bmi1) and nucleophosphin 1 (Npm1), was significantly increased in all experimental groups. This study provides detailed information regarding the genetic changes in the testicular transcriptome of the mouse in response to modulation of non-canonical oestrogen receptor activity.

Towards understanding leydigioma: do G protein-coupled estrogen receptor and peroxisome proliferator-activated receptor regulate lipid metabolism and steroidogenesis in Leydig cell tumors?

Leydig cell tumors (LCT) are the most common type of testicular stromal tumor. Herein, we investigate the G protein-coupled estrogen receptor (GPER) and peroxisome proliferator-activated receptor (PPAR) implication in regulation of lipid homeostasis including the expression of steroidogenesis-controlling molecules in clinical specimens of LCTs and tumor Leydig cells (MA-10). We showed the general structure and morphology of LCTs by scanning electron and light microscopy. In LCTs, mRNA and protein analyses revealed increased expression of GPER and decreased expression of PPARα, ß, and γ. Concomitantly, changes in expression pattern of the lutropin receptor (LHR), protein kinase A (PKA), perilipin (PLIN), hormone sensitive lipase (HSL), steroidogenic acute regulatory protein (StAR), translocator protein (TSPO), HMG-CoA synthase, and reductase (HMGCS, HMGCR) were observed. Using MA-10 cells treated with GPER and PPAR antagonists (alone and in combination), we demonstrated GPER-PPAR-mediated control of estradiol secretion via GPER-PPARα and cyclic guanosine monophosphate (cGMP) concentration via GPER-PPARγ. It is assumed that GPER and PPAR can crosstalk, and this can be altered in LCT, resulting in a perturbed lipid balance and steroidogenesis. In LCTs, the phosphatidylinositol-3-kinase (PI3K)-Akt-mTOR pathway was disturbed. Thus, PI3K-Akt-mTOR with cGMP can play a role in LCT outcome and biology including lipid metabolism.

Leydig cell tumorigenesis - implication of G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and xenoestrogen exposure. In vivo and in vitro appraisal.

The etiology and molecular characteristics of Leydig cell tumor (LCT) are scarcely known. From the research data stems that estrogen can be implicated in LCT induction and development, however it is not investigated in detail. Considering the above, herein we analyzed the relation between G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and insulin-like family peptides (insulin-like 3 peptide; INSL3 and relaxin; RLN) expressions as well as estrogen level with impact of xenoestrogen (bisphenol A; BPA, tetrabromobisphenol A; TBBPA, and tetrachlorobisphenol A; TCBPA). While in our previous studies altered GPER-PPAR partnership was found in human LCT being a possible cause and/or additionally effecting on LCT development, here mouse testes with experimentally induced LCT and mouse tumor Leydig cell (MA-10) treated with BPA chemicals were examined. We revealed either diverse changes in expression or co-expression of GPER and PPAR in mouse LCT as well as in MA-10 cells after BPA analogues when compared to human LCT. Relationships between expression of INSL3, RLN, including co-expression, and estrogen level in human LCT, mouse LCT and MA-10 cells xenoestrogen-treated were found. Moreover, involvement of PI3K-Akt-mTOR pathway or only mTOR in the interactions of examined receptors and hormones was showed. Taken together, species, cell of origin, experimental system used and type of used chemical differences may result in diverse molecular characteristics of LCT. Estrogen/xenoestrogen may play a role in tumor Leydig cell proliferation and biochemical nature but this issue requires further studies. Experimentally-induced LCT in mouse testis and MA-10 cells after BPA exposure seem to be additional models for understanding some aspects of human LCT biology.

Do G-protein coupled estrogen receptor and bisphenol A analogs influence on Leydig cell epigenetic regulation in immature boar testis ex vivo?

Organotypic culture of testicular fragments from 7-day-old male pigs (Polish White Large) was used. Tissues were treated with an antagonist of G-protein coupled estrogen receptor (GPER) (G-15; 10 nM), and bisphenol A (BPA), and its analogs (TBBPA, TCBPA; 10 nM) alone or in combination and analyzed using electron and light (stainings for collagen fibers, lipid droplet and autophagy markers) microscopes. In addition, mRNA and protein abundances and localization of molecules required for miRNA biogenesis and function (Drosha, Exportin 5; EXPO5, Dicer, and Argonaute 2; AGO2) were assessed together with calcium ion (Ca2+) and estradiol concentrations. Regardless of GPER blockade and/or treatment with BPA, TBBPA and TCBPA, there were no changes in Leydig cell morphology. Also, there were no changes in lipid droplet content and distribution but there were changes in lipid and autophagy protein abundance. In the interstitial tissue, there was an increase of collagen content, especially after treatment with BPA analogs and G-15 + BPA. Independent of the treatment, there was downregulation of EXPO5 and Dicer genes but the Drosha and AGO2 genes were markedly upregulated as a result of treatment with G-15 + BPA and TCBPA, respectively. There was always a lesser abundance of EXPO5 and AGO2 proteins regardless of treatment. There was markedly greater abundances of Drosha after G-15 + BPA treatment, and this also occurred for Dicer after treatment with G-15 + TCBPA. Immunolocalization of miRNA proteins indicated there was a cytoplasmic-nuclear pattern in control and treated cells. There was an increase of Ca2+ concentrations after treatment with G-15 and BPA analogs. Estradiol secretion decreased after antagonist and chemical treatments when these were administered alone, however, there was an increase in estradiol secretion after treatment with combinations of these compounds.

