IVIg treatment increases thrombin activation of platelets and thrombin generation in paediatric patients with immune thrombocytopenia.

Clinical manifestations and laboratory parameters of haemostasis were investigated in 23 children with newly diagnosed immune thrombocytopenia (ITP) before and after intravenous immunoglobulin (IVIg) treatment. ITP patients with platelet counts of less than 20 × 109 /L and mild bleeding symptoms, graded by a standardized bleeding score (BS), were compared with healthy children with normal platelet counts and children with chemotherapy-related thrombocytopenia. Markers of platelet activation and platelet apoptosis in the absence and presence of platelet activators were analysed by flow cytometry; thrombin generation in plasma was determined. ITP patients at diagnosis presented with increased proportions of platelets expressing CD62P and CD63 and activated caspases, and with decreased thrombin generation. Thrombin-induced activation of platelets was reduced in ITP compared with controls, while increased proportions of platelets with activated caspases were observed. Children with a higher BS had lower proportions of CD62P-expressing platelets compared with those with a lower BS. IVIg treatment increased the number of reticulated platelets, the platelet count to more than 20 × 109 /L and improved bleeding in all patients. Decreased thrombin-induced platelet activation, as well as thrombin generation, were ameliorated. Our results indicate that IVIg treatment helps to counteract diminished platelet function and coagulation in children with newly diagnosed ITP.

Platelets express adaptor proteins of the extrinsic apoptosis pathway and can activate caspase-8.

BACKGROUND: Apoptotic pathways in platelets are important for their survival and function. Platelet apoptosis may be involved in the pathogenesis of immune thrombocytopenia (ITP), an autoimmune-mediated disease. In contrast to the intrinsic apoptosis pathway, not much is known about the extrinsic pathway mechanisms in platelets. OBJECTIVES: To investigate the expression of proteins involved in the extrinsic apoptosis pathway, including the death receptors, adaptor and regulator proteins in human platelets. To determine a possible trigger of the extrinsic apoptosis pathway in platelets. METHODS: To investigate the expression of key markers of the extrinsic pathway we used targeted immunofluorescence and flow cytometry assays. To study their expression and interaction we performed Western blotting and co-immunoprecipitation. Treated platelets with different apoptosis triggers were subjected to flow cytometry. RESULTS: We could identify the protein expression of the pro-apoptotic proteins TRADD (Tumor Necrosis Factor Receptor type 1- Associated DEATH Domain protein), TRAF2/5, (TNF Associated Factor) and DEDAF (Death Effector Domain- Associated Factor), FADD (Fas-Associated protein with death domain) as well as the anti-apoptotic proteins DJ-1 (Deglycase 1) and c-FLIP in human platelets. ABT-737 treatment induced a disruption in the co-localization of DJ-1 with FADD. Platelets treated with ABT-737 showed an activation in caspase-3 and -8. The exposure to TNF (Tumor Necrosis Factor), FasL (Fas ligand), and TWEAK or to plasma derived from ITP patients, did not lead to changes in caspase-3 and -8 activation in platelets. CONCLUSIONS: Human platelets express some proteins of the extrinsic apoptosis pathway which can be modulated only by ABT-737 treatment. However so far, no other apoptosis trigger or interaction with an external receptor have been yet identified.

Increased levels of IL-10 and IL-1Ra counterbalance the proinflammatory cytokine pattern in acute pediatric immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disease which leads to accelerated platelet clearance. We investigated the plasma cytokine, chemokine and growth factor signatures and their clinical significance in pediatric ITP patients during acute, chronic and follow-up stages as well as the effects of intravenous immunoglobulin (IVIg) treatment, by using the Multiplex technology. In acute ITP before and/or after IVIg treatment we found significantly increased plasma levels of the pro- (tumour necrosis factor-α (TNF-α), interleukin IL-15) and anti- (IL-1 receptor antagonist (Ra), IL-10 and the growth factor interferon γ-induced protein (IP-10)) inflammatory cytokines, compared to healthy controls. Except for IL1-Ra, these cytokines decreased to normal levels in chronic patients. In contrast, growth-regulated α protein (GRO) and soluble CD40 ligand (sCD40L), known as platelet-derived molecules, were found to be significantly decreased in acute and increased in chronic ITP patients compared to healthy controls. GRO levels positively correlated with the platelet counts in the follow-up and chronic cohort. Monocyte counts showed a significant positive correlation only with IP-10 levels in acute ITP after IVIg treatment and follow-up patients. Expression levels of mRNAs for macrophage inflammatory protein MIP1-ß, IL-1Ra and GRO determined in peripheral blood mononuclear cells (PBMCs) were significantly reduced in both acute and chronic ITP compared to controls. Our findings suggest that the different clinical presentation of acute and chronic pediatric ITP and to a lesser extent the IVIg treatment effects are characterized overall by a counterbalanced cytokine, chemokine and growth factor pattern response that might exert a pathogenic role in this disease.

