Differential expression of IGF-binding protein-3 in normal and malignant colon and its influence on apoptosis.

IGF-binding protein-3 (IGFBP-3) has been reported to exert a protective influence on the pathogenesis of colorectal cancer. This may reflect its modulation of IGF-I bioactivity as well as IGF-I-independent effects on cell proliferation and apoptosis. Although local expression of IGF-I in the colon is increasingly recognised as having important regulatory consequences, the role of locally expressed IGFBP-3 remains unknown. The aims of the present study were: (i) to quantify and localise the expression of IGFBP-3 in human normal and malignant colon; (ii) to relate this expression to that of other components of the IGF-I axis; and (iii) to investigate the effects of IGFBP-3 on colonic epithelial cell proliferation and apoptosis. RNA was extracted from 46 paired samples of normal and malignant colonic tissue. IGFBP-3, IGF-I, IGF-I receptor and GH receptor mRNA levels were quantified using real-time RT-PCR. Laser-capture microdissection of the same samples was used to isolate mRNA from epithelium and stromal components and localise mRNA expression. Expression was confirmed at a protein level by immunohistochemistry. Human colorectal cancer HT-29 and CaCo-2 cells were cultured with IGFBP-3 (200 ng/ml), +/- IGF-I (20 ng/ml), +/- sodium butyrate (5 mM). Cell number was assessed by an MTS assay (a modification of the MTT assay), and apoptosis assessed by cell morphology and FACS analysis using both annexin and propidium iodide staining. UO146, a MAP kinase inhibitor, and wortmannin, an inhibitor of the phosphatidylinositol 3-kinase (PI-3K) pathway, were used to determine the contribution of these signalling pathways on the effects of IGFBP-3. IGFBP-3 mRNA was detected in all samples (mean copy number/mug total RNA in normal colon, 2.6 x 10(6) compared with 1.3 x 10(7) in the cancers; P < 0.0001). Immunohistochemistry confirmed the expression and showed it to be equally distributed between epithelial and stromal components in normal tissue, but to be mainly restricted to the stromal component of malignant tissue. This differential expression was confirmed by RT-PCR of RNA from laser-capture microdissected samples. IGF-I mRNA was detected in 31 samples of normal colon; mean IGFBP-3 copy number was higher in the IGF-I-positive samples compared with IGF-I-negative samples. IGFBP-3 on its own induced apoptosis in HT-29 cells (P < 0.001). Co-incubation of 200 ng/ml IGFBP-3 with butyrate (5 mM) resulted in the potentiation of its apoptosis (P < 0.0001), which was not rescued by co-incubation with IGF-I (P < 0.0001). The addition of UO126 caused a decrease in cell number and increased the effects of IGFBP-3. IGFBP-3 is differentially expressed between stromal and epithelial components of normal and malignant colon, which may reflect its pro-apoptotic, IGF-I-independent effect on colonic epithelial cells. These effects are mediated in part by the PI-3K pathway in contrast to the MAP kinase pathway used by IGF-I.

The expression and function of IGFBP-3 in normal and malignant breast tissue.

The mitogenic and anti-apoptotic effects of insulin-like growth factor-I (IGF-I) are regulated by a family of insulin-like growth factor binding proteins (IGFBPs), particularly IGFBP-3. Little is known about the IGF-independent role of IGFBP-3 in breast cancer and the mechanisms regulating its production. The expression of IGFBP-3 in paired malignant and adjacent normal (n=53), and healthy normal (n=17) breast tissue samples was investigated using RT-PCR, immunohistochemistry and ELISA. We compared IGFBP-3 expression with other members of the IGF-I axis, other known tumorigenic genes and clinicopathological parameters. We also developed a novel tissue explant system using fresh normal and malignant breast tissue, with which we examined the in vitro effects of IGFBP-3 alone and in combination with known apoptotic agent, doxorubicin (n=6), on tissue viability and apoptosis. We demonstrated a high level of expression of IGFBP-3 mRNA in all samples. 96% of samples also expressed IGFBP-3 protein. No significant correlation was seen between IGFBP-3 expression and other clinicopathological parameters. The in vitro tissue explant system demonstrated that IGFBP-3 had little effect by itself on apoptosis. However, when used in combination with doxorubicin, increased apoptosis was seen in tumours. In contrast, less apoptosis was seen in normal tissue suggesting a protective effect. These divergent effects suggest a potential novel chemotherapeutic approach in the treatment of breast cancer. These findings suggest that IGFBP-3 may play a role in tumorigenesis, and that IGFBP-3 levels could be used in the future in cancer risk assessment/prevention or as markers of response to cancer treatments.

[Efficacy of the vaccination of C57BL/6 mice against infection with different species of Leishmania]. / Efficacité de la vaccination de la Souris C 57 BL/6 contre l'infection par différentes espèces de Leishmania.

