Specific retrograde transduction of spinal motor neurons using lentiviral vectors targeted to presynaptic NMJ receptors.

To understand how receptors are involved in neuronal trafficking and to be able to utilize them for specific targeting via the peripheral route would be of great benefit. Here, we describe the generation of novel lentiviral vectors with tropism to motor neurons that were made by coexpressing onto the lentiviral surface a fusogenic glycoprotein (mutated sindbis G) and an antibody against a cell-surface receptor (Thy1.1, p75(NTR), or coxsackievirus and adenovirus receptor) on the presynaptic terminal of the neuromuscular junction. These vectors exhibit binding specificity and efficient transduction of receptor positive cell lines and primary motor neurons in vitro. Targeting of each of these receptors conferred to these vectors the capability of being transported retrogradely from the axonal tip, leading to transduction of motor neurons in vitro in compartmented microfluidic cultures. In vivo delivery of coxsackievirus and adenovirus receptor-targeted vectors in leg muscles of mice resulted in predicted patterns of motor neuron labeling in lumbar spinal cord. This opens up the clinical potential of these vectors for minimally invasive administration of central nervous system-targeted therapeutics in motor neuron diseases.

Venezuelan equine encephalitis virus glycoprotein pseudotyping confers neurotropism to lentiviral vectors.

We have produced high-titre HIV-1 green fluorescent protein-expressing lentiviral (LV) vectors pseudotyped with strain 3908 Venezuelan equine encephalitis virus glycoprotein (VEEV-G) and used them to study transduction of: (1) rat embryonic motor neuron (MN) and striatal neuron primary cultures, (2) differentiated MN cell line NSC-34 and (3) adult rat striatum. In primary neuronal cultures, transduction with VEEV-G-pseudotyped LV was more efficient and more neuronal than with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped LV. In NSC-34 cells clear retrograde transport of VEEV-G vector particles was observed. In the striatum at the injection site, transduction with the VEEV-G vectors driven by cytomegalovirus or phosphoglycerate kinase promoters exhibited a distinct neuronal tropism with no microglial and only a minor astroglial component, superior to that obtained with VSV-G-pseudotyped LV, irrespective of the promoter used. Neuronal transduction efficiency increased over time. Distal to the injection site transduction of mitral cells in the olfactory bulb, thalamic neurons and dopaminergic neurons in the substantia nigra pars compacta was detected. This, together with observations of retrograde axonal trafficking in vitro indicates that these vectors also possess low level of retrograde neuronal transduction capability in vivo. In this study, we demonstrate both strong neurotropism as well as sustainability of expression and minimal host immune response in vivo, making the VEEV-G-pseudotyped LV vectors potentially useful for gene therapy of neurodegenerative diseases.

Advances in the topical treatment of diabetic foot ulcers.

The diabetic foot remains a major cause of morbidity worldwide. Even though considerable progress has been achieved over the past years, there is still an urgent need for improvement. While established therapeutic modalities (revascularization, casting and debridement) remain the mainstay of management, there is, therefore, continuous development of new treatment options. This review provides an outlook of advances in topical treatment, including bioengineered skin substitutes (such as Dermagraft, Apligraf, HYAFF, OASIS and Graftjacket), extracellular matrix proteins (such as Hyalofill and E-matrix), as well as miscellaneous further therapeutic adjuncts. Although promising, new therapies should not, for the time being, constitute the basis of management, since clinical experience has not yet confirmed their effectiveness in hard-to-heal diabetic foot ulcers. Furthermore, their cost-effectiveness merits further investigation. Instead, they should only be considered in combination with established treatments or be attempted when these have not been successful. Moreover, we should not be oblivious to the fact that established and emerging treatments need to be practised in the setting of multidisciplinary foot clinics to reduce the number of amputations.

The role of the osteoprotegerin/RANKL/RANK system in diabetic vascular disease.

