A novel mutation in GCK gene: Beware of SGA child with diabetic mother.

MODY is a monogenic, autosomal dominant form of diabetes mellitus. MODY can be caused by mutations in several genes; glucokinase (GCK) accounts for 30-50% of the cases. The diagnosis can be suspected in early-onset diabetes with atypical features for type 1/type 2. Treatment is usually not recommended. A 5-year-old girl came to our attention for occasional episodes of hyperglycaemia. She was born at term, her birth weight was small for gestational age. At the beginning of her pregnancy, her mother was already on insulin therapy for impaired fasting glucose levels, detected before conception and confirmed in the first weeks of gestation. She was treated with insulin until the childbirth without further investigations. The patient was asymptomatic and in good clinical condition. Basal blood tests have shown a fasting plasma glucose of 125 mg/dl, an HbA1c of 6.5%. Antibodies against islet cells, anti-GAD and anti-ZNT8 antibodies were all negative. A 2-h oral glucose tolerance test was performed and underlined an impaired glucose tolerance. HLA haplotypes were screened, excluding susceptibility. GCK Sanger Sequencing identified a novel heterozygous variant. It is not described as a classical mutations. The analysis has been extended to the parents, finding out the same variant in her mother. To our knowledge this mutation has not been described previously; we believe that this variant is responsible for MODY2 due to FBG and Hb1Ac of all the affected members of family. We suggest high suspicion of an underlying GCK variant in SGA children with hyperglycaemia born to a diabetic mother.

Preliminary profiling of blood transcriptome in a rat model of hemorrhagic shock.

Hemorrhagic shock is a leading cause of morbidity and mortality worldwide. Significant blood loss may lead to decreased blood pressure and inadequate tissue perfusion with resultant organ failure and death, even after replacement of lost blood volume. One reason for this high acuity is that the fundamental mechanisms of shock are poorly understood. Proteomic and metabolomic approaches have been used to investigate the molecular events occurring in hemorrhagic shock but, to our knowledge, a systematic analysis of the transcriptomic profile is missing. Therefore, a pilot analysis using paired-end RNA sequencing was used to identify changes that occur in the blood transcriptome of rats subjected to hemorrhagic shock after blood reinfusion. Hemorrhagic shock was induced using a Wigger's shock model. The transcriptome of whole blood from shocked animals shows modulation of genes related to inflammation and immune response (Tlr13, Il1b, Ccl6, Lgals3), antioxidant functions (Mt2A, Mt1), tissue injury and repair pathways (Gpnmb, Trim72) and lipid mediators (Alox5ap, Ltb4r, Ptger2) compared with control animals. These findings are congruent with results obtained in hemorrhagic shock analysis by other authors using metabolomics and proteomics. The analysis of blood transcriptome may be a valuable tool to understand the biological changes occurring in hemorrhagic shock and a promising approach for the identification of novel biomarkers and therapeutic targets. Impact statement This study provides the first pilot analysis of the changes occurring in transcriptome expression of whole blood in hemorrhagic shock (HS) rats. We showed that the analysis of blood transcriptome is a useful approach to investigate pathways and functional alterations in this disease condition. This pilot study encourages the possible application of transcriptome analysis in the clinical setting, for the molecular profiling of whole blood in HS patients.

A diagnostic flowchart, including TCD, Xe-CT and angiography, to improve the diagnosis of vasospasm critically affecting cerebral blood flow in patients with subarachnoid haemorrhage, sedated and ventilated.

The aim of this study was to prospectively evaluate a clinical protocol including transcranial doppler (TCD), Xenon-CT (Xe-CT) and angiography, for the detection of vasospasm leading to critical reductions of regional cerebral blood flow (rCBF) in both ventilated and sedated SAH patients, i.e. patients in whom clinical evaluation was not possible. Seventy-six patients were prospectively included in a surveillance protocol for daily TCD vasospasm monitoring. When TCD showed a V(mean) above 120 cm/sec in the middle cerebral artery (MCA), patients underwent Xe-CT study. If rCBF in the MCA was reduced to below 20 ml/100 g/min or if there was a reduction in the rCBF with significant asymmetry between the two MCAs, angiography was performed. Conversely, further Xe-CT and angiography were not obtained unless the TCD V(mean) values reached values above 160 cm/sec. In 35 patients, V(mean) attained values above 120 cm/sec, but only in five of them, rCBF was suggestive of vasospasm, and angiography confirmed the diagnosis in four. The protocol suggests that in sedated and ventilated patients, detection of a critical rCBF reduction due to vasospasm is possible to allow for more specific treatment and to reduce undue medical complications.