Antigens were isolated from lysates of promastigotes of Leishmania infantum by electro-elution from polyacrylamide gels. Antigens with respective molecular weights for F2 = 94-67 kd; F5 = 30-20 kd and F6 below 20 kd, were injected intravenously in C 57 BL/6 mice. The immune sera were studied by indirect immunofluorescence; an in vivo test showed their inhibitory effect on the life cycle of several Leishmania species from the Old and the New world. Furthermore, mice immunized with F2, F5 or F6 were protected against an infection by Leishmania major. These results demonstrate that vaccination is efficient in mice differing genetically for either susceptibility (BALB/c) or partial resistance (C 57 BL/6) to Leishmania infections. Recently, we reported that a single monoclonal antibody raised against Leishmania infantum can prevent the development of Leishmania major and Leishmania mexicana amazonensis. Indeed, BALB/c mice injected subcutaneously with promastigotes pre-treated with this monoclonal antibody, did not present any cutaneous lesions over a period of 3 months. Using a mouse in vivo model--intraperitoneal injection of TG 180 mouse sarcoma cells along with monoclonal antibody pre-treated promastigotes--such an antibody-mediated inhibition was also observed against Old and New World Leishmania species. Protective monoclonal antibodies recognized by immunoblotting technique three antigenic fractions (40, 70 and 113 kd) common to several Leishmania species. Antigenic preparations from Leishmania infantum extracts were isolated by electro-elution from polyacrylamide gels.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmodium falciparum exoprotein stimulation of human T-lymphocytes unsensitized to malaria.

The effects of Plasmodium falciparum proteins released in asexual blood stage culture supernatants on human T-lymphocytes from malaria non-immune donors were examined. Supernatants from several plasmodial strains stimulated both CD+4 and CD+8 T-lymphocytes to proliferate and secrete interferon gamma in vitro. Active moieties were predominantly released during the final stages of the parasite cycle. They were enriched by gel filtration and were further purified by anion-exchange and Superose 12 column fast protein liquid chromatography. Three active fractions of apparent 250, 70 and 18 kilodaltons were identified. The parasitic origin of the predominant 70-kilodaltons protein(s) was shown by biosynthesis experiments with radioactive amino acid precursors and was also demonstrated by in vitro translation of parasitic mRNA species. Interestingly, antibodies to the 70-kilodalton exoprotein(s) also reacted to a schizont protein of similar molecular weight.

Interferon inducibility and sensitivity of human teratocarcinoma-derived cell lines.

Three cell lines tera I, tera II, and PA1, derived from human teratocarcinomas were tested for their capacity to produce interferon (IFN) and for their sensitivity to both human IFN-alpha and IFN-beta. When treated with Newcastle disease virus or Sendai virus, or a synthetic polyribonucleotide, poly(rI):poly(rC), tera I cells produced no IFN and the 2',5'-oligoadenylate (2-5A) synthetase enzymatic pathway was not activated, although there was an increase in protein kinase. In contrast, tera II and PA1 cells produced IFN and both enzymatic activities were detected. IFN treatment has no effect on the growth of any of the cell lines. Tera I and PA1 cells did not develop resistance to challenge with vesicular stomatitis virus or encephalomyocarditis virus, but the growth of a type-C baboon retrovirus was inhibited. Tera II cells were protected against all three viruses. It appears that human teratocarcinoma cell lines can thus differ greatly in their ability to produce IFN and to respond to it.

Immunological selection for Leishmania (Viannia) braziliensis antigens.

Comparative ELISA and selective immunoblotting procedures were used in attempts to identify differential serological indicators of infection with the Leishmania (Viannia) braziliensis complex, infection with the L. braziliensis species, and therapeutic cure of localized or mucocutaneous leishmaniasis (LCL or MCL). Although mean ELISA absorbance values were significantly higher for MCL sera than for LCL sera, absorbance could not be used as a reliable indicator of the clinical form of disease. Immunoblotting profiles were similar with sera from MCL and LCL. Pre-adsorption with heterologous trypanosomatid antigens indicated that recognition of antigens of about 56, 60, 66, 72, 88 and 110 kDa might be specific to the subgenus Viannia. In two-colour, sequential, dual ELISA-based immunoblotting, no antigens recognized only by sera from MCL patients were detected. After glucantime therapy, immunoblotting profiles with LCL sera were reduced both in intensity and in the range of antigens detected; a 104-kDa antigen was newly detected with post-treatment LCL sera. Overall, the results show the value of differential immunological detection strategies and support the close relationship between species of the subgenus Viannia but fail to indicate a prognostic antigen for MCL.

Effects of human recombinant alpha and gamma and of highly purified natural beta interferons on simian Spumavirinae prototype (simian foamy virus 1) multiplication in human cells.

