Clinical and molecular detection of inherited colorectal cancers in northeast Italy: a first prospective study of incidence of Lynch syndrome and MUTYH-related colorectal cancer in Italy.

The reported incidence of hereditary colorectal cancers (CRCs) is widely variable. The principal aim of the study was to prospectively evaluate the incidence of familial CRCs in a region of northern Italy using a standardized method. Consecutive CRC patients were prospectively enrolled from October 2002 to December 2003. Patients underwent a structured family history, the microsatellite instability (MSI) test and a screen for MUTYH mutations. Following family history patients were classified as belonging to high, moderate and mild risk families. Immunohistochemistry for MLH1, MSH2, MSH6 and PMS2 proteins and investigation for MLH1/MSH2 mutations, for MLH1 promoter methylation and for the V600E hotspot BRAF mutation were performed in high MSI (MSI-H) cases. Of the 430 patients enrolled, 17 (4%) were high risk [4 hereditary non-polyposis colorectal cancer (HNPCC), 12 suspected HNPCC and 1 MUTYH-associated adenomatous polyposis coli (MAP)], 53 moderate risk and 360 mild risk cases. The MSI test was performed on 393 tumours, and 46 (12%) of them showed MSI-H. In these patients, one MLH1 pathogenetic mutations and two MSH2 pathogenetic mutations were found. Thirty-two (70%) MSI-H cases demonstrated MLH1 methylation and/or BRAF mutation: None of them showed MLH1/MSH2 mutation. Two biallelic germline MUTYH mutations were found, one with clinical features of MAP. A strong family history of CRC was present in 4% of the enrolled cases; incidence of MLH1/MSH2 or MUTHY mutations was 1.3% and of MSI-H phenotype was 12%. MLH1 methylation and BRAF mutation can exclude 70% of MSI-H cases from gene sequencing.

Soft tissue sarcoma and the hereditary non-polyposis colorectal cancer (HNPCC) syndrome: formulation of an hypothesis.

Hereditary non-polyposis colorectal cancer (HNPCC) is a genetic disorder caused by mutation in one of the mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2) which predisposes to colorectal cancer and other malignances, that not yet include sarcomas. For sustaining that soft tissue sarcomas could be HNPCC related malignances, we report on a HNPCC patient with leiomyosarcoma and review the English literature. Overall, we report on eleven cases of soft tissue malignant tumors involving HNPCC patients, with a mean age of 34 years at diagnosis of sarcomas. In the majority of these tumors loss of MSH2 expression can be found at immunohistochemistry (IHC) and in 10 patients a germline mutation in one of the MMR genes was found (7 cases were MSH2 defective and 3 cases MLH1 defective). Data for supporting our hypothesis are also experimental, epidemiologic, histopathological: excess of sarcomas in PMS2 defective mice; sporadic soft tissue sarcomas are rare, with mean age at onset of 56 years and normal IHC for MMR proteins. In conclusion, the data collected support the hypothesis that soft tissue sarcomas could be included in the spectrum of tumors that, even if rarely, depend on MMR genes deficiency.

T cell mapping of one epitope from thyroglobulin inducing experimental autoimmune thyroiditis (EAT).

These data collect the advance made in the last few years in our laboratory in defining one epitope from the thyroglobulin (Tg) molecule (660 KDa) inducing Experimental Autoimmune thyroiditis (EAT) in CBA/J mice. We achieved the characterization of one EAT-inducer Tg peptide by combining "in vitro" biochemical and immunological approaches and "in vivo" studies. Since T cells recognize degraded forms of the antigen and since endogenous antigens preferentially activate class I-restricted T cells, we hypothesized that one cytotoxic T cell hybridoma, named HTC2, which prevents further EAT induction in mice injected with Tg would be specific for one EAT inducer peptide. In order to identify one Tg epitope inducing EAT, enzymatic treatment of the protein by trypsin, HPLC purification and sequence analysis were performed. Simultaneously, tryptic digests were used to pulse CBA/J macrophages and tested for their ability to be recognized by HTC2 cells. Lastly, when digests were recognized by HTC2 cells their capacity to induce EAT in CBA/J mice was evaluated. To further assess the pathogenicity of the sequenced Tg peptide, one synthetic peptide was made and its capacity to induce EAT verified. By this procedure we identified for the first time one 40 amino-acid peptide from human thyroglobulin inducing EAT in CBA/J mice.

