Basic Medical Sciences

Abstracts of the 4th Congress of ECCO the European Crohn's and Colitis Organisation S99 7.3 (3.4 17.5) mmol/L vs. 10.5 (4.9 22.5) mmol/L in women and 7.4 (4.0 23.9) mmol/L vs. 12.9 (6.7 36.1) mmol/L in men, respectively (p < 0.0001 for both analyses). Furthermore, each 1 mM increase in serum bilirubin was associated with 15% decrease of odds for subsequent CD occurrence (OR [95% CI] = 0.85 [0.80 0.90], p < 0.001). This relationship remained stable even after adjustment for age and gender (OR [95% CI] = 0.87 [0.82 0.93], p < 0.001). Simultaneously, the odds of unconjugated hyperbilirubinemia (>17.1 mM, a phenotypic sign of Gilbert syndrome) was more than four times lower in CD patients as compared to controls (OR [95% CI] = 0.24 [0.11 0.56]; p < 0.001). The distribution of UGT1A1 (TA)n genotypes did not differ significantly between CD and controls (p = 0.53). However, due to the limited number of subjects, milder effect (OR 2.6) of UGT1A1 polymorphism might not be revealed. Conclusion: CD is associated with lower serum levels of bilirubin. We suggest, this is not due to the genetic predisposition, but rather due to an increased consumption of bilirubin in the early stage of the disease or even before the clinical manifestation of CD. Further analysis of UGT1A1 polymorphism in a larger CD cohort is needed to confirm its role in pathogenesis of CD.

Y chromosomal haplogroup J as a signature of the post-neolithic colonization of Europe Abstract In order to attain a finer reconstruction of the peopling of southern and central-eastern Europe from the Levant, we determined the frequencies of eight lineages internal to the Y chromosomal haplogroup J, defined by biallelic markers, in 22 population samples obtained with a fine-grained sampling scheme. Our results partially resolve a major multifurcation of lineages within the haplogroup. Analyses of molecular variance show that the area covered by haplogroup J dispersal is characterized by a significant degree of molecular radiation for unique event polymorphisms within the haplogroup, with a higher incidence of the most derived sub-haplogroups on the northern Mediterranean coast, from Turkey westward; here, J diversity is not simply a subset of that present in the area in which this haplogroup first originated. Dating estimates, based on simple tandem repeat loci (STR) diversity within each lineage, confirmed the presence of a major population structuring at the time of spread of haplogroup J in Europe and a punctuation in the peopling of this continent in the post-Neolithic, compatible with the expansion of the Greek world. We also present here, for the first time, a novel method for comparative dating of lineages, free of assumptions of STR mutation rates.

In this work we focus on a microsatellite-defined Y-chromosomal lineage (network 1n2) identified by us and reported in previous studies, whose geographic distribution and antiquity appear to be compatible with the Neolithic spread of farmers. Here, we set network 1.2 in the Y-chromosomal phylogenetic tree, date it with respect to other lineages associated with the same movements by other authors, examine its diversity by means of tri-and tetranucleotide loci and discuss the implications in reconstructing the spread of this group of chromosomes in the Mediterranean area. Our results define a tripartite phylogeny within HG 9 , with the deepest branching defined by alleles T (Haplogroup Eu10) or G (Haplogroup Eu9) at M172 , and a subsequent branching within Eu9 defined by network 1n2. Population distributions of HG 9 and network 1n2 show that their occurrence in the surveyed area is not due to the spread of people from a single parental population but, rather, to a process punctuated by at least two phases. Our data identify the wide area of the Balkans, Aegean and Anatolia as the possible homeland harbouring the largest variation within network 1n2. The use of recently proposed tests based on the stepwise mutation model suggests that its spread was associated to a population expansion, with a high rate of male gene flow in the Turkish-Greek area.

Chronic idiopathic neutropenia (CIN) has been well recognized as a granulocytic disorder not associated with increased risk to malignant transformation. Four cases, however, of acute myeloid leukemia have been recently reported in patients with CIN. In the current paper, we report on a CIN patient who developed acute myeloid/natural killer (NK) precursor cell leukemia 11 years after diagnosis and 4 months after initiation of treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF). Leukemic cells had trisomy 4 as the sole cytogenetic abnormality and, also, a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSFR) leading to truncated protein with a loss of 36 amino acids. There was no evidence that this receptor transmitted signals even in the presence of high doses of rhG-CSF in the cultures. We consider that CIN may be a preleukemic condition, at least in a subset of patients, and that rhG-CSF administration is unlikely to be involved in the leukemic transformation in this patient, although such a possibility could not be completely ruled out.

