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Biomedical subjects

R Heller

Publications and source records attributed to R Heller.

At least 19 recordsLinked to original sources
PubMed2006

Optimization of cutaneous electrically mediated plasmid DNA delivery using novel electrode.

The easy accessibility of skin makes it an excellent target for gene transfer protocols. To take advantage of skin as a target for gene transfer, it is important to establish an efficient and reproducible delivery system. Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo. A critical component of this technique is the electrode configuration. Electroporation parameters were optimized for transgene expression with minimal tissue damage with a novel electrode. The highest transgene expression and efficiency of individual cell transformation with minimal damage was produced with eight 150 ms pulses at field strength of 100 V/cm. This electrode design offers the potential for easier and more reproducible electrically mediated cutaneous plasmid delivery than the simple electrodes currently commercially available. This electrode can be a valuable tool in determining the applicability of electrically mediated cutaneous gene transfer.

Animals↗
PubMed2006

Regression of subcutaneous B16 melanoma tumors after intratumoral delivery of an IL-15-expressing plasmid followed by in vivo electroporation.

In vivo electroporation has been used to efficiently deliver drugs and 'therapeutic' genes to tumors, including melanoma lesions. This study reports on the effect of intratumoral delivery of an optimized DNA plasmid expressing interleukin-15 (pIL-15) on established murine melanoma tumors. IL-15 has been demonstrated to have a pivotal role in the function of memory CD8+ T cells and natural killer cells, which are critical for tumor immunosurveillance. In this study, C57BL/6 mice were injected with B16.F10 melanoma cells and randomized into different experimental groups: untreated (P-V-E-), treated with pIL-15 (P+) or backbone plasmid (V+), with or without electroporation (E+ or E-). Treatment was performed intratumorally with 50 microg of plasmid on days 0, 4 and 7 and tumor volume/size, tumor regression and long-term survival were measured. At day 100 after initiation of treatment, the percentage of mice surviving with complete tumor regression in the P-V+E+, P+V-E-, P+V-E+ and P-V-E- treatment groups were 0, 12.5, 37.5 and 0%, respectively. These results demonstrate the ability of pIL-15 to mediate B16 melanoma regression, with the effect being significantly enhanced by electroporative delivery. This is the first description of the ability of a naked DNA plasmid expressing IL-15 to alone mediate complete regression of B16 melanoma tumors and underscores the potential clinical use of these plasmids for the treatment of malignant tumors when delivered with in vivo electroporation.

Animals↗
PubMed2006

Mildly affected patients with spinal muscular atrophy are partially protected by an increased SMN2 copy number.

Spinal muscular atrophy (SMA) is a recessive neuromuscular disorder caused by loss of the SMN1 gene. The clinical distinction between SMA type I to IV reflects different age of onset and disease severity. SMN2, a nearly identical copy gene of SMN1, produces only 10% of full-length SMN RNA/protein and is an excellent target for a potential therapy. Several clinical trials with drugs that increase the SMN2 expression such as valproic acid and phenylbutyrate are in progress. Solid natural history data for SMA are crucial to enable a correlation between genotype and phenotype as well as the outcome of therapy. We provide genotypic and phenotypic data from 115 SMA patients with type IIIa (age of onset <3 years), type IIIb (age of onset >3 years) and rare type IV (onset >30 years). While 62% of type IIIa patients carry two or three SMN2 copies, 65% of type IIIb patients carry four or five SMN2 copies. Three type IV SMA patients had four and one had six SMN2 copies. Our data support the disease-modifying role of SMN2 leading to later onset and a better prognosis. A statistically significant correlation for > or =4 SMN2 copies with SMA type IIIb or a milder phenotype suggests that SMN2 copy number can be used as a clinical prognostic indicator in SMA patients. The additional case of a foetus with homozygous SMN1 deletion and postnatal measurement of five SMN2 copies illustrates the role of genotypic information in making informed decisions on the management and therapy of such patients.

Adult↗
PubMed2005

Balanced translocation in a patient with craniosynostosis disrupts the SOX6 gene and an evolutionarily conserved non-transcribed region.