Lutropin receptor and thyrotropin receptor share a common epitope.

In the present work, using an immunological approach, we have investigated the existence of common epitopes between two receptors of the glycoprotein hormone family, lutropin (LH) and thyrotropin (TSH) receptors. We have immunized high responder mice with purified porcine LH receptors obtained by successive affinity chromatographies on agarose-human chorionic gonadotropin (hCG) gels. From one fusion of splenocytes with the murine myeloma NSC1, secreting hybridomas were tested for their anti-LH receptor specificities. During sequential selection for this activity including direct recognition of the purified LH receptors in dot-blot assays and displacement experiments of 125I-pLH and 125I-hCG binding to different sources of receptors, we performed a parallel investigation of their anti-porcine TSH receptor activities. Purified immunoglobulins from two of them showed a TSH-like activity on the iodide metabolism of porcine thyroid cell, this activity being related to the phosphoinositide breakdown pathway; moreover, these antibodies obtained after immunization with porcine LH receptors were able to immunopurify human TSH receptors. The double selection process led us to characterize three groups of immunoglobulins: exclusive specificities for lutropin receptors or thyrotropin receptors and cross-reactive specificities. Our results demonstrate the possibility of sequence homologies at the protein and the gene levels between the receptors for the glycoprotein hormone family supporting the hypothesis of a common origin in evolution.

Trans-resveratrol, a grapevine-derived polyphenol, blocks hepatocyte growth factor-induced invasion of hepatocellular carcinoma cells.

We have shown that liver myofibroblasts stimulate in vitro invasion of hepatocellular carcinoma cell lines through a hepatocyte growth factor/urokinase-dependent mechanism. Resveratrol, a grapevine-derived polyphenol, has been shown to inhibit cellular events associated with tumor initiation, promotion and progression. The aim of this study was to evaluate the effects of trans-resveratrol on invasion of the human hepatoma cell line HepG2. Cell invasion was assessed using a Boyden chamber assay. Activation of the HGF signal transduction pathways was evaluated by Western blot with phospho-specific antibodies. Urokinase expression was measured by RT-PCR and zymography. Trans-resveratrol decreased hepatocyte growth factor-induced cell scattering and invasion. It also decreased cell proliferation without evidence for cytotoxicity or apoptosis. Trans-resveratrol did not decrease the level of the hepatocyte growth factor receptor c-met and did not impede the hepatocyte growth factor-induced increase in c-met precursor synthesis. Moreover, trans-resveratrol did not decrease hepatocyte growth factor-induced c-met autophosphorylation, or Akt-1 or extracellular-regulated kinases-1 and -2 activation. Finally, it did not decrease urokinase expression and did not block the catalytic activity of urokinase. In conclusion, our results demonstrate that trans-resveratrol decreases hepatocyte growth factor-induced HepG2 cell invasion by an as yet unidentified post-receptor mechanism.

Ig VH gene family usage in spleen cells of CBA/J mice immunized with experimental autoimmune thyroiditis (EAT) inducer antigens.

