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J Wahlfors

Publications and source records attributed to J Wahlfors.

At least 19 recordsLinked to original sources
PubMed2006

Efficacy of gene transfer through the round window membrane: an in vitro model.

The viral vector-transgene soaked gelatin-sponge method has been shown to be successful in mediating transgene expression across an intact round window membrane (RWM) in mouse in vivo. However, there are many confounding factors which make it difficult to evaluate the role of the RWM in gene transfer. We have created an in vitro model to test the feasibility of gene delivery through an intact RWM. The round window including the bony niche of a CD1 mouse was removed under an operating microscope and fixed with adhesive on the base of a petri dish through which a hole had been drilled. Toluidine blue was injected into the niche containing a hyaluronic acid ester sponge against the round window membrane. The niche was closed with a fascia. A plastic tube containing PBS was fixed on the opposite side, from where the samples were collected at different time points. The concentration of toluidine blue was evaluated spectrophotometrically. An adenoviral vector containing green fluorescent protein (GFP) marker gene was injected into the niche. Samples were collected from the opposite side at different time points. The presence of the vector was studied with GFP PCR. We also modulated the permeability of the RWM by treating it with clinically applicable detergents, histamine or silver nitrate. Silver nitrate and trichloracetic acid caused destruction of the surface epithelium of the RWM as shown by light microscopy. Both toluidine blue and adenoviral vectors passed through the RWM in a time-dependent fashion. RWM cells expressed GFP after Ad-GFP treatment. The permeability of the RWM was decreased after treatment with different detergents, histamine or silver nitrate. RWM offers an atraumatic route to the inner ear. Compared with more invasive gene delivery methods, this technique represents a safer and clinically more viable route of cochlear gene delivery.

Animals↗
PubMed2003

The effect of photodynamic therapy on rat urinary bladder with orthotopic urothelial carcinoma.

OBJECTIVE: To assess the effect of whole-bladder photodynamic therapy (PDT) on a rat model with orthotopic superficial bladder cancer, as PDT is an alternative intravesical therapy for treating superficial bladder cancer, based on an interaction between a photosensitizer and light energy to induce oxygen radicals that destroy tissue by lipid peroxidation. MATERIALS AND METHODS: In all, 76 female Fischer F344 rats were inoculated intravesically with AY-27 tumour cells. After establishing superficial tumour, 24 rats were treated with PDT using aminolaevulinic acid (ALA)-induced protoporphyrin IX as a photosensitizer, and a continuous-wave argon pumped-dye laser (638 nm). At 4 h after intravenous (300 mg/kg) or intravesical (100 mg/mL) administration of ALA the bladders were intravesically exposed to a 40 J/cm(2) light dose; 12 rats received no ALA but were exposed to the same light dose. Before administering ALA, urine cytology samples were taken for analysis. At 3 or 21 days the treated rats were killed and morphological changes in the bladder walls analysed by light microscopy. Forty rats served as controls to examine the presence of tumour. RESULTS: The tumour established in 33 of 40 rats (83%) in the controls, but after PDT with intravesical ALA there was carcinoma in only in one of 12 (P < 0.001, Pearson's chi(2) test). After PDT with intravenous ALA there was carcinoma in five of 11 rats (P = 0.063, Pearson's chi2 test). In the control group of 12 rats receiving only light energy there was carcinoma in three (P = 0.001, Pearson's chi(2) test). Histologically, at 3 days after PDT there was only mild superficial damage in all six rats treated intravesically. Bladder wall destruction reached the muscular layer, with an abscess in one of six rats treated intravenously. After 3 weeks of PDT there was muscular necrosis with perforation and abscess from catheterization two of six rats treated intravesically and in three the bladder wall totally recovered. In the intravenous group the bladder walls were normal or had only mild superficial damage. Cytology of the urine sediment failed to detect half the tumours in the treatment groups. CONCLUSION: These results support the use of PDT with intravesical ALA-induced protoporphyrin X for treating superficial bladder carcinoma. Intravesical was better than intravenous ALA in eradicating bladder carcinoma with PDT.

Administration, Intravesical↗
PubMed2001

Green fluorescent protein (GFP) fusion constructs in gene therapy research.

The history of green fluorescent protein (GFP) as a marker is less than 10 years old, but it has already made a major impact on many areas of natural sciences, especially on cell biology and histochemistry. GFP can be detected in living cells without selection or staining and it can be fused to other proteins to yield fluorescent chimeras. The potential of GFP has also been recognised by gene therapy researchers and various GFP-tagged therapeutic proteins have been constructed. These chimeric proteins have been used to determine the expression level, site and time course of the therapeutic gene, or the correlation between gene transfer rate and therapeutic outcome. This review summarises the status of the applications of GFP fusions in gene therapy research.

