Prognostic bioindicators in severe COVID-19 patients.

BACKGROUND: Severe acute respiratory syndrome caused by novel coronavirus 2 (SARS-CoV-2) emerged in Wuhan (China) in December 2019. Here we evaluated a panel of biomarkers to phenotype patients and to define the role of immuno-inflammatory mediators as biomarkers of severity. MATERIALS AND METHODS: Serum samples were obtained from 24 COVID-19 patients on admission to hospital, before any treatment or infusion of intravenous steroids or invasive ventilation. KL-6 IL-6 and C-peptide were measured by chemiluminescent enzyme immunoassay. IL-6 assay was validated for accuracy and precision. The validity of variables used to distinguish severe from mild-to-moderate patients was assessed by areas under curves (AUC) of the receiver operating characteristic (ROC) and logistic regression was performed to combine parameters of the two groups. RESULTS: In the severe group, IL-6, CRP and KL-6 concentrations were significantly higher than in mild-to-moderate patients. KL-6, IL-6 and CRP concentrations were directly correlated with each other. ROC curve analysis of the logistic regression model including IL-6, KL-6 and CRP showed the best performance with an AUC of 0.95. CONCLUSIONS: Besides corroborating previous reports of over-expression of IL-6 in severe COVID-19 patients requiring mechanical ventilation, analytical determination of other mediators showed that IL-6 concentrations were correlated with those of KL-6 and CRP. The combination of these three prognostic bioindicators made it possible to distinguish severe COVID-19 patients with poor prognosis from mild-to-moderate patients.

Diagnostic application of a capture based NGS test for the concurrent detection of variants in sequence and copy number as well as LOH.

Whole exome sequencing (WES) has made the identification of causative SNVs/InDels associated with rare Mendelian conditions increasingly accessible. Incorporation of softwares allowing CNVs detection into the WES bioinformatics pipelines may increase the diagnostic yield. However, no standard protocols for this analysis are so far available and CNVs in non-coding regions are totally missed by WES, in spite of their possible role in the regulation of the flanking genes expression. So, in a number of cases the diagnostic workflow contemplates an initial investigation by genomic arrays followed, in the negative cases, by WES. The opposite workflow may also be applied, according to the familial segregation of the disease. We show preliminary results for a diagnostic application of a single next generation sequencing panel permitting the concurrent detection of LOH and variations in sequences and copy number. This approach allowed us to highlight compound heterozygosity for a CNV and a sequence variant in a number of cases, the duplication of a non-coding region responsible for sex reversal, and a whole-chromosome isodisomy causing reduction to homozygosity for a WFS1 variant. Moreover, the panel enabled us to detect deletions, duplications, and amplifications with sensitivity comparable to that of the most widely used array-CGH platforms.

von Willebrand disease type 1 mutation p.Arg1379Cys and the variant p.Ala1377Val synergistically determine a 2M phenotype in four Italian patients.

INTRODUCTION: We characterized five patients affected with von Willebrand disease (VWD) carrying the p.Arg1379Cys mutation. One was diagnosed as VWD type 1 and four as type 2M. The 2M patients also have the variant p.Ala1377Val in cis with p.Arg1379Cys. AIM: To evaluate the role of p.Ala1377Val and p.Arg1379Cys von Willebrand factor (VWF) variants to explain patients' phenotype. METHODS: Conventional phenotype tests were used to evaluate patients' plasma and platelets. Direct sequence analysis of exon 28 was carried out. The allele frequency of p.Ala1377Val was evaluated using online database. pcDNA3.1-VWF-WT and mutant (A1377V, R1379C and A1377V-R1379C) expression vectors were transiently transfected in HEK293 cells. The capacity of WT and mutant recombinant (r)VWF (along with patients' plasma VWF) to bind glycoprotein Ibα (GpIbα) were evaluated, using two ELISA assays. One with a wild-type (WT) recombinant (r)GpIbα at increasing ristocetin concentrations (from 0 to 1.50 mg mL-1 ) and the other with a gain-of-function mutant rGpIbα (VWF:GPIbM). RESULTS: The substitution c.4130C>T (p.Ala1377Val) was reported as rare variant in online databases. At 0.25 mg mL-1 of ristocetin, WT, A1377V and R1379C showed 6, 7.5 and 12-fold increased binding to rGpIbα, respectively. A1377V-R1379C rVWF showed no increased binding to rGpIbα at the same ristocetin concentration and reached the highest binding, of only 3-fold increased, at 1.50 mg mL-1 of ristocetin. The VWF:GPIbM showed strongly reduced values for the A1377V-R1379C rVWF and the 2M patients' plasma. CONCLUSION: Our study showed that the presence of both p.Ala1377Val and p.Arg1379Cys mutations (synergistic effect) abolishes the binding of rVWF to rGpIbα, explaining patients' 2M phenotype.

