Playing with Biophysics: How a Symphony of Different Electromagnetic Fields Acts to Reduce the Inflammation in Diabetic Derived Cells.

Several factors, such as ischemia, infection and skin injury impair the wound healing process. One common pathway in all these processes is related to the reactive oxygen species (ROS), whose production plays a vital role in wound healing. In this view, several strategies have been developed to stimulate the activation of the antioxidative system, thereby reducing the damage related to oxidative stress and improving wound healing. For this purpose, complex magnetic fields (CMFs) are used in this work on fibroblast and monocyte cultures derived from diabetic patients in order to evaluate their influence on the ROS production and related wound healing properties. Biocompatibility, cytotoxicity, mitochondrial ROS production and gene expression have been evaluated. The results confirm the complete biocompatibility of the treatment and the lack of side effects on cell physiology following the ISO standard indication. Moreover, the results confirm that the CMF treatment induced a reduction in the ROS production, an increase in the macrophage M2 anti-inflammatory phenotype through the activation of miRNA 5591, a reduction in inflammatory cytokines, such as interleukin-1 (IL-1) and IL-6, an increase in anti-inflammatory ones, such as IL-10 and IL-12 and an increase in the markers related to improved wound healing such as collagen type I and integrins. In conclusion, our findings encourage the use of CMFs for the treatment of diabetic foot.

Intensive Risk Factor Management and Cardiovascular Autonomic Neuropathy in Type 2 Diabetes: The ACCORD Trial.

OBJECTIVE: The effects of preventive interventions on cardiovascular autonomic neuropathy (CAN) remain unclear. We examined the effect of intensively treating traditional risk factors for CAN, including hyperglycemia, hypertension, and dyslipidemia, in individuals with type 2 diabetes (T2D) and high cardiovascular risk participating in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial. RESEARCH DESIGN AND METHODS: CAN was defined as heart rate variability indices below the fifth percentile of the normal distribution. Of 10,251 ACCORD participants, 71% (n = 7,275) had a CAN evaluation at study entry and at least once after randomization. The effects of intensive interventions on CAN were analyzed among these subjects through generalized linear mixed models. RESULTS: As compared with standard intervention, intensive glucose treatment reduced CAN risk by 16% (odds ratio [OR] 0.84, 95% CI 0.75-0.94, P = 0.003)-an effect driven by individuals without cardiovascular disease (CVD) at baseline (OR 0.73, 95% CI 0.63-0.85, P < 0.0001) rather than those with CVD (OR 1.10, 95% CI 0.91-1.34, P = 0.34) (P interaction = 0.001). Intensive blood pressure (BP) intervention decreased CAN risk by 25% (OR 0.75, 95% CI 0.63-0.89, P = 0.001), especially in patients ≥65 years old (OR 0.66, 95% CI 0.49-0.88, P = 0.005) (P interaction = 0.05). Fenofibrate did not have a significant effect on CAN (OR 0.91, 95% CI 0.78-1.07, P = 0.26). CONCLUSIONS: These data confirm a beneficial effect of intensive glycemic therapy and demonstrate, for the first time, a similar benefit of intensive BP control on CAN in T2D. A negative CVD history identifies T2D patients who especially benefit from intensive glycemic control for CAN prevention.

MicroRNA-21/PDCD4 Proapoptotic Signaling From Circulating CD34+ Cells to Vascular Endothelial Cells: A Potential Contributor to Adverse Cardiovascular Outcomes in Patients With Critical Limb Ischemia.

