Autologous vascular smooth muscle cell-based myocardial gene therapy to induce coronary collateral growth

For therapeutic angiogenesis to achieve clinical relevance, it must be effective, with minimal side effects to other end organ systems. We developed a cardiac-specific gene delivery mechanism by transfecting autologous vascular smooth muscle cells (VSMC) with VEGF and administering these cells via intracoronary injection. We evaluated the efficacy of this protocol by its ability to stimulate angiogenesis in the presence of a subthreshold stimulus for collateralization. A modified canine repetitive coronary occlusion model was utilized in these experiments with left anterior descending coronary artery occlusions for 2 min every 2 h four times per day for 21 days. An intramyocardial catheter in the perfusion territory of the left anterior descending coronary artery measured proteins in the myocardial interstitial fluid. VSMC from jugular vein explants were isolated, amplified in culture for 3 wk, and transfected with a plasmid expressing VEGF-165 and/or enhanced green fluorescent protein. Cells were injected before commencement of occlusions. VEGF levels in myocardial interstitial fluid were significantly higher in VEGF-transfected animals than in sham (repetitive occlusions without cell transplantation) and control (repetitive occlusions with enhanced green fluorescent protein-transfected cells) animals at the onset of occlusions (P < 0.05). In the VEGF group, collateral flow was increased at day 7 and remained higher than in sham and control groups thereafter. We found that intracoronary administration of VEGF-transfected autologous VSMC effectively promotes collateral development. This approach may provide a way to confine delivery of a gene to a specified organ, thus minimizing complications related to gene transfection in nontargeted organ systems.     

Overlapping and selective roles of endothelial intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 in lymphocyte trafficking

The integrin LFA-1 interacts with a variety of ligands termed ICAMs. ICAM-1 and ICAM-2 are both expressed on endothelium and serve as counterreceptors during lymphocyte trafficking. In this study, we analyzed their relative contribution to lymphocyte recirculation through lymph nodes and to recruitment into lung and inflamed skin by blocking mAbs against ICAM-1 and ICAM-2 and mice deficient for ICAM-1. The entry of lymphocytes into peripheral and mesenteric lymph nodes was found to be unaffected by the functional deletion of either ICAM-1 or ICAM-2. However, when both pathways were blocked, recirculation through lymph nodes was strongly reduced. Trapping of lymphocytes in the lung after i.v. injection is partly mediated by LFA-1/ICAM interactions; the data presented in this study show an exclusive role of ICAM-1 in LFA-1-dependent lung trapping. Similarly, ICAM-1, but not ICAM-2, was required for the migration of T effector cells into the inflamed skin. These results indicate that ICAM-1 and ICAM-2 have redundant functions in lymphocyte recirculation through lymph nodes, but ICAM-1 is unique in supporting migration into inflamed sites and trapping within the lung.     

Experience with insulin analogues in children

Current data on rapid and long-acting insulin analogues in the paediatric age group is limited. While several studies indicate a benefit in reducing hypoglycaemia, particularly at night, with rapid or long-acting insulin analogue treatment, the effect on long-term glycaemic control remains controversial. The continuous glucose monitoring system offers a new option for tailoring treatment with insulin analogues to achieve optimal glycaemia. In 29 adolescents with diabetes this approach confirmed the non-inferiority of postprandial rapid-acting analogue administration compared to preprandial regular insulin, but revealed significant mealtime differences, with increased analogue requirement at breakfast and dinner. Although rapid- and long-acting insulin analogues may offer potential benefits for problems frequently encountered in paediatric diabetology, their value for the individual child still has to be tested in long-term observations in daily clinical practice.     