Genetic Therapy↗
PubMed2001

Human prostate carcinoma cells as targets for herpes simplex virus thymidine kinase-mediated suicide gene therapy.

To evaluate human prostate carcinoma cells as targets for herpes simplex virus thymidine kinase (HSV-TK) -mediated gene therapy, we tested the utility of different viral vectors on three human cell lines DU-145, LNCaP, and PC-3. Our viral vectors were carrying a fusion gene of HSV-TK and green fluorescent protein for accurate determination of the gene transfer rate and its contribution to the treatment outcome in each case. We observed that adenoviral and lentiviral vectors were efficient vehicles for all the cell lines, whereas Semliki Forest virus and Sindbis virus vectors yielded only a few percent of transgene-positive cells. Despite sufficient gene transfer rates (25-45%) in the ganciclovir (GCV) sensitivity experiment, only DU-145 cells were efficiently destroyed under clinically relevant GCV concentrations. This was shown to be due to low level of "bystander effect" in PC-3 and LNCaP cells. Our data demonstrate that human prostate tumors can be good targets for adenovirus- or lentivirus-mediated HSV-TK/GCV gene therapy, but each tumor should be investigated for gene transfer rate and bystander effect to warrant a sufficient treatment result.

Adenoviridae↗
PubMed2000

Adenovirus-mediated gene transfer into an experimental pancreatic tumour.

BACKGROUND AND AIMS: Gene therapy has been suggested as a novel approach against pancreatic cancer, a disease with a grim prognosis with current modes of therapy. Despite recent advances in in vitro and experimental in vivo studies, there is no data available concerning gene transfer efficiency in intrapancreatic tumours in immunocompetent animal models. MATERIAL AND METHODS: In in vitro studies rat pancreatic carcinoma cells (DSL-6A/C1) were transduced with replication-deficient adenovirus carrying Escherichia Coli beta-galactosidase (lacZ) gene. Gene transfer efficacy was assessed at different multiplicities of infection (MOIs). Pancreatic tumours were induced by inoculating cultured DSL-6A/C1 cancer cells into Lewis rat pancreases. Established tumours were transduced and three days post-transduction, pancreatic tumours as well as other intra-abdominal organs were harvested and processed for histological analyses, including staining for marker gene expression. RESULTS AND CONCLUSIONS: In vitro assays showed that DSL-6A/C1 cells were transduced efficiently, even at low MOIs. In vivo gene transfer was successful in all animals, and all pancreatic samples showed reporter gene expression. Positive cells were detected in the peritumoural areas as well as to a lesser extent within the tumours. The transgene activity was not evenly distributed and the gene transfer efficiency varied from a few detectable blue cells to 11% per field. Our studies demonstrated safe in vivo gene transfer into intrapancreatic tumours, suggesting that pancreatic tumours are potential targets for in vivo delivery of therapeutic genes.

Adenoviruses, Human↗
PubMed1998

Azaserine-induced rat pancreas tumor model with transplantable cultured cells.

The purpose of this study was to describe the inoculation technique and patterns of growth as well as to characterize typical histological features of Lewis rat subcutaneous and intrapancreatic tumors, induced by inoculation of cultured pancreatic cancer cells (DSL-6A/C1). Subcutaneous inoculation of cultured cells produced a solid tumor that was a locally invasive, well- to moderately differentiated ductal adenocarcinoma. Tumor take was 100% in animals 5 weeks of age; tumor growth was consistent and predictable and a tumor volume of approximately 1 cm3 was reached in 8 weeks. After intrapancreatic transplantation the tumors showed the same histological features as subcutaneous tumors. During inoculation carcinoma cells easily spread around the injected area, and after 2 weeks both pancreatic tumors and superficially infiltrating carcinomas were found in the liver and spleen and around the peritoneum. Tumor take was 60% and tumor growth was somewhat indefinite and unpredictable in the pancreas. However, by reducing the injected carcinoma cell volume and solving the technical problems, 100% tumor take was achieved. The tumor volume reached 2 mm3 during 2 weeks and larger tumors showed a tendency for invasion. According to our results, subcutaneous as well as intrapancreatic tumor induction with cultured cells offers a model for pancreatic cancer studies.

Adenocarcinoma↗
PubMed1998

Herpes simplex virus thymidine kinase-green fluorescent protein fusion gene: new tool for gene transfer studies and gene therapy.