Experimental autoimmune thyroiditis (EAT) is an autoimmune disorder of the thyroid gland induced in susceptible strains of mice by thyroglobulin (Tg). We recently showed that low Mr (< 10 kDa) Tg tryptic fragments and a 40 amino-acid peptide (F40D) from Tg could induce EAT as well as native Tg. Because it has been reported that autoantibodies (A-Abs) express VH families preferentially located in the D-proximal VH gene segment, we investigated whether A-Abs specific for one pathogenic peptide from Tg were also skewed towards D proximal VH gene segment. In that respect, we immunized CBA/J mice with EAT inducer antigens of decreasing sizes: Tg (660 M(r)), < 10 kDa Tg trypic fragments or F40D peptide (4.9 kDa M(r)) from Tg. The VH gene segments utilized by immune spleen cells were determined by hybridization to total spleen cell RNA previously deposited onto nylon membranes and densitometric scans. This study was conducted on days 7 and 9 after determination of the maximum amounts of mRNA coding for immunoglobulins and on day 28 when A-Ab levels are the highest. Results were compared to VH gene segment expression both in normal and adjuvant-injected mice. We found that immunization of CBA/J mice with EAT inducer antigens stimulate B cells the restriction of which, in terms of VH family usage, depends on the size of the immunizing antigen: the larger the antigen, the higher the numbers of VH families used. Moreover, we found that B cell stimulation consecutive to immunization with the peptidic antigen inducing EAT occurs in VH Q52 family, a VH encoded by D-proximal gene segment.

Modifications of the sinusoidal barrier in a model of postsinusoidal hypertension in the rat. An ultrastructural study of endothelial cells.

Sinusoids and sinusoidal cells were examined by light and electron microscopy, using a rat model of postsinusoidal hypertension. One month after partial ligation of the vena cava (PLVC) above the hepatic veins, subcapsular hemorrhagic areas were visible with proliferation of hepatic veins; in non hemorrhagic areas, sinusoidal congestion was found. Postsinusoidal hypertension led to a significant increase in sinusoidal volume and to major abnormalities of the endothelium such as endothelial processes and pouches with numerous diaphragmed fenestrae; some red blood cells could be seen in these pouches. Endothelial cells sent out processes in between hepatocytes. Complete and incomplete pseudo-neolumens were found near sinusoids. Numerous Kupffer cells were located either in the sinusoidal barrier or infiltrating the Disse space close to extravasated red blood cells and perisinusoidal cell processes. 18 months after PLVC, lesions were much the same except for the presence of red blood cells in the Disse space.

Multivariate analysis approach to the serum peptide profile of morbidly obese patients.

BACKGROUND: Obesity is currently epidemic in many countries worldwide and is strongly related to diabetes and cardiovascular disease. Mass spectrometry, in particular matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) is currently used for detecting different pattern of expressed protein. This study investigated the differences in low molecular weight (LMW) peptide profiles between obese and normal-weight subjects in combination with multivariate statistical analysis. MATERIALS: Serum samples of 60 obese patients and 10 healthy subjects were treated by cut-off membrane (30000 Da) to remove the most abundant proteins. The filtrates containing the LMW protein/peptides were analyzed by MALDI-TOF mass spectrometry. Dataset was elaborated to align and normalize the spectra. We performed cluster analysis and principal component analysis to detect some ionic species that could characterize and classify the subject groups. RESULTS: We observed a down-expression of ionic species at m/z 655.94 and an over-expression of species at m/z 1518.78, 1536.77, 1537.78 and 1537.81 in obese patients. Furthermore we found some ionic species that can distinguish obese patients with diabetes from those with normal glucose level. CONCLUSION: Serum peptide profile of LMW associate with multivariate statistical approach was revealed as a promising tool to discriminate and characterize obese patients and it was able to stratify them in relation to comorbidity that usually are associated with this disease. Further research involving a larger sample will be required to validate these findings.

Circulating cell-free DNA: a promising marker of regional lymphonode metastasis in breast cancer patients.

