Array-CGH in the investigation of karyotype changes of CD34+ haematopoietic stem cells in lymphoma and multiple myeloma patients who underwent to autologous transplantation.

Hematopoietic stem cell transplantation (HSCT) represents an effective treatment strategy for a variety of hematologic and not hematologic malignancies. In particular, autologous transplantation of haematopoietic stem cells (ASCT) from bone marrow of patients with hematologic malignancies is feasible and has low treatment-related mortality (Gribben JG, 2009). However, literature assessed late mortality in 29.4% of individuals who had survived 2 or more years after autologous haematopoietic cell transplantation (Burns, L.J., 2009; Bhatia S. and al, 2005). This could be due to the purified stem cells which might carry a mutation on a chromosome predisposing to the disease and lead to the risk of pathology recurrence. Lymphoma and myeloma are perfect candidates for autologous transplantation after G-CSF stimulation and bone marrow ablation trough chemotherapy. However, in this case too, pathology relapse or secondary malignancies are found in a high percentage of patients. The aim of this project is to verify the existence of detectable imbalanced chromosome anomalies in stem cells before any ablative treatment for HSCT or developed after G-CSF stimulation or chemotherapy. A cohort of 24 lymphoma and myeloma patients have been analyzed trough array-CGH to identify significant imbalanced chromosome anomalies also present in low percentage of mosaicism. The result showed anomalies in 8/24 patients: one patient affected by Hodgkin Lymphoma (HL) revealed a deletion of chromosome 2 in p16.1, where the REL gene is located and in part deleted; the amplification of chromosome 11 in q12.2q13.4 containing CCND1 gene (this patient was investigated both before and after transplantation) was found in one patient with multiple myeloma (MM); alterations of chromosome 14 in q32.31-33, where genes for variable chain of immune globulin are located, were found in five patients with Hodgkin and non Hodgkin lymphomas (HL/NHL). FISH on interphase nuclei has been used to confirm a-CGH data. A short-time (36 months) clinical and haematological follow-up examination did not show a different trend between patients with chromosome imbalances and without but a long-term follow-up is needed to definitely correlate the imbalances with the clinical evolution and to have the indications of global survival of the considered population. Work in progress is the extension of clinical and haematological observation to obtain evidence of a difference statistically significant and to reach the final goal of suggesting a possible protocol to candidate patients to purging treatments before the CD34+ cells re-infusion

Comparative analysis of retroviral and native promoters driving expression of β1,3-galactosyltransferase β3Gal-T5 in human and mouse tissues

β1,3-Galactosyltransferase β3Gal-T5 is highly expressed in the colons of humans and certain primates due to a retroviral long terminal repeat (LTR) acting as a strong promoter. Because this promoter is inactive in other human tissues or mice, we attempted to understand how adoption of a retrotransposon allowed the gene to acquire tissue-specific expression. We identified three novel 5′-UTRs of 3Gal-T5 mRNA, types A, B, and C, and found wide-spread expression of the type A transcript at much lower levels than the LTR transcript, the expression of which is restricted to organs of the gastrointestinal tract. Expression of the type C 5′-UTR transcript was mostly restricted to the ileum, where it was expressed at high levels. We cloned the 5′-flanking regions of both types A and B 5′-UTRs, found deletion constructs functionally active as promoters, and identified CCAAT-binding factor (CBF) and hepatocyte nuclear factor 1 (HNF-1) as the principal nuclear factors controlling the promoters of types A and B 5′-UTR transcripts, respectively. The CCAAT-binding factor binding site and the entire downstream sequence driving the expression of type A transcripts in humans are structurally and functionally conserved in mice, where they constitute a unique β3Gal-T5 promoter that appears to be the ancestral promoter of the gene. The HNF-1 binding motif of the second human promoter is identical to the HNF-1/Cdx binding motif of the LTR promoter but is in the antisense orientation, resulting in much lower binding affinity and promoter strength. These data may explain the successful insertion of the transposon during evolution

CA19.9 antigen circulating in the serum of colon cancer patients: Where is it from?