Do estrogens regulate lipid status in testicular steroidogenic Leydig cell?

In this study mouse Leydig cell (MA-10) were treated with G-protein coupled membrane estrogen receptor antagonist (G-15; 10 nM). Cells were analyzed by Western blotting for expression of estrogen-related receptors (ERRα, ß and γ), steroidogenic markers (lutropin receptor; LHR and 3ß-hydroxysteroid dehydrogenase; 3ß-HSD) and lipid droplet markers (perilipin; PLIN and microtubule-associated protein 1 A/1B-light chain 3; LC3). Concomitantly, microscopic analyses by light microscope (immunofluorescent staining for lipid droplets, PLIN and LC3) as well as by electron microscope (for lipid droplet ultrastructure) were utilized. For analysis of cholesterol content, cAMP level and progesterone secretion, G-15, estrogen receptor (ER) antagonist (ICI 182,780; 10 µM), 17ß-estradiol (10 mM) and, bisphenol A (BPA; 10 nM) were used alone or in combinations. We revealed no changes in ERRs expression but alterations in ERRß and γ localization in G-15-treated cells when compared to control. Partial translocation of ERRß and γ from the cell nucleus to cytoplasm was observed. Decreased expression of LHR, 3ß-HSD, PLIN and LC3 was detected. Moreover, in treated cells large lipid droplets and differences in their distribution were found. Very strong signal of co-localization for PLIN and LC3 was found in treated cells when compared to control. In ultrastructure of treated cells, degenerating lipid droplets and double membrane indicating on presence of lipophagosome were observed. We found, that only (i) BPA and G-15 did not effect on cholesterol content, (ii) BPA, G-15 and ICI did not effect on cAMP level and (iii) BPA, ICI alone and in combination, and BPA with G-15 did not modulate progesterone secretion. These findings showed complex and diverse estrogen effects on mouse Leydig cells at various steps of steroid hormone production (cholesterol storage, release and processing). Lipid homeostasis and metabolism in these cells were affected by endogenous and exogenous estrogen, interactions of receptors (GPER, ER and ERR) and GPER and ER antagonists.

Regulation of steroidogenic function of mouse Leydig cells: G-coupled membrane estrogen receptor and peroxisome proliferator-activated receptor partnership.

We tested whether G-coupled membrane estrogen receptor (GPER) and peroxisome proliferator activated receptor (PPAR) partnership exists and whether this interaction regulates mouse Leydig cell function. Mature and aged mice were treated with the antagonist of GPER (G-15; 50 µg/kg b.w). Leydig cells (MA-10) were treated with G-15 (10 nM) alone or in combination with peroxisome proliferator-activated receptor α or γ antagonists, respectively (PPARα, 10 µM; PPARγ, 10 µM). GPER blockage affected testis steroidogenic status via changes in lutropin and cholesterol levels as well as protein expression alterations of the lutropin receptor, acute steroidogenesis activating protein, translocator protein, and protein kinase A in mouse Leydig cells both in vivo and in vitro. Inactivation of both GPER and PPAR in vitro revealed expressional modulation of other steroidogenesis-controlling molecules acting on various steps of lipid homeostasis e.g. cytochrome P450scc, perilipin, hormone sensitive lipase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase. Concomitantly, microscopic analysis of cells treated with antagonists showed changes in morphology, migration competences and cytoskeleton structure. In the above processes, the action of GPER and PPARα was regulated through the PI3K/Akt pathway, while PPARγ was mediated by the Ras/Raf pathway. In addition, GPER and PPARs specifically controlled individual signaling proteins. For the first time, we report here the importance of GPER-PPARα and -PPARγ 'neopartnership' in maintenance of Leydig cell morpho-functional status.

The role of G-protein-coupled membrane estrogen receptor in mouse Leydig cell function-in vivo and in vitro evaluation.