thalassemias constitute hereditary blood disorders characterized by reduced or absence of-globin synthesis resulting in mild to severe anemia, depending on the genotype. More than 200 mutations in the-globin gene are responsible for their specific features leading to a very heterogeneous phenotype. Current therapies for-thalassemia include blood transfusions, usually along with iron chelation and in selected cases with bone marrow transplantation (BMT) of HLA-matched hematopoietic stem cells (HSCs). However, these approaches are limited by factors, such as iron overload and donor availability, respectively. Since 2000, when globin lentiviral vectors (LVs) were first successfully tested for transfer efficiency of the therapeutic transgene, which led to disease amelioration in murine models, attention was drawn towards the improvement of such vectors for-thalassemia gene therapy. Constantly improving vector design and efficient HSC manipulation led recently to the first successful clinical trial in France, which further proved that this genetic approach can be curative. Furthermore, improved new efficient vectors and methods to safely monitor integration sites and therapeutic transgene position effects, promise a new era for-thalassemia gene therapy, with more and safer clinical trials yet to come.

The development of oncoretrovirus vectors for human g-globin has been hampered by problems of low expression and gene silencing. In order to address these problems, we investigated an enhancer element identified from individuals with deletional hereditary persistence of fetal hemoglobin 2 (HPFH2), a genetic condition characterized by elevated levels of g-globin in adults. Plasmid transfection studies in erythroid MEL (murine erythroleukemia) cells demonstrated the HPFH2 element could function synergistically with the b-globin locus control region to enhance the expression of an A g-globin gene with a truncated À382 bp promoter. A series of oncoretrovirus vectors were subsequently generated that contain an expression cassette for A g-globin linked to various combinations of the HPFH2 enhancer, the a-globin HS40 enhancer, and several versions of the promoter from A g-globin or b-globin. Expression analysis in transduced MEL cell clones revealed very high levels of promoterautonomous silencing that was at least partially abrogated by the HPFH2 enhancer. The vector containing a combination of a À201 bp A g-globin gene promoter with the Greek HPFH À117 point mutation and both the HPFH2 and HS40 enhancers exhibited no signs of vector silencing and was expressed at 248799% per copy of mouse a-globin (62% of total a-globin). This represents a significant improvement over previously reported oncoretrovirus vectors for A g-globin, and demonstrates the capacity of the HPFH2 enhancer to abrogate sequence-autonomous silencing of the A g-globin promoter in the context of a gene transfer vector.

The intravenous administration of 548,ug of killed Corynebocterium parvum (C. porvum) Into C57BL mice leads to a significant decrease In the number of bone-marrow colony-forrning-unlts in spleen (CFUs) as early as 12 h after the injection of the bacterium. This decrease persisted in varying degrees for 3 weeks. After an initial fall at 24 h, the splenic CFUs exhibited a rapid expansion and reached values 10 times higher than the control range on the ninth day. A significant rise in the number of circulating CFUs, reaching aflrst peak at 2 h and a second one on the fifth day, was also observed. The proliferative status of femoral CFUs was increased a t 48 and 72 h, while that of splenic CFUs presented a significant increase only 48 h after the injection of C. parvurn. The sequence of events which were observed in these experiments indicates that an accelerated migration of hemopoietic stem cells from bone marrow to spleen via the blood clrculation has to take place.

Background and objective: Azathioprine (AZA) and 6-mercaptopurine (6MP) are used in the treatment of paediatric inflammatory bowel disease (IBD). Genetic variations in thiopurine S-methyltranfarase (TPMT) gene have been correlated with enzyme activity and with the occurrence of adverse events to AZA and 6MP. The aim of the present study was to investigate the frequency of the functional TPMT polymorphisms and their association with the occurrence of adverse events during azathioprine therapy in a paediatric IBD cohort. Methods: Ninety-seven thiopurine-treated paediatric IBD patients (41AE24% boys and 58AE76% girls) with a mean age 11AE25 years (range 3-16), were assessed for TPMT polymorphisms and adverse events. Results: Of the 97 patients enrolled in the study, 18 (18AE56%) were heterozygous mutated; two (2AE06%) were homozygous for a mutated TPMT gene. Ten patients (10AE31%) developed adverse effects, and four of them (40%) had one of the variant alleles. Conclusions: In this small cohort of subjects, no association was found between TPMT polymorphisms and the occurrence of thiopurines-related adverse events.

