Pericytes resident in postnatal skeletal muscle differentiate into muscle fibres and generate satellite cells.

Skeletal muscle fibres form by fusion of mesoderm progenitors called myoblasts. After birth, muscle fibres do not increase in number but continue to grow in size because of fusion of satellite cells, the postnatal myogenic cells, responsible for muscle growth and regeneration. Numerous studies suggest that, on transplantation, non-myogenic cells also may contribute to muscle regeneration. However, there is currently no evidence that such a contribution represents a natural developmental option of these non-myogenic cells, rather than a consequence of experimental manipulation resulting in cell fusion. Here we show that pericytes, transgenically labelled with an inducible Alkaline Phosphatase CreERT2, but not endothelial cells, fuse with developing myofibres and enter the satellite cell compartment during unperturbed postnatal development. This contribution increases significantly during acute injury or in chronically regenerating dystrophic muscle. These data show that pericytes, resident in small vessels of skeletal muscle, contribute to its growth and regeneration during postnatal life.

Partial dysferlin reconstitution by adult murine mesoangioblasts is sufficient for full functional recovery in a murine model of dysferlinopathy.

Dysferlin deficiency leads to a peculiar form of muscular dystrophy due to a defect in sarcolemma repair and currently lacks a therapy. We developed a cell therapy protocol with wild-type adult murine mesoangioblasts. These cells differentiate with high efficiency into skeletal muscle in vitro but differ from satellite cells because they do not express Pax7. After intramuscular or intra-arterial administration to SCID/BlAJ mice, a novel model of dysferlinopathy, wild-type mesoangioblasts efficiently colonized dystrophic muscles and partially restored dysferlin expression. Nevertheless, functional assays performed on isolated single fibers from transplanted muscles showed a normal repairing ability of the membrane after laser-induced lesions; this result, which reflects gene correction of an enzymatic rather than a structural deficit, suggests that this myopathy may be easier to treat with cell or gene therapy than other forms of muscular dystrophies.

Cerebellar neurons and glial cells are transducible by lentiviral vectors without decrease of cerebellar functions.

Due to the profuse connections of the cerebellum to the rest of the central nervous system, cerebellar dysfunction impacts tremendously on movement coordination, maintenance of equilibrium, muscle tone and motor memory. Efficient gene transfer of therapeutic genes to this central nervous system structure would constitute a relevant step ahead the design of treatments to ameliorate cerebellar dysfunction. Lentiviral vectors (LVs) have been used as efficient vehicles to integrate transgenes into dividing and non-dividing cells, such as postmitotic adult neurons, with minimal toxicity and immune response. This study aimed to use LVs carrying green fluorescent protein (GFP) cDNA for transduction of cerebellar cells in vivo without compromising neurological cerebellar functions. Our results indicate that LVs, injected in the lobulus simplex, transduced different cerebellar neurons including stellate, Purkinje cells, granular neurons and glial cells such as astrocytes, oligodendrocytes, and that this gene transfer approach was not accompanied by cerebellar deficits.

Design and optimization of lentiviral vectors for transfer of GALC expression in Twitcher brain.

BACKGROUND: Demyelination in globoid cell leukodystrophy (GLD) is due to a deficiency of galactocerebrosidase (GALC) activity. Up to now, in vivo brain viral gene transfer of GALC showed modest impact on disease development in Twitcher mice, an animal model for GLD. Lentiviral vectors, which are highly efficient to transfer the expression of therapeutic genes in neurons and glial cells, have not been evaluated for direct cerebral therapy in GLD mice. METHODS: Lentiviral vectors containing the untagged cDNA or the hemagglutinin (HA)-tagged cDNA for the full-length mouse GALC sequence were generated and validated in vitro. In vivo therapeutic efficacy of these vectors was evaluated by histology, biochemistry and electrophysiology after transduction of ependymal or subependymal layers in young Twitcher pups. RESULTS: Both GALC lentiviral vectors transduced neurons, oligodendrocytes and astrocytes with efficiencies above 75% and conferred high levels of enzyme activity. GALC accumulated in lysosomes of transduced cells and was also secreted to the extracellular medium. Conditioned GALC medium was able to correct the enzyme deficiency when added to non-transduced Twitcher glial cultures. Mice that received intraventricular injections of GALC vector showed accumulation of GALC in ependymal cells but no diffusion of the enzyme from the ependymal ventricular tree into the cerebral parenchyma. Significant expression of GALC-HA was detected in neuroglioblasts when GALC-HA lentiviral vectors were injected in the subventricular zone of Twitcher mice. Life span and motor conduction in both groups of treated Twitcher mice were not significantly ameliorated. CONCLUSIONS: Lentiviral vectors showed to be efficient for reconstitution of the GALC expression in Twitcher neural cells. GALC was able to accumulate in lysosomes as well as to enter the secretory pathway of lysosomal enzymes, two fundamental aspects for gene therapy of lysosomal storage diseases. Our in vivo results, while showing the capacity of lentiviral vectors to transfer expression of therapeutic GALC in the Twitcher brain, did not limit progression of disease in Twitchers and highlight the need to evaluate other routes of administration.

