New species of Colletotrichum from wild Poaceae and Cyperaceae plants in Iran.

Twenty-two Colletotrichum strains were isolated from anthracnose symptoms or leaf spots on leaves of various wild Poaceae and Cyperaceae plants collected in three provinces of Iran and tentatively identified as belonging to the Graminicola species complex based on morphology. All strains were studied via a polyphasic approach combining colony characteristics, morphology and phylogeny inferred from multi-locus sequences, including the nuc rDNA ITS1-5.8S-ITS2 (ITS), partial sequences of the ß-tubulin (tub2), actin (act), manganese superoxide dismutase 2 (sod2), DNA lyase 2 (apn2) genes, a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (gapdh), and the intergenic spacer between the apn2 gene and the mat1 idiomorph (apn2/mat1). Six species were distinguished, including three new species, namely C. caspicum, C. persicum, and C. sacchari, and three previously described species, C. cereale, C. nicholsonii and C. sublineola. Comprehensive morphological descriptions and illustrations are provided for all species. Furthermore, this study provided new insights into the distribution and host range of known species.

T-helper immune phenotype may underlie 'paradoxical' tumour necrosis factor-α inhibitor therapy-related psoriasiform dermatitis.

BACKGROUND: Therapeutics targeting tumour necrosis factor (TNF)-α are effective for psoriasis; however, in patients treated for other disorders, psoriasis may worsen and psoriasiform dermatitis (PsoD) may arise. T helper (Th) cytokines in psoriasis upregulate keratin (K)17, which modulates TNF-α transduction, leading to vascular adhesion molecule upregulation and lymphocytic extravasation. AIM: We investigated Th phenotype and expression of K17, intercellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1 in psoriasis and anti-TNF-α-related PsoD. METHODS: Skin biopsies from patients with psoriasis unresponsive to TNF-α inhibitor therapy (n = 11), PsoD-related to TNF-α inhibition (n = 9), untreated psoriasis (n = 9) or atopic dermatitis (AD; n = 9) were immunohistochemically analysed for Th1, Th2, Th17 and Th22. Expression of K17, ICAM-1 and VCAM-1 was also examined. RESULTS: Anti-TNF-α-unresponsive psoriasis and anti-TNF-α-related PsoD showed decreased Th1 : Th2 raio and increased Th17 : Th1 ratio compared with untreated psoriasis. Anti-TNF-α-unresponsive psoriasis had significantly fewer Th1 (4% vs. 12%) and more Th17 (51% vs. 20%) cells than untreated psoriasis. No difference in Th22 cells was identified. K17 was present in all cases of untreated psoriasis and anti-TNF-α-related PsoD, 91% of anti-TNF-α-unresponsive psoriasis, and only 22% of AD. VCAM-1 and ICAM-1 in anti-TNF-α-related PsoD was akin to untreated psoriasis, but decreased in anti-TNF-α-unresponsive psoriasis. CONCLUSIONS: These findings further the current understanding of the anti-TNF-α-related psoriasiform phenotype and support a rationale for therapeutic targeting of interleukin-17 and TNF-α in combination.

Delayed type IV hypersensitivity reaction to porcine acellular dermal matrix masquerading as infection resulting in multiple debridements.

INTRODUCTION: Delayed type IV hypersensitivity reactions are well established in the surgical setting with respect to external exposure via topical antibiotics and internal exposure via synthetic materials. In contrast, biologic matrix is derived from decellularized human or animal tissues and is consequently believed to elicit a minimal host inflammatory response. OBJECTIVE: We report a case of delayed type IV hypersensitivity reaction secondary to a biologic comprised of porcine-derived acellular dermal matrix, [Strattice™]. CONCLUSIONS: While biologic matrix is often preferred over synthetic mesh due to its decreased risk for infection, this case emphasizes that potential for hypersensitivity to the material persists. Type IV hypersensitivity reactions should be included in the differential diagnosis for suspected post-operative infections.

The dysbindin-containing complex (BLOC-1) in brain: developmental regulation, interaction with SNARE proteins and role in neurite outgrowth.