Diagnostic impact of the spectrum of ischemic cerebral blood flow thresholds in sedated subarachnoid hemorrhage patients.

OBJECTIVE: Ischemia is the main cause of secondary damage in subarachnoid hemorrhage (SAH). Cerebral blood flow (CBF) measurement is useful to detect critical values. We analyzed the diagnostic impact of CBF ischemic thresholds to predict a new low attenuation area on computed tomography (CT) due to failure of large vessel perfusion. METHODS: We analyzed 48 xenon CT (Xe-CT) studies from 10 patients with SAH. CBF measurements were obtained by means of Xe-CT and cortical regions of interest (ROls). The ROIs which appeared in a hypoattenuation area were recorded. Cortical CBF was tested for specificity and sensitivity as a predictor of hypoattenuation by means of a receiver operating characteristic curve. RESULTS: Mean age was 58 (SD +/- 12.4) years. The median Fisher score and Hunt and Hess scale were 2 and 3, respectively. The area under the receiver operating characteristic curve was 0.912 (CI 0.896 to 0.926). The cut-off value for best accuracy was 6 mL/ 100 g/min, with a likelihood ratio of 37. CONCLUSION: The present study suggests a threshold of 6 mL/100 g/ min as a predictor of a new low attenuation area. However, each clinician should choose the most useful threshold according to pre-test probability and the cost/effectiveness ratio of the applied therapies.

Reperfusion of low attenuation areas complicating subarachnoid hemorrhage.

Hypoattenuation areas shown on brain CT scans after subarachnoid hemorrhage (SAH) are believed to be associated with persistent ischemia. The aim of this study was to evaluate regional cerebral blood flow (rCBF) in hypoattenuation areas and its evolution over time by means of Xenon CT (Xe-CT). We enrolled 16 patients with SAH who developed a hypoattenuation area in the middle cerebral artery territory. Patients were studied at time zero (the first Xe-CT), within 24 to 96 hours, and 96 hours after the initial Xe-CT. We analyzed 19 hypoattenuation areas caused by vascular distortion, vasospasm, or post-surgical embolization in 48 Xe-CT studies. Areas of hypoattenuation were divided in 2 groups according to initial rCBF. In the first group (n = 15), rCBF was initially above 6 mL/100 gr/min but only 2 were still ischemic (rCBF < 18 mL/ 100 gr/min) 96 hours after the first Xe-CT, while 7 (58%) were hyperemic. Conversely, in the second group with severe ischemia (rCBF < 6 mL/100 gr/min; n = 4) mean rCBF increased (p = 0.08) but still remained below the ischemic threshold. In severely ischemic lesions, rCBF reperfusion occurs but is probably marginally relevant. Conversely, in lesions not initially severely ischemic, residual CBF gradually improved and frequently became hyperemic. The functional recovery of these zones remains to be evaluated.

A systematic review of brain injury epidemiology in Europe.

BACKGROUND: The world's literature on traumatic brain injury (TBI) grows annually including new reports on epidemiologic findings from many regions. With the wide variety of reports emphasizing various factors it is useful to compile these findings, hence the objective of this report. Thus, we describe epidemiological factors from European studies largely published in the last 20 years. METHOD: The Medline was searched for TBI related articles from about 1980 to 2003 including terms such as "epidemiology", "head injury", "brain injury" and others. From the research reports identified, we checked references for additional relevant reports and from those reports we abstracted data on TBI incidence, severity, external cause, gender, mortality, prevalence, cost and related factors. RESULTS: Twenty three European reports met inclusion criteria and included findings from national studies from Denmark, Sweden, Finland, Portugal, Germany, and from regions within Norway, Sweden, Italy, Switzerland, Spain, Denmark, Ireland, the U.K. and France. An aggregate hospitalized plus fatal TBI incidence rate of about 235 per 100,000 was derived. Prevalence rate data were not reported from any European country. An average mortality rate of about 15 per 100,000 and case fatality rate of about 11 per 100 were derived. The TBI severity ratio of hospitalized patients was about 22:1.5:1 for mild vs. moderate vs. severe cases, respectively. The percentages of TBI from external causes varied considerably and several reports reported an association of alcohol use with TBI. Outcome or disability findings were mixed and inconsistent. INTERPRETATION: It was difficult to reach a consensus on all epidemiological findings across the 23 published European studies because of critical differences in methods employed across the reports. We highly recommend the development of research guidelines to standardize definitional, case finding, and data reporting parameters to help establish a more precise description and hence utility of the epidemiology of TBI in Europe.