Pericytes in Primary Familial Brain Calcification.

Pericytes are perivascular cells along capillaries that are critical for the development of a functional vascular bed in the central nervous system and other organs. Pericyte functions in the adult brain are less well understood. Pericytes have been suggested to mediate functional hyperemia at the capillary level, regulate the blood-brain barrier and to give rise to scar tissue after spinal cord injury. Furthermore, pericyte loss has been suggested to precede cognitive decline in mouse models of Alzheimer's disease. Despite this observation, there is no convincing causality between pericyte loss and the pathogenesis of Alzheimer's disease. However, recent loss-of-function mutations in PDGFB and PDGFRB genes have implicated pericytes as the principle cell type affected in primary familiar brain calcification (PFBC), a neuropsychiatric disorder with dominant inheritance. Here we review the role of the PDGFB/PDGFRB signaling pathway in pericyte development and briefly discuss homeostatic functions of pericytes in the brain. We provide an overview of recent studies with mouse models of PFBC and discuss suggested pathogenic mechanisms for PFBC with special reference to pericytes.

Right and Left Ventricular Strain Patterns After the Atrial Switch Operation for D-Transposition of the Great Arteries-A Magnetic Resonance Feature Tracking Study.

Introduction: Adult survivors of the atrial switch operation for transposition of the great arteries present with a systemic morphologic right ventricle and a subpulmonary morphologic left ventricle. This physiology can be considered a model for the effects of long-term right ventricular pressure overload and of decreased left ventricular afterload. We aimed to determine the impact of these chronically altered loading conditions on myocardial deformation of the ventricles. Materials and methods: Two-dimensional steady state free precession cine images of 29 patients after atrial repair (age 29 ± 7 years) and 19 controls (24 ± 10 years; n.s.) were post-processed with feature tracking software (TomTec 2D CPA). Volumes, ejection fractions, global and free wall longitudinal and circumferential strains of both ventricles were compared between both groups. Results: Systemic right ventricular global longitudinal strain was decreased in patients compared to controls (-12.9 ± 3.3% vs. -18.9 ± 4.6%, p < 0.001), while right ventricular circumferential strain was unchanged (-15.8 ± 3.4% vs. -15.1 ± 5%; n.s.). Left ventricular longitudinal strain was similar in both groups (-17 ± 5.6% vs. -17.5 ± 4.6%; n.s.), but global left ventricular circumferential strain was lower in patients (-20.7 ± 4.1% vs. -27.3 ± 4.5%, p < 0.001). The systemic right ventricle, compared to the systemic left ventricle, showed decreased global longitudinal (p < 0.001) and circumferential strain (p < 0.001). The subpulmonary left ventricle, compared to the subpulmonary right ventricle, demonstrated similar longitudinal (p = 0.223) but higher circumferential strain (p < 0.001). Conclusions: In patients after atrial switch repair for transposition of the great arteries, the systemic right ventricle shows poor longitudinal strain, but maintains normal right ventricular circumferential strain. The left ventricle shows higher circumferential strain than the right ventricle, in both systemic and subpulmonary positions.

Satellite cells fail to contribute to muscle repair but are functional in Pompe disease (glycogenosis type II).

Pompe disease, which is due to acid alpha-glucosidase deficiency, is characterized by skeletal muscle dysfunction attributed to the accumulation of glycogen-filled lysosomes and autophagic buildup. Despite the extensive tissue damages, a failure of satellite cell (SC) activation and lack of muscle regeneration have been reported in patients. However, the origin of this defective program is unknown. Additionally, whether these deficits occur gradually over the disease course is unclear. Using a longitudinal pathophysiological study of two muscles in a Pompe mouse model, here, we report that the enzymatic defect results in a premature saturating glycogen overload and a high number of enlarged lysosomes. The muscles gradually display profound remodeling as the number of autophagic vesicles, centronucleated fibers, and split fibers increases and larger fibers are lost. Only a few regenerated fibers were observed regardless of age, although the SC pool was preserved. Except for the early age, during which higher numbers of activated SCs and myoblasts were observed, no myogenic commitment was observed in response to the damage. Following in vivo injury, we established that muscle retains regenerative potential, demonstrating that the failure of SC participation in repair is related to an activation signal defect. Altogether, our findings provide new insight into the pathophysiology of Pompe disease and highlight that the activation signal defect of SCs compromises muscle repair, which could be related to the abnormal energetic supply following autophagic flux impairment.

MxB is an interferon-induced restriction factor of human herpesviruses.