Profile of cholesterol-related sterols in aged amyloid precursor protein transgenic mouse brain

Cholesterol is implicated to play a role in Alzheimer disease pathology. Therefore, the concentrations of cholesterol, its precursors, and its degradation products in brain homogenates of aging wild-type and beta-amyloid precursor protein transgenic mice carrying the Swedish mutation (APP23) were analyzed. Among the sterols measured, lanosterol is the first common intermediate of two different pathways, which use either desmosterol or lathosterol as the predominant precursors for de novo synthesis of brain cholesterol. In young mice, cholesterol is mainly synthesized via the desmosterol pathway, while in aged mice, lathosterol is the major precursor. 24S-hydroxycholesterol (cerebrosterol), which plays a key role in the removal of cholesterol from the brain, modestly increased during aging. No differences in the levels of cholesterol, its precursors, or its metabolites were found between wild-type and APP23 transgenic mice. Moreover, the levels of the exogenous plant sterols campesterol and sitosterol were significantly elevated in the brains of APP23 animals at age 12 and 18 months. This time point coincides with abundant plaque formation.     

Effect of phorbol esters on Ca2+ sensitivity and myosin light-chain phosphorylation in airway smooth muscle

We studied in -escin-permeabilized canine tracheal smoothmuscle (CTSM) the effect of the protein kinase C (PKC) agonist phorbol12,13-dibutyrate (PDBu) on isometric force at a constant submaximalCa²⁺ concentration (i.e., theeffect on Ca²⁺ sensitivity) andregulatory myosin light-chain (rMLC) phosphorylation. PDBuincreased Ca²⁺sensitivity, an increase associated with a concentration-dependent, sustained increase in rMLC phosphorylation. PDBu altered therelationship between rMLC phosphorylation and isometric force such thatthe increase in isometric force was less than that expected for the increase in rMLC phosphorylation observed. The effect of four PKCinhibitors [calphostin C, chelerythrine chloride, apseudosubstrate inhibitor for PKC, PKC peptide-(1931) (PSSI), andstaurosporine] on PDBu-inducedCa²⁺ sensitization as well as theeffect of calphostin C and PSSI on rMLC phosphorylation weredetermined. Whereas none of these compounds prevented or reversed thePDBu-induced increase in Ca²⁺sensitivity, the PDBu-induced increase in rMLC phosphorylation wasinhibited. We conclude that PDBu increases rMLC phosphorylation byactivation of PKC but that the associated PDBu-induced increases inCa²⁺ sensitivity are mediated bymechanisms other than activation of PKC in permeabilized airway smoothmuscle.

     

Entry of Amphotropic and 10A1 Pseudotyped Murine Retroviruses Is Restricted in Hematopoietic Stem Cell Lines

Although transduction with amphotropic murine leukemia virus (MLV) vectors has been optimized successfully for hematopoietic differentiated progenitors, gene transfer to early hematopoietic cells (stem cells) is still highly restricted. A similar restriction to gene transfer was observed in the mouse stem cell line FDC-Pmix compared with transfer in the more mature myeloid precursor cell line FDC-P1 and the human erythroleukemia cell line K562. Gene transfer was not improved when the vector was pseudotyped with gp70^SU of the 10A1 strain of MLV, which uses the receptor of the gibbon ape leukemia virus (Pit1), in addition to the amphotropic receptor (Pit2). Although 10A1 and amphotropic gp70^SU bound to FDC-P1, K562, and fibroblasts, no binding to FDC-Pmix cells was detected. This indicates that FDC-Pmix cells lack functional Pit2 and Pit1 receptors. Pseudotyping with the vesicular stomatitis virus G protein improved transduction efficiency in FDC-Pmix stem cells by 2 orders of magnitude, to fibroblast levels, confirming a block to retroviral infection at the receptor level.     