CA19.9 antigen is a glycoprotein present in human serum and found elevated in various diseases. It is intensively studied since long time as a potential marker for managing cancers of the gastrointestinal tract, but its reliability is widely accepted only for pancreatic cancers. Here, we focused on the tetrasaccharide epitope (NeuAc\u3b12-3Gal\u3b21-3[Fuc\u3b11-4]GlcNAc) sialyl-Lewis a studying the biosynthesis, expression, and secretion in colon cancers and related cancer cell lines. We found that the \u3b21,3 galactosyltransferase \u3b23Gal-T5, responsible for sialyl-Lewis a synthesis, is dramatically reduced in colon adenocarcinomas, in terms of both transcript and enzyme activity levels. Moreover, no or very faint antigen is detectable in colon cancer homogenates, by dot-blot or enzyme immunoassay, while it is commonly evident in sera from different patients. In cancer cell lines synthesizing CA19.9, the amount of antigen secreted is proportional to that expressed on the cell surface, and depends on appreciable levels of \u3b23Gal-T5, which appear much higher than those measured in colon cancer specimens. Since colon cancers appear unable to synthesize relevant amount of CA19.9, we suggest that the antigen circulating in the serum of colon cancer patients may have a different and more complex origin than expected so far

APP1 Transcription Is Regulated by Inositol-phosphorylceramide Synthase 1-Diacylglycerol Pathway and Is Controlled by ATF2 Transcription Factor in Cryptococcus neoformans

Inositol-phosphorylceramide synthase 1 (Ipc1) is a fungal-specific enzyme that regulates the level of two bioactive molecules, phytoceramide and diacylglycerol (DAG). In previous studies, we demonstrated that Ipc1 regulates the expression of the antiphagocytic protein 1 (App1), a novel fungal factor involved in pathogenicity of Cryptococcus neoformans. Here, we investigated the molecular mechanism by which Ipc1 regulates App1. To this end, the APP1 promoter was fused to the firefly luciferase gene in the C. neofor-mans GAL7:IPC1 strain, in which the Ipc1 expression can be modulated, and found that the luciferase activity was indeed regulated when Ipc1 was modulated. Next, using the luciferase reporter assay in both C. neoformans wild-type and GAL7:IPC1 strains, we investigated the role of DAG and sphingolipids in the activation of the APP1 promoter and found that treatment with 1,2-dioctanoylglycerol does increase APP1 transcription, whereas treatment with phytosphingosine or ceramides does not. Two putative consensus sequences were found in the APP1 promoter for ATF and AP-2 transcription factors. Mutagenesis analysis of these sequences revealed that they play a key role in the regulation of APP1 transcription: ATF is an activator, whereas AP-2 in a negative regulator. Finally, we identified a putative Atf2 transcription factor, which is required for APP1 transcription and under the control of Ipc1-DAG pathway. These studies provide novel regulatory mechanisms of the sphingolipid pathway involved in the regulation of gene transcription of C. neoformans

Chromosome anomalies in bone marrow as primarycause of aplastic or hypoplastic conditions andperipheral cytopenia: disorders due to secondaryimpairment of RUNX1 and MPL genes