PURPOSE: We undertook the current study with untreated breast cancer to (1) role the variations in the plasma levels of cfDNA and the size distribution in early stage, (2) determine the frequency in plasma of methylation of three candidate genes, RASSF1A, MAL, and SFRP1, and (3) to determine whether detection of cfDNA variations and methylation changes in plasma might have specific clinical utility. METHODS AND MATERIALS: Thirty-nine patients woman patients (median age 64 years; range, 36-90 years) who underwent surgery for primary BR and 49 healthy females' subjects (control group without any breast lesion) were evaluated. The cfDNA levels were analyzed using quantitative real-time polymerase chain reaction of ß-globin. Based on the ALU repeats, the cfDNA was considered as either total (fragments of 115 bp, ALU115) or tumoral (fragments of 247 bp, ALU247). The association between the levels of the ALU247, ALU115 repeat, and ALU 247/115and the pathologic tumor characteristics was analyzed. Used methylight qPCR method, cfDNA from plasma samples of healthy donors and patients with breast cancer were evaluated for the diagnotic value of the methylation status of three genes (RASSF1A, MAL, SFRP1) frequently methylated in breast cancer. RESULTS: The baseline levels of cfDNA were significantly higher in the patients with cancer, and the level of ALU247 was the most accurate circulating cfDNA marker in discriminating the cancer from non-cancer subjects. A high statistical significance was found by considering the T stage and patients with regional LN metastasis positive cancers showed significantly higher cfDNA level of ALU247. Moreover, patients with methylation of at least one of the gene under investigate showed a higher quantity of cfDNA ALU115 (p< 0.0001) and ALU247 level (p< 0.0001). CONCLUSIONS: We observed that necrosis could be a potential source of circulating tumour-specific cfDNA ALU247; and that cfDNA ALU247 and methylated cfDNA (RASSF1A, MAL and SFRP1) are both a phenotypic feature of tumour biology.

Prevention of experimental autoimmune thyroiditis through the anti-idiotypic network.

The central goal in the therapy of autoimmune diseases is to develop potent tools able to exert specific control of the immune response to self Ag. Anti-Id may provide such specific immunodulators because the relevance of the idiotypic network in autoimmunity is well documented. We now describe the protective immunity against experimental autoimmune thyroiditis induced exclusively by a thyroglobulin (Tg)-specific cytotoxic T cell clone and show that this down-regulation occurs through the generation of anti-Id antibodies (Ab) (Ab2Beta) which recognize the paratope of a anti-Tg mAb (Ab1) specific to the pathogenic epitope of the Tg molecule. We further analyze the various steps of the Ab responses (Ab1, Ab2, and Ab3) in terms of poly-, mono-, and autospecificities for the pathogenic epitope of the Tg molecule and for the idiotope of the related Ab.

Molecular heterogeneity of antigen- or idiotype-induced anti-thyroglobulin monoclonal autoantibodies.

To define the molecular basis of the cognitive interaction in experimental autoimmune thyroiditis (EAT), we sequenced the variable regions of monoclonal autoantibodies to thyroglobulin (Tg), specific or not for the F40D peptide, a Tg peptide capable of inducing EAT in CBA/J mice. Three MoAbs were obtained by immunization with syngeneic Tg of CBA/J (3B8G9, 2F6F2) or C57Bl/6 (4D11F4) mice. 3B8G9 was specific for F40D peptide, whereas 2F6F2 and 4D11F4 were not. Two others were raised in CBA/J mice by manipulation of idiotypic pathways: B12 resulted from the immunization with one Ab2 beta, bearing the internal image of one F40D epitope, and TA2 from the immunization with F40D-specific cytotoxic HTC2 T cells. B12 and TA2 were both specific for F40D. All hybridomas expressed different members of the J558 VH family, except 3B8G9 which expressed a Q52 VH gene segment. These data led us to hypothesize that regulatory anti-id autoantibodies used members of one VH family located in the 5'-end of the VH locus, whereas EAT-associated autoantibodies used a member of one of the most D-proximal VH family. As expected, no homologies were found when anti-F40D monoclonal autoantibodies were compared with two other monoclonal autoantibodies displaying a different epitopic specificity. Among the anti-F40D monoclonal autoantibodies, one histidine residue located in position 35 of the CDR1 region was constantly found. Moreover, TA2 and B12 exhibited two common amino acids in their CDR3 regions, one glycine and one tyrosine, in positions 98 and 99, respectively. Striking homologies were found between TA2 and one anti-polyGAT MoAb, and between 3B8G9 and some anti-phenyloxazolone (phOx) monoclonal autoantibodies. Lastly, the VK sequence from 4D11F4 was identical at the amino acid level to the VK sequence from another monoclonal autoantibody, 81B1, which was previously raised towards syngeneic Tg in CBA/J mice. Our data imply that anti-idiotypic regulatory circuits in EAT might be generated by a heterogeneous population of B cells rather than obtained by a single dominant B cell population.