Estimated cerebral respiratory quotient and arteriovenous differences of CO2 in the ultra early detection of global ischemia in severe head injury.

The specificity of jugular bulb saturation (SjO2) and arteriovenous oxygen difference (AVDO2) to detect global cerebral ischemia remains controversial. An absolute increase in the arteriovenous difference of carbon dioxide tension (AVDpCO2) and, more specifically, the estimated respiratory quotient (eRQ = AVDpCO2/AVDO2) may indicate anaerobic CO2 production. We compared these variables with SjO2 to predict global cerebral ischemia. We selected 36 patients from a cohort of 69 consecutive patients suffering from severe traumatic brain injury. All patients had jugular bulb sampling within 6 hours after injury. Brain death at 48 hours was used as a surrogate index of irreversible ischemia to build a receiver operating characteristics (ROC) curve analysis. The mean (+/- standard deviation) eRQ in the 13 patients who died early (3.7 +/- 3.2 mmHg/ml/dl) was higher than the survivors (1.78 +/- 0.45 mmHg/ml/dl, P = 0.03). There was no differences in SjO2 between groups. The area under the ROC curves for eRQ, but not that of AVDpCO2, was greater (P = 0.04) than that of SjO2. The eRQ, more than AVDpCO2, appears to be a potentially more informative index of global cerebral ischemia than SjO2.

Cerebral blood flow in mean cerebral artery low density areas is not always ischemic in patients with aneurysmal subarachnoid hemorrhage--relationship with neurological outcome.

Aneurysmal subarachnoid hemorrhage (SAH) can be complicated by reduction of regional cerebral blood flow (rCBF) from large conductance vessels leading to focal edema appearing as an area of hypoattenuation on CT. In this study we included 29 patients with SAH due to aneurysmal rupture, having 36 CT low density areas within the middle cerebral artery territory in whom a total of 56 Xenon-CT (Xe-CT) studies were performed. Collectively, we evaluated 70 hypoattenuated areas. rCBF levels were measured in two different regions of interest drawn manually on the CT scan, one in the low density area and the other in a corresponding contralateral area of normal-appearing brain tissue. In the low density area (22.6 +/- 22.7 ml/100 gr/min) rCBF levels were significantly lower than in the contralateral area (32.8 +/- 17.1 7 ml/100 gr/min) (p = 0.0007). In the injured areas deep ischemia (CBF < 6 ml/ 100 g/min) was present in only 25.7% of Xe-CT studies, suggesting that hypodense areas are not always ischemic, whereas in 43.7% of the lesions/Xe-CT studies we found hyperemic values. Patients with a better outcome had hyperemic lesions, suggesting brain tissue recovery in injured areas.

Cerebral blood flow mapping in two different subtypes of intraparenchymal hemorrhagic traumatic lesions.

The pathogenesis and the viability of edematous tissue may be different in traumatic hematomas and traumatic contusions. We tested the hypothesis that mapping of regional Cerebral Blood Flow (rCBF) was different in these two subtypes of traumatic intraparenchymal lesions. We evaluated rCBF by means of Xenon-enhanced computerized tomography (Xe-CT) in 59 traumatic intracerebral lesions from 43 patients with severe head injury. One-hundred-nine intracerebral lesions/Xe-CT CBF measurements were obtained. The rCBF was measured in the hemorrhagic core, in the intralesional oedematous low density area and in a 1 cm rim of apparently normal perilesional parenchyma of both lesion subtypes. Not statistically significant lower rCBF levels were found in the edematous area of traumatic contusions. In traumatic hematomas rCBF levels were lower in the core than in the low density area, suggesting that rCBF in edematous area is marginally involved in the initial traumatic injury and that edema is probably influenced by the persistence of the hemorrhagic core. Conversely, in the traumatic contusions a difference in rCBF values was found between core, low density area and perilesional area, indicating that rCBF of the low density area is related to a concentrical distribution of the initial injury.

