Sandfly surveillance and investigation of Leishmania spp. DNA in sandflies in Kosovo.

In the past decade, leishmaniasis seems to be re-emerging in Balkan countries. There are serious implications that Kosovo is a visceral leishmaniasis endemic region with autochthonous transmission; nevertheless, surveillance of vectors, reservoirs or the disease is not yet established. Gaining knowledge about sandfly vector species is a prerequisite for the development of a monitoring and control plan in the future. After a long gap in research of over 70 years, sandfly studies in Kosovo were resumed in 2014. During this presence/absence study, nine sandfly species were detected: Phlebotomus papatasi, Ph. perfiliewi, Ph. tobbi, Ph. neglectus, Ph. simici, Ph. balcanicus, Ph. alexandri, Ph. mascittii and Sergentomyia minuta. Three species are new with regard to the fauna of Kosovo - Ph. alexandri, Ph. balcanicus and Ph. mascittii. Besides increased diversity, changes in the number of collected specimens and distribution range of species were recorded, with Ph. neglectus being the most dominant species with the widest distribution. Testing of randomly chosen females for Leishmania spp. DNA resulted the in detection of L. tropica in a specimen of Ph. neglectus. The presence of numerous vector species in the sandfly fauna of Kosovo pose a threat for the re-emergence of vector-borne diseases. Therefore, continuous surveillance is recommended with regular updates on vector distribution and abundance.

Biocompatibility and biodegradability of Mg-Sr alloys: the formation of Sr-substituted hydroxyapatite.

Magnesium is an attractive material for use in biodegradable implants due to its low density, non-toxicity and mechanical properties similar to those of human tissue such as bone. Its biocompatibility makes it amenable for use in a wide range of applications from bone to cardiovascular implants. Here we investigated the corrosion rate in simulated body fluid (SBF) of a series of Mg-Sr alloys, with Sr in the range of 0.3-2.5%, and found that the Mg-0.5 Sr alloy showed the slowest corrosion rate. The degradation rate from this alloy indicated that the daily Sr intake from a typical stent would be 0.01-0.02 mg day⁻¹, which is well below the maximum daily Sr intake levels of 4 mg day⁻¹. Indirect cytotoxicity assays using human umbilical vascular endothelial cells indicated that Mg-0.5 Sr extraction medium did not cause any toxicity or detrimental effect on the viability of the cells. Finally, a tubular Mg-0.5 Sr stent sample, along with a WE43 control stent, was implanted into the right and left dog femoral artery. No thrombosis effect was observed in the Mg-0.5 Sr stent after 3 weeks of implantation while the WE43 stent thrombosed. X-ray diffraction demonstrated the formation of hydroxyapatite and Mg(OH)2 as a result of the degradation of Mg-0.5 Sr alloy after 3 days in SBF. X-ray photoelectron spectroscopy further showed the possibility of the formation of a hydroxyapatite Sr-substituted layer that presents as a thin layer at the interface between the Mg-0.5 Sr alloy and the corrosion products. We believe that this interfacial layer stabilizes the surface of the Mg-0.5 Sr alloy, and slows down its degradation rate over time.

Effect of Si and Fe doping on calcium phosphate glass fibre reinforced polycaprolactone bone analogous composites.