Background Chromosome changes in the bone marrow (BM) of patients with persistent cytopenia are often considered diagnostic for a myelodysplastic syndrome (MDS). Comprehensive cytogenetic evaluations may give evidence of the real pathogenetic role of these changes in cases with cytopenia without morphological signs of MDS. Results Chromosome anomalies were found in the BM of three patients, without any morphological evidence of MDS: 1) an acquired complex rearrangement of chromosome 21 in a boy with severe aplastic anaemia (SAA); the rearrangement caused the loss of exons 2-8 of the RUNX1 gene with subsequent hypoexpression. 2) a constitutional complex rearrangement of chromosome 21 in a girl with congenital thrombocytopenia; the rearrangement led to RUNX1 disruption and hypoexpression. 3) an acquired paracentric inversion of chromosome 1, in which two regions at the breakpoints were shown to be lost, in a boy with aplastic anaemia; the MPL gene, localized in chromosome 1 short arms was not mutated neither disrupted, but its expression was severely reduced: we postulate that the aplastic anaemia was due to position effects acting both in cis and in trans, and causing Congenital Amegakaryocytic Thrombocytopenia (CAMT). Conclusions A clonal anomaly in BM does not imply per se a diagnosis of MDS: a subgroup of BM hypoplastic disorders is directly due to chromosome structural anomalies with effects on specific genes, as was the case of RUNX1 and MPL in the patients here reported with diagnosis of SAA, thrombocytopenia, and CAMT. The anomaly may be either acquired or constitutional, and it may act by deletion/disruption of the gene, or by position effects. Full cytogenetic investigations, including a-CGH, should always be part of the diagnostic evaluation of patients with BM aplasia/hypoplasia and peripheral cytopenias

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Independent and combined effects of improved water, sanitation, and hygiene, and improved complementary feeding, on child stunting and anaemia in rural Zimbabwe: a cluster-randomised trial.

BACKGROUND: Child stunting reduces survival and impairs neurodevelopment. We tested the independent and combined effects of improved water, sanitation, and hygiene (WASH), and improved infant and young child feeding (IYCF) on stunting and anaemia in in Zimbabwe. METHODS: We did a cluster-randomised, community-based, 2 × 2 factorial trial in two rural districts in Zimbabwe. Clusters were defined as the catchment area of between one and four village health workers employed by the Zimbabwe Ministry of Health and Child Care. Women were eligible for inclusion if they permanently lived in clusters and were confirmed pregnant. Clusters were randomly assigned (1:1:1:1) to standard of care (52 clusters), IYCF (20 g of a small-quantity lipid-based nutrient supplement per day from age 6 to 18 months plus complementary feeding counselling; 53 clusters), WASH (construction of a ventilated improved pit latrine, provision of two handwashing stations, liquid soap, chlorine, and play space plus hygiene counselling; 53 clusters), or IYCF plus WASH (53 clusters). A constrained randomisation technique was used to achieve balance across the groups for 14 variables related to geography, demography, water access, and community-level sanitation coverage. Masking of participants and fieldworkers was not possible. The primary outcomes were infant length-for-age Z score and haemoglobin concentrations at 18 months of age among children born to mothers who were HIV negative during pregnancy. These outcomes were analysed in the intention-to-treat population. We estimated the effects of the interventions by comparing the two IYCF groups with the two non-IYCF groups and the two WASH groups with the two non-WASH groups, except for outcomes that had an important statistical interaction between the interventions. This trial is registered with ClinicalTrials.gov, number NCT01824940. FINDINGS: Between Nov 22, 2012, and March 27, 2015, 5280 pregnant women were enrolled from 211 clusters. 3686 children born to HIV-negative mothers were assessed at age 18 months (884 in the standard of care group from 52 clusters, 893 in the IYCF group from 53 clusters, 918 in the WASH group from 53 clusters, and 991 in the IYCF plus WASH group from 51 clusters). In the IYCF intervention groups, the mean length-for-age Z score was 0·16 (95% CI 0·08-0·23) higher and the mean haemoglobin concentration was 2·03 g/L (1·28-2·79) higher than those in the non-IYCF intervention groups. The IYCF intervention reduced the number of stunted children from 620 (35%) of 1792 to 514 (27%) of 1879, and the number of children with anaemia from 245 (13·9%) of 1759 to 193 (10·5%) of 1845. The WASH intervention had no effect on either primary outcome. Neither intervention reduced the prevalence of diarrhoea at 12 or 18 months. No trial-related serious adverse events, and only three trial-related adverse events, were reported. INTERPRETATION: Household-level elementary WASH interventions implemented in rural areas in low-income countries are unlikely to reduce stunting or anaemia and might not reduce diarrhoea. Implementation of these WASH interventions in combination with IYCF interventions is unlikely to reduce stunting or anaemia more than implementation of IYCF alone. FUNDING: Bill & Melinda Gates Foundation, UK Department for International Development, Wellcome Trust, Swiss Development Cooperation, UNICEF, and US National Institutes of Health.The SHINE trial is funded by the Bill & Melinda Gates Foundation (OPP1021542 and OPP113707); UK Department for International Development; Wellcome Trust, UK (093768/Z/10/Z, 108065/Z/15/Z and 203905/Z/16/Z); Swiss Agency for Development and Cooperation; US National Institutes of Health (2R01HD060338-06); and UNICEF (PCA-2017-0002)