Synthetic glycopeptide-based delivery systems for systemic gene targeting to hepatocytes.

PURPOSE: To design, synthesize, and test synthetic glycopeptide-based delivery systems for gene targeting to hepatocytes by systemic administration. METHODS: All peptides were synthesized by the solid phase method developed using Fmoc chemistry on a peptide synthesizer. The binding of galactosylated peptides to HepG2 cells and accessibility of the galactose residues on particle surface was demonstrated by a competition assay using 125I-labeled asialoorosomucoid and RCA lectin agglutination assay, respectively. DNA plasmid encoding chloramphenicol acetyl transferase (CAT) gene was complexed with a tri-galactosylated peptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in the presence of an endosomolytic peptide (GM225.1) or endosomolytic lipopeptide (GM227.3) to obtain DNA particles of 100-150 nm in size. The plasmid/peptide complexes were added to HepG2 cell cultures or intravenously administered by tail vein injection into normal mice or rats. Plasmid uptake and expression was quantified by qPCR and ELISA, respectively. RESULTS: Multiple antennary glycopeptides that have the ability to condense and deliver DNA plasmid to hepatocytes were synthesized and complexed with DNA plasmid to obtain colloidally stable DNA/peptide complexes. Addition of DNA/GM245.3/GM225.1 peptide complexes (1:3:1 (-/+/-)) to HepG2 cell cultures yielded CAT expression in transfected cells. The transfection efficiency was significantly reduced in the absence of galactose ligand or removal of endosomolytic peptide. Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5 (-/+)) into the tail vein of normal rats yielded DNA uptake in the liver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3 resulted in more stable DNA particles, and a 10-fold enhancement in liver plasmid uptake. CAT expression was detectable in liver following intravenous administration of DNA/GM246.3 complexes. Addition of endosomolytic lipopeptide GM227.3 into the complexes (DNA/ GM246.3/GM227.3 (1:0.5:1 (-/+/-))) yielded a 5-fold increase in CAT expression. Liver expression was 8-fold and 40-fold higher than lung and spleen, respectively, and localized in the hepatocytes only. The transfection efficiency in liver was enhanced by increasing DNA dose and injection volume. The plasmid uptake and expression in liver using DNA/GM246.3/GM227.3 complexes was 100-200-fold higher than DNA formulated in glucose. Tissue examination and serum biochemistry did not show any adverse effect of the DNA/GM246.3/ GM227.3 (1:0.5:1 (-/+/-)) complexes after intravenous delivery. CONCLUSIONS: Gene targeting to hepatocytes was achieved by systemic administration of a well-tolerated synthetic glycopeptide-based delivery system. The transfection efficiency of this glycopeptide delivery system was dependent on peptide structure, endosomolytic activity, colloidal particle stability, and injection volume.

Selection, establishment and characterization of cell lines derived from a chemically-induced rat mammary heterogeneous tumor, by flow cytometry, transmission electron microscopy, and immunohistochemistry.

In order to isolate, characterize, and establish culture cell lines with different diagnostic and prognostic significance, derived from multiclonal neoplasms, a ductal infiltrating mammary tumor was induced in rats by 7,12-dimethylbenz[a]anthracene. Clones with different DNA/protein content, being the DI of 1.16, 1.30, and 1.60, respectively, were observed in the primary tumor. Biparametric flow cytometry suggested that the clone at 1.30 is made up of two subpopulations with different protein and slightly different DNA contents. The culture, after a few passages, exhibited the presence of aneuploid cells and the absence of diploid components, demonstrating that only tumor cells survived. The limiting dilution method gave rise to four lines with DI of 1.16, 1.25, 1.30, and 1.50; a mean chromosome number of 45, 46, 47, and 88, respectively; and different morphological and ultrastructural features. These characteristics were stable during the experimental procedure, that is, for about 20 passages. Conversely, the detection of cytoskeletal proteins indicated that the tumor epithelial cells underwent early dedifferentiation into sarcoma-like cells showing markers of stromal cell type and thus exhibiting phenotypic instability in vitro, a feature reported in many advanced human breast cancers in vivo. In conclusion, this cellular model represents the in vivo situation and appears suitable for in vitro studies of tumor cell characteristics and might be used to predict clinical behavior.

