Modification of collagen matrices for enhancing angiogenesis

The vascularization of engineered tissues in many cases does not keep up with the ingrowth of cells. Nutrient and oxygen supply are not sufficient, which ultimately leads to the death of the invading cells. The enhancement of the angiogenic capabilities of engineered tissues therefore represents a major challenge in the field of tissue engineering. The immobilization of angiogenic growth factors may be useful for enhancing angiogenesis. The most potent angiogenic growth factor specific to endothelial cells, vascular endothelial growth factor (VEGF), occurs in several splice variants. The variant with 165 amino acids both has a high angiogenic activity and a high affinity for heparin. We therefore incorporated heparin molecules into collagen matrices by covalently cross-linking them to amino functions on the collagen. Physical binding of VEGF to the heparin may then prevent a rapid clearance from the implant, while the release rate may become coupled to the degradation of the collagen matrix. The modified matrices were characterized by determination of the extent of the heparin immobilization, the in vitro degradation rate by collagenase. For testing the angiogenic properties, non-modified and heparinized collagen specimens were--either loaded with VEGF or non-loaded--subcutaneously implanted on the back of rats. Specimens were explanted after varying periods of implantation, the dry weights and the hemoglobin contents, as well as immunostained histological sections were evaluated: heparinized collagen matrices loaded with VEGF are vascularized to a substantially higher extent as compared to non-modified matrices.     

In vivo and in vitro effects of imidacloprid on sheep keds (Melophagus ovinus): a light and electron microscopic study

The effects of imidacloprid (Advantage) on sheep keds (Melophagus ovinus Linné 1758) were studied in vivo and in vitro by means of direct observation (monitored on video tape) and by light and electron microscopy. It was found that: 1. Imidacloprid acted rapidly on all motile stages of the sheep keds. Within 3–4 min after exposure they became immobile and their legs and the abdomen started tetanic trembling movements for 15–30 min, leading to death. 2. The compound is apparently taken up by the body, since it also acted on those sheep keds that had been exclusively exposed to imidacloprid-contaminated filter papers. 3. The compound is available and active for more than 1 month in the wool of sheep; even rainfall does not reduce its efficacy. Body contact between treated mother sheep and their lambs protects them from infestation with these ectoparasites. 4. The compound initiates an ultimately lethal destruction of the ganglia, nerve chords and related muscle fibers, as can be seen in electron micrographs. Received: 7 October 2000 / Accepted: 18 October 2000     

The fluorolysis assay,a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers

We have developed a highly sensitive cytolysis test, the fluorolysis assay, as a simple nonradioactive and inexpensive alternative to the standard 51Cr-release assay. P815 cells were stably transfected with a plasmid expressing the enhanced green fluorescent protein (EGFP) gene. These target cells were coated with or without cognate peptide or anti-CD3 Ab and then incubated with CD8(+) T cells to allow antigen-specific or nonspecific lysis. The degree of target cell lysis was measured using flow cytometry to count the percentage of viable propidium iodide(-) EGFP(+) cells, whose numbers were standardized to a reference number of fluorochrome-linked beads. By using small numbers of target cells (200-800 per reaction) and extended incubation times (up to 2 days), the antigen-specific cytolytic activity of one to two activated CD8(+) T cells of a CTL line could be detected. The redirected fluorolysis assay also measured the activity of very few (> or =6) primary CD8(+) T cells following polyclonal activation. Importantly, antigen-specific lysis by small numbers (> or =25) of primary CD8(+) T cells could be directly measured ex vivo. This exquisite sensitivity of the fluorolysis assay, which was at least 8-33-folds higher than an optimized 51Cr-release assay, allows in vitro and ex vivo studies of immune responses that would otherwise not be possible due to low CTL numbers or frequencies.     

Expression of inducible nitric oxide synthase in skin lesions of patients with american cutaneous leishmaniasis

Cytokine-inducible (or type 2) nitric oxide synthase (iNOS) is indispensable for the resolution of Leishmania major or Leishmania donovani infections in mice. In contrast, little is known about the expression and function of iNOS in human leishmaniasis. Here, we show by immunohistological analysis of skin biopsies from Mexican patients with local (LCL) or diffuse (DCL) cutaneous leishmaniasis that the expression of iNOS was most prominent in LCL lesions with small numbers of parasites whereas lesions with a high parasite burden (LCL or DCL) contained considerably fewer iNOS-positive cells. This is the first study to suggest an antileishmanial function of iNOS in human Leishmania infections in vivo.     

