Platelet activation markers in patients with venous thromboembolism without predisposing factors

A constant in vitro hypersensitivity of platelets (adenosine diphosphate) has been suggested as a risk factor for arterial and even venous thrombosis. Our aim was to determine phenotypic and functional alterations of platelets by flow cytometry as potential prothrombotic risk factors in patients with a history of unexplained spontaneous venous thrombosis. Forty-nine patients with a history of spontaneous venous thrombosis and no inherited or acquired thrombophilic risk factors were compared with a reference group of 39 healthy volunteers. Flow cytometry (FACS) was used to analyze the surface expression of CD62 (P-selectin) and CD63 in nonactivated platelets and after in vitro stimulation with adenosine diphosphate and thrombin receptor activator peptide 6. Mean fluorescence intensity of CD62 and CD63 surface expression as well as percentage of CD62 and CD63 positive cells and binding index differed in patients with a history of thrombosis compared with the reference group, but failed to reach statistical significance. Similar results were observed after in vitrostimulation with adenosine diphosphate and thrombin receptor activator peptide 6. In conclusion, the expression of CD62 and CD63 of resting and in vitro activated platelets could not be established as a risk factor for spontaneous venous thromboembolism.     

A sensitive bioassay for chicken interleukin-18 based on the inducible release of preformed interferon-gamma

Conventional tests for measuring the biological activity of chicken interleukin (IL)-18 require primary chicken spleen cells. We now describe a sensitive bioassay that is based on interleukin-18-induced release of interferon (IFN)-gamma by a permanent chicken cell line. In B19-2D8 cells, cytoplasmically stored interferon-gamma is quickly secreted in response to interleukin-18 exposure.     

Chorioallantoic membrane angiogenesis model for tissue engineering: a new twist on a classic model

Tissue-engineering (TE) applications include the isolation, culture, and seeding of cells into a suitable matrix or scaffold before in vivo transplantation. After transplantation, vascularization of the scaffold is a principal limiting factor for cell viability for the first 6-8 days posttransplantation. A model for systematic analysis of this process has been developed. Fertilized White Leghorn eggs were incubated (at 37.8 degrees C in 60% relative humidity) and opened on day 3 of incubation. Preadipocyte-seeded fibrin constructs were implanted in a specially designed plastic cylinder and placed through the opening on the surface of the chorioallantoic membrane (CAM) on day 8 of incubation. Vascularization of the constructs by chorioallantoic blood vessels was assessed for up to 8 days posttransplantation. The survival rate for embryos receiving transplanted constructs was about 90%. Histology confirmed transplant cell viability at day 4 posttransplantation and vascularization of the constructs by avian endothelial cells began at this time. A new in vivo model to study the effect of angiogenesis in TE constructs, including assessments of viability, proliferation, and differentiation of transplanted cells and biomaterial properties, is presented. Advantages include easy access to the vascular network of the CAM, lack of immunocompetence, low costs, and avoidance of animal experiments.     

Dopamine in the orbitofrontal cortex regulates operant responding under a progressive ratio of reinforcement in rats

Prefrontocortical dopamine (DA) plays an essential role in the regulation of cognitive functions and behavior. The orbitofrontal cortex (OFC) receives a dopaminergic projection from the ventral tegmental area and is particularly important for goal-directed appetitive behaviors and for the neural representation of reward value. We here examined the effects of DA receptor blockers locally infused into the OFC, on instrumental behavior under a progressive schedule of reinforcement. After continuous reinforcement training (lever pressing for casein pellets) rats received bilateral intra-OFC-infusions of the DA D1-receptor antagonist SCH23390 (3 μg/0.5 μl), the DA D2-receptor antagonist sulpiride (3 μg/0.5 μl), or phosphate buffered saline through chronically indwelling cannulae. Immediately after infusion they were tested under a time-constrained progressive ratio schedule of reinforcement (3, 6, 9, 12, … lever presses for 1 casein pellet within 180 s). Both SCH23390 and sulpiride led to a significant reduction of the break point (cessation to respond to the increasing criterion of instrumental effort) compared to vehicle infusions. A food preference test revealed no drug effects on the amount of consumed pellets and on the preference of casein pellets over laboratory chow. Leftward shifts of the break point in progressive ratio tasks indicate a disturbance of the mechanisms that translate motivation into appetitive behavior under conditions of increasing instrumental effort. Therefore, our data indicate that orbitofrontal dopamine is necessary for reward-related instrumental behavior.     

