Stavros Bashiardes

Intrathecal gene therapy rescues a model of demyelinating peripheral neuropathy.

Significance Inherited demyelinating peripheral neuropathies are progressive incurable diseases caused by mutations in a variety of genes expressed by myelinating Schwann cells. A major challenge in developing effective gene therapy is to gain access to multiple nerves for cell-specific expression. Our study demonstrates for the first time, to our knowledge, that intrathecal injection of a lentiviral vector with a myelin-specific promoter can achieve targeted expression in adult myelinating Schwann cells in a widespread distribution throughout the peripheral nervous system. Furthermore, this translatable approach restored the expression of a neuropathy-associated gene and led to a phenotypic, functional, and pathological rescue of a neuropathy model. These results have important implications for further preclinical and clinical testing in this and other types of inherited demyelinating neuropathies. Inherited demyelinating peripheral neuropathies are progressive incurable diseases without effective treatment. To develop a gene therapy approach targeting myelinating Schwann cells that can be translatable, we delivered a lentiviral vector using a single lumbar intrathecal injection and a myelin-specific promoter. The human gene of interest, GJB1, which is mutated in X-linked Charcot–Marie–Tooth Disease (CMT1X), was delivered intrathecally into adult Gjb1-null mice, a genetically authentic model of CMT1X that develops a demyelinating peripheral neuropathy. We obtained widespread, stable, and cell-specific expression of connexin32 in up to 50% of Schwann cells in multiple lumbar spinal roots and peripheral nerves. Behavioral and electrophysiological analysis revealed significantly improved motor performance, quadriceps muscle contractility, and sciatic nerve conduction velocities. Furthermore, treated mice exhibited reduced numbers of demyelinated and remyelinated fibers and fewer inflammatory cells in lumbar motor roots, as well as in the femoral motor and sciatic nerves. This study demonstrates that a single intrathecal lentiviral gene delivery can lead to Schwann cell-specific expression in spinal roots extending to multiple peripheral nerves. This clinically relevant approach improves the phenotype of an inherited neuropathy mouse model and provides proof of principle for treating inherited demyelinating neuropathies.

Intraneural GJB1 gene delivery improves nerve pathology in a model of X-linked Charcot-Marie-Tooth disease.

X‐linked Charcot–Marie–Tooth disease (CMT1X) is a common inherited neuropathy caused by mutations in the GJB1 gene encoding the gap junction protein connexin32 (Cx32). Clinical studies and disease models indicate that neuropathy mainly results from Schwann cell autonomous, loss‐of‐function mechanisms; therefore, CMT1X may be treatable by gene replacement.

Prevalence of Helicobacter pylori cagA and vacA genes in Cypriot patients

INTRODUCTION The prevalence of H. pylori varies with geographic locations. To date there are no epidemiological data on its prevalence in Cyprus; therefore, we determined the prevalence and molecular characteristics of H. pylori infection in Cypriot patients. METHODOLOGY DNA extracted from 103 gastric biopsies was analyzed for the presence of H. pylori by PCR using primers for ureA. H. pylori-positive biopsies were characterized by PCR using specific primers for cagA and vacA genes. The presence of clarithromycin-associated resistant mutations such as A2143G, A2142G, A2142C in 23S rRNA gene of H. pylori-positive patients was determined using a real-time PCR allelic discrimination assay. RESULTS H. pylori was detected in 41 (39.8%) biopsies and, out of these, 17 (41.5%) tested positive for the cagA gene. The vacA alleles m1, m2, s1a, s1b, and s2 were detected in 7 (17.1%), 34 (82.9%), 12 (29.3%), 2 (4.9%), and 22 (53.7%) isolates, respectively. One (2.4%) biopsy was vacA s1a and s2-positive while one (2.4%) was positive for vacA s1a, s1b, and s2. Three (7.3%) biopsies were untypable for vacA s1, s1b, and s2. The majority (35; 85.4%) of strains were susceptible to clarithromycin while two (4.9%) had the A2143G mutation. Three (7.3%) had a mixture of an A2143G point mutant and susceptible strains while one (2.4%) had a mixture of an A2142G point mutant and susceptible strains. CONCLUSIONS The distribution of the virulence factors cagA and vacA in the Cypriot strains resembled that of strains circulating in Middle Eastern countries geographically close to Cyprus.