Reinforcing biodegradable polymers with phosphate-based glass fibres (PGF) is of interest for bone repair and regeneration. In addition to increasing the mechanical properties, PGF can also release bioinorganics, as they are water soluble, a property that may be controllably translated into a fully degradable composite. Herein, the effect of Si and Fe on the solubility of calcium-containing phosphate-based glasses (PG) in the system (50P(2)O(5)-40CaO-(10-x)SiO(2)-xFe(2)O(3), where x=0, 5 and 10 mol.%) were investigated. On replacing SiO(2) with Fe(2)O(3), there was an increase in the glass transition temperature and density of the PG, suggesting greater crosslinking of the phosphate chains. This significantly reduced the dissolution rates of degradation and ion release. Two PG formulations, 50P(2)O(5)-40CaO-10Fe(2)O(3) (Fe10) and 50P(2)O(5)-40CaO-5Fe(2)O(3)-5SiO(2) (Fe5Si5), were melt drawn into fibres and randomly incorporated into polycaprolactone (PCL). Initially, the flexural strength and modulus significantly increased with PGF incorporation. In deionized water, PCL-Fe(5)Si(5) displayed a significantly greater weight loss and ion release compared with PCL-Fe10. In simulated body fluid, brushite was formed only on the surface of PCL-Fe(5)Si(5). Dynamic mechanical analysis in phosphate buffered saline (PBS) at 37°C revealed that the PCL-Fe10 storage modulus (E') was unchanged up to day 7, whereas the onset of PCL-Fe(5)Si(5)E' decrease occurred at day 4. At longer-term ageing in PBS, PCL-Fe(5)Si(5) flexural strength and modulus decreased significantly. MC3T3-E1 preosteoblasts seeded onto PCL-PGF grew up to day 7 in culture. PGF can be used to control the properties of biodegradable composites for potential application as bone fracture fixation devices.

Identification and functional characterization of thromboxane A2 receptors in Schwann cells.

Previous reports have demonstrated the presence of functional thromboxane A2 (TP) receptors in astrocytes and oligodendrocytes. In these experiments, the presence and function of TP receptors in primary rat Schwann cells (rSC) and a neurofibrosarcoma-derived human Schwann cell line (T265) was investigated. Immunocytochemical and immunoblot analyses using polyclonal anti-TP receptor antibodies demonstrate that both cell types express TP receptors. Treatment with the stable thromboxane A2 mimetic U46619 (10 microM) did not stimulate intracellular calcium mobilization in rSC, whereas T265 cells demonstrated a calcium response that was inhibited by prior treatment with TP receptor antagonists. U46619 also stimulated CREB phosphorylation on Ser133 in T265 cells and, to a lesser extent, in rSC. To identify potential mechanisms of CREB phosphorylation in rSC, we monitored intracellular cAMP levels following U46619 stimulation. Elevated levels of cAMP were detected in both rSC (20-fold) and T265 (15-fold) cells. These results demonstrate that TP receptor activation specifically stimulates CREB phosphorylation in T265 cells, possibly by a calcium- and/or cAMP-dependent mechanism. In contrast, TP receptor activation in rSC stimulates increases in cAMP and CREB phosphorylation but does not elicit changes in intracellular calcium.

Role of NMDA receptors in adult primate cortical somatosensory plasticity.

We have previously shown that most of the reorganization that typically follows median nerve transection in adult squirrel monkeys is dependent on normally functioning N-methyl-D-aspartate (NMDA) receptors. Here, we have evaluated two additional hypotheses: (1) is the immediate "unmasking" found after median nerve transection NMDA receptor-dependent? and (2) are NMDA receptors necessary for both the initiation and maintenance of the second phase of reorganizational changes, or only the former? To address these issues, we implanted osmotic minipumps subcutaneously to deliver an NMDA receptor antagonist (3-((+/-)-2- carboxypiperazin-4-yl)propyl-1-phosphonic acid, CPP) systemically either before examining the immediate effects of median nerve transection, or after reorganization had presumably occurred. For the first set of experiments, NMDA receptor blockade was initiated either 1 or 4 weeks prior to multi-unit mapping in area 3b followed by transection of the median nerve and remapping of the cortex. In the second set of experiments, median nerve transection was followed 4 weeks later by either 1 or 4 weeks of NMDA receptor blockade prior to terminal mapping. We report that the immediate unmasking of new receptive fields after acute nerve injury is not prevented by NMDA receptor blockade; nor are completely reorganized cortical maps dependent upon NMDA receptors for their maintenance. We conclude that the immediate changes in cortical topography are not due to an NMDA receptor-dependent mechanism, but more likely due to release from tonic inhibition. Furthermore, the later phase of reorganization, as for some forms of hippocampal long-term potentiation (LTP), is dependent on normally functioning NMDA receptors for its initiation, but not for its maintenance.