Microcirculatory changes in children undergoing cardiac surgery: a prospective observational study.

BACKGROUND: The effects of cardiac surgery on the microcirculation of children are unknown. The aim of this study was to assess the microcirculatory changes in children undergoing surgery for correction of congenital heart disease. METHODS: We used a videomicroscope (Sidestream Dark Field, SDF) in a convenience sample of 24 children

Genetic background and risk of postpartum haemorrhage: results from an Italian cohort of 3219 women.

Postpartum haemorrhage (PPH) is a leading cause of maternal mortality, particularly in the developing countries, and of severe maternal morbidity worldwide. To investigate the impact of genetic influences on postpartum haemorrhage, in association with maternal and intrapartum risk factors, using a candidate gene approach. All women (n = 6694) who underwent a vaginal delivery at the Obstetric Unit of a large University hospital in Milan (Italy) between July 2007 and September 2009 were enrolled. The first consecutive 3219 women entered the genetic study. Postpartum haemorrhage was defined as ≥500 mL blood loss. Eight functional polymorphisms in seven candidate genes were chosen because of their potential role in predisposing to or protecting from haemorrhagic conditions: tissue factor (F3), factor V (F5), tissue factor pathway inhibitor (TFPI), platelet glycoprotein Ia/IIa (ITGA2), prothrombin (F2), platelet glycoproteins Ibα (GP1BA) and angiotensin-converting enzyme (ACE). After correction for the already known PPH risk factors, only the promoter polymorphism of the tissue factor gene (F3 -603A>G) showed a significant association with PPH, the G allele exerting a protective effect (P = 0.00053; OR = 0.79, 95% CI = 0.69-0.90). The protective effect against PPH of the TF -603A>G polymorphism is biologically plausible since the G allele is associated with an increased protein expression and Tissue Factor is strongly represented in the placenta at term, particularly in decidual cells of maternal origin.

RoboticAssisted (RATS) versus Video-Assisted (VATS) lobectomy: A monocentric prospective randomized trial.

INTRODUCTION: The study aim is to compare Video-Assisted (VATS) and Robotic-Assisted (RATS) lobectomy in the effort to identify advantages and limits of robotic procedures considering the high costs and specific surgeon training. MATERIALS AND METHODS: This is a monocentric prospective randomized trial in which patients suitable for mini-invasive lobectomy were randomized 1:2 in two groups: Group A, RATS (25 patients), and Group B, VATS (50 patients). The two groups were compared in terms of perioperative and postoperative results with a mean follow up of 37.9 (±10.9) months. RESULTS: We observed a significant reduction of pleural effusion on day 1 (140 ml vs 214, p = 0.003) and day 2 (186 vs 321, p = 0.001) for group A. The Visual Analogue Scale (VAS) showed significantly lower pain in the 1st p.o. day in group A (0,92 vs 1,17, p = 0,005). Surgery time in Group B was significantly lower (160 min vs 180, p = 0.036), but had a higher onset of atrial fibrillation and other cardiac arrhythmias (0/25 vs 9/50, p = 0.038). The OS and DFS were similar between the two groups (95.5 % vs 93.1 %, and 95.5 % vs 89.7 %, respectively). Furthermore, no statistical difference in the evaluation of quality of life during follow-up was found. CONCLUSIONS: The RATS approach, although burdened by higher surgical costs, constitutes a valid alternative to VATS; as it determines a lower inflammatory insult, with a consequent reduction in pleural effusion, less post-operative pain and cardiological comorbidities for the patient, it can potentially determine the shortening in hospitalization. In addition, RATS allows accurate lymph node dissection, which permit to reach results that are not inferior to VATS in terms of long-term outcomes.