Myelin deterioration in Twitcher mice: motor evoked potentials and magnetic resonance imaging as in vivo monitoring tools.

We have used magnetic resonance imaging (MRI) and motor evoked potentials (MEPs) for monitoring disease progression within the CNS of the Twitcher mouse, the murine model for globoid cell leukodystrophy (GLD). GLD is a lysosomal storage disorder, resulting from galactocerebrosidase deficiency, causing central and peripheral myelin impairment, leading to death, usually during early infancy. Neuroradiological, electrophysiological, and pathological parameters of myelin maturation were evaluated in Twitcher mice between postnatal days 20 and 45. Healthy controls showed a gradual-appearance MRI T2-weighted hypointensity of the corpus callosum (CC) starting at about P30 and ending at about P37, whereas MRI of age-matched Twitcher mice showed a complete loss of the CC-related MRI signal. MEPs allowed the functional assessment of myelin maturation within corticospinal motor pathways and showed a progressive deterioration of MEPs in Twitcher mice with increased central conduction time (CCT; 5.12 +/- 0.49 msec at P27 to 6.45 +/- 1.96 msec at P32), whereas physiological CCT shortening was found in healthy controls (3.01 +/- 0.81 msec at P27 to 2.5 +/- 0.27 msec at P32). These findings were not paralleled by traditional histological stainings. Optical observation of Bielchowsky and Luxol fast blue-PAS stainings showed mild axonal/myelin deterioration of the Twitcher brain within this time frame. Our results demonstrate that serial MRI and MEP readings are sensitive evaluation tools for in vivo monitoring of dysmyelination in Twitcher mice and underscore their potential use for longitudinal evaluation of the therapeutic impact of gene and cell therapies on these animals.

Analysis of galactocerebrosidase activity in the mouse brain by a new histological staining method.

Gene therapy of galactocerebrosidase (GALC) deficient mice (Twitcher mutants) requires a fast and sensitive assay to detect transduced cells in vitro and in vivo. We have developed a new rapid histochemical method that specifically detects GALC activity in situ in neural cells using 5-Br-3Cl-beta-galactopiranoside (X-Gal) in the presence of taurodeoxycholic and oleic acids to enhance suspension of the substrate at low pH. Using this method, we observed robust X-Gal staining in diverse neuronal populations and interfascicular oligodendrocytes in sections from normal mouse brain. In contrast, sections of Twitcher brain did not show a specific staining pattern in neurons or glial cells. The availability of this new sensitive and rapid in situ detection assay is fundamental for the follow-up of Twitcher mice under gene or cellular therapies to correct central GALC deficiency.

Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan polymerase chain reaction.

Here we present a novel methodology to quantitate bovine cytokines and growth factors contributing to immunity against bacterial infections of the mammary gland in cattle. Real-time TaqMan PCR systems were developed to overcome limitations of conventional quantitative PCR methods. The TaqMan method is based on the cleavage of fluorescent dye-labeled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by an automated spectrophotometer integrated in a sequence detection system (Applied Biosystems, Foster City, CA). The bovine-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation between complementary DNA (cDNA) and genomic DNA (gDNA) amplification products. Quantitative analysis of cytokine cDNA was performed in comparison to bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in different numbers of cells in the starting material, in the efficiencies of RNA extraction and reverse transcription. With this method, high-throughput analysis of large numbers of samples was possible within a short time. In addition, decreasing the numbers of working steps shortened the time for analysis and increased accuracy. Profiles of cytokines (interleukin (IL)-2, IL-6, IL-8, IL-12 p40, TNF-alpha, IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF) were established in normal lactating cattle. Differences of cytokine profiles obtained with the real-time TaqMan PCR system and conventional methods are discussed.