Myelin basic protein (MBP) and MBP peptides are mitogens for cultured astrocytes.

After CNS demyelination, astrogliosis interferes with axonal regeneration and remyelination. We now provide evidence that myelin basic protein (MBP) can contribute to this observed astrocyte proliferation. We found that astrocytes grown in either serum-containing or serum-free medium proliferate in response to MBP. The mitogenic regions of MBP in both media were MBP(1-44), MBP(88-151) and MBP(152-167). The mitogenic effect of these individual peptides was potentiated by simultaneous treatment with microglia conditioned media (CM). MBP-induced proliferation was inhibited by suramin at concentrations known to block the fibroblast growth factor receptor (FGFR), whereas neither MBP(1-44), MBP(88-151) nor MBP(152-167) were affected. Cholera toxin B, that binds to ganglioside GM(1), inhibited the mitogenicity of MBP(1-44) and had no significant effect on the mitogenicity of MBP, MBP(88-151) or MBP(152-167). Treatment of astrocytes with MBP and MBP(152-167) caused a modest and transitory elevation of intracellular calcium, whereas treatment with MBP(1-44) resulted in a substantial and sustained increase in intracellular calcium. These results suggest that for cultured astrocytes 1) FGFR and extracellular calcium play a major role in MBP mitogenicity; 2) MBP(1-44), MBP(88-151) and MBP(152-167) are the mitogenic regions of MBP; 3) MBP(1-44) and MBP(152-167) interact with ganglioside GM(1) and FGFR, respectively; 4) Component(s) present in microglial CM potentiate the mitogenicity of MBP(1-44), MBP(88-151) and MBP(152-167). These data support the hypothesis that MBP related peptides in conjunction with microglial secreted factors may stimulate astrogliosis after demyelination in vivo.

Expression of Kit in neurofibromin-deficient human Schwann cells: role in Schwann cell hyperplasia associated with type 1 neurofibromatosis.

Type 1 Neurofibromatosis (NF1) is characterized by the formation of neurofibromas, benign tumors composed mainly of Schwann cells, which can turn malignant to form neurofibrosarcomas. Neurofibromin, the protein product of the Nf1 gene, is believed to act as a tumor suppressor, accelerating the conversion of the oncogene Ras to its inactive form. The absence of neurofibromin could therefore lead to higher Ras activity in Schwann cells, resulting in uncontrolled growth through a cascade of events not yet elucidated. We describe the abnormal expression of high levels of the Kit tyrosine kinase receptor in both NF1-derived Schwann cell lines and tissue, as compared to primary Schwann cells or schwannoma-derived cells. High levels of Kit expression in the neurofibrosarcoma-derived Schwann cells correlate with a decrease in neurofibromin expression. Using inhibitors of tyrosine kinase receptors, we found that proliferation of the neurofibrosarcoma-derived cells is dependent upon activation of a subclass of tyrosine-kinase receptors. The proliferation of these cells is not dependent upon an autocrine loop involving typical Schwann cell mitogens. Finally, the proliferation of the neurofibrosarcoma-derived Schwann cells can be increased by stimulation with Kit ligand. These data implicate Kit as one of the components leading to the Schwann cell hyperplasia observed in NF1.

Somatotopic consolidation: a third phase of reorganization after peripheral nerve injury in adult squirrel monkeys.

It has previously been demonstrated that the central somatosensory topographic reorganization within deprived cortex that follows peripheral nerve injury in adult monkeys occurs in at least two stages: an immediate unmasking period; and a more prolonged period where deprived areas of cortex come to express new receptive fields in a topographically arranged manner. In the present experiments, we have compared cortical topography many months after combined median and ulnar nerve transection with "complete" reorganization evident at relatively short (i.e., 2-5 months) survival times. We find further reorganizational changes in cortical topography with longer survival times. That is, the roughly somatotopic, generally multiple-digit receptive fields frequently observed at the shorter survival times are generally sharpened to more distinct, single-digit receptive fields at longer survival times. We hypothesize that the early crudely topographic maps reflect all available inputs while the refined map is the outcome of an extraction process where only the most useful subset of available inputs is expressed. It is further suggested that this distillation process is a use-dependent phenomenon.