Expression of bta-miR-222 and LHCGR in bovine cultured granulosa cells: Impact of follicle deviation and regulation by FSH/insulin in vitro.

Final antral follicle development and future ovulation are mediated by gonadotropin-induced changes with spatio-temporally regulated expression of genes. Here, we aimed to quantify the relative mRNA abundance of bta-miR-222 and its predicted target, LHCGR, in granulosa cells (GCs) from follicles, after follicle deviation, as well as from GCs cultured in vitro with follicle stimulating hormone (FSH) and/or insulin. Thus, to study the impact of follicle deviation, Nelore heifers (n = 10; Bos taurus indicus) were hormonally synchronized and slaughtered 3 days after ovulation. Then, GCs from the dominant follicle (DF) and its respective subordinate follicle (SF) were recovered for RT-qPCR. For in vitro analysis, small follicles (2-5 mm) were dissected from bovine ovaries collected from a local abattoir. The GCs were isolated and cultured in serum-free medium, or treated with insulin (1 ng/mL or 10 ng/mL) alone or in combination with human recombinant FSH (1 ng/mL), for 6 days. Our findings showed that the relative mRNA abundance of LHCGR in GCs was higher in the DF compared to the SF (p = 0.01). Inversely, bta-miR-222 expression was lower in the DF compared to the SF (p = 0.01). Furthermore, GCs cultured with FSH and insulin together resulted in a higher abundance of LHCGR and a lower abundance of bta-miR-222 (p ≤ 0.05) when compared to GCs cultured with insulin alone. In conclusion, we found that the LHCGR upregulation in GCs from the DF is inversely related to bta-miR-222 expression. We also suggest the involvement of FSH in bta-miR-222 suppression in healthy bovine GCs.

Comparison between an uncalibrated pulse contour method and thermodilution technique for cardiac output estimation in septic patients.

BACKGROUND: The purpose of this study was to evaluate the reliability of a new uncalibrated pulse contour method, the MostCare, in determining cardiac output (CO) in septic patients. METHODS: Thirty patients with septic shock admitted to an intensive care unit, receiving a norepinephrine infusion and requiring haemodynamic monitoring with a pulmonary artery catheter, were prospectively enrolled. Thermodilution measurements of CO (ThD-CO) were considered as the 'gold standard'. MostCare was connected to the monitoring system of the radial arterial pressure waveform to obtain a continuous CO calculation (MostCare-CO). ThD-CO and MostCare-CO measurements were recorded at three different haemodynamic states: baseline (T1), after raising mean arterial pressure (MAP) to 90 mm Hg by increasing the norepinephrine infusion (T2), and after returning the MAP to baseline value by decreasing vasopressor therapy (T3). A Bland-Altman and linear regression analyses were performed. RESULTS: A total of 90 paired ThD-CO and MostCare-CO measures were obtained (range 4.1-13.9 litre min(-1) for ThD-CO and 4.5-13.5 litre min(-1) for MostCare-CO). A good correlation between ThD-CO and MostCare-CO was observed (R = 0.93). The mean bias between the two techniques was -0.26 litre min(-1) (sd 0.98 litre min(-1)) and the 95% limits of agreement were -2.22 to 1.70 litre min(-1). The percentage of error was 25%. Pearson's R was 0.94, 0.92, and 0.93 at T1, T2, and T3, respectively. CONCLUSIONS: MostCare-CO and ThD-CO showed a good agreement at each time of the study. The reliability of the MostCare system was not affected by the vascular tone changes produced by a norepinephrine infusion.