The PAR1 (YAP1/SNQ3) gene of Saccharomyces cerevisiae,ac-jun homologue,is involved in oxygen metabolism

Summary The PAR1/SNQ3 gene of S. cerevisiae, which increases resistance to iron chelators in multi-copy transformants, is identical to the YAP1 gene, a yeast activator protein isolated as a functional homologue of the human c-jun oncogene by binding specifically to the AP-1 consensus box. The observed H₂O₂-sensitivity of par1 mutants has been attributed to an increased sensitivity to reduced oxygen intermediates. Accordingly, par1 mutants did not survive an elevated oxygen pressure and were very sensitive to menadione and methylviologene, two chemicals enhancing the deleterious effects of oxygen. The specific activities of enzymes involved in oxygen detoxification, such as superoxide dismutase, glucose 6-phosphate dehydrogenase and glutathione reductase, were decreased in par1 mutants and increased after PAR1 over-expression. As in the case of oxygen detoxification enzymes, the cellular levels of glutathione were similarly affected. These observations indicate that PAR1/YAP1/SNQ3 is involved in the gene regulation of certain oxygen detoxification enzymes. The finding that H₂O₂ promotes DNA-binding of human c-jun is consistent with a similar function for PAR1/YAP1/SNQ3 and c-jun in cellular metabolism.Dedicated to Professor Dr. R. W. Kaplan on the occasion of his 80th birthday     

Gradual and reversible central vestibular reorganization in frog after selective labyrinthine nerve branch lesions

Postlesional reorganization of vestibular afferent and commissural inputs onto second-order vestibular neurons was studied in the isolated brain after unilateral section of the N.VIII, of the ramus anterior (RA) of N.VIII, of the utricular (UT) or of the anterior vertical and horizontal canal nerves in combination. RA nerve section eliminated the inputs from utricular, anterior vertical and horizontal canal organs. In the first set of experiments we recorded field potentials on the operated side of the vestibular nuclei 2 months after RA nerve section. These responses were evoked by electrical stimulation of the RA nerve or of the posterior vertical canal nerve on the operated or on the intact side. The amplitudes of afferent field potentials evoked by stimulation of the spared posterior vertical canal nerve were increased. The amplitudes of afferent field potentials evoked by stimulation of the axotomized RA nerve remained unaltered. After N.VIII section the commissural, but not the afferent, field potentials increased significantly on the operated side following stimulation of N.VIII on the intact and on the operated side, respectively. After UT nerve section no change in commissural but an increase in the amplitude of afferent field potentials from each of the three intact canal nerves was observed on the operated side. In the context of earlier results these findings imply that second-order vestibular neurons, disfacilitated due to afferent nerve section, became receptive to additional, excitatory synaptic inputs, preferentially from intact vestibular nerve afferent fibers. The reduced excitation via afferent nerve inputs was thereby replaced by other afferent nerve inputs from spatially inadequate vestibular end-organs. The synaptic terminals of inactivated afferent nerve fibers were maintained and not repressed. The process of central reorganization after vestibular nerve lesion was activity related, the expansion of signals restricted to inputs from intact fibers, its extent graded and its onset delayed with respect to the onset of corresponding spinal changes and to the onset of postural recovery after the same type of nerve lesion. After the section of RA nerve or of an individual nerve branch the labyrinthine end-organs remained intact and were not removed as after unilateral labyrinthectomy (UL). Peripheral reinnervation of the end-organs was thus excluded after UL, but expected after one of the former types of lesion. Functional reinnervation of the utricular macula was mirrored behaviorally by the reappearance of severe postural deficits following a second RA nerve section. These lesion-induced postural deficits began to reappear if the repeated RA nerve section was delayed with respect to the first by about 3 months. We therefore studied postlesional reorganization in the brainstem 3 months after the first RA nerve section. Reinnervation of the utricular macula was accompanied by a rapid decline of the increased amplitudes of afferent and commissural vestibular field potentials towards control values, suggesting the reversibility of the lesion-induced central reorganization. Electronic Publication     

Mesenteric lymph nodes are critical for the induction of high-dose oral tolerance in the absence of Peyer's patches