Denervation-induced sprouting of intact peripheral afferents into the cuneate nucleus of adult rats.

In adult monkeys with dorsal rhizotomies extending from the second cervical (C2) to the fifth thoracic (T5) vertebrae, cortex deprived of its normal inputs regained responsiveness to inputs conveyed by intact peripheral afferents from the face [T.P. Pons, P.E. Garraghty, A.K. Ommaya, J.H. Kaas, E. Taub, M. Mishkin, Massive reorganization of the primary somatosensory cortex after peripheral sensory deafferentation, Science 252 (1991) 1857-1860]. It has been suggested that the extent of this massive topographic reorganization may be due to the establishment of novel connections between intact afferents and neurons denervated after dorsal rhizotomy [P.E. Garraghty, D.P. Hanes, S.L. Florence, J.H. Kaas, Pattern of peripheral deafferentation predicts reorganizational limits in adult primate somatosensory cortex, Somatosens. Motor Res. 11 (1994) 109-117]. Using adult rats with comparably extensive dorsal rhizotomies, we employed anatomical tracing techniques to address this possibility. Subcutaneous hindpaw injections of horseradish peroxidase conjugated to either wheat germ agglutinin or cholera toxin subunit B revealed aberrant expansions of gracile projections into the cuneate and, in one case, external cuneate nucleus within three months of the deafferentation. It seems plausible that such modest sprouting of ascending projections at the level of the brainstem may form functional connections which, through divergence, ultimately drive a larger population of neurons in cortex. This new growth may well account for both the substantial cortical reorganization observed in the 'Silver Spring monkeys' [T.P. Pons, P.E. Garraghty, A.K. Ommaya, J.H. Kaas, E. Taub, M. Mishkin, Massive reorganization of the primary somatosensory cortex after peripheral sensory deafferentation, Science 252 (1991) 1857-1860] and the 'referred sensation' phenomena (see J.P. Donoghue, Plasticity of adult sensorimotor representations, Curr. Opin. Neurobiol., 5 (1995) 749-754 for review) reported to follow proximal limb amputations in humans.

NMDA receptors and plasticity in adult primate somatosensory cortex.

Topographic maps in adult primate somatosensory cortex are capable of dramatic reorganizations after peripheral nerve injuries. In the present experiments, we have deprived a circumscribed portion of the hand map in somatosensory cortex of our adult squirrel monkeys by transecting the median nerve to one hand, and evaluated the hypothesis that N-methyl-d-aspartate (NMDA) glutamatergic receptors are necessary for the reorganization that follows within four weeks. In one monkey, we confirm previous results demonstrating that the deprived cortex has regained responsiveness in its expanse four weeks after median nerve transection. However, in three monkeys in which NMDA receptors were concurrently blocked, most of the deprived cortex remained unresponsive. Thus, much of the cortical "recovery" that typically follows peripheral nerve injury in adult monkeys is apparently dependent on NMDA receptors and may well be due to Hebbian-like changes in synaptic strength. Perhaps the elimination of the normally dominant inputs to "median nerve cortex" permits the gradual strengthening of correlations between the activity of the formally impotent presynaptic and deprived postsynaptic elements. These enhanced correlations may also have been made possible by reductions in intracortical inhibition as a necessary but not sufficient condition.

Possible use-dependent changes in adult primate somatosensory cortex.

Topographic changes in adult primate somatosensory cortical maps have been reported to follow well-regulated manipulations in the animals' experience. We report briefly here cortical mapping data from one monkey which arrived at our laboratory with a chronic paralytic condition in one hand that resulted in a unique pattern of skin surface stimulation. Isolated receptive fields across the mediolateral extent of cortical area 3b were highly unusual with respect to normal topography, but they were completely consistent with the hypothesis that the correlated activation of peripheral afferents acts to shape expressed cortical receptive fields.