CT and MRI of Wernicke's encephalopathy.

The purpose of this pictorial essay is to present the computed tomography (CT) and magnetic resonance imaging (MRI) findings of Wernicke's encephalopathy, a rare, severe, acute neurological syndrome due to thiamine (vitamin B1) deficiency, associated with high morbidity and mortality. The classical clinical triad, which includes ocular signs, altered consciousness and ataxia, can be found in only one-third of patients. Although chronic alcoholic patients are the most commonly affected, Wernicke's encephalopathy may complicate malnutrition conditions in nonalcoholic patients, in whom it is greatly underestimated. CT and above all MRI of the brain play a fundamental role in diagnosing the condition and ruling out other diseases. MRI is the most sensitive technique and is required in all patients with a clinical suspicion of Wernicke's encephalopathy. Medial thalami, mamillary bodies, tegmentum, periaqueductal region, and tectal plate are typical sites of abnormal MRI signal. The dorsal medulla, red nuclei, cranial nerve nuclei, cerebellum, corpus callosum, frontal and parietal cerebral cortex are less common sites of involvement although they are more frequently affected in nonalcoholic patients. Paramagnetic contrast material may help to identify lesions not otherwise visible.

A Metabolic Intravascular Platform to Study FDG Uptake in Vascular Injury.

PURPOSE: Metabolic alterations underlie many pathophysiological conditions, and their understanding is critical for the development of novel therapies. Although the assessment of metabolic changes in vivo has been historically challenging, recent developments in molecular imaging have allowed us to study novel metabolic research concepts directly in the living subject, bringing us closer to patients. However, in many instances, there is need for sensors that are in close proximity to the organ under investigation, for example to study vascular metabolism. METHODS: In this study, we developed and validated a metabolic detection platform directly in the living subject under an inflammatory condition. The signal collected by a scintillating fiber is amplified using a photomultiplier tube and decodified by an in-house tunable analysis platform. For in vivo testing, we based our experiments on the metabolic characteristics of macrophages, cells closely linked to inflammation and avid for glucose and its analog 18F-fluorodeoxyglucose (18F-FDG). The sensor was validated in New Zealand rabbits, in which inflammation was induced by either a) high cholesterol (HC) diet for 16 weeks or b) vascular balloon endothelial denudation followed by HC diet. RESULTS: There was no difference in weight, hemodynamics, blood pressure, or heart rate between the groups. Vascular inflammation was detected by the metabolic sensor (Inflammation: 0.60 ± 0.03 AU vs. control: 0.48 ± 0.03 AU, p = 0.01), even though no significant inflammation/atherosclerosis was detected by intravascular ultrasound, underscoring the high sensitivity of the system. These findings were confirmed by the presence of macrophages on ex vivo aortic tissue staining. CONCLUSION: In this study, we validated a tunable very sensitive metabolic sensor platform that can be used for the detection of vascular metabolism, such as inflammation. This sensor can be used not only for the detection of macrophage activity but, with alternative probes, it could allow the detection of other pathophysiological processes.

Partial luteolysis during early diestrus in cattle downregulates VEGFA expression and reduces large luteal cell and corpus luteum sizes and plasma progesterone concentration.