We have previously demonstrated the loss of oral tolerance (OT) in lymphotoxinalpha-/- (LTalpha-/-) and TNFalpha / lymphotoxinalpha deficient (TNFalpha / LTalpha-/-) mice which have defective Peyer's patches (PP) and lymph node (LN) development. We have now studied OT in BALB / c mice with differential defects of the gut-associated lymphoid tissue (GALT) caused by inhibition of LTbetaR signaling during fetal development. Treatment of pregnant mice with LTbetaR-IgG (LTbetaRIgG) and TNFR I55-IgG (TNFR55IgG) abrogates the formation of PP (LTbetaRIgG) or of PP and mesenteric LN (MLN) (LTbetaRIgG / TNFRIgG) without genetically deleting the respective cytokine pathways. OT was readily induced in mice without PP but retaining MLN (PP null / LN +). In contrast, OTcould not be induced in mice lacking both MLN and PP (PP null / MLN null) as shown by the inability of these mice to suppress IFN-gamma secretion or DTH reactions. We next assessed OT in 129 x B6 LTalpha-/- mice with and without MLN. Timed treatment of pregnant LTalpha-/- mice with an agonist anti-LTbetaR mAb induces formation of MLN but not of PP in LTalpha-/- mice. LN + LTalpha-/- mice developed OT while LN LTalpha-/- mice were resistant to OT induction. Taken collectively, the data show that in the presence of MLN PP are not required for OT induction and that the presence of MLN is sufficient for OT induction in the LTalpha-/- model.     

Autoimmune diseases in myelodysplastic syndrome favors patients survival: A case control study and literature review

Background

We conducted a monocentric retrospective study of patients with myelodysplastic syndromes (MDS) and autoimmune or inflammatory disorders (AIMs) and a literature review. We analyzed the association with subgroups of the WHO 2016 MDS classification and patient's survival in a case control study. Risk factors associated with survival were analyzed by uni- and multivariate analysis.

Moraxella catarrhalis--infected alveolar epithelium induced monocyte recruitment and oxidative burst

The recruitment of monocytes appears to be a crucial factor for inflammatory lung disease. Alveolar epithelial cells contribute to monocyte influx into the lung, but their impact on monocyte inflammatory capacity is not entirely clear. We thus analyzed the modulation of monocyte oxidative burst by A549 and isolated human alveolar epithelial cells. Epithelial infection with Moraxella catarrhalis induced monocyte adhesion, transepithelial migration, and superoxide generation, whereas stimulation with lipopolysaccharide, tumor necrosis factor-alpha, interleukin-1beta, or interferon-gamma induced adhesion or transmigration, but failed to initiate monocyte burst. The effect of microbial challenge was mimicked by phorbol myristate acetate and inhibited by the protein kinase C inhibitor bisindoylmaleimide. Furthermore, evidence for a role of platelet-activating factor-signaling in monocytes is presented. Monocyte burst was neither induced by supernatant nor affected by fixation of A549 cells, excluding the contribution of epithelium-derived soluble factors but emphasizing the mandatory role of intercellular contact. The employment of blocking antibodies, however, denied a role for the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, or CD11b/CD18 and CD49d/CD29. In essence, infection of alveolar epithelial cells with M. catarrhalis might amplify the inflammatory capacity of invading monocytes eliciting their superoxide production. The epithelial response to this microbial challenge thus clearly differed from that to proinflammatory cytokines.     

Association of the -141C Del variant of the dopamine D2 receptor (DRD2) with positive family history and suicidality in German alcoholics.

Several lines of evidence indicate an involvement of the dopaminergic system in alcoholism, withdrawal, suicidality, and attention-deficit hyperactivity disorder (ADHD). The functionally relevant -141C Ins/Del polymorphism located upstream to exon 1 in the 5'-region of the dopamine D2 receptor (DRD2) gene might be an interesting candidate gene. We investigated a sample of 1,126 well-characterized, primary chronic alcoholics of German descent according to a phenotype-genotype strategy, i.e., alcoholics suffering from severe withdrawal complications such as seizure or delirium, family history positive (FH+) alcoholics, alcoholics with an antisocial personality disorder (ASPD), alcoholics with an ADHD, and type 1 or type 2 alcoholics according to Cloninger's typology. Compared to the control subjects, there was a significant excess of the -141C Del allele in alcoholics with a paternal and grandpaternal history of alcoholism and in alcoholic subgroups with suicidality or without a history of withdrawal symptoms. There were no significant differences in allele frequency between the entire group or subgroups of alcoholics and healthy controls. Therefore, the -141C Del variant of the DRD2 might be a protective factor against the development of withdrawal symptoms. However, it might also be a risk factor in a highly burdened subgroup of alcoholics with a paternal and grandpaternal history of alcoholism and it might contribute to the substantially higher likelihood of suicide in alcoholics.