OBJECTIVE: In patients with type 2 diabetes (T2D) and critical limb ischemia (CLI), migration of circulating CD34+ cells predicted cardiovascular mortality at 18 months after revascularization. This study aimed to provide long-term validation and mechanistic understanding of the biomarker. RESEARCH DESIGN AND METHODS: The association between CD34+ cell migration and cardiovascular mortality was reassessed at 6 years after revascularization. In a new series of T2D-CLI and control subjects, immuno-sorted bone marrow CD34+ cells were profiled for miRNA expression and assessed for apoptosis and angiogenesis activity. The differentially regulated miRNA-21 and its proapoptotic target, PDCD4, were titrated to verify their contribution in transferring damaging signals from CD34+ cells to endothelial cells. RESULTS: Multivariable regression analysis confirmed that CD34+ cell migration forecasts long-term cardiovascular mortality. CD34+ cells from T2D-CLI patients were more apoptotic and less proangiogenic than those from control subjects and featured miRNA-21 downregulation, modulation of several long noncoding RNAs acting as miRNA-21 sponges, and upregulation of the miRNA-21 proapoptotic target PDCD4. Silencing miR-21 in control CD34+ cells phenocopied the T2D-CLI cell behavior. In coculture, T2D-CLI CD34+ cells imprinted naive endothelial cells, increasing apoptosis, reducing network formation, and modulating the TUG1 sponge/miRNA-21/PDCD4 axis. Silencing PDCD4 or scavenging reactive oxygen species protected endothelial cells from the negative influence of T2D-CLI CD34+ cells. CONCLUSIONS: Migration of CD34+ cells predicts long-term cardiovascular mortality in T2D-CLI patients. An altered paracrine signaling conveys antiangiogenic and proapoptotic features from CD34+ cells to the endothelium. This damaging interaction may increase the risk for life-threatening complications.

Can Nurse-Based Management Screening Ensure Adequate Outcomes in Patients With Gestational Diabetes? A Comparison of 2 Organizational Models.

BACKGROUND: Gestational diabetes mellitus (GDM) is an impaired glucose tolerance with onset or first recognition during pregnancy. The purpose of this study is to evaluate the clinical outcomes of a blood glucose monitoring protocol implemented by nurses and dietitians in a diabetes team to the previously established protocol of direct monitoring of GDM patients by a diabetologist. METHODS: Two groups of patients were formed: The first group was based on a traditional protocol (P1: 230 patients) with patients' blood glucose constantly checked by a diabetologist. In the second structured group (P2: 220 patients) patients were referred to a diabetologist only if they required insulin therapy. RESULTS: The number of medical visits (P2: 1.28 ± 0.70 vs P1: 3.27 ± 1.44; P < .001) and the percentage of patients with hypoglycemia (P2: 6.8% vs P1: 15.2%; P < .006) were found to be lower in group P2 than in group P1. In both groups, a direct relationship was found between a parental history of diabetes and the risk of GDM (odds ratio [OR]: P1 = 2.2 [1.17-4.12]; P2 = 2.5 [1.26-5.12]). In group P1, it was observed that hyperweight gain in patients who were already overweight before becoming pregnant significantly increased the risk of macrosomia (OR: 3.11 [1.39-25.7]), whereas this was not detected in patients in group P2. In group P2, a correlation was found between macrosomia and insulin therapy (OR: 0.066 vs 0.34). In group P1 and group P2, a correlation was observed between insulin therapy and a family history of diabetes (OR: 2.20 vs 2.27), and a body mass index of greater than 30 kg/m in group P2 (OR: 3.0 vs 1.47). CONCLUSIONS: The data we collected show that creating a structured protocol for GDM management reduces the number of medical visits required by patients without increasing the risk of hypoglycemia, macrosomia, or hyperweight gain during pregnancy.

Proinsulin-expressing dendritic cells in type 2 neuropathic diabetic patients with and without foot lesions.