Our objectives were to investigate potential changes in the size of steroidogenic large luteal cells (LLC) during partial luteolysis induced by a sub-dose of cloprostenol in early diestrus and to determine transcriptional variations in genes involved in corpus luteum (CL) functions. Cows were subjected to an Ovsynch protocol, with the time of the second GnRH treatment defined as Day 0 (D0). On D6, cows were randomly allocated into three treatments: Control (2 mL saline, im; n = 10), 2XPGF (two doses of 500 µg of cloprostenol, im, 2 h apart; n = 8) or 1/6PGF (single dose of 83.3 µg of cloprostenol, im; n = 10). Before treatments and every 8 h during the 48-h experimental period, blood samples were collected and CL volumes measured. Furthermore, two CL biopsies were obtained at 24 and 40 h post-treatment. The 1/6PGF treatment caused partial luteolysis, characterized by sudden decreases in plasma progesterone (P4) concentrations, luteal volume and LLC size, followed by increases (to pretreatment values) in P4 and luteal volume at 24 and 40 h post-treatment, respectively. However, at the end of the study, P4, luteal volume and LLC size were all significantly smaller than in Control cows. Temporally associated with these phenotypes, there was a lower mRNA abundance of VEGFA at 24 and 40 h, and ABCA1 at 24 h (P < 0.05). In conclusion, a sudden reduction in CL size during partial luteolysis induced by a sub-dose of PGF2α analog on day 6 of the estrous cycle was attributed to a reduction in LLC size, although these changes did not account for the entire phenomenon. In addition to its involvement in reducing CL size, decreased VEGFA mRNA abundance impaired CL development, resulting in a smaller luteal gland and lower plasma P4 concentrations compared to Control cows.

Expression of fibroblast growth factor 22 (FGF22) and its receptor, FGFR1B, during development and regression of bovine corpus luteum.

The aim of this study was to determine the expression of fibroblast growth factor 22 (FGF22) in the bovine corpus luteum (CL) and to investigate the effects of in vivo total or partial cloprostenol-induced luteolysis on the mRNA abundance of FGF22 and its receptor, FGFR1B. Corpora lutea at different stages of development were then dissected from abattoir ovaries (n = 10/stage); a portion of the tissue samples was fixed in paraformaldehyde and the remaining samples were homogenized and subjected to total RNA extraction. To assess mRNA abundance of target genes during induced luteolysis, nineteen cows were synchronized and then randomly assigned to a Latin square design as follows: Control; 2 administrations of prostaglandin F2α (PGF2α, total luteolysis; 2 × 250 µg of cloprostenol sodium) and 1/6PGF2α (partial luteolysis; 83.33 µg of cloprostenol sodium). FGF22 and FGFR1B expression levels were measured by RT-qPCR, and FGF22 protein expression was detected by immunohistochemistry. In summary, FGF22 mRNA was detected at all stages of CL development, and FGF22 protein was also detected in luteal tissue. FGF22 mRNA expression was lower at stage IV than at stage III (P < 0.05), and the same pattern was observed in luteal immunoreactivity. Furthermore, cloprostenol-induced luteolysis, both total and partial, increased FGFR1B mRNA abundance in luteal tissue (P < 0.05), but did not affect FGF22 mRNA abundance. In conclusion, these data suggest a potential role for the FGF22-FGFR1B system during development and regression of bovine CL.

Effect of superstimulation on the expression of microRNAs and genes involved in steroidogenesis and ovulation in Nelore cows.

To better understand the impact of ovarian superstimulation on bovine follicular microenvironment, Nelore cows (Bos taurus indicus) were subjected to ovarian superstimulation with follicle stimulating hormone (FSH, n = 10; P-36 protocol) or FSH combined with eCG (n = 10; P-36/eCG protocol). Follicular fluid was analyzed for cholesterol concentration. Granulosa cells were analyzed by RT-qPCR to assess the expression of genes involved in steroidogenic and ovulatory and expression of microRNAs involved in final follicular development and luteinizing hormone/choriogonadotropin receptor (LHCGR) expression. Plasma concentration of estradiol was also measured. Follicular fluid from the P-36 group showed higher concentration of cholesterol than that of control (non-superstimulated) cows. Plasma concentration of estradiol was higher in the P-36/eCG group. Abundance of STAR and FSHR mRNAs were lower in granulosa cells from the P-36/eCG group. In contrast, LHCGR mRNA abundance was higher in superstimulated granulosa cells from the P-36 group and showed a pattern opposite to that of miR-222 expression. Ovarian superstimulation did not affect the expression of other markers (mmu-miR-202-5p, has-miR-873, has-miR-144, and their target genes, CREB, TGFBR2, and ATG7) of antral follicle development. However, the mRNA expression of VEGF pathway components was modulated by P-36 treatment. Taken together, these results demonstrate that superstimulatory protocols modify steroidogenic capacity, increase plasma estradiol, and regulate the abundance of VEGF system, LHCGR mRNA and suppress the expression of miR-222 in bovine granulosa cells.