The present study demonstrates the inhibitory effect of human recombinant interferons (r-Hu-IFN) alpha and gamma, and that of highly purified natural human interferon beta on the replication of simian foamy virus type 1 (SFV1) in human AV3-cell cultures. All IFN led to strong inhibition of the SFV1 cytopathic effect. Electron microscopy showed a 70 to 95% decrease in viral particles. Significant inhibition of virus-associated reverse transcriptase activity was found in supernatant fluids of infected IFN-treated cultures. Metabolic labelling of the virus confirmed the inhibition of extracellular release of SFV1. PAGE analysis of immunoprecipitates indicated a reduction in viral-specific protein bands. Altogether, these results indicate that the mechanism of inhibition of Spumavirinae infection by interferon differs from that described for the other Retroviridae, and particularly for types B, C and D viruses. Our data is of therapeutic interest since Spumavirinae have been linked to pathological processes such as de Quervain thyroiditis.

Pancreatitis in fibrocalculous pancreatic diabetes mellitus is not associated with common mutations in the trypsinogen gene.

BACKGROUND: A distinct type of pancreatitis associated with diabetes, termed fibrocalculous pancreatic diabetes (FCPD), has been reported in tropical developing countries including Bangladesh. The molecular basis for autosomal dominant hereditary pancreatitis (HP) has recently been attributed to mutations in exons 2 and 3 of the trypsinogen gene. We have investigated the hypothesis that mutations in the aforementioned exons of this gene might also predispose to FCPD. METHODS: Seventy Bangladeshi and 50 South Indian unrelated FCPD patients and seven South Indian families with FCPD probands were studied. Pancreatic calcification was confirmed by abdominal X-ray, ultrasound and/or ERCP. Established mutations of exons 2 and 3 of the trypsinogen gene were studied in these subjects by PCR-RFLP analysis and DNA sequencing. RESULTS: The mutations found in hereditary pancreatitis were not observed in this collection of FCPD subjects, and complete DNA sequencing of exons 2 and 3 of the fourth cationic trypsinogen gene also excluded any new mutations. CONCLUSIONS: These results indicate that chronic pancreatitis of FCPD is unlikely to be caused by common mutations in the trypsinogen gene.

hTERT expression in human breast cancer and non-cancerous breast tissue: correlation with tumour stage and c-Myc expression.

Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. hTERT (human telomerase reverse transcriptase) gene is the rate-limiting determinant of telomerase reactivation. The present study aims to quantitatively measure the expression of hTERT mRNA in human breast cancer, adjacent non-cancerous tissue (ANCT) and benign breast lesions, examine the association between hTERT and the clinicopathological characteristics of the cancer specimens and to explore the relationship between c-Myc and hTERT expressions. RNA was extracted from 49 breast carcinomas, 46 matched ANCT, and eight fibroadenomas. hTERT and c-Myc mRNA expressions were estimated by reverse transcriptase-PCR (RT-PCR) and Taqman methodology. hTERT mRNA was present in all of the cancerous and most of ANCT specimens with levels being much higher in the cancerous tissue than in ANCT. The ratio of hTERT mRNA in tumour to that in ANCT was 2011 (95% confidence interval 373-10,853, P < 0.0001). There was no significant association between tumour hTERT expression and patient's age, tumour size, grade, nodal metastasis, estrogen receptor (ER) positivity, lymphovascular (LVI) or c-Myc expression. However, there was a weak but significant negative correlation between hTERT expression and progesterone receptor (PR) status (p = 0.04) in tumours. hTERT mRNA expression was also significantly higher in carcinomas (median = 2.61 x 10(6)) than in fibroadenomas (median = 424).We conclude that hTERT mRNA expression is significantly higher in human breast cancer than in non-cancerous breast tissue suggesting that hTERT has a potential role in breast cancer diagnosis. The hTERT mRNA levels in tumour do not seem to be associated with the patient's age or advanced tumour stage. Furthermore, hTERT mRNA expression does not correlate with c-Myc mRNA expression in breast cancer.

Family association studies of markers on chromosome 2q and Type 1 diabetes in subjects from South India.

BACKGROUND: Several Type 1 diabetes susceptibility loci have been located to chromosome 2q12-21. However, results have not always been consistent and this may reflect study design and the population analysed. We have used a family-based design to look for an association between Type 1 diabetes and markers located to 2q12-21. METHODS: Ninety-one South Indian families consisting of subjects with Type 1 diabetes and their parents were genotyped for eight polymorphic markers localised to 2q12-21, which includes the interleukin-1 gene cluster. Radiation hybrid mapping was used to localise the map position of D2S308 and D2S363 on 2q12-21. The extended transmission disequilibrium test was used for statistical analysis. RESULTS: No associations were found between Type 1 diabetes and markers located in and around the interleukin-1 gene cluster or the interleukin-1 Type 1 receptor. In contrast, a suggestive association was found between Type 1 diabetes and two closely-linked markers telomeric of the interleukin-1 gene cluster (D2S308 and D2S363, separated by 3.3 cR) (p=0.004 and p=0.002, respectively). CONCLUSION: This preliminary study suggests that a locus close to D2S308 and D2S363 is involved in the aetiology of Type 1 diabetes in the South Indian population.