Hypertrophy of biliary epithelium in rats with portacaval shunt.

Bile ducts of rats with 3-month-old portacaval shunt were shown by light microscopy to present hypertrophy of the biliary epithelium. This hypertrophy could be linked to the increased hepatic arterial flow following portacaval shunt.

Failure to induce selective cholestasis in the rat after long-term extrahepatic selective biliary obstruction.

Forty-eight hours after extra-hepatic selective biliary obstruction (SBO), there is evidence of cholestasis in the obstructed lobes (OL). However, some major ultrastructural features of cholestasis are missing. The aim of this work was to investigate the long-term effect of SBO. One month after surgery, and in comparison with sham-operated rats, bile flow, liver weight, and liver weight ratio of obstructed/nonobstructed lobes were normal. Furthermore, there was no evidence of cholestasis in OL by light and electron microscopy. Bile duct communications between obstructed and non-obstructed lobes were evidenced by Indian ink injection. In sham-operated rats, bile duct communications between ducts of the different lobes were involved in bile drainage. It appears, therefore, that the main reason for the lack of cholestasis 1 month after SBO is the drainage of bile from OL through accessory bile ducts.

Hepatocyte ultrastructure in the rat after long term portacaval anastomosis: a morphometric study.

Hepatocyte ultrastructure was studied in rats after long term portacaval anastomosis (PCS). Compared to controls rats of the same age, in one year PCS rats, the liver atrophy was 24% of the mass. Hepatocytes area was decreased (-16%) but their architecture was grossly normal. The rough endoplasmic reticulum appeared well developed. The morphometric study revealed that: the rough endoplasmic surface density was significantly increased in the two zones of the liver acinus, the smooth endoplasmic reticulum surface density was significantly decreased in zone 1 only and the peroxisomes volume density was increased 2.5 times. These findings differ from data obtained shortly after surgery and probably indicate an adaptation to important functions such as protein synthesis, drug metabolism etc. Increased number of peroxisomes could be possibly linked to the abnormal uric acid metabolism observed in rats with portacaval shunts.

Sinusoidal cells in rats with portacaval shunt: a morphometric study.

Sinusoidal cells were studied in rats 2 weeks after portacaval shunt. Livers were perfused with glutaraldehyde via the aorta. Morphometric analysis was performed in periportal and centrolobular zones of the liver acinus. Liver atrophy was the main consequence of portal blood derivation. Per unit of hepatic parenchyma the number of hepatocytes and Kuppfer cells counted on 1 micron section was comparable in portacaval shunt and sham operated rats. 2 weeks after the shunt, sinusoidal cell ultrastructure was nearly normal. Morphometric analysis showed sinusoids slightly enlarged and Kupffer cell volume density increased in both zones. Kupffer cell lysosomal-like structures such as electron-lucent vacuoles and phagolysosomes had larger volume density. Ito cells number and volume density slightly increased after portacaval shunt. These findings suggest that endotoxemia which could occur after portal blood shunting might be better related to shunting than to depression of the reticuloendothelial system.

Bile canaliculi morphology after bile duct ligation in the rat with portacaval shunt: a time course study.

Besides the spontaneous occurrence of portacaval shunts in chronic liver diseases and the performance of shunt to cure portal hypertension, shunts are now proposed for the treatment of some metabolic diseases, especially in children. The portacaval shunt model in the rat, known to induce liver atrophy related to hepatocyte loss and atrophy, was used to investigate morphological liver changes occurring in cholestasis. Two weeks after portacaval or sham portacaval shunt, the bile duct was ligated and livers were examined by light and electron microscopy at 5 h, 20 h, 4 days and 8 days. Portal inflammatory reaction and proliferation of bile ducts were identical but areas of patchy hepatic necrosis were more numerous in shunted rats. Hepatocyte size decreased in the sham group but increased in the other group. Hepatocyte ultrastructural changes were similar in the two groups. At 5 h, the number of bile canaliculi sections increased (X2) and 95% of them were normal. As time elapsed, the ratio of dilated bile canaliculi without microvilli and of bile canaliculi filled with cytoplasmic blebs increased but in no case reached 50% of the total. These results show that in the rat, cholestasis induced by bile duct ligation has approximately the same characteristics in control or shunted animals.