Prematurity, ventricular septal defect and dysmorphisms are independent predictors of pathogenic copy number variants: a retrospective study on array-CGH results and phenotypical features of 293 children with neurodevelopmental disorders and/or multiple congenital anomalies.

BACKGROUND: Since 2010, array-CGH (aCGH) has been the first-tier test in the diagnostic approach of children with neurodevelopmental disorders (NDD) or multiple congenital anomalies (MCA) of unknown origin. Its broad application led to the detection of numerous variants of uncertain clinical significance (VOUS). How to appropriately interpret aCGH results represents a challenge for the clinician. METHOD: We present a retrospective study on 293 patients with age range 1 month - 29 years (median 7 years) with NDD and/or MCA and/or dysmorphisms, investigated through aCGH between 2005 and 2016. The aim of the study was to analyze clinical and molecular cytogenetic data in order to identify what elements could be useful to interpret unknown or poorly described aberrations. Comparison of phenotype and cytogenetic characteristics through univariate analysis and multivariate logistic regression was performed. RESULTS: Copy number variations (CNVs) with a frequency < 1% were detected in 225 patients of the total sample, while 68 patients presented only variants with higher frequency (heterozygous deletions or amplification) and were considered to have negative aCGH. Proved pathogenic CNVs were detected in 70 patients (20.6%). Delayed psychomotor development, intellectual disability, intrauterine growth retardation (IUGR), prematurity, congenital heart disease, cerebral malformations and dysmorphisms correlated to reported pathogenic CNVs. Prematurity, ventricular septal defect and dysmorphisms remained significant predictors of pathogenic CNVs in the multivariate logistic model whereas abnormal EEG and limb dysmorphisms were mainly detected in the group with likely pathogenic VOUS. A flow-chart regarding the care for patients with NDD and/or MCA and/or dysmorphisms and the interpretation of aCGH has been made on the basis of the data inferred from this study and literature. CONCLUSION: Our work contributes to make the investigative process of CNVs more informative and suggests possible directions in aCGH interpretation and phenotype correlation.

Mitomycin C and etoposide in advanced colorectal carcinoma. A clinical and in vitro experience that focuses the problem of schedule dependence in combination therapy.

BACKGROUND: Aim of this study was to evaluate the activity of a combination regimen of chemotherapy containing mitomycin C (MMC) and etoposide (ETO) in advanced colorectal carcinoma. METHODS: Fourteen pretreated patients received MMC 2 mg/m2 and ETO 60 mg/m2, days 1-5 every 28 days. The clinical study was interrupted since no clinical response was observed in 14 patients following four courses of chemotherapy. An in vitro study was then performed on HTC-8 cell line. The cytotoxic activity of the MMC/ETO combination was tested by sulforhodamine B assay and the type of drug interaction was assessed using the method of Chou and Talalay. Cell cycle perturbations and apoptosis were evaluated by flow cytometry. RESULTS: While MMC and ETO were singularly active, the simultaneous exposure of cells to both drugs and the sequence MMC-->ETO ensued in antagonistic interaction at all levels of killed cell fraction. Conversely, the sequence ETO-->MMC produced a synergistic interaction. CONCLUSIONS: These results suggest that the activity of the MMC/ETO combination is highly schedule-dependent and that the experimental drug associations should be based on a preclinical rationale before clinical trials are designed.

Lipid profiles of follicular fluid from cows submitted to ovarian superstimulation.