Diabetic neuropathy is the most common complication of diabetes and is frequently associated with foot ischemia and infection, but its pathogenesis is controversial. We hypothesized that proinsulin expression in peripheral blood mononuclear cells is a process relevant to this condition and could represent a link among hyperglycemia, nerve susceptibility, and diabetic foot lesions. We assessed proinsulin expression by using flow cytometry in dendritic cells from control participants and patients with type 2 diabetes with or without peripheral neuropathy or accompanied by diabetic foot. Among 32 non-neuropathic and 120 neuropathic patients with type 2 diabetes, we performed leg electromyography and found average sensory sural nerve conduction velocities of 48 ± 4 and 30 ± 4 m/s, respectively ( P < 0.03). Of those with neuropathy, 42 were without lesions, 39 had foot lesions, and 39 had neuroischemic foot lesions (allux oximetry <30 mmHg). In this well-defined diabetic population, but not in nondiabetic participants, a progressively increasing level of peripheral blood dendritic cell proinsulin expression was detected, which directly correlated with circulating TNF-α levels ( P < 0.002) and multiple conduction velocities of leg nerves ( P < 0.05). These results are consistent with the hypothesis that, in type 2 diabetes, proinsulin-expressing blood cells, possibly via their involvement in innate immunity, may play a role in diabetic peripheral neuropathy and foot lesions.-Sambataro, M., Sambado, L., Trevisiol, E., Cacciatore, M., Furlan, A., Stefani, P. M., Seganfreddo, E., Durante, E., Conte, S., Della Bella, S., Paccagnella, A., dei Tos, A. P. Proinsulin-expressing dendritic cells in type 2 neuropathic diabetic patients with and without foot lesions.

Dabigatran antagonizes growth, cell-cycle progression, migration, and endothelial tube formation induced by thrombin in breast and glioblastoma cell lines.

Thrombin activates its G-coupled seven transmembrane protease-activated receptor (PAR-1) by cleaving the receptor's N-terminal end. In several human cancers, PAR1 expression and activation correlates with tumor progression and metastatization. This provides compelling evidence for the effectiveness of an appropriate antithrombin agent for the adjuvant treatment of patients with cancer. Dabigatran is a selective direct thrombin inhibitor that reversibly binds to thrombin. In this study, we aimed to explore if dabigatran may affect mechanisms favoring tumor growth by interfering with thrombin-induced PAR-1 activation. We confirmed that exposure of tumor cells to thrombin significantly increased cell proliferation and this was coupled with downregulation of p27 and concomitant induction of cyclin D1. Dabigatran was consistently effective in antagonizing thrombin-induced proliferation as well as it restored the baseline pattern of cell cycle protein expression. Thrombin significantly upregulated the expression of proangiogenetic proteins like Twist and GRO-α in human umbilical vascular endothelial cells (HUVEC) cells and their expression was significantly brought down to control levels when dabigatran was added to culture. We also found that the chemoattractant effect of thrombin on tumor cells was lost in the presence of dabigatran, and that the thrombin antagonist was effective in dampening vascular tube formation induced by thrombin. Our data support a role of thrombin in inducing the proliferation, migration, and proangiogenetic effects of tumor cells in vitro. Dabigatran has activity in antagonizing all these effects, thereby impairing tumor growth and progression. In vivo models may help to understand the relevance of this pathway.

Low-Dose Paclitaxel Reduces S100A4 Nuclear Import to Inhibit Invasion and Hematogenous Metastasis of Cholangiocarcinoma.

Nuclear expression of the calcium-binding protein S100A4 is a biomarker of increased invasiveness in cholangiocarcinoma, a primary liver cancer with scarce treatment opportunities and dismal prognosis. In this study, we provide evidence that targeting S100A4 nuclear import by low-dose paclitaxel, a microtubule-stabilizing agent, inhibits cholangiocarcinoma invasiveness and metastatic spread. Administration of low-dose paclitaxel to established (EGI-1) and primary (CCA-TV3) cholangiocarcinoma cell lines expressing nuclear S100A4 triggered a marked reduction in nuclear expression of S100A4 without modifying its cytoplasmic levels, an effect associated with a significant decrease in cell migration and invasiveness. While low-dose paclitaxel did not affect cellular proliferation, apoptosis, or cytoskeletal integrity, it significantly reduced SUMOylation of S100A4, a critical posttranslational modification that directs its trafficking to the nucleus. This effect of low-dose paclitaxel was reproduced by ginkolic acid, a specific SUMOylation inhibitor. Downregulation of nuclear S100A4 by low-dose paclitaxel was associated with a strong reduction in RhoA and Cdc42 GTPase activity, MT1-MMP expression, and MMP-9 secretion. In an SCID mouse xenograft model, low-dose metronomic paclitaxel treatment decreased lung dissemination of EGI-1 cells without significantly affecting their local tumor growth. In the tumor mass, nuclear S100A4 expression by cholangiocarcinoma cells was significantly reduced, whereas rates of proliferation and apoptosis were unchanged. Overall, our findings highlight nuclear S100A4 as a candidate therapeutic target in cholangiocarcinoma and establish a mechanistic rationale for the use of low-dose paclitaxel in blocking metastatic progression of cholangiocarcinoma. Cancer Res; 76(16); 4775-84. ©2016 AACR.