Infantile hemangioma is a proliferation of beta 4-negative endothelial cells adjacent to HLA-DR-positive cells with dendritic cell morphology

Although hemangioma is referred as to the most common tumor in infancy, the underlying pathogenetic events and the biologic origin of this benign vascular neoplasm have remained obscure. By using immunohistochemistry on frozen sections of infantile hemangiomas, we show here that proliferating endothelial cells abundantly expressed alpha(v)beta(3) but lacked beta(4) integrins. Instead, regressing and involuting infantile hemangiomas due to treatment with IFN-alpha showed positive staining of beta(4) integrin, which might point to the angiogenic significance of beta(4) integrin in infantile hemangiomas. Moreover, immunofluorescence analysis revealed the existence of HLA-DR(+), mostly CD68(+) and partly DC-SIGN/CD209(+) cells with dendritic cell morphology in the intimate vicinity of hemangiomatous vessels. Such cells were also detected in the dermal microvascular unit in normal skin. The coupled occurrence of vascular structures and perivascular cells that were stained positive with markers of monocyte or macrophage or dendritic cells might suggest that the development of infantile hemangioma is a result of vasculogenesis, that is, the formation of primitive blood vessels from angioblasts, rather than of angiogenesis, that is, the sprouting of capillaries from preexisting vessels.     

Influence of calcium and ionophore 23187 on tubular phosphate reabsorption

In previous studies it has been demonstrated that a decline of plasma calcium concentration accounts for the decrease of phosphate reabsorption in thyroparathyroidectomized (TPTX) rats undergoing phosphate loading.Microinfusion studies were performed in TPTX rats in order to discriminate between a systemic effect of calcium an a direct renal effect.Thyroparathyroidectomized animals were infused with a phosphate solution continuously. When plasma calcium concentration fell below 1.30 mmol/l, proximal convoluted tubules were microinfused with a phosphate tracer solution for 42 min. After 18 min a calcium chloride-containing solution was applied superficially (superfused) to the area of the microinfused tubule. This elevation of peritubular calcium concentration led to an immediate increase of phosphate reabsorption up to 12% of the microinfused phosphate load within 24 min.In another series of experiments, the calcium specific ionophore A 23187 — a substance which is known to increase intracellular calcium — was superfused on the microinfused tubule. This resulted again in an increase of fractional phosphate reabsorption of about 15% after 24 min. In contrast, when calcium chloride-free as well as ionophore-free solutions were superfused fractional phosphate reabsorption decreased (7%).From these data we conclude that 1. calcium has a direct renal effect on phosphate reabsorption in the absence of parathyroid hormone and 2. intracellular calcium appears to be a major parameter in the regulation of renal phosphate transport under these conditions.This study was supported by Dr. Legerlotz StiftungParts of this study were presented at the fall meeting of the Nephrologische Gesellschaft in Bonn, 1977 and at the spring meeting of the Deutsche Physiologische Gesellschaft in Göttingen, 1978     

Indolent systemic mastocytosis with elevated serum tryptase, absence of skin lesions, and recurrent severe anaphylactoid episodes

BACKGROUND: In contrast to aggressive mastocytosis, patients with indolent systemic mastocytosis (ISM) usually present with urticaria pigmentosa-like skin lesions. In those who lack skin lesions, mastocytosis is often overlooked or confused with endocrinologic, allergic, or other internal disorders. CASE REPORT AND RESULTS: We report on a 33-year-old male patient in whom severe hypotensive episodes occurred after contact with ants or yellow jackets. Since no specific IgE was detected, the serum tryptase concentration was measured and found to be clearly elevated (70 ng/ml). Consecutive staging and examination of the bone marrow revealed ISM. The patient was advised to circumvent insect contact, to take antihistamines on demand, and to carry an epinephrine self-injector for emergency events. In a retrospective analysis of 40 patients seen between 1988 and 2003, only 2 had a life-threatening mediator-related episode before ISM was diagnosed. CONCLUSIONS: Our report confirms the diagnostic value of tryptase in patients with suspected mastocytosis. In addition, the report suggests that the lack of typical skin lesions does not exclude an indolent form of mastocytosis even if the serum tryptase is clearly elevated. Finally, our case further shows that mastocytosis can be an important differential diagnosis to be considered in patients with unexplained anaphylactoid or other mediator-related symptoms.     

Synergistic convergence of microbiota-specific systemic IgG and secretory IgA

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Comparison of a shiga toxin enzyme-linked immunosorbent assay and two types of PCR for detection of shiga toxin-producing Escherichia coli in human stool specimens

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of sporadic cases of disease as well as serious outbreaks worldwide. The spectrum of illnesses includes mild nonbloody diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. STEC produces one or more Stxs, which are subdivided into two major classes, Stx1 and Stx2. The ingestion of contaminated food or water, person-to-person spread, and contact with animals are the major transmission modes. The infective dose of STEC may be less than 100 organisms. Effective prevention of infection is dependent on rapid detection of the causative bacterial pathogen. In the present study, we examined 295 stool specimens for the presence of Stx-producing E. coli by three different methods: an Stx enzyme-linked immunosorbent assay, a conventional PCR assay, and a LightCycler PCR (LC-PCR) assay protocol recently developed by our laboratory at the Institute of Medical Microbiology at Hannover Medical School. Our intent was to compare these three methods and to examine the utility of the STEC LC-PCR protocol in a clinical laboratory. The addition of a control DNA to each sample to clearly discriminate inhibited specimens from negative ones enhanced the accuracy of the LC-PCR protocol. From our results, it can be concluded that LC-PCR is a very useful tool for the rapid and safe detection of STEC in clinical samples.