Ovarian superstimulation with exogenous gonadotropins has been extensively used to produce in vivo-derived embryos for embryo transfer in cattle. This process modifies the antral follicle microenvironment and affects oocyte and embryo quality as well the differentiation of granulosa cells. Lipids play significant roles in the cell, such as energy storage, cell structure, and fine-tuning of the physical properties and functions of biological membranes. The phospholipid (PL) contents as well as the effects of superstimulatory treatments on the PL profile of follicular fluid from cows, however, remain unknown. Therefore, to gain insight into the effects of superstimulation with follicle-stimulating hormone (FSH; P-36 protocol) or FSH combined with equine chorionic gonadotropin (eCG; P-36/eCG protocol) on the profile and abundance of PL from cows submitted or not submitted to superstimulatory protocols, were treated with these two superstimulatory protocols. As a control, non-superstimulated cows were only submitted to estrous synchronization. The follicular fluid was aspirated, the remaining cells removed and the follicular fluid stored at -80 °C until extraction. The lipid screening was performed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and this technique allowed the identification of sphingomyelins (SM) and phosphatidylcholines (PC) and phosphoethanolamines (PE). The relative abundance of the ions observed in the three experimental groups was analyzed by multivariate and univariate statistical models. The phospholipid SM (16:0) and PC (36:4) and/or PC (34:1) were less (P < 0.05) abundant in the P-36 group compared to the control or P-36/eCG groups. However, the PC (34:2) was more (P < 0.05) abundant in both group of superstimulated cows compared to the control. In summary, ovarian superstimulation seems to modulate the PL content of bovine follicular fluid with a significant increase in PC (34:2), which jointly with others PC and SM, seems to offer a suitable biomarker involved with reproductive processes successful as ovary superstimulation response and embryo development.

Anophthalmos with limb anomalies (Waardenburg opththalmo-acromelic syndrome): report of a new Italian case with renal anomaly and review.

Anophthalmos with limb anomalies (Waardenburg Opththalmo-Acromelic Syndrome) is a very rare autosomal recessive multiple congenital anomaly syndrome, first described by Waardenburg et al. in 1961 (MIM 206920). It is characterized by mono or more often bilateral anophthalmia/microphthalmia and foot malformations, which can be observed in 91% of the patients. The most common anomaly of the feet is the presence of four toes. The hands are affected bilaterally in 77% of the cases. The most characteristic anomaly is the synostosis of the fourth and fifth metacarpals. To date, 33 cases from 19 families have been reported. We present an Italian case of anophthalmia with limb anomalies and a renal malformation, which has never been described in the literature.

Modulation of 5-fluorouracil in mice using uridine diphosphoglucose.

Uridine diphosphoglucose (UDPG) is a precursor of uridine that can be used as a rescuing agent from 5-fluorouracil (5FU) toxicity. Four doses of UDPG (2000 mg/kg i.p. or p.o. at 2, 6, 24, and 30 h after 5FU bolus) allowed the escalation of a weekly bolus of 5FU from 100 mg/kg (5FU100) to 150 mg/kg (5FU150) in healthy and tumor-bearing BALB/c, C57/BI, and CD8F1 (BALB/c x DBA/8) mice. 5FU150 without rescuing agents is not tolerated by the animals. When followed by UDPG, on the contrary, it is possible to increase the dose of 5FU even when it is modulated by leucovorin. Toxicity was the same for 5FU100 and 5FU150 + UDPG, and the nadir values (expressed as a percentage of pretreatment values) were 83 and 85% for weight, 45 and 45% for hematocrit, and 45 and 61% for leukocytes, respectively. Platelets were not affected by treatment. A protective effect was also shown for the gastrointestinal tract. The enzymes thymidine kinase, maltase, and sucrase were measured in the intestinal mucosa at different times after 5FU treatment with or without UDPG rescue. Even if the nadir values in enzyme activities were similar in mice receiving or not receiving UDPG, the pattern of recovery showed that cell repopulation was more rapid in the group treated with UDPG. 5FU150 + UDPG had enhanced antitumor activity against CD8F1 mammary carcinoma and against the resistant tumor Colon 26 (tumor doubling time 1.9 days for controls, 8.5 days for 5FU100, 13.7 days for 5FU150 + UDPG, and 15.9 days for 5FU150 + leucovorin + UDPG). We demonstrated that UDPG administered at 2, 24, and 30 h after 5FU100 does not reduce the antitumor activity of 5FU in two sensitive tumors (Colon 38 and Colon 26-10). In conclusion, UDPG is a promising rescuing agent for 5FU; it reduces the toxic side effects and increases the therapeutic index.