A family with factor X deficiency from Argentina: a compound heterozygosis because of the combination of a new mutation (Gln138Arg) with an already known one (Glu350Lys).

The objective was to investigate a family from Argentina. The proposita was a 51-year-old woman who had a moderate bleeding tendency. Some of her children showed a mild bleeding tendency. Her mother and the husband were asymptomatic. Clotting, immunological and molecular biology techniques were used. Partial thromboplastin, prothrombin, Russell Viper venom-clotting times were moderately prolonged in the proposita, whereas they were slightly prolonged in the children and in her mother. Factor X (FX) activity was about 2-3% of normal in all assay systems. FX antigen was less than 5%. Other clotting factors and platelet were normal. Genetic analysis showed a compound heterozygosis: combination of a 'new' mutation (Gln138Arg) with an already known mutation (Glu350Lys). The children had intermediate FX levels (35-63% of normal) and were carriers of one of the two mutations present in the proposita. This is the first observation of a FX deficiency in Argentina.

Enteral nutrition at home and in nursing homes: an 11-year (2002-2012) epidemiological analysis.

BACKGROUND: Home enteral nutrition (HEN) is a well-established extra-hospital therapy that can reduce the risk of malnutrition, ensure the rapid discharge of patients from hospital and significantly reduce health care expenditure. The data reported in this study allow us to understand the relationships between mortality, the place of treatment either at patients' homes (PH) or in nursing homes (NHR) and nutritional status. METHODS: Patients were analyzed according to age, gender, underlying disease, the Karnofsky Index, type of enteral access device (nasogastric tube or percutaneous endoscopic gastrostomy), weight and Body Mass Index (BMI). The duration of HEN therapy was then calculated and the outcome was established on patient mortality or survival. RESULTS: Over an 11-year period, 3246 subjects were administered HEN therapy. The mean duration of HEN therapy was equal to 312±487 days at PH and 398±573 in NHR. The mean incidence is 406±58 patients/million inhabitants/year at PH and 319±44 in NHR (mean prevalence rate: 464±129 cases/million inhabitants at PH compared to 478±164 in NHR). Analysis of variance was used for continuous variables. The study reveals that >8% (8.6% at PH; 8.5% in NHR) of patients die within 10 days of starting HEN therapy. CONCLUSIONS: The study shows a progressive increase in HEN therapy and highlights clinical, organizational and ethical issues, which also need to be analyzed in relation to the progressively aging population.

Prevalence of hypertension and its complications in congenital prekallikrein deficiency: analysis of all reported cases and clinical significance.

The extra coagulation effects of prekallikrein and of the other factors of the contact phase of blood clotting have received great attention in the past few years.The clinical observation that hypertension was present in two families with congenital prekallikrein deficiency prompted a survey of all reported cases of this disorder.Altogether, 89 cases of proven prekallikrein deficiency have been described in the literature. Hypertension or vascular complications of it were found in 21 patients (12 men and nine women). If the analysis is limited to patients over 25 years of age, the number becomes 21 out of 64 cases (38.2%).This prevalence is much higher than that seen for other conditions occasionally found in patients with prekallikrein deficiency, namely hyperthyroidism, lupus erythematosus, chronic lymphocytic leukemia, kidney malformation, peptic ulcer, and myelofibrosis (1-2%).These results indicate the need to investigate further the relation between prekallikrein deficiency and hypertension.