In vitro proliferation and in vivo malignancy of cell lines simultaneously derived from a chemically-induced heterogeneous rat mammary tumor.

Identification of clones in primary tumors responsible for proliferation, invasion, and metastasis was carried out. Four different aneuploid established cell lines derived from a ductal infiltrating mammary rat tumor induced by 7,12-dimethylbenz[a]anthracene were studied for proliferative and growth features in vitro and for tumorigenic and metastatic potential in vivo in nude mice. Clones, named RM1, RM2, RM3, and RM4, were characterized by different proliferative activity. Clone RM1 showed the highest proliferative activity by both tritiated thymidine incorporation and S-phase flow cytometry, followed by clone RM4. Conversely, clones RM2 and RM3 showed a lower proliferation rate. Growth-promoting activity, tested on 3T3 Swiss cells, was high in all clones, although RM1 showed significantly lower growth factors-releasing activity. Nude mice tumorigenesis demonstrated a strong tumor induction of line RM1 (100% of the mice after 47 +/- 7 d) and a slightly lower tumor induction of line RM4 (70% of the mice after 69 +/- 9 d). Line RM3 showed tumor induction in 40% of the mice after 186 +/- 16 d. Lines RM2 showed no tumor induction. Metastasis occurred in mice treated with line RM1 only. Therefore, tumorigenesis and metastasis correlate with proliferation but not with the release of growth factors. In conclusion, flow cytometry monitoring of clones from heterogeneous primary tumors proved to be a suitable model for the study of in vivo malignancy and in vitro proliferation.

Biodistribution and gene expression of lipid/plasmid complexes after systemic administration.

The objectives of this study were to investigate the influence of physicochemical properties of lipid/plasmid complexes on in vivo gene transfer and biodistribution characteristics. Formulations based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and novel biodegradable cationic lipids, such as ethyl dioleoyl phosphatidylcholine (EDOPC), ethyl palmitoyl myristyl phosphatidylcholine (EPMPC), myristyl myristoyl carnitine ester (MMCE), and oleyl oleoyl L-carnitine ester (DOLCE), were assessed for gene expression after tail vein injection of lipid/plasmid complexes in mice. Gene expression was influenced by cationic lipid structure, cationic lipid-to-colipid molar ratios, plasmid-to-lipid charge ratios, and precondensation liposome size. Detectable levels of human growth hormone (hGH) in serum, human factor IX (hFIX) in plasma, and chloramphenicol acetyltransferase (CAT) in the lung and liver were observed with positively charged lipid/plasmid complexes prepared from 400-nm extruded liposomes with a cationic lipid-to-colipid ratio of 4:1 (mol/mol). Intravenous administration of lipid/CAT plasmid complexes resulted in distribution of plasmid DNA mainly to the lung at 15 min after injection. Plasmid DNA accumulation in the liver increased with time up to 24 hr postinjection. There was a 10-fold decrease in the amount of plasmid DNA in the lung at 15 min after injection, when the lipid/plasmid complex charge ratio was decreased from 3:1 to 0.5:1 (+/-). Bright fluorescent aggregates were evident in in vivo-transfected lung with the positively charged pCMV-CAT/DOLCE:dioleyl phosphatidylethanolamine (DOPE) (1:1, mol/mol) complexes, while more discrete punctate fluorescence was observed with a 4:1 molar ratio of cationic lipid:colipid formulations. Preinjection of polyanions such as plasmid, dextran sulfate, polycytidic acid, and polyinosinic acid decreased hGH expression, whereas the preinjection of both positively charged and neutral liposomes had no effect on hGH serum levels. Of the cationic lipids tested, DOLCE was found to be the most effective potentially biodegradable cationic lipid. A correlation between gene expression and cationic lipid:colipid ratios and lipid-to-plasmid charge ratio was also observed for DOTMA- and DOLCE-based formulations.