Intraspecific morphological variation in late Miocene Bolboforma, and implications for their classification, ecology, and biostratigraphic utility

A preliminary morphological study of large, well preserved bolboform populations from the late Miocene section of DSDP Site 593, in the southern Tasman Sea, reveals a relationship between test size and intraspecific morphological variation interpreted as a gradual transformation in test shape and surface ornamentation. The succession begins with small, smooth, thin-walled subspheroidal tests. As test size increases, rudimentary ornaments become more common and the shape more globose. With further size increase, the walls thicken and ornaments become more structured, but specimens still retain a rounded globose shape. In still larger tests, the ornament becomes more prominent and tests develop a squatter shape with flatter aboral surfaces. The transformation generally ends with large, thick-walled, highly ornate forms with squat globose tests, robust ornaments, and relatively flat aboral surfaces. Although the size of the test increases notably through the transformation, there appears to be no corresponding change in the internal chamber size from its initial value, and so the transformation is largely due to the addition of calcite to the outer surface of the test wall. This appears to be an inherent biological feature and does not involve calcite dissolution/reprecipitation or other retrograde processes. The detail of the transformation varies between species and is presented here for Bolboforma subfragoris s.l., B. gruetzmacheri n. sp., and B. metzmacheri s.s., which exhibit different types of ornament — spinose, irregularly ridged, and cancellate, respectively.When different morphological forms of a single species occur in the same sample, they have the potential to be interpreted as different species. Taxonomic confusion is further compounded by the morphological similarity of many small, smooth-walled, and weakly ornamented forms of different species. These features are common in bolboform populations and have contributed to the misinterpretation of some species. Differences in test shape and ornament are most notable in large ornate forms and taxonomic interpretations should be based wherever possible on such forms. Once the taxonomy is clear, the biostratigraphic utility of the late Miocene bolboforms from Site 593 has been in the recognition of single species (or at most two) in a given sample. This provides an excellent biostratigraphic framework for a period of time that lacks significant alternative microfossils for age control in New Zealand sediments.The distribution of bolboforms in (mainly) southern hemisphere marine sediments appears to track the palaeocirculation of Southern Component Intermediate Water (SCIW). Hence addition of calcite to the outer surface of the test wall probably occurred while the bolboforms were entrained in subsurface flows of SCIW. In this respect, the increased complexity of ornamentation may have been a morphological adaptation to counter the loss of buoyancy as calcite was added to the outer surface of the test wall. This would have presumably contributed to the entrainment and dispersal of bolboforms in the flow of subsurface water masses, and it suggests that the tests of some bolboforms at least may represent a dispersal phase in the life cycle of this enigmatic group of microfossils.     

Dystroglycan Binding to α-Neurexin Competes with Neurexophilin-1 and Neuroligin in the Brain

α-Neurexins (α-Nrxn) are mostly presynaptic cell surface molecules essential for neurotransmission that are linked to neuro-developmental disorders as autism or schizophrenia. Several interaction partners of α-Nrxn are identified that depend on alternative splicing, including neuroligins (Nlgn) and dystroglycan (αDAG). The trans-synaptic complex with Nlgn1 was extensively characterized and shown to partially mediate α-Nrxn function. However, the interactions of α-Nrxn with αDAG, neurexophilins (Nxph1) and Nlgn2, ligands that occur specifically at inhibitory synapses, are incompletely understood. Using site-directed mutagenesis, we demonstrate the exact binding epitopes of αDAG and Nxph1 on Nrxn1α and show that their binding is mutually exclusive. Identification of an unusual cysteine bridge pattern and complex type glycans in Nxph1 ensure binding to the second laminin/neurexin/sex hormone binding (LNS2) domain of Nrxn1α, but this association does not interfere with Nlgn binding at LNS6. αDAG, in contrast, interacts with both LNS2 and LNS6 domains without inserts in splice sites SS#2 or SS#4 mostly via LARGE (like-acetylglucosaminyltransferase)-dependent glycans attached to the mucin region. Unexpectedly, binding of αDAG at LNS2 prevents interaction of Nlgn at LNS6 with or without splice insert in SS#4, presumably by sterically hindering each other in the u-form conformation of α-Nrxn. Thus, expression of αDAG and Nxph1 together with alternative splicing in Nrxn1α may prevent or facilitate formation of distinct trans-synaptic Nrxn·Nlgn complexes, revealing an unanticipated way to contribute to the identity of synaptic subpopulations.