Complex history of the discovery and characterization of congenital factor X deficiency.

Factor X (FX) plays a pivotal role in blood coagulation. FX represents the point where all coagulation systems converge and, once activated, it converts prothrombin into thrombin. The discovery and definition of FX are based on the description between 1956 and 1957 about three patients and their families with a peculiar defect later demonstrated to be almost identical. These patients were an American (Mr. Stuart), a British (Ms. Prower), and a Swiss with Italian background (infant Delia B). We stated "almost identical" because immunological and molecular biology studies subsequently revealed that even though the basic clotting defect was identical, the FX protein level and the mutation were different in each case. Mr. Stuart had no FX protein in his plasma and the mutation was Val298Met (homozygote). Ms. Prower instead had a normal level of FX protein and the mutation was Arg287Trp + Asp282Asn (compound heterozygote). Unfortunately, the status of the Swiss patient in this regard is not known. Subsequent studies described a few major variants (FX Friuli, FX Melbourne, FX Padua, and other similar patients), which showed peculiar activation patterns (FX Friuli had a normal Russell viper venom clotting time; FX Melbourne was defective only in the intrinsic coagulation system; FX Padua, on the contrary, was defective only in the extrinsic coagulation system). All these studies have informed on the great heterogeneity and complexity of the FX defect. The story of the discovery and classification of FX deficiency has contributed considerably to our understanding of blood coagulation. The three original families and the families of the major variants, together with the researchers that discovered them, should be remembered with deep respect and gratitude.

Myocardial infarctions and other acute coronary syndromes in rare congenital bleeding disorders: a critical analysis of all reported cases.

OBJECTIVE: To investigate the occurrence of myocardial infarction or other acute coronary syndromes in rare congenital bleeding disorders. PATIENTS: All patients with factor I (FI), factor II (FII), factor V (FV), factor VII (FVII), factor X (FX), factor XI (FXI), or factor XIII (FXIII) deficiency or abnormality reported to have presented a myocardial infarction or another acute coronary syndrome were investigated. The condition had to be demonstrated by objective means, including a coronary/angiography. Cases of stable angina were excluded. RESULTS: A total of 53 patients (4 had FI, 2 had FV, 2 had FVII, 36 had FXI, 1 had FXIII deficiency, and 8 patients had platelet disorders) met the inclusion criteria . No patient with FII or FX deficiency and acute coronary disease met the inclusion criteria. In the majority of patients, common risk factors were present, namely hypertension, hypercholesterolemia, smoking, and diabetes. Replacement therapy was involved in 5 cases. CONCLUSION: The congenital hypocoagulability present in these patients was unable to allow a protection from acute coronary diseases. The significance of the findings is discussed.

Venous thrombosis in von Willebrand disease as observed in one centre and as reported in the literature.

The aim of this article was to investigate the prevalence of venous thrombosis in patients with von Willebrand disease. Personal records on 486 patients were reevaluated together with a time unlimited PubMed search. The venous thrombotic event had to be proven by objective means. Only cases of congenital von Willebrand disease were taken into consideration and all types of the diseases were included. No case of venous thrombosis was reported in our cohort of patients. On the contrary, 33 patients with proven venous thrombosis were gathered from the literature (17 cases of deep venous thrombosis with or without pulmonary embolism; isolated pulmonary embolism was seen in seven instances, superficial veins or portal system thrombosis was present in the remaining cases). Associated risk factors, mainly replacement therapy, were present in 26 cases. Therapeutic approach was usually based on heparin and Coumadin. Overall results were fair or good, as no fatalities occurred.