Previous studies have implicated DTNBP1 as a schizophrenia susceptibility gene and its encoded protein, dysbindin, as a potential regulator of synaptic vesicle physiology. In this study, we found that endogenous levels of the dysbindin protein in the mouse brain are developmentally regulated, with higher levels observed during embryonic and early postnatal ages than in young adulthood. We obtained biochemical evidence indicating that the bulk of dysbindin from brain exists as a stable component of biogenesis of lysosome-related organelles complex-1 (BLOC-1), a multi-subunit protein complex involved in intracellular membrane trafficking and organelle biogenesis. Selective biochemical interaction between brain BLOC-1 and a few members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) superfamily of proteins that control membrane fusion, including SNAP-25 and syntaxin 13, was demonstrated. Furthermore, primary hippocampal neurons deficient in BLOC-1 displayed neurite outgrowth defects. Taken together, these observations suggest a novel role for the dysbindin-containing complex, BLOC-1, in neurodevelopment, and provide a framework for considering potential effects of allelic variants in DTNBP1--or in other genes encoding BLOC-1 subunits--in the context of the developmental model of schizophrenia pathogenesis.

Case report. Appearance of nephrogenic fibrosing dermopathy on a bone scan.

We report a case of nephrogenic systemic fibrosis involving the thighs bilaterally in a patient who received multiple MRI scans with gadolinium. A recent bone scan demonstrated uptake of radiotracer in a region that correlates with CT and dermatopathological findings.

Two components of actin-based retrograde flow in sea urchin coelomocytes.

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase-specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a "push-pull" mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.

Steady state hemodynamic and energetic characterization of the Penn State/3M Health Care Total Artificial Heart.

Total Artificial Heart (TAH) development at Penn State University and 3M Health Care has progressed from design improvements and manufacturing documentation to in vitro and in vivo testing to characterize the system's hemodynamic response and energetic performance. The TAH system is completely implantable and intended for use as an alternative to transplantation. It includes a dual pusher plate pump and rollerscrew actuator, welded electronics and battery assembly, transcutaneous energy transmission system, telemetry, and a compliance chamber. In vitro testing was conducted on a Penn State mock circulatory loop with glycerol/water solution at body temperature. Tests were performed to characterize the preload and afterload response, left atrial pressure control, and power consumption. A sensitive preload response was demonstrated with left atrial pressure safely maintained at less than 15 mm Hg for flow rates up to 7.5 L/min. Variations in aortic pressure and pulmonary vascular resistance were found to have minimal effects on the preload sensitivity and left atrial pressure control. In vivo testing of the completely implanted system in its final configuration was carried out in two acute studies using implanted temperature sensors mounted on the electronics, motor, and energy transmission coil in contact with adjacent tissue. The mean temperature at the device-tissue interface was less than 4 degrees C above core temperature.

Temporal kinetics and cellular phenotype of TNF p55/p75 receptors in experimental allergic encephalomyelitis.

TNF-alpha and LT-alpha are thought to be involved in the immunopathology of CNS demyelinating diseases. Both cytokines induce cellular effects through 55-kDa type-1 receptors (R1) and 75-kDa type-2 receptors (R2). To date, no study has specifically identified the various cell populations that express TNF receptors (TNFR) in the inflammatory and demyelinating mouse model, EAE. Phenotyping the TNFR positive cells is important in determining when and where the ligands may be acting and playing a role in disease pathology. We observed an upregulation of TNF R1 and R2 mRNA in high endothelial venules (HEVs) in the lymph node and CNS before the onset of EAE (preclinical phase). This upregulation of TNFR expression in HEVs was followed by a rapid increase in leukocytes within the CNS after the onset of clinical disease. The temporal kinetics of these data suggest that HEVs become activated early, probably through the release of pro-inflammatory cytokines originating from circulating leukocytes. An increase in TNFR on HEVs would make these cells more susceptible to TNF-induced changes, such as increasing cellular adhesion molecules, thereby further facilitating the trafficking of leukocytes into the CNS parenchyma.

Expression of green fluorescent protein in oligodendrocytes in a time- and level-controllable fashion with a tetracycline-regulated system.

Developments in transgenic technology have greatly enhanced our ability to understand the functions of various genes in animal models and relevant human diseases. The tetracycline (tet)-regulated transactivation system for inducing gene expression allowed us to control the expression of exogenous genes in a temporal and quantitative way. The ability to manipulate a cell-specific promoter enabled us to express one particular protein in a single type of cell. The combination of a tetracycline system and a tissue-specific promoter has led us to the development of an innovative gene expression system, which is able to express genes in a cell type-specific and time- and level-controllable fashion. An oligodendrocyte-specific myelin basic protein (MBP) gene promoter controls the reversed tet-inducible transactivator. The green fluorescent protein (GFP) gene was placed under the control of the human cytomegalovirus (CMV) basic promoter in tandem with seven tet-responsive elements (TRE), binding sites for the activated transactivator. Upon the addition of doxycycline (DOX, a tetracycline derivative), tet transactivators became activated and bound to one or more TRE, leading to the activation of the CMV promoter and the expression of GFP in oligodendrocytes. We have successfully expressed GFP and luciferase at high levels in oligodendrocytes in a time- and dose-dependent fashion. In the absence of DOX, there was almost no GFP expression in oligodendroglial cultures. Graded levels of GFP expression were observed after induction with DOX (0.5 to 12.5 microg/ml). Our data indicate that this inducible gene expression system is useful for the study of gene function in vivo and for the development of transgenic animal models relevant to human diseases such as multiple sclerosis.