Evaluation of an heterogeneous group of patients with von Willebrand disease using an assay alternative to ristocetin induced platelet agglutination.

BACKGROUND: Diagnosis of von Willebrand disease (VWD) type 2 usually relies on the discrepancy between the von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) and VWF antigen (VWF:Ag). Type 2B patients can be discriminated from other qualitative VWD variants by using ristocetin-induced platelet agglutination (RIPA) test. The major limitation of RIPA is the requirement of fresh blood sample. OBJECTIVES: In this study, we evaluated the VWF gain-of-function mutant GPIb binding (VWF:GPIbM) and VWF:RCo assays to investigate whether the VWF:GPIbM/VWF:RCo ratio was able to identify the type 2B variant among an heterogeneous VWD population, previously characterized following the ISTH-SSC guidelines. PATIENTS/METHODS: Seventy-six VWD patients and 31 healthy subjects were evaluated by using VWF:Ag, VWF:RCo, and VWF:GPIbM assays. RESULTS: The mean (minimum-maximum values) VWF:GPIbM/VWF:RCo ratio was higher in type 2B patients (2.53, 0.84-6.11) than in healthy controls (1.05, 0.87-1.34), type 1 (0.85, 0.51-1.15), 2A (1.20, 0.36-2.82), and 2M (1.07, 0.91-1.38) (P < 0.0001). Type 2B variants were divided into four groups (A, B, C, and D) according to their different multimeric patterns. The mean value of the VWF:GPIbM/VWF:RCo ratio in the four groups showed an increasing trend from group A (1.08) to D (3.69), proportional to the loss of high molecular weight multimers. Among 32 type 2B patients, previously diagnosed with RIPA, 8 (mainly with a type I New York/Malmö phenotype) were not confirmed using the VWF:GPIbM/VWF:RCo ratio. CONCLUSIONS: Whenever the RIPA test is not feasible, the VWF:GPIbM/VWF:RCo ratio might help to identify severe type 2B VWD patients.

Endogenous dopamine (DA) and DA2 receptors: a mechanism limiting excessive sympathetic-adrenal discharge in humans.

We studied the effects of presynaptic dopamine (DA) 2 receptor blockade on the sympathetic-adrenal response to graded exercise in seven normal men. DA2 receptor blockade was achieved by means of domperidone (DMP) administration. The exercise consisted of progressive cycling activity, from 30-80% of the predetermined maximal oxygen consumption for each man. Systolic, diastolic, and mean arterial pressures; heart rate; and plasma norepinephrine (NE), epinephrine (E), PRL, glucose, lactate, FFA, sodium, potassium, cortisol, and PRA were measured at rest, during exercise, and during recovery after placebo or DMP administration. Graded exercise caused significant increases in systolic and mean arterial pressures and plasma NE, E, lactate, sodium, potassium, FFA, cortisol, and PRA. DMP administration before exercise caused a significant increase in plasma PRL (P = 0.0009), a greater increase in plasma NE at the end of the exercise (P = 0.002), and an overall increase in plasma E (P = 0.02) and FFA (P = 0.02) concentrations. These results strongly suggest that endogenous DA limits catecholamine release during sympathetic-adrenal stimulation by activating DA2 receptors.