Comparison of three different immunoassays in the diagnosis of heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is caused by platelet activating antibodies that recognize platelet factor 4/heparin (PF4/H) complexes. Laboratory testing plays a key role in the diagnosis of HIT. As functional assays are unfeasible for most clinical laboratories, antigen binding assays are commonly used in routine testing. However, their low specificity leads to overdiagnosis of HIT. Therefore, it is advisable to improve screening tests in this setting. METHODS: Blood samples from 114 patients in whom HIT was suspected were investigated using a chemiluminescence test (HemosIL® AcuStar HIT-IgG), a PF4/H IgG enzyme immunoassay (Lifecodes PF4 IgG), an IgG-specific lateral flow immunoassay heparin-induced thrombocytopenia (LFI-HIT, STic Expert® HIT) and the heparin-induced platelet aggregation (HIPA) test. RESULTS: Twenty-nine (25.4%) out of 114 subjects with suspected HIT had a positive HIPA test. None of patients with a 4Ts score <4 were positive at HIPA. HemosIL® AcuStar HIT-IgG showed the best performance in term of sensitivity and specificity when used as single test. Receiver operating characteristic (ROC) analysis showed optimization of sensitivity and specificity using a cut-off of 1.13 U/mL (0.95 and 0.98, respectively). As an alternative approach, a strategy based on screening samples by STic Expert® HIT and then retesting positive results by Lifecodes PF4 IgG (cut-off 1 OD) or HemosIL® AcuStar HIT-IgG (cut-off 1.3 U/mL) showed a performance compared to a single test approach by HemosIL® AcuStar HIT-IgG. CONCLUSIONS: The HemosIL® AcuStar HIT or a combinatorial approach with the STic Expert® HIT and the PF4/H IgG enzyme immunoassay provide an accurate diagnosis of immune HIT.

The old and the new in prekallikrein deficiency: historical context and a family from Argentina with PK deficiency due to a new mutation (Arg541Gln) in exon 14 associated with a common polymorphysm (Asn124Ser) in exon 5.

Prekallikrein (PK) is one of the clotting factors involved in the contact phase of blood. PK has an important historical role as its deficiency state represents the second instance of a clotting defect without bleeding manifestations, the first one being factor XII deficiency. PK deficiency is a rare clotting disorder. Moreover, only 11 patients have been investigated so far by molecular biology techniques. In this article, we briefly review some of the history around PK and also present some recent data on a newly identified family from Argentina suffering from PK deficiency. Two patients are homozygous whereas other family members are heterozygous. PK activity and antigen are 1% of normal in the homozygotes and around 60 to 70% of normal in the heterozygotes. As expected, all patients are asymptomatic of bleeding or thrombosis presentations. However, the two homozygotes showed essential hypertension. The PK deficiency in this family is due to a new mutation (Arg541Gln) in exon 14. The defect segregates together with a known polymorphism, Asn124Ser, in exon 5. The significance of the presence of hypertension in the two homozygotes is discussed in view of the extra coagulation effects of PK on vasodilation, vessel permeability, and the control of blood pressure. Structure function analysis indicates that the substitution of Arg with Gln probably impedes the transmembrane diffusion of the molecule, which therefore cannot be secreted in the homozygotes. The presence of hypertension in patients with PK deficiency has been previously reported in some but not all patients. Future research activities will probably concentrate on the effect of PK and other contact phase factors on the vascular system.

Serum Thrombopoietin and cMpl Expression in Thrombocytopenia of Different Etiologies.

The relationship between thrombopoietin (TPO) and its receptor cMpl in thrombocytopenic conditions has not been entirely clarified. To elucidate this interplay may expand the spectrum of indications of TPO mimetics. In this study we have explored the relationship between TPO and cMpl in platelets and megakaryocytes of 43 patients with thrombocytopenia due to idiopathic thrombocytopenic purpura (ITP), bone marrow hypoplasia, myelodysplastic syndromes (MDS), and familial thrombocytopenia. Data were compared to cMpl and TPO in patients with a normal platelet count and in patients with thrombocytosis due to essential thrombocythemia (ET). All but familial patients showed higher TPO compared to controls. All thrombocytopenic states were invariably associated with increased expression of platelet cMPL compared to healthy controls. ET patients showed normal TPO and a trend toward a reduced cMpl expression. Immunofluorescence of bone marrow sections from patients with ITP and MDS failed to show a peculiar pattern compared to controls. Multiple mechanisms regulate TPO and cMpl in thrombocytopenic conditions.