Background: In recent years, the therapeutic application of the anti-tumour necrosis factor (TNF)-a antibody, infliximab (IFX) has expanded for both inducing and maintaining remission in young Crohn's disease (CD) patients. However, there are very few outcome reports from Japan in this clinical setting. With this in mind, we became interested to evaluate the clinical efficacy and safety of IFX in paediatric CD patients. Methods: In a single centre setting, we enrolled 15 paediatric CD patients, age 11 17yr, with mild to severe disease, CD activity index (CDAI) 256.3±88.7, and CD duration 4.1±2.5yr. All patients had received 5-aminosalicylic acid based medications, but none had received an immunomodulator (IM) like azathioprine or 6-mercaptopurine. The primary and secondary end points were weeks 10 and 54 following the first IFX infusion. Clinical response was defined as a decrease in CDAI by at least 70 points, while clinical remission was defined as CDAI <150. Further, loss of response was defined as the need for escalating IFX dose to maintain clinical remission. Results: At the primary end point, 12 of 15 patients (80%) responded, of these, 10 patients (66.7%) achieved clinical remission (Remission group). The age at CD diagnosis was shorter in the non-remission group vs remission group, 15.2±1.5 vs 12.8±1.5. At the secondary end point, 6 of 15 patients (40%) had maintained clinical remission (IFX effective group). Nine of 15 patients (60%) had lost response to IFX (IFX failure group). Patients' weight at first IFX infusion was smaller in the IFX failure group vs IFX effective group, 42.5±11.7 vs 53.4±7.2. Additionally, the L2 type (disease location, colon) and high serum C-reactive protein (CRP) before the first IFX infusion were associated with IFX failure. Only one patient developed mild dyspnoea at the 15th IFX infusion. No other serious adverse event or opportunistic infection during our observation time. Conclusions: The short-term response and remission rate in the present paediatric cohort are similar to the levels in the REACH study and also the study by Colombel JF, et al., while the long-term outcome is less impressive. Likewise, IFX-failure rate was higher in our study as compared with the aforementioned studies. We believe that IFX mono-therapy (without IM) s one factor for weaker long-term efficacy in our cohort vs earlier studies. Accordingly, IFX therapy in paediatric CD patients may be influenced by concomitant IM, CD refractoriness, poor nutrition, and disease location.

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We have developed a method that permits rapid identification of the consequences of mutations that alter I3-globin RNA processing in erythroid cells. SI nuclease mapping techniques were used to analyze total bone marrow RNA obtained from 15 patients who are clinically homozygous for ,B-thalassemia and from 5 patients with erythroid hyperplasia from other causes. This analysis was facilitated by the use of single-stranded uniformly labeled DNA probes of high specific activity that were prepared by using recombinant phage M13-f-globin DNA templates. Two abnormalities of RNA processing were found to occur with high frequency in these patients. Nine thalassemic patients were found to have increased levels ofan RNA species that retains all sequences transcribed from intervening sequence 1, implying the presence of mutations that decrease the correct splicing of this intron. Seven of 15 thalassemic patients were found to have an abnormally processed RNA species that retains 19 nucleotides transcribed from the 3' end of intron 1; this abnormality is caused

An intravenous injection of 548 ,ug of killed Corynebacterium parvum (C. parvum) into C57BL mice led to a significant increase in the number of both committed stem cells (GM-CFC) and proliferating cells of bone marrow granulocytic series. This increase appeared 24 h after the injection of the bacterium, persisted in varying levels until the 4th week and was accompanied by a marked and prolonged granulocytosis. The proportion of GM-CFC in DNA synthesis was found to be increased 1, 2, and 5 d after injection of C. parvum. Increased serum lysozyme levels were observed on the 9th and 15th d. These findings suggest that C. parvum can stimulate granulopoiesis in the animal system. This nroperty of the bacterium should be considered when a combined chemo-immunotherapy schedule has to be applied.

The basic role of vascular endothelial growth factor (VEGF) in cancer is underscored by the approval of bevacizumab for first-line treatment of cancer patients. Recent anticancer therapeutics based on active tumor targeting by conjugating tumor-specific antibodies has become of great interest in oncology. Current progress in nanomedicine has exploited the possibility of designing tumor-targeted nanocarriers able to deliver specific molecule payloads in a selective manner to improve the efficacy and safety of cancer imaging and therapy. We herein aimed to determine the targeting ability of bevacizumab-conjugated quantum dots (QDs) in vitro and in vivo. We used QDs labeled with bevacizumab, in various in vitro experiments using cell lines derived from colorectal cancer (CRC) and breast cancer (BC). For a competition study of QD-bevacizumab complex and bevacizumab, the cells were pre-treated with bevacizumab (100 nmol/L) for 24 h before exposure to the QD-bevacizumab complex. The breas...

An i.v. injection of 548 microgram of killed Corynebacterium parvum into C57B1 mice leads to significant changes in serum lysozyme (muramidase) levels. After an initial fall at 24 h, the activity of the enzyme increased progressively, reached a peak on the 9th day and returned to control range after the 15th day.