Transferrin is an essential factor for myelination.

It has been established that oligodendrocytes, the myelin forming cells, participate in iron homeostasis through the synthesis and secretion of transferrin. Here we investigated whether a correlation exists between myelination, the commonly studied function of oligodendrocytes, and that of transferrin synthesis and secretion. We used a proteolipid protein mutant, the myelin deficient rat, whose condition is characterized by severe hypomyelination. We compared the ontogenic profile for transferrin gene expression in mutants with that of unaffected rat pups through northern blot analysis and in situ hybridization. Surprisingly, transferrin synthesis was null in mutant oligodendrocytes. Next, we demonstrated that a single apo-transferrin intraparenchymal injection administered to P5 rat pups enabled mutant oligodendrocytes to synthesize myelin basic protein and to myelinate axons, indicating that transferrin effects mutant oligodendrocyte maturation regardless of its source. Thus, transferrin availability is essential for oligodendrocyte maturation and function, and oligodendrocytes are most vulnerable to transferrin deficiency during the premyelinating stage.

Thioredoxin peroxidase (natural killer enhancing factor) regulation of activator protein-1 function in endothelial cells.

Thioredoxin peroxidase-1 (TxP-1), originally cloned as natural killer enhancing factor-B, belongs to a highly conserved antioxidant family. Tumor necrosis factor-alpha (TNF) activates the expression of activator protein-1 (AP-1) responsive genes. We show here that over-expression of TxP-1 blocks TNF-induced AP-1 activation in endothelial ECV304 cells, which was demonstrated by three independent experimental protocols: electromobility shift assay with AP-1 oligonucleotide probe; reporter gene expression with AP-1 binding site, and interleukin-8 production, which is dependent on AP-1. These results are consistent with the role of TxP-1 as an antioxidant and the previous reports that TNF-induced reactive oxygen species were responsible for AP-1 activation.

Ciliary neurotrophic factor activates JAK/Stat signal transduction cascade and induces transcriptional expression of glial fibrillary acidic protein in glial cells.

In recent reports, ciliary neurotrophic factor (CNTF) has been implicated as an injury factor involved in regulating astrogliosis in the CNS. In this study, we used a rat oligodendroglial progenitor cell line that is highly responsive to CNTF to examine CNTF-induced alterations that may play a role in activation of the glial fibrillary acidic protein (GFAP) gene. We determined that CNTF induces the transient translocation of Stat1 alpha/p91 to the nucleus. This nuclear translocation was followed by GFAP promoter activation and an up-regulation of GFAP mRNA and protein. Level of CNTF-alpha receptor mRNA, however, were unaffected by addition of the ligand. Transfection studies using an upstream 5'-flanking, 1.9-kb rat GFAP promoter linked to a luciferase reporter gene revealed CNTF-induced transcriptional activation within 1 h of ligand exposure. Moreover, serial-deleted constructs identified a distal (-1,857 to -1,546 bp) and a proximal (-384 to -106 bp) region as being important for CNTF-induced GFAP promoter activation. These two regions showed a strong degree of overlap for CNTF- and serum-induced activation of the GFAP gene. Analysis of the two regions revealed several cis-elements that are thought to be involved in GFAP regulation and/or the regulation of other genes by members of the interleukin-6 family of cytokines. Moreover, we are the first to report the presence of several putative CNTF-responsive elements within our identified distal and proximal regions in the GFAP gene promoter.

Transplantation of CG4 oligodendrocyte progenitor cells in the myelin-deficient rat brain results in myelination of axons and enhanced oligodendroglial markers.