Discrepant ratios of arterial versus venous thrombosis in hemophilia A as compared with hemophilia B.

The occurrence of thrombosis in patients with congenital bleeding disorders represents an exceptional event. Hemophilia A and hemophilia B patients have been showed to present both arterial and venous thrombosis (85 cases of arterial thrombosis and 34 cases of venous thrombosis). The great majority of arterial thrombosis are myocardial infarction or other acute coronary syndromes, whereas the majority of venous thrombosis are deep vein thrombosis and/or pulmonary embolisms. However there are discrepancies in the proportion of arterial and venous thrombosis seen in hemophilia A versus hemophilia B. The ratio of arterial versus venous thrombosis in hemophilia A is 3.72 whereas that for hemophilia B is 1.12. This indicates that arterial thrombosis is more frequent in hemophilia A as compared to hemophilia B and the opposite is true for venous thrombosis. The potential significance of this discrepancy is discussed.

A family with factor-XI deficiency due to a compound heterozygosis between Gln 47 Pro (new mutation) in exon 3 and Leu 619 Pro in exon 15.

A new factor XI mutation (Gln 47 Pro) has been found in combination with another known mutation (Leu 619 Pro) in a female patient with FXI deficiency and a moderate bleeding tendency. FXI activity and antigen in the proposita were 2% activity and less than 5% of normal, respectively. The parents are not consanguineous and are asymptomatic. The father is heterozygote for the new mutation whereas the mother is heterozygote for the known mutation. Other family members are heterozygotes for either one of the two mutations. The new mutation is not a polymorphism as it was not found in the population of the area. The geographical area, north-east of Italy, of the present family is the same area where a cluster of another new mutation (Ile 436 Lys) was recently reported. No relation was found between the present family and those with the previous mutation.

Bleeding manifestations apparently unrelated to coagulation or other organic disorders: A tentative classification and diagnostic clues.

OBJECTIVE: To study the features of bleeding conditions apparently not associated with vascular, platelet, or clotting dysfunctions. METHOD: Conditions that may meet these criteria are: Münchausen syndrome factitious or fictitious, suicidal or homicidal bleeding, bleeding due to self-punishment, stigmatization, the battered child syndrome, and psychogenic bleeding. RESULTS: The importance of these variegate conditions is not trivial in clinical practice. Differential diagnosis may be difficult and involve other specialists besides hematologists. Occasionally, invasive procedures are involved. DISCUSSION: The occurrence of bleeding in patients, without a clotting defect or a systemic disorder and a negative family history for bleeding represents a diagnostic challenge. A careful examination of the physical and psychological status of the patient and an appropriate evaluation of the environment in which bleeding occurs, is always needed.

Platelet-derived growth factor-D and Rho GTPases regulate recruitment of cancer-associated fibroblasts in cholangiocarcinoma.

UNLABELLED: Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer-associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial-mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)-expressing human male CCA cell line (EGI-1) after xenotransplantation into severe-combined-immunodeficient mice, (3) expression of platelet-derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF-D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF-D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α-SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α-SMA-expressing CAFs, which were negative for EGFP and the human Y-probe, but positive for the murine Y-probe. CCA cells were strongly immunoreactive for PDGF-A and -D, whereas CAFs expressed PDGF receptor (PDGFR)ß. PDGF-D, a PDGFRß agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF-D and CCA conditioned medium and was significantly inhibited by PDGFRß blockade with Imatinib and by silencing PDGF-D expression in CCA cells. In fibroblasts, PDGF-D activated the Rac1 and Cdc42 Rho GTPases and c-Jun N-terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF-D-induced fibroblast migration. CONCLUSION: CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF-D, which stimulates fibroblast migration through PDGFRß and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF-D pathway may offer a novel therapeutic approach.