Financial analysis of alternative treatments for clinical mastitis associated with environmental pathogens.

For two large California dairy herds with twice daily milking, 171 infected quarters of lactating cows with mild clinical mastitis were randomly assigned to one of three treatment groups. Group A (50 cows) was treated with 62.5 mg of intramammary amoxicillin every 12 h for three milkings. Group C (50 cows) was treated with 200 mg of intramammary cephapirin every 12 h for two milkings. Group O (71 cows) was treated with 100 U of intramuscular oxytocin every 12 h for three milkings. Clinical cure rates did not differ among treatment groups. The cost per episode of clinical mastitis was higher ($54.47) for group C than for groups A ($38.53) or O ($34.88). Group O had a higher incidence of relapse (41%), and a greater percentage of group O cows (65%) experienced an additional mastitic event in the enrolled lactation than did cows in groups A and C. Treatment had no effect on the mean number of nonsalable milkings (24.7) associated with mastitis in the lactation interval subsequent to enrollment on the trial. Twenty-two percent of the cows accrued more than 30 nonsalable milkings and produced 55% of the total nonsalable milkings associated with mastitis. There was no treatment effect on total milk production, fat production, or time to removal of the enrolled cows from the herd.

Performance of various tests used to screen antibiotic residues in milk samples from individual animals.

The 10-point Milk and Dairy Beef Quality Assurance Program was developed collaboratively by the National Milk Producers Federation and the American Veterinary Medical Association and is designed to promote and document the responsible use of antibiotics in the dairy industry. One area of emphasis in this program is testing of individual animals for antibiotic residues after a specified post-treatment withdrawal time. We examined the performance of various assay systems on milk samples from individual cows. These assays are used at present on bulk tank milk samples by regulatory agencies, processing plants, producers, and veterinarians to detect the presence of beta-lactam antibiotics. A high proportion of false-positive results was obtained for both the pretreatment milk samples from cows with clinical mastitis and the milk samples obtained 21 days after initial therapy (nonantibiotic and antibiotic) for the treatment of mastitis. A high proportion of false-positive outcomes was obtained from the milk of clinically normal cows that had not received any medication for at least 30 days prior to evaluation. The results indicate a serious problem in the use of some assays that were designed to evaluate residues bulk tank milk samples to analyze samples from individual cows. This error in assay specificity results in the unjustifiable discarding of milk that meets regulatory standards and may be misused to accuse the producer or veterinarian of not adhering to regulatory guidelines. Maintaining a safe, high-quality milk supply is a constant goal of the dairy industry, which must be provided the appropriate tools and techniques to meet this challenge.

Evaluation of milk antibiotic residue screening tests in cattle with naturally occurring clinical mastitis.

Milk from 172 commercial cows with mild to moderate clinical mastitis was tested with five antibiotic residue detection assay systems. One hundred cows were treated with one of two intramammary beta-lactam antibiotics, and the remaining 72 cows were treated with intramuscular oxytocin. Milk samples were collected pretreatment, twice after therapy, and again 21 d following the initiation of treatment. Presumptive false-positive assay results were tabulated from all pretreatment and 21-d milk samples and from samples collected following oxytocin therapy. The percentage of false-positive results was 43.6, 37.7, 81.7, 2.6, and 18.8% for the CITE probe (beta-lactam), Delvotest-P, Charm Farm, LacTek (beta-lactam), and Bacillus stearothermophilus var. calidolactis disk assay, respectively. In four of the assay systems, average SCC were significantly higher in samples yielding false-positive results than in those with negative results. Specificity and sensitivity were estimated for each assay system, and, based on these estimates, positive and negative predictive value curves were graphed as the prevalence of milk samples containing detectable concentrations of exogenous antibiotic residues in the sample population was varied from 0 to 100%.