In this study, G-coupled estrogen receptor (GPER) was inactivated, by treatment with antagonist (G-15), in testes of C57BL/6 mice: immature (3 weeks old), mature (3 months old) and aged (1.5 years old) (50 µg/kg bw), as well as MA-10 mouse Leydig cells (10 nM/24 h) alone or in combination with 17ß-estradiol or antiestrogen (ICI 182,780). In G-15-treated mice, overgrowth of interstitial tissue was found in both mature and aged testes. Depending on age, differences in structure and distribution of various Leydig cell organelles were observed. Concomitantly, modulation of activity of the mitochondria and tubulin microfibers was revealed. Diverse and complex GPER regulation at the mRNA level and protein of estrogen signaling molecules (estrogen receptor α and ß; ERα, ERß and cytochrome P450 aromatase; P450arom) in G-15 Leydig cells was found in relation to age and the experimental system utilized (in vivo and in vitro). Changes in expression patterns of ERs and P450arom, as well as steroid secretion, reflected Leydig cell heterogeneity to estrogen regulation throughout male life including cell physiological status.We show, for the first time, GPER with ERs and P450arom work in tandem to maintain Leydig cell architecture and supervise its steroidogenic function by estrogen during male life. Full set of estrogen signaling molecules, with involvement of GPER, is crucial for proper Leydig cell function where each molecule acts in a specific and/or complementary manner. Further understanding of the mechanisms by which GPER controls Leydig cells with special regard to male age, cell of origin and experimental system used is critical for predicting and preventing testis steroidogenic disorders based on perturbations in estrogen signaling.

Interplay between estrogen-related receptors and steroidogenesis-controlling molecules in adrenals. In vivo and in vitro study.

Estrogen-related receptors (ERRs) α, ß and γ appear to be novel molecules implicated in estrogen signaling. We blocked and activated ERRs in mouse (C57BL/6) adrenals and adrenocortical cells (H295R) using pharmacological agents XCT 790 (ERRα antagonist) and DY131 (ERRß/γ agonist), respectively. Mice were injected with XCT 790 or DY131 (5 µg/kg bw) while cells were exposed to XCT 790 or DY131 (0.5 µg/L). Irrespectively of the agent used, changes in adrenocortical cell morphology along with changes in lutropin, cholesterol levels and estrogen production were found. Diverse and complex ERRs regulation of multilevel-acting steroidogenic proteins (perilipin; PLIN, cytochrome P450 side-chain cleavage; P450scc, translocator protein; TSPO, steroidogenic acute regulatory protein; StAR, hormone sensitive lipase; HSL and HMG-CoA reductase; HMGCR) was revealed. Blockage of ERRα decreased P450scc, StAR and TSPO expressions. Activation of ERRß/γ increased P450scc, StAR and HMGCR while decreased HSL expressions. PLIN expression increased either after XCT 790 or DY131 treatment. Additionally, treatment with both XCT 790 or DY131 decreased activity of Ras/Raf, Erk and Akt indicating their involvement in control of morphology and steroidogenic function of cortex cells. ERRs are important in maintaining morpho-function of cortex cells through action in specific, opposite, or common manner on steroidogenic molecules.

Does signaling of estrogen-related receptors affect structure and function of bank vole Leydig cells?

To get a deeper insight into the function of estrogen-related receptors (ERRs) and dissect underlying mechanism in Leydig cells, ERRs (type α, ß and γ) were blocked or activated in testes of adult bank voles (Myodes glareolus) which show seasonal changes in the intratesticular sex hormones level. Both actively reproducing animals (long day conditions; LD) and those with regression of the reproductive system (short day conditions; SD) received intraperitoneal injections of selective ERRα antagonist 3-[4-(2,4-Bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide (XCT 790) or selective ERRß/ERRγ agonist N-(4-(Diethylaminobenzylidenyl)-N'-(4-hydroxybenzoyl)-hydrazine (DY131) (50 µ/kg bw; six doses every other day). Markedly more, XCT 790 (P < 0.05) but also DY131 affected interstitial tissue histology whose volume increased in both LD and SD males while seminiferous epithelium structure was untouched. Ultrastructure analysis revealed alterations in mitochondria number as well as endoplasmic reticulum and Golgi complexes volume and structure especially after ERRα blockage. Diverse and complex ERRs regulation at mRNA level and protein expression (P < 0.05; P < 0.01 and P < 0.001) of steroidogenic (lutropin receptor (LHR), translocator protein (TSPO), steroidogenic acute regulatory protein (StAR)) and secretory (insulin-like protein 3 (INSL3) and relaxin (RLN)) molecules were revealed in relations to endogenous estrogen level in treated males. Notably, immunolocalization of ERRs and above proteins, exclusively in Leydig cells, indicated their involvement in Leydig cell function control based on interactions with endogenous estrogen level and/or estrogen signaling via ERRs. Treatment with XCT 790 or DY131 significantly decreased (P < 0.05; P < 0.01 and P < 0.001) intratesticular estrogens concentration, with exception in SD DY131 males. In addition, androgens level was decreased, but not in LD DY131 voles. Similarly, ERRßγ activation significantly reduced (P < 0.05; P < 0.01 and P < 0.001) cAMP and calcium ions (Ca2+) concentrations particularly in DY131 voles. Overall, for the first time, we have shown that ERRs are involved in maintenance of Leydig cell architecture and supervision of its steroidogenic and secretory activity that is closely related to endogenous estrogen status in the testis. Further understanding of mechanism(s) by which individual types of ERRs can control Leydig cell function is relevant for predicting and preventing steroidogenic and spermatogenic disorders.