Craniosynostosis is a congenital developmental disorder involving premature fusion of cranial sutures, which results in an abnormal shape of the skull. Significant progress in understanding the molecular basis of this phenotype has been made for a small number of syndromic craniosynostosis forms. Nevertheless, in the majority of the approximately 100 craniosynostosis syndromes and in non-syndromic craniosynostosis the underlying gene defects and pathomechanisms are unknown. Here we report on a male infant presenting at birth with brachycephaly, proptosis, midfacial hypoplasia, and low set ears. Three dimensional cranial computer tomography showed fusion of the lambdoid sutures and distal part of the sagittal suture with a gaping anterior fontanelle. Mutations in the genes for FGFR2 and FGFR3 were excluded. Standard chromosome analysis revealed a de novo balanced translocation t(9;11)(q33;p15). The breakpoint on chromosome 11p15 disrupts the SOX6 gene, known to be involved in skeletal growth and differentiation processes. SOX6 mutation screening of another 104 craniosynostosis patients revealed one missense mutation leading to the exchange of a highly conserved amino acid (p.D68N) in a single patient and his reportedly healthy mother. The breakpoint on chromosome 9 is located in a region without any known or predicted genes but, interestingly, disrupts patches of evolutionarily highly conserved non-genic sequences and may thus led to dysregulation of flanking genes on chromosome 9 or 11 involved in skull vault development. The present case is one of the very rare reports of an apparently balanced translocation in a patient with syndromic craniosynostosis, and reveals novel candidate genes for craniosynostoses and cranial suture formation.

Amino Acid Sequence↗
PubMed2005

Comparison of processes and intermediate outcomes between South Asian and European patients with diabetes in Blackburn, north-west England.

BACKGROUND: Diabetes and its complications are more prevalent among South Asians than people of European origin and there is some evidence that patients of South Asian origin with diabetes receive poorer quality care. METHODS: Longitudinal study of patients with diabetes in Blackburn, UK. Processes of care indicators [measurement of blood pressure (BP), cholesterol and glycosolated haemoglobin (HbA1c)] and values of these intermediate outcomes were extracted for all patients registered on a diabetes information system for the period 1995-2001. Differences in processes of care indicators and intermediate outcomes between ethnic groups were estimated after adjusting for the potential confounding factors of sex, age and socioeconomic status (SES). Generalized estimating equations were used to model trends and to test for differences in trends over time. RESULTS: Process of care was similar in South Asian and European patients. Mean BP and cholesterol concentration fell during the study period. South Asians had a higher level of HbA1c throughout the study period. South Asians had lower levels of BP and cholesterol in 1995 but the differences diminished or were abolished over time. SES did not explain differences between ethnic groups. Analyses stratified by baseline levels of intermediate outcomes (above or within target) demonstrated improvements among above target patients were greater among European patients. CONCLUSIONS: Processes of care indicators are similar in diabetic patients of South Asian and European origin, irrespective of SES. However, there are worrying differences in improvements over time in the intermediate outcomes, and glycaemic control remained poorer among patients of South Asian origin patients throughout.

Adult↗
PubMed2002

[Cerebrotendinous xanthomatosis, a treatable metabolic disorder].

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disorder of bile acid synthesis which can be clinically diagnosed and specifically treated. It is an underdiagnosed disorder worldwide.Here,we describe two women who were diagnosed with CTX during their forties after symptoms had already developed 15 years earlier. Both patients showed gait ataxia, spastic paraparesis, polyneuropathy, bilateral premature cataracts, tendon xanthomas, and cognitive deficits. One of the patients had also chronic diarrhea. The deficiency of the mitochondrial enzyme sterol 27-hydroxylase results in a virtual absence of chenodeoxycholic acid. This leads to excessive production of cholestanol and cholesterol and accumulation of these sterols in many tissues, especially the eye lens, central nervous system,and tendons. The determination of a high cholestanol serum level allows the diagnosis,which can be confirmed through genetic analysis. Early diagnosis of CTX is important, since an effective therapy is available. Long-term therapy with chenodeoxycholic acid is effective for CTX, mainly in prevention of further deterioration.

Adult↗
PubMed2002

Hypertension control and medication increase in primary care.