Protective interactive noncondensing (PINC) polymers for enhanced plasmid distribution and expression in rat skeletal muscle.

We have developed protective interactive noncondensing (PINC) polymers, such as poly(N-vinyl pyrrolidone) (PVP) and poly(vinyl alcohol) (PVA), to protect plasmids from extracellular nuclease degradation while allowing the flexible complex to diffuse throughout the muscle tissue. Molecular modeling, zeta potential modulation, and ethidium bromide intercalation studies were performed to assess the mechanism of interaction between PVP and plasmid. The effect of salt concentration, pH, and polymer-plasmid ratios were investigated. We have correlated these variables with beta-galactosidase (beta-gal) expression after intramuscular administration to rats. PVP can form hydrogen bonds with the base pairs within the major groove of DNA at pH 4.0. The PVP-plasmid interaction results in a complex that is more hydrophobic (less negatively charged) than the uncomplexed plasmid due to the vinyl backbone of PVP. Up to a ten-fold enhancement in gene expression in rat muscle over the use of 'naked' DNA has been demonstrated using these systems. A linear structure-activity relationship (SAR) was found between the percent vinyl pyrrolidone monomer content in poly (vinyl pyrrolidone-covinyl acetate) polymers and beta-gal expression in muscle. Modulation of the interaction between PINC polymers and plasmid directly impacts the levels of gene expression in vivo. The linear SAR is being used to design novel PINC polymers with enhanced binding affinity to plasmids.

[Immunotherapy of solid tumors. Current status and prospects]. / Immunoterapia dei tumori solidi. Stato attuale e prospettive.

Immunotherapy is the most recent therapeutic strategy in the treatment of cancer. It has not yet achieved an elevated curative efficiency and a wide clinical application. Nevertheless the possibilities of improvement seem very promising. The knowledge of the immune response mechanisms and the first clinical trials have determined a more efficient immunotherapy. Here we will critically analyze current immunotherapeutic strategies by reviewing the latest and the most important experimental works. The latest protocols of immunotherapy have been aimed to be more integrated in the physiological immune response schemes. The orientation of the experimental works have been changed from non specific immunotherapy using lymphokines to immunotherapy with specific lymphocytes expanded in vitro and, finally, the active specific immunotherapy in vivo by modificated tumoral vaccines or by variously manipulated tumoral antigens.

[Immunotherapy of solid tumors. Clinical studies]. / Immunoterapia dei tumori solidi. Studi clinici.

In this study we analyze the major clinical trials of immunotherapy for solid tumors. Much progress have been made in reducing the side effects and the percentage of patients which respond has increased. In immunotherapy with lymphokines the innovative orientation consist in the administration of low doses or decreasing doses and by alternative ways as regards infusion and systemically. The use of immunotherapy to stimulate the specific immune response seems to represent the most promising field from a therapeutic point of view. Studies in the field of in vitro expansion of immunocompetent cells have obtained results in the simplification of the technique and in an increase of its efficiency; moreover, at the moment, many clinical trials are involving specific immunotherapy using autologous neoplastic cells altered with adjuvant substance and the results are promising with very few side effects. In the near future immunotherapy with specific tumor antigens is sure it will play a major role.

Systemic specific active immunotherapy for solid tumors. An overview about cancer vaccinetherapy.

Augmentation of specific immunity is one of the most promising immunotherapeutical approaches against solid tumors. Protocols using autologous tumor cells or tumor associated antigens are easily performed and not charged by severe side effects. Recently some clinical trials suggested good results from immunotherapeutical protocols applied as an adjuvant to surgery in terms of disease free interval, survival and progression time in different stages. In this review the authors report the results of the most important clinical trials of vaccinetherapy in solid tumors. Little is known about the possibility of this new approach to oncology since we are at the real beginning of a new clinical treatment but in the considered trial its effectiveness seems to suggest a future wider application.