Array-CGH in the investigation of karyotype changes of CD34+ haematopoietic stem cells in lymphoma and multiple myeloma patients who underwent to autologous transplantation.

Hematopoietic stem cell transplantation (HSCT) represents an effective treatment strategy for a variety of hematologic and not hematologic malignancies. In particular, autologous transplantation of haematopoietic stem cells (ASCT) from bone marrow of patients with hematologic malignancies is feasible and has low treatment-related mortality (Gribben JG, 2009). However, literature assessed late mortality in 29.4% of individuals who had survived 2 or more years after autologous haematopoietic cell transplantation (Burns, L.J., 2009; Bhatia S. and al, 2005). This could be due to the purified stem cells which might carry a mutation on a chromosome predisposing to the disease and lead to the risk of pathology recurrence. Lymphoma and myeloma are perfect candidates for autologous transplantation after G-CSF stimulation and bone marrow ablation trough chemotherapy. However, in this case too, pathology relapse or secondary malignancies are found in a high percentage of patients. The aim of this project is to verify the existence of detectable imbalanced chromosome anomalies in stem cells before any ablative treatment for HSCT or developed after G-CSF stimulation or chemotherapy. A cohort of 24 lymphoma and myeloma patients have been analyzed trough array-CGH to identify significant imbalanced chromosome anomalies also present in low percentage of mosaicism. The result showed anomalies in 8/24 patients: one patient affected by Hodgkin Lymphoma (HL) revealed a deletion of chromosome 2 in p16.1, where the REL gene is located and in part deleted; the amplification of chromosome 11 in q12.2q13.4 containing CCND1 gene (this patient was investigated both before and after transplantation) was found in one patient with multiple myeloma (MM); alterations of chromosome 14 in q32.31-33, where genes for variable chain of immune globulin are located, were found in five patients with Hodgkin and non Hodgkin lymphomas (HL/NHL). FISH on interphase nuclei has been used to confirm a-CGH data. A short-time (36 months) clinical and haematological follow-up examination did not show a different trend between patients with chromosome imbalances and without but a long-term follow-up is needed to definitely correlate the imbalances with the clinical evolution and to have the indications of global survival of the considered population. Work in progress is the extension of clinical and haematological observation to obtain evidence of a difference statistically significant and to reach the final goal of suggesting a possible protocol to candidate patients to purging treatments before the CD34+ cells re-infusion

Cytogenetic Monitoring in Shwachman-Diamond Syndrome: A Note on Clonal Progression and a Practical Warning

We analyzed the results of periodic chromosome analyses performed on bone marrow of 22 patients with Shwachman-Diamond syndrome (SDS), 8 directly observed and 14 from the literature, selected because of changes in the cytogenetic picture during the course of the disease. This study points out some features of the cytogenetic evolution in SDS relevant for prognostic evaluation but never noted in the literature. In particular, the lack of any clonal progression and the frequent appearance of independent clones with chromosomal changes different from the one initially discovered, with possible severe prognostic implications, are reported