The type I interferon (IFN) system plays an important role in controlling herpesvirus infections, but it is unclear which IFN-mediated effectors interfere with herpesvirus replication. Here we report that human myxovirus resistance protein B (MxB, also designated Mx2) is a potent human herpesvirus restriction factor in the context of IFN. We demonstrate that ectopic MxB expression restricts a range of herpesviruses from the Alphaherpesvirinae and Gammaherpesvirinae, including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), and Kaposi's sarcoma-associated herpesvirus (KSHV). MxB restriction of HSV-1 and HSV-2 requires GTPase function, in contrast to restriction of lentiviruses. MxB inhibits the delivery of incoming HSV-1 DNA to the nucleus and the appearance of empty capsids, but not the capsid delivery to the cytoplasm or tegument dissociation from the capsid. Our study identifies MxB as a potent pan-herpesvirus restriction factor which blocks the uncoating of viral DNA from the incoming viral capsid.

Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1.

Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate.IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time-controlled windows for HSV-1 replication. High Rep protein levels in S/G2 phase support AAV2 replication and inhibit HSV-1 replication. Conversely, low Rep protein levels in G1 phase permit HSV-1 replication but are insufficient for AAV2 replication. This allows both viruses to productively replicate in distinct sets of dividing cells.

Atrial Function after the Atrial Switch Operation for Transposition of the Great Arteries: Comparison with Arterial Switch and Normals by Cardiovascular Magnetic Resonance.

OBJECTIVES: The atria serve as reservoir, conduit, and active pump for ventricular filling. The performance of the atrial baffles after atrial switch repair for transposition of the great arteries may be abnormal and impact the function of the systemic right ventricle. We sought to assess atrial function in patients after atrial repair in comparison to patients after arterial switch repair (ASO) and to controls. METHODS: Using magnetic resonance imaging, atrial volumes and functional parameters were measured in 17 patients after atrial switch repair, 9 patients after ASO and 10 healthy subjects. RESULTS: After the atrial switch operation, the maximum volume of the pulmonary venous atrium was significantly enlarged, but not of the systemic venous atrium. In both patients groups, independently from the surgical technique used, the minimum atrial volumes were elevated, which resulted in a decreased total empting fraction compared with controls (P < .01). The passive empting volume was diminished for right atrium, but elevated for left atrium after atrial switch and normal for left atrium after ASO; however, the passive empting fraction was diminished for both right atrium and left atrium after both operations (P < .01). The active empting volume was the most affected parameter in both atria and both groups and active empting fractions were highly significantly reduced compared with controls. CONCLUSION: Atrial function is abnormal in all patients, after atrial switch and ASO repair. The cyclic volume changes, that is, atrial filling and empting, are reduced when compared with normal subjects. Thus, the atria have lost part of their capacity to convert continuous venous flow into a pulsatile ventricular filling. The function of the pulmonary venous atrium, acting as preload for the systemic right ventricle, after atrial switch is altered the most.

Laboratory diagnosis of ruminant abortion in Europe.

Abortion in ruminants is a major cause of economic loss worldwide, and the management and control of outbreaks is important in limiting their spread, and in preventing zoonotic infections. Given that rapid and accurate laboratory diagnosis is central to controlling abortion outbreaks, the submission of tissue samples to laboratories offering the most appropriate tests is essential. Direct antigen and/or DNA detection methods are the currently preferred methods of reaching an aetiological diagnosis, and ideally these results are confirmed by the demonstration of corresponding macroscopic and/or histopathological lesions in the fetus and/or the placenta. However, the costs of laboratory examinations may be considerable and, even under optimal conditions, the percentage of aetiological diagnoses reached can be relatively low. This review focuses on the most commonly occurring and important abortifacient pathogens of ruminant species in Europe highlighting their epizootic and zoonotic potential. The performance characteristics of the various diagnostic methods used, including their specific advantages and limitations, are discussed.

Mammographic and ultrasonographic comparison between intraoperative radiotherapy (IORT) and conventional external radiotherapy (RT) in limited-stage breast cancer, conservatively treated.

OBJECTIVE: Assess mammographic and echographic modifications in mild cases of breast cancer (suitable for conservative surgery) after intraoperatory radio treatment (IORT) as opposed to conventional post-operative radiotherapy (RT). MATERIALS AND METHODS: We report data from 45 patients in each group (IORT and RT). All patients were examined using the same mammographic and ecographic equipment at 6, 12 and 24 months after treatment. We focused on structural alterations, edema and others, and quantified them using pre-established (unbiased) protocols. Both patient examination and subsequent assessment of the results were performed by radiologists with exepertise in breast cancer evaluation. RESULTS: At 6 months, IORT patients showed slightly more pronounced structural distortions and oedema than RT patients; these differences became more apparent at 12 months, with the addition of fat necrosis and/or calcifications. These alterations were evident and consistent under both mammographic and ecographic examination, and became even more pronounced at 24 months. At this stage, RT patients showed minimal alterations of the tissue (apart from normal post-surgical scarring), whereas IORT patients showed virtually no improvement over the preceding 12-month period. CONCLUSION: We show radiological alterations in post-operative breast cancer are significantly more pronounced in patients treated with IORT as opposed to RT.