Autoantibody specific for a thyroglobulin epitope inducing experimental autoimmune thyroiditis or its anti-idiotype correlates with the disease.

In order to elucidate the role of anti-thyroglobulin (Tg) autoantibodies (A-Ab) in experimental autoimmune thyroiditis (EAT), a kinetic study was conducted in EAT-susceptible (CBA/J) and non-susceptible (C57BL/6) strains of mice. From day 0 to 70 post-Tg immunization, titers of A-Ab to Tg and to the linear 5-10-kDa Tg tryptic fragment inducing EAT as well as anti-idiotypic A-Ab representing the internal image of the thyroidogeneic antigen were measured. EAT onset, development and recovery correlate with the presence of two subsets of A-Ab only in susceptible strains of mice. First, with the presence of anti-Tg A-Ab to one determinant borne by the linear 5-10-kDa Tg tryptic fragment, and second with the presence of anti-idiotypic A-Ab specific for the monoclonal anti-Tg A-Ab (3B8G9) paratope which binds to Tg determinant inducing EAT.

Transformation of sinusoids into capillaries in a rat model of selenium-induced nodular regenerative hyperplasia: an immunolight and immunoelectron microscopic study.

The oral administration of selenium (Se) to young rats induces, over a 2-month period, the formation of nodular regenerative hyperplasia with sinusoidal damage around nodules. Perinodular areas located in zone 1 comprise atrophic hepatocytes and capillarized sinusoids without fibrosis. We used this unique model of capillarization without fibrosis to investigate the temporal relationship between the process of capillarization and changes occurring in the deposition of components of the extracellular matrix. After 2 weeks of intoxication, type III collagen and fibronectin were stable, but laminin and type IV collagen had increased in zone 1, resulting in the formation of septae between portal tracts. Even at 8 weeks, these two components still formed the principal deposits in perinodular zones. Electron microscopy showed already at 1 week in zone 1 that part of the endothelial wall had detached from hepatocytes. Sinusoidal endothelial cells progressively acquired certain of the characteristics of a vascular endothelium, some proliferated, and perisinusoidal cells transformed into myofibroblasts, surrounded by deposits of laminin and type IV collagen. These results indicate that both laminin and type IV collagen are involved in capillarization without fibrosis and in angiogenesis; fibronectin would not seem to play a role.

Protection from experimental autoimmune thyroiditis conferred by a monoclonal antibody to T cell receptor from a cytotoxic hybridoma specific for thyroglobulin.

A clonotypic mAb, AG7, has been prepared from splenocytes of CBA/J mice immunized with a cytotoxic T cell hybridoma, HTC2, specific for a pathogenic epitope of the thyroglobulin molecule in association with class I MHC Ag. AG7 binds to HTC2 cells but not to the other T cell hybridomas tested. Moreover, when used in functional studies, AG7 blocks the HTC2 capacity of specific target lysis. It also reacts with a determinant that comodulates with the CD3 Ag present on the surface of HTC2 cells. Immunoprecipitation of 125I-labeled solubilized HTC2 membranes demonstrated two bands located at 90 and 72 kDa under nonreducing conditions, which became a 46-kDa band under reducing conditions. Finally, when AG7 is injected into CBA/J mice, on day -1 before immunization with the pathogenic tryptic fragments of the thyroglobulin molecule, experimental autoimmune thyroiditis is abrogated. Thus, one of the multiple potential mechanisms of the protective immunity against EAT induced by HTC2 cells that we previously proposed, i.e., the generation of anti-clonotypic antibodies to HTC2 TCR, seems apparent.