Over half of treated patients with hypertension are not well controlled. However, little is known about physicians' prescribing behaviour for these patients. Our objective was to clarify whether physicians increase antihypertensive medication in patients with poorly controlled hypertension and what characteristics are predictors of medication increase. This was a retrospective cohort study by surveying medical records in primary care clinics in Tochigi, Japan. Twenty-nine of 79 randomly selected physicians agreed to select 20 consecutive hypertensive patients. This resulted in 547 patients (women 60%; mean (s.d.) age, 68 (12) years) who had blood pressure measurements taken in 1998 and prescription of antihypertensive medication in 1998 and 1999. Mean (s.d.) systolic/diastolic blood pressure was 142 (12)/81 (9) mm Hg and the percentage of patients in good control (<140/90 mm Hg), fair (140-159/90-94) and poor (> or =160/95) were 42%, 47%, and 11%, respectively. Physicians increased medication in 28% of poorly controlled patients (95% confidence interval (CI), 17-41%), which was more than those in fair (12%, 95%CI 8-16%) or good control (7%, 95%CI 4-12%). Multivariate logistic regression analysis showed that systolic and diastolic blood pressures were positively, and the number of kinds of antihypertensive medications and the age of the physician were negatively, associated with an increase in medication. In conclusion, primary care physicians did not increase antihypertensive medication adequately for patients with uncontrolled hypertension. Attempts to understand and to change physicians' prescription behaviour could reduce the burden of uncontrolled hypertension among treated hypertensive patients.

Aged↗
PubMed2002

Dialister pneumosintes associated with human brain abscesses.

In this report, we review two cases of brain infection due to Dialister pneumosintes in previously healthy patients. The bacterium was isolated from the first patient by blood culture and directly from a brain abscess in the second patient. In both cases, the infection was suspected to be of nasopharyngeal or dental origin. The patients had favorable outcomes following surgical debridement and antibiotic treatment. After in vitro amplification and partial sequencing of the 16S rRNA gene, two strains were classified as D. pneumosintes. However, traditional biochemical tests were not sufficient to identify the bacteria. In addition to causing periodontal and opportunistic infections, D. pneumosintes, contained in mixed flora, may behave as a clinically important pathogen, especially in the brain. In addition to phenotypic characterization, 16S rRNA partial sequencing was used to identify D. pneumosintes definitively.

Adolescent↗
PubMed2001

The influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease.

Multiple sclerosis is a demyelinating disease, characterized by inflammation in the brain and spinal cord, possibly due to autoimmunity. Large-scale sequencing of cDNA libraries, derived from plaques dissected from brains of patients with multiple sclerosis (MS), indicated an abundance of transcripts for osteopontin (OPN). Microarray analysis of spinal cords from rats paralyzed by experimental autoimmune encephalomyelitis (EAE), a model of MS, also revealed increased OPN transcripts. Osteopontin-deficient mice were resistant to progressive EAE and had frequent remissions, and myelin-reactive T cells in OPN-/- mice produced more interleukin 10 and less interferon-gamma than in OPN+/+ mice. Osteopontin thus appears to regulate T helper cell-1 (TH1)-mediated demyelinating disease, and it may offer a potential target in blocking development of progressive MS.

Animals↗
PubMed2001

Distinct gene expression profiles of human type 1 and type 2 T helper cells.

BACKGROUND: The development and activation of CD4+ helper T cell (Th) subsets with distinct patterns of unbalanced production of cytokines play an important part in infectious, allergic and autoimmune diseases. Human neonatal cord blood CD4+ Th cells can be polarized into type 1 or type 2-like effector cells in vitro by culturing them in the presence of interleukin (IL)-12 or IL-4, respectively. We have exploited this experimental system to identify marker genes that are differentially expressed by polarized Th1 and Th2 cells. An oligonucleotide microarray specifically designed to screen for inflammation-related candidate genes was used and the differential expression was further validated with a quantitative real-time RT-PCR method. RESULTS: In addition to the previously described marker genes of Th cells, we report subtle changes in the expression of several other genes that represent growth factors, receptors and other signaling molecules in polarized Th1 and Th2 cell subsets. Additionally, we describe a novel set of genes as Th1/Th2 differentiation markers for cells activated by anti-CD3 and anti-CD28 antibodies. CONCLUSIONS: This study demonstrates the power of the targeted use of microarrays in combination with quantitative real-time RT-PCR in identifying and validating new marker genes for gene expression studies.

CD4 Antigens↗
PubMed2001

Epidemiology of Bartonella infection in domestic cats in France.