Over the last years our knowledge on the mechanisms involved in the pathogenesis of cardiovascular disease has been enriched by the discovery of new molecules emerging as novel risk factors. Osteoprotegerin (OPG) is a soluble glycoprotein, member of the tumor necrosis factor (TNF)-related superfamily, involved in bone resorption. It was first described as a key regulator of bone homeostasis and vascular calcification in mice. Clinical studies have suggested that serum OPG is associated with vascular calcification in humans. The role of OPG in the development of macroangiopathy in diabetes is not yet clear. It is possible that the increased OPG levels in diabetes reflect a compensatory response to arterial injury and that it is not involved in the pathogenesis of atherosclerosis. Whether harmful or not, determination of serum OPG levels has been suggested as a prognostic biomarker of cardiovascular disease. In addition, increased OPG levels have been reported in diabetic patients with microvascular complications. The potential of OPG administration for therapeutic reasons is challenging for future investigators. This review summarizes the current knowledge on the association between OPG and macrovascular as well microvascular complications of diabetes.

Acute hyperhomocysteinemia impairs endothelium function in subjects with type 2 diabetes mellitus.

The objective of the present study was to examine the effect of acute, methionine-induced hyperhomocysteinemia (HHCY) on endothelial function and indices of arterial stiffness in subjects with type 2 diabetes mellitus (T2DM). A total of 30 subjects with T2DM, free of macrovascular disease were examined in a crossover study. L-methionine (M) (0.1 g/kg) and water (W) load were given in random order with an interval of about 1 week in between. Endothelial function was assessed by flow-mediated vasodilation (FMD). Arterial stiffness was assessed by determination of augmentation index (AI). Measurements were performed in the fasting state, 1, 2 and 3 h after the M or the W load. Total plasma homocysteine (HCY) levels did not change after W administration, while M administration resulted in a significant increase in HCY concentrations at 3 h. FMD throughout the experiment expressed as area under the curve (AUC) was significantly lower after the M than after the W load. Consistent with impairment in endothelial function, the AUC of AI was significantly higher after the M than after the W administration. Acute HHCY impairs endothelial function and increases arterial stiffness in patients with T2DM. This effect is probably mediated by a reduction of nitric oxide bioavailability in endothelium.

Peripheral arterial disease in diabetes--a review.

Diabetic patients are at high risk for peripheral arterial disease (PAD) characterized by symptoms of intermittent claudication or critical limb ischaemia. Given the inconsistencies of clinical findings in the diagnosis of PAD in the diabetic patient, measurement of ankle-brachial pressure index (ABI) has emerged as the relatively simple, non-invasive and inexpensive diagnostic tool of choice. An ABI < 0.9 is not only diagnostic of PAD even in the asymptomatic patient, but is also an independent marker of increased morbidity and mortality from cardiovascular diseases. With better understanding of the process of atherosclerosis, avenues for treatment have increased. Modification of lifestyle and effective management of the established risk factors such as smoking, dyslipidaemia, hyperglycaemia and hypertension retard the progression of the disease and reduce cardiovascular events in these patients. Newer risk factors such as insulin resistance, hyperfibrinogenaemia, hyperhomocysteinaemia and low-grade inflammation have been identified, but the advantages of modifying them in patients with PAD are yet to be proven. Therapeutic angiogenesis, on the other hand, represents a promising therapeutic adjunct in the management of PAD in these patients. Outcomes after revascularization procedures, such as percutaneous transluminal angioplasty and surgical bypasses in diabetic patients, are poorer, with increased perioperative morbidity and mortality compared with that in non-diabetic patients. Amputation rates are higher due to the distal nature of the disease. Efforts towards increasing awareness and intensive treatment of the risk factors will help to reduce morbidity and mortality in diabetic patients with PAD.

The effects of medications used for the management of dyslipidemia on postprandial lipemia.

Postprandial lipemia has emerged as an independent risk factor for coronary artery disease. In this systematic review we examined the effect of the medications used for the management of dyslipidemia on postprandial lipemia. Statins, beyond their effects on fasting lipid levels, reduce also postprandial lipemia mainly by inhibiting the production of apoB containing lipoproteins from the liver and thus increasing the clearance of triglyceride-rich lipoproteins of either liver or intestinal origin. Fibrates decrease fasting triglyceride and increase high density lipoprotein cholesterol levels. Besides, fibrates are particularly potent drugs in the reduction of postprandial lipemia; they decrease the production or triglyceride-rich lipoproteins and increase their clearance by enhancing the activity of lipoprotein lipase.