Green fluorescent protein (GFP) and herpes simplex virus type-I thymidine kinase (TK) are commonly used markers in gene transfer studies. The latter gene has also proven to be an effective tool in cancer "suicide" gene therapy. To facilitate rapid and reliable selection of cells expressing TK, we constructed a plasmid expressing a TK-green fluorescent protein fusion gene (TK-GFP). In this fusion gene, the expression of each component is coupled to one another, permitting accurate determination of the percentage of cells expressing TK by detecting the green fluorescence produced by GFP. Transfection of the fusion plasmid to mammalian cells revealed that the construct is fully functional, making the cells both fluorescent and sensitive to ganciclovir.

Animals↗
PubMed1997

Identification of Bacteroides forsythus in subgingival dental plaque with the aid of a rapid PCR method.

Bacteroides forysthus has been shown to be prevalent among patients with periodontitis. Conventional microbiological methods used to identify this bacterium, however, are laborious and time-consuming and are therefore not well-suited for screening purposes. We have developed a polymerase chain-reaction (PCR) method which is rapid, specific, and simple to perform and does not require other sample pre-treatment except a brief centrifugation. This method was applied to the detection of B. forsythus in subgingival plaque of 58 periodontitis patients. When compared with the results of conventional culturing, the PCR method always confirmed the culture-positive results, while none of the PCR negative samples was shown to be culture-positive. The PCR method appeared to give more than double the number of samples positive for B. forsythus than culturing (89.7% vs. 37.9%). The analysis requires less than 4 hrs to perform, and is specific only to B. forsythus and sensitive enough to detect fewer than 5 bacteria.

Adult↗
PubMed1996

Hypermethylation of calcitonin gene regulatory sequences in human breast cancer as revealed by genomic sequencing.

DNA methylation has been studied intensively during the past years in order to elucidate its role in the regulation of gene expression, gene imprinting and cancer progression. Earlier studies have shown that a general genomic under-methylation is associated with chronic lymphocytic leukemia and metastatic prostate cancer. Site-specific methylation changes, as revealed by the use of methylation-sensitive restriction enzymes, have been reported to occur in the promotor region of the calcitonin gene in chronic myeloid leukemia as it progresses from the chronic phase to blast crisis, in non-Hodgkin's lymphoid neoplasms and in non-lymphocytic leukemia. We have now explored possible methylation changes associated with benign and malignant breast tumors. Two approaches were employed: (i) chemical determination of general genomic methylation status and (ii) base-specific analysis of the methylation changes in the promoter of the calcitonin gene with the aid of genomic sequencing. The results did not reveal any changes of total DNA 5-methylcytosine content in ductal carcinoma of breast in comparison with benign tumors. There was a small, yet significant, increase in 5-methylcytosine content in lobular carcinoma. Genomic sequencing of the promoter region of the calcitonin gene, however, revealed a striking hypermethylation at or around the transcription start site of the gene in ductal carcinomas. In benign tumors and lobular carcinomas, this region was either entirely unmethylated or only slightly methylated. The latter changes may reflect a regional hypermethylation of the short arm of chromosome 11, which harbors, in addition to the calcitonin gene, a number of putative or established tumor-suppressor genes. Our results demonstrate that genomic sequencing in its present form can be used for a reliable and precise DNA methylation analysis of primary human tumors.

5-Methylcytosine↗
PubMed1995

Life-long over-expression of ornithine decarboxylase (ODC) gene in transgenic mice does not lead to generally enhanced tumorigenesis or neuronal degeneration.

Ornithine decarboxylase, the rate-controlling enzyme of the biosynthetic pathway of the polyamines, is one of the most inducible mammalian enzymes showing many of the features common to the oncoproteins. Ornithine decarboxylase activity is likewise strongly induced in response to various neurotoxic stimuli, and the enhanced enzyme activity is believed to be causally related to neuronal damage. We have generated several transgenic mouse lines over-expressing human ornithine decarboxylase gene. We have now subjected the animals to a long-term survival study in order to assess whether constitutively over-expressed ornithine decarboxylase would predispose them to enhanced tumorigenesis and neuronal degeneration. When the transgenic animals were 2 years old, their tissue ornithine decarboxylase activity was 20- to 50-fold that in their syngenic littermates. Macroscopic and microscopic examination of organs revealed no differences between syngenic and transgenic animals as regards spontaneous tumor incidence or age-related changes in brain.

Animals↗
PubMed1995

Long-term reduction of amplified ornithine decarboxylase sequences in human myeloma cells.