Blood samples were collected between February and June 1996 from a convenience sample of 436 domestic French cats living in Paris and its environs and were tested for Bartonella bacteremia and seropositivity. Seventy-two cats (16.5%) were Bartonella bacteremic, of which 36 cats (50%) were infected with Bartonella henselae type II (B.h. II) only, 15 cats (21%) were infected with Bartonella clarridgeiae (B.c.) only, and 11 cats (15%) were infected with B. henselae type I (B.h. I) only. Eight cats (11%) were co-infected with B. henselae and B. clarridgeiae (B.h. II/B.c.: five cats; B.h. I/B.c.: three cats). Two cats (2.8%) were concurrently bacteremic with B. henselae types I and II. Risk factors associated with bacteremia included ownership for <6months (prevalence ratio (PR)=1.80; 95% confidence interval (CI)=1.13-2.85), adoption from the pound or found as a stray (PR=1.67, 95% CI=1.05-2.65), and cohabitation with one or more cats (PR=1.60, 95% CI=1.01-2.53). Bartonella antibodies to either B. henselae or B. clarridgeiae were detected in 179 cats (41.1%). Risk factors associated with seroposivity paralleled those for bacteremia, except for lack of association with time of ownership. Prevalence ratios of bacteremic or seropositive cats increased with the number of cats per household (p=0.02). The lack of antibodies to B. henselae or B. clarridgeiae was highly predictive of the absence of bacteremia (predictive value of a negative test=97.3%). Multiple logistic regression analysis indicated that bacteremia, after adjustment for age and flea infestation, and positive serology, after adjustment for age, were associated with origin of adoption and number of cats in the household. Flea infestation was associated with positive serology.

Animals↗
PubMed2001

Hypochlorite-modified low density lipoprotein inhibits nitric oxide synthesis in endothelial cells via an intracellular dislocalization of endothelial nitric-oxide synthase.

Hypochlorous acid/hypochlorite, generated by the myeloperoxidase/H(2)O(2)/halide system of activated phagocytes, has been shown to oxidize/modify low density lipoprotein (LDL) in vitro and may be involved in the formation of atherogenic lipoproteins in vivo. Accordingly, hypochlorite-modified (lipo)proteins have been detected in human atherosclerotic lesions where they colocalize with macrophages and endothelial cells. The present study investigates the influence of hypochlorite-modified LDL on endothelial synthesis of nitric oxide (NO) measured as formation of citrulline (coproduct of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) upon cell stimulation with thrombin or ionomycin. Pretreatment of human umbilical vein endothelial cells with hypochlorite-modified LDL led to a time- and concentration-dependent inhibition of agonist-induced citrulline and cGMP synthesis compared with preincubation of cells with native LDL. This inhibition was neither due to a decreased expression of endothelial NO synthase (eNOS) nor to a deficiency of its cofactor tetrahydrobiopterin. Likewise, the uptake of l-arginine, the substrate of eNOS, into the cells was not affected. Hypochlorite-modified LDL caused remarkable changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the Golgi location without altering myristoylation or palmitoylation of the enzyme. In contrast, cyclodextrin known to deplete plasma membrane of cholesterol and to disrupt caveolae induced only a disappearance of eNOS from the plasma membrane that was not associated with decreased agonist-induced citrulline and cGMP formation. The present findings suggest that mislocalization of NOS accounts for the reduced NO formation in human umbilical vein endothelial cells treated with hypochlorite-modified LDL and point to an important role of Golgi-located NOS in these processes. We conclude that inhibition of NO synthesis by hypochlorite-modified LDL may be an important mechanism in the development of endothelial dysfunction and early pathogenesis of atherosclerosis.

Antioxidants↗
PubMed2001

L-ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin.

Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time- and concentration-dependent fashion without affecting NO synthase (NOS) expression or l-arginine uptake. The present study investigates if the underlying mechanism is related to the NOS cofactor tetrahydrobiopterin. Pretreatment of human umbilical vein endothelial cells with ascorbate (1 microm to 1 mm, 24 h) led to an up to 3-fold increase of intracellular tetrahydrobiopterin levels that was concentration-dependent and saturable at 100 microm. Accordingly, the effect of ascorbic acid on Ca(2+)-dependent formation of citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) was abolished when intracellular tetrahydrobiopterin levels were increased by coincubation of endothelial cells with sepiapterin (0.001-100 microm, 24 h). In contrast, ascorbic acid did not modify the pterin affinity of endothelial NOS, which was measured in assays with purified tetrahydrobiopterin-free enzyme. The ascorbate-induced increase of endothelial tetrahydrobiopterin was not due to an enhanced synthesis of the compound. Neither the mRNA expression of the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I, nor the activities of either GTP cyclohydrolase I or 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the de novo synthesis pathway, were altered by ascorbate. Our data demonstrate that ascorbic acid leads to a chemical stabilization of tetrahydrobiopterin. This was evident as an increase in the half-life of tetrahydrobiopterin in aqueous solution. Furthermore, the increase of tetrahydrobiopterin levels in intact endothelial cells coincubated with cytokines and ascorbate was associated with a decrease of more oxidized biopterin derivatives (7,8-dihydrobiopterin and biopterin) in cells and cell supernatants. The present study suggests that saturated ascorbic acid levels in endothelial cells are necessary to protect tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis.