Estimating the contribution of occupational solar ultraviolet exposure to skin cancer.

BACKGROUND: Exposure to solar ultraviolet (UV) radiation is the main causative factor for skin cancer. Outdoor workers are at particular risk because they spend long working hours outside, may have little shade available and are bound to take their lunch at their workplace. Despite epidemiological evidence of a doubling in risk of squamous cell carcinoma (SCC) in outdoor workers, the recognition of skin cancer as an occupational disease remains scarce. OBJECTIVES: To assess occupational solar UV doses and their contribution to skin cancer risk. METHODS: A numerical model (SimUVEx) was used to assess occupational and lunch break UV exposure, and to characterize exposure patterns and anatomical distribution. Risk of SCC was estimated from an existing epidemiological model. RESULTS: Horizontal body locations received 2.0-2.5 times more UV than vertical locations. The dose associated with having lunch outdoors every day was similar to that from doing outdoor work 1 day per week, but only half that of a seasonal worker. Outdoor work is associated with an increased risk of SCC and also with frequent acute episodes. CONCLUSIONS: Occupational solar exposure contributes greatly to overall lifetime UV dose, resulting in an excess risk of SCC. The magnitude of the estimated excess in risk supports the recognition of SCC as an occupational disease.

Anatomical exposure patterns of skin to sunlight: relative contributions of direct, diffuse and reflected ultraviolet radiation.

BACKGROUND: The dose-response between ultraviolet (UV) exposure patterns and skin cancer occurrence is not fully understood. Sun-protection messages often focus on acute exposure, implicitly assuming that direct UV radiation is the key contributor to the overall UV exposure. However, little is known about the relative contribution of the direct, diffuse and reflected radiation components. OBJECTIVE: To investigate solar UV exposure patterns at different body sites with respect to the relative contribution of the direct, diffuse and reflected radiation. METHODS: A three-dimensional numerical model was used to assess exposure doses for various body parts and exposure scenarios of a standing individual (static and dynamic postures). The model was fed with erythemally weighted ground irradiance data for the year 2009 in Payerne, Switzerland. A year-round daily exposure (08:00-17:00 h) without protection was assumed. RESULTS: For most anatomical sites, mean daily doses were high (typically 6.2-14.6 standard erythemal doses) and exceeded the recommended exposure values. Direct exposure was important during specific periods (e.g. midday during summer), but contributed moderately to the annual dose, ranging from 15% to 24% for vertical and horizontal body parts, respectively. Diffuse irradiation explained about 80% of the cumulative annual exposure dose. Acute diffuse exposures were also observed during cloudy summer days. CONCLUSIONS: The importance of diffuse UV radiation should not be underestimated when advocating preventive measures. Messages focused on avoiding acute direct exposures may be of limited efficiency to prevent skin cancers associated with chronic exposure.

Role of tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103:H2.

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.

Structure/antigenicity relationship of cyclic and linear peptides mimicking the V3 loop of HIV2 envelope glycoprotein.