Gene delivery targeted to oligodendrocytes using a lentiviral vector

Most leukodystrophies result from mutations in genes expressed in oligodendrocytes that may cause autonomous loss of function of cell structural proteins. Therefore, effective gene delivery to oligodendrocytes is necessary to develop future treatments.

An in-house method for the detection and quantification of HCV in serum samples using a TaqMan assay real time PCR approach.

Abstract Background: Hepatitis C virus (HCV) is one of the most common viral infections worldwide causing major human chronic pathology. Viral RNA can be detected 1–3 weeks after infection and in cases where HCV RNA is still detectable after 6 months, the patient is considered to be chronically infected. Early detection is crucial in preventing loss of treatment opportunities. Methods: Here, we present an in-house real time PCR TaqMan® probe based screening method for the detection and quantification of the six recognized HCV genotypes and evaluate its efficiency with the use of quality control for molecular diagnostics HCV quantification panels. Results and conclusions: The quantification method presented performed well with all quality control panels and is therefore an attractive approach for the clinical setting. Clin Chem Lab Med 2008;46:1729–31.

2005 Poliovirus eradication: poliovirus presence in Cyprus 2 years after

The last case of polio in Cyprus caused by a wild-type poliovirus occurred in 1995. Since then Cyprus belongs to the countries considered poliovirus-free by the WHO. The aim of this study was to confirm the absence of any circulating wild-type polioviruses and to monitor vaccine-derived polioviruses in Cyprus by analysis of sewages. During the course of this study no wild-type polioviruses were identified, although the identification of viable oral poliovirus vaccine isolates confirmed the presence and circulation of poliovirus vaccine strains in sewage in Cyprus.

Evaluation of whole-genome amplification using multiple-displacement amplification of a limited number of cells.

The issue of limited amounts of DNA or depleting existing DNA stocks is a major problem in genetic analyses. The prospect of efficiently generating (through amplification) adequate quantities of genomic DNA for subsequent analyses from as little as a single cell would be extremely relevant and important to such applications. In this study, we investigated the feasibility of using multiple-displacement amplification (MDA) for wholegenome amplification (WGA) of genetic material from as little as a single cell, and report the problems associated with the use of such quantities of starting genetic material. We carried out WGA using MDA from a single cell and 10 cells, and compared these amplified products to unamplified genomic DNA to determine the efficiency and uniformity of amplification throughout the genome, the level of allele dropout, and consequently the feasibility of using MDA in subsequent clinical applications. Lymphocyte cells from five individuals with normal karyotypes designated P (46,XY), G (46,XY), S (46,XY), E (46,XX) and A (46,XX) were isolated from whole peripheral blood by centrifugation over Ficoll and resuspended in magnesium-free phosphate-buffered saline (PBS) with 15 mg/mL polyvinylpyrrolidone. Single lymphocytes and 10-lymphocyte aliquots were accurately isolated under an inverted microscope (Nikon, Tokyo, Japan) and micromanipulator controllers (Narishige, Tokyo, Japan) using assisted hatching pipettes with a 5-mm opening (Cook, Brisbane, Australia). Isolated cells were first placed in 5-mL PBS microdrops under oil, washed again in a second 5-mL PBS microdrop under oil and then placed in a 0.2-mL PCR tube on ice containing 3.5 mL of PBS. This methodology and technology provided a very accurate and sensitive procedure for true 1and 10-cell isolation.

Acute hepatitis and myositis associated with Erythema infectiosum by Parvovirus B19 in an adolescent

BackgroundErythema infectiosum is the most common clinical manifestation of Parvovirus B19 infection although it has also been associated with rheumatologic diseases and various types of systemic vasculitides. Acute hepatitis and benign myositis however are rarely reported in association with Parvovirus B19 infection.Case presentationHere we report a 14-year old male, who developed acute hepatitis and benign myositis associated with erythema infectiosum following Parvovirus B19 infection.ConclusionParvovirus B19 infection has rarely been associated with acute hepatitis and exceptionally rarely with benign myositis. Parvovirus B19 should be considered in the differential diagnosis of acute non-A to E hepatitis and in the case of acute benign myositis presenting with a rash especially in children.