Ascorbic Acid↗
PubMed2001

Immunomodulation by electrically enhanced delivery of plasmid DNA encoding IL-12 to murine skeletal muscle.

Recent reports have shown the successful delivery of plasmid DNA to skeletal muscle by in vivo electroporation. However, most of these reports have used what has been characterized as low-voltage, millisecond pulses. One of the purposes of this study was to examine a wide range of parameters to establish a better understanding of the effects of field strength and pulse length. Two electroporation conditions, one utilizing low-voltage millisecond pulses and the other high-voltage microsecond pulses, were compared for their ability to deliver a molecule that can be measured systemically, IL-12. Not only was this molecule efficiently delivered, but one of the molecules it induces (IFN-gamma) was also measured systemically, with expression peaking at greater than 200 pg/ml serum levels on day 7 and still as high as 40 pg/ml on day 21 when using electrical parameters of 100 V/cm, 20-ms pulses. Of the two types of parameters tested, low-voltage, millisecond pulses resulted in higher, prolonged expression of plasmid DNA than high-voltage, microsecond pulses. In summary, this report provides evidence that in vivo electroporation is an efficient method for the delivery of plasmid DNA, and its use in immunotherapy protocols should be furthered examined.

Animals↗
PubMed2001

Sleep deprivation as a predictor of response to light therapy in major depression.

BACKGROUND: While the majority of depressed patients benefit from total sleep deprivation (TSD), light therapy is regarded as a first-line treatment only for seasonal affective disorder (SAD). The results of light therapy in nonseasonal major depressive disorder have been non-conclusive. We examined the correlation of TSD response and light therapy response in major depressed patients. METHODS: 40 inpatients with major depressive disorder (seven with seasonal pattern, 33 without seasonal pattern) were deprived of a night's sleep. The TSD responders, as well as the TSD nonresponders, were randomly assigned to receive adjunct light therapy either with bright white light (2500 lux) or dim red light (50 lux) during 2 weeks beginning on the third day after TSD. RESULTS: The 20 TSD responders improved significantly better under the light therapy than the 20 TSD nonresponders (according to the Hamilton Depression Rating Scale and the self-rating depression scale Bf-S; v. Zerssen). LIMITATIONS: No significant difference could be found between the two light intensities. Since the patients were additionally treated with medication an interaction with the two adjunctive therapies cannot be excluded. CONCLUSION: Our results indicate that a positive TSD response in major depressed patients can be predicative of beneficial outcome of subsequent light therapy.

Adult↗
PubMed2001

Effects of electrochemotherapy with bleomycin on normal liver tissue in a rat model.

Preliminary studies that used electric pulses in vivo to facilitate entry of chemotherapeutic agents into tumour cells resulted in a 69% complete response rate for hepatocellular carcinoma in rats. This success motivated a focused investigation to define the adverse effects of this treatment on normal liver tissue. Bleomycin doses ranging from 0.5 to 2.5 U and electric fields from 500 to 2250 V/cm were investigated. Electrical treatment was administered using an array of six needles arranged in a circular pattern. Necrosis and four other histological parameters were examined 14 and 56 days after treatment. Results indicated that treatment effects were localised to the volume of treated tissue. These parameters, at both time points, were not significantly altered for liver tissue that was treated with all drug doses and electric fields of 1250 V/cm and below. Only the combination of more intense electric pulses with bleomycin produced adverse histological events in the form of localised liver necrosis at day 14. These effects were not visible at day 56. Liver function was normal through all of the treatment except for an elevation of several enzymes 1 day post-treatment.

Animals↗