We report the structure and antigenicity of the third variable region (V3) of the HIV2 envelope glycoprotein by the use of linear and cyclic peptides. To this end, a peptide mimicking this region was synthesized and purified, both as an iodoacetamidated linear peptide and a disulphide-bridged cyclic peptide. The cross-reactivity of three monoclonal antibodies (mAbs) produced against the envelope glycoprotein gp140 with the linear and cyclic peptides was tested with ELISA. The results showed that the cyclic peptide is a better ligand for the 3 mAbs 125-F, 125-J and 125-K. The avidity of the mAb/peptide interaction was further analysed by determining the concentration of linear or cyclic peptide leading to 50% inhibition of mAb-peptide complex formation (K0.5). The K0.5 value of mAb 125-F, which displayed the best reactivity with gp140, was estimated to be 5 times higher for the linear (K0.5 = 1.5 x 10(-6) M) than for the cyclic peptide (K0.5 = 3 x 10(-7) M). This indicates a higher affinity of mAb 125-F for the cyclic peptide. mAb 125-J, which exhibited a lower avidity for the gp140 compared to mAb 125-F, had a similar affinity for the cyclic and the linear peptides (K0.5 = 3 x 10(-7) M). mAb 125-K had the lowest reactivity with gp140 and its binding to adsorbed peptide could not be inhibited by the soluble linear or cyclic peptide used up to 10(-5) M. These results suggest that cyclic peptides may have a higher propensity for adopting a native-like structure for the peptide/antibody interaction. Nuclear magnetic resonance experiments at 25 degrees C in phosphate buffer pH 5.4, however, showed that neither peptide displayed a well-defined structure.

Enteropathogenic Escherichia coli O103 from rabbit elicits actin stress fibers and focal adhesions in HeLa epithelial cells, cytopathic effects that are linked to an analog of the locus of enterocyte effacement.

Escherichia coli O103, a major agent of weaned-rabbit diarrhea in Western Europe, was previously shown to produce diarrhea and attaching-and-effacing intestinal lesions in experimentally infected rabbits and to possess a homolog of the eaeA gene of enteropathogenic E. coli (EPEC). In the present study, we have shown that although negative in the fluorescent-actin staining test on HeLa cells, prototype rabbit E. coli O103 strain B10 was able to induce an original cytopathic effect (CPE) in the same interaction model. This CPE was characterized by a generalized reorganization of the actin cytoskeleton and the formation of focal adhesions on the entire surface of the target cells. These effects amplified with time, leading to cell death about 5 days after the interaction. They were produced by all rabbit E. coli O103 strains tested, by rabbit-infecting E. coli RDEC-1, and also by two human EPEC isolates. We localized genes associated with CPE by using TnphoA insertion mutagenesis in strain B10. In all five independent CPE-negative mutants that we were able to generate, the insertion was located in a region of the genome homologous to the 35-kb locus of enterocyte effacement (LEE locus) of EPEC E2348/69. The mutants concurrently lost the ability to secrete four major supernatant proteins of 25, 37, 39, and 40 kDa, which were shown by immunoprecipitation to share antigenic determinants with secreted proteins of human EPEC E2348/69. The virulence of one of these mutants (strain B10/CA1) was compared with that of the parental strain in the weaned-rabbit diarrhea model. The mutant was totally deprived of virulence, although it colonized the intestine as efficiently as the parental strain did. This study points to a new pathogenic trait of EPEC strains, which is associated with the LEE locus and, possibly, with in vivo virulence.

Characterization of a myxoma virus-encoded serpin-like protein with activity against interleukin-1 beta-converting enzyme.

A genomic library of myxoma virus (MV) DNA, a leporipoxvirus that causes myxomatosis, was constructed and screened by in vitro transcription-translation. A clone was selected on the basis of its strong reactivity with MV antiserum. Analysis of the corresponding DNA sequence and the deduced amino acid sequence revealed an open reading frame coding for a 34-kDa protein with strong homologies to members of the serpin superfamily. The gene encoding this new protein, called serp2, was localized on the MV genome. Interestingly, this gene is deleted in an attenuated strain. We constructed a baculovirus vector to produce recombinant Serp2 protein and raised specific antisera that allowed the characterization of Serp2 expression during the MV cycle. The biological relevance of this new serpin from MV was monitored, and it was shown that Serp2 could inhibit human interleukin-1 beta-converting enzyme activity.

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.

Gene technology-based antimetabolite design: the use of an in vitro protein expression system to facilitate antimetabolite design for virally-induced human diseases and malignant conditions.

A precondition for the chemotherapeutic treatment of a variety of virally-induced human diseases and malignant conditions is a highly selective interaction of the drug molecule to be used with it's biological target. To ensure the development of novel, effective drugs, it is essential that the biological target is well characterised with regard to it's structure and activity. Such characterisation relies upon adequate amounts of pure target being available. One of the most important enzymatic importers for antimetabolites is the enzyme thymidine kinase. In this article an in vitro protein expression system is described which facilitates the production of milligram amounts of pure and biologically active thymidine kinase, from a number of important biological sources. Results have shown that the in vitro produced enzyme has the exact biochemical propeties of the in vivo enzyme. Thus the in vitro protein expression system is an ideal vechicle to facilitate an in depth investigation of the enzyme's biological properties.