Transplantation of oligodendrocyte (Ol) progenitor cells into the central nervous system is a promising approach for the treatment of myelin disorders. This approach requires providing adequate numbers of healthy cells with myelinating potential. We recently showed the successful transplantation of Ol progenitors into the myelin-deficient (md) rat brain. In the present work, CG4 cells, a cell line with properties of Ol progenitors, were labeled with fast blue and grafted into P3-P5 pups born to carrier mothers. Examination of host brains 2 weeks posttransplant indicated that CG4 cells display a much more extensive migration capacity than their wild-type counterparts. These cells synthesized myelin components. In addition, ultrastructural analysis showed myelin formation along axons of md hosts in various brain regions, including corpus callosum, cerebellum, and brainstem. Furthermore, in situ hybridization studies performed on sagittal sections revealed extensive expression of transferrin-mRNA within the md host parenchyma. The high survival and functional features displayed by CG4 cells after transplantation, together with their striking wide distribution within the host parenchyma, as assessed by the presence of myelinated fibers in mutant hosts, emphasizes the importance of using highly motile and proliferative Ol progenitor cells. Strategies to improve the condition and life span of md rat pups are currently under investigation.

Recent improvements in a completely implanted total artificial heart.

The total artificial heart under development by the Pennsylvania State University and 3M Health Care has undergone a number of design improvements to improve reliability, manufacturability, implantability, and performance. These improvements are nearing completion in preparation for formal durability testing. The redesigned implanted electronics canister, consisting of a welded titanium shell with hermetic connectors, contains the control, telemetry, and energy transmission electronics, as well as a 9 cell, 800 mAhr Ni-Cd battery pack. Functional changes include a reduction in the battery recharge time from 14 hours to 4 hours, and a new inductive telemetry system. The energy transmission system operating frequency has been increased from 160 kHz to 200 kHz. Electromagnetic interference filters and a more efficient control mode have also been implemented. The energy converter has been modified to incorporate a new motor with integral Hall effect position sensors, and new cable, and compliance chamber conduit fittings. High flex life cable is now used for the motor and coil cables. Two prototype durability mock circulatory loops have been built and are being tested. Substantial progress has been made in the completion of manufacturing documentation, and in the implementation of a quality system.

In vivo testing of a completely implanted total artificial heart system.

The authors performed 14 implants of a completely implanted total artificial heart (TAH) system in calves. The system consisted of a dual pusher plate rollerscrew energy converter, two sac type blood pumps, an implanted electronic control and battery package, and a transcutaneous energy transmission system. Ten of the implants included a percutaneous lead for monitoring of the implant; the remainder made use of wireless two way telemetry between the implant and the outside. Three animals survived the perioperative period. These calves survived for 98 to 118 days, and one was still alive at 150 days. Causes for termination of the 98 and 118 day cases were abdominal pocket sepsis originating at a monitoring line, and systemic sepsis acquired perioperatively. Death or termination in the shorter cases was mainly due to respiratory complications or bleeding. The TAH system proved capable of providing adequate cardiac outputs at modest atrial pressures. Wireless monitoring and wireless intervention for weaning from cardiopulmonary bypass were readily achieved. All organ systems functioned normally in the presence of the device. Once recovery from implantation in these very young animals was achieved, the system proved its ability to reliably support these animals until body mass exceeded its cardiac output capabilities.

A completely implantable total artificial heart system.

The authors developed, built, and tested in vivo a completely implanted total artificial heart (TAH) system. The system used a reduced size version of a roller screw energy converter and mating sac blood pumps. The motor drive, pumps, and a compliance chamber were implanted intrathoracically. A canister containing controlling electronics and an emergency battery was implanted in the abdomen. The secondary coil of an inductive energy transmission and telemetry system was placed over the ribs. The system was implanted in three calves, that survived 0.5-13 days with the system. The system maintained safe left atrial pressures and adequate cardiac outputs during each animal's entire course.

Digital signal processing of ultrasonic signals for blood flow measurement.

This paper describes the application of advanced digital signal processing techniques in a noninvasive ultrasonic Doppler flowmeter used to measure extracorporeal blood flow during open heart surgery. The use of ultrasound to determine blood flow rates started in the 1950's with much of this work focused on measurement of blood flow in a patient by a variety of means, both invasive and noninvasive. Although the use of ultrasonics to measure blood flow is not in itself a new concept, the application of advanced digital signal processing techniques in the system being described has resulted in a unique product for accurate and reliable blood flow measurements. The flowmeter system is intended for use with a centrifugal blood pump and will measure blood flow in the flexible tubing used during surgery to an accuracy of better than +/- 10%. This paper describes the development and implementation of the digital flowmeter and its application to flow measurement.