(1) Human myeloma cell line Sultan, resistant to 20 mM difluoro-methylornithine (DFMO) owing to ornithine decarboxylase (ODC) gene amplification, was grown in the absence of DFMO for a period of 10 months. The gene copy number and methylation status of the ODC gene were monitored after withdrawal of DFMO. Moreover, levels of ODC mRNA, immunoreactive ODC protein, ODC activity and polyamine levels were recorded recurrently during the course of the study. (2) The results revealed that ODC gene copy number started to decrease after 4 weeks growth without DFMO, to a final level of less than 30% of the original gene dosage. The methylation status of the ODC gene, however, remained almost unaltered, displaying only a modest increase in methylation after 10 months without DFMO. The amount of ODC message dropped very rapidly to 75% of the original value, then started to decrease in a gene copy-number-dependent manner. The amount of ODC protein closely followed the levels of mRNA during the study, whereas the ODC activity, after a transient increase during the first week, decreased to half of the original level after 4 weeks. Between 6 and 16 weeks ODC activity stabilized to a fifth of the original value and no more changes were detected during the subsequent period of observation. (3) Due to the grossly elevated ODC enzyme activity, levels of putrescine and spermidine first peaked and then stabilized at 6 weeks after DFMO withdrawal. The final spermidine level was comparable with that of the parental Sultan cell line with only one copy of active ODC gene. However, putrescine content was strikingly elevated, being stabilized to a level that was 20 times higher than in parental cells. Spermine concentration was practically unchanged during the study. (4) According to the results obtained in this study, the abnormal level of ODC expression in human myeloma cells is suppressed partially at the level of transcription or post-transcriptionally, but it is not due to increased methylation of the gene. The major regulatory mechanism to compensate for a highly elevated ODC expression was modulation of the enzyme activity. After 10 months without DFMO, the cells still displayed about 20 times higher ODC activity and putrescine concentration than the myeloma cell line with a single copy of the ODC gene. They did not, however, show any signs of growth retardation or other features different from the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Development of a rapid PCR method for identification of Helicobacter pylori in dental plaque and gastric biopsy specimens.

A rapid and simple polymerase chain reaction (PCR) method was developed to detect Helicobacter pylori in gastric biopsy specimens and dental plaque samples. The primers were targeted to the 16S rRNA sequence of Helicobacter pylori strain ATCC 43504. The system was found to have a theoretical detection level of 0.5 to 5 Helicobacter pylori cells in a 5 microliters sample of dental plaque. In the absence of plaque, the detection level was even better: theoretically, 0.05 to 0.5 Helicobacter pylori cells were detected in water suspension. However, this appeared to be due to the presence of free bacterial DNA in the culture used for the sensitivity determination. Thus, the actual sensitivity of the system was found to be fewer than five Helicobacter pylori cells, irrespective of the type of sample used. The method was then used to analyse 29 dental plaque and gastric biopsy specimens collected from patients with a history of recurrent peptic ulcer disease. Fourteen stomach specimens were positive for Helicobacter pylori when tested with the PCR method, while the respective figures with culture, histological examination and the urease test were 11, 12 and 9. No positive dental plaque samples were observed.

Adult↗
PubMed1995

Molecular cloning of a cDNA encoding human spermine synthase.

We have isolated and sequenced cDNA clones that encode human spermine synthase (EC 2.5.1.22). The total length of the sequenced cDNA was 1,612 nucleotides, containing an open reading frame encoding a polypeptide chain of 368 amino acids. All of the previously sequenced peptide fragments of human and bovine spermine synthase proteins could be located within the coding region derived from the cDNA. An unusual sequence of AATTAA apparently signaled the initiation of polyadenylation. Sequence comparisons between human spermine synthase and spermidine synthases from bacterial and mammalian sources revealed a nearly complete lack of similarity between the primary structures of these two enzymes catalyzing almost identical reactions. A modest similarity found was restricted to a relatively short peptide domain apparently involved in the binding of decarboxylated S-adenosylmethionine, the common substrate for both enzymes. The apparent lack of an overall similarity may indicate that spermine synthase, the enzyme found only in eukaryotes, and spermidine synthase with more universal distribution, although functionally closely related, have evolved separately.

Amino Acid Sequence↗
PubMed1995

Simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis by a rapid PCR method.

The identification of periodontal pathogens by conventional methods is time-consuming and difficult. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.) and Porphyromonas gingivalis (P.g.) was developed for rapid and easy determination of these risk-indicator bacteria in human periodontal disease. The PCR primers were designed to hybridize to various regions of 16S rRNA genes, and a hot-start technique was used to obtain maximum sensitivity and specificity. This method can detect both of these bacteria in subgingival plaque samples at concentrations as low as 5 to 50 cells per sample. The sensitivity, however, was even 10 times better when the bacteria were analyzed in a water suspension. Since the only step between sample collection and the actual analysis is a brief centrifugation of the patient sample, the detection can be readily carried out in four hours. The performance of the method was studied with 36 patient samples. The results showed that the PCR method detected A.a. (44% vs. 25%, respectively) and P.g. (56% vs. 42%, respectively) more often than the conventional culture in plaque samples. Thus, our multiplex PCR method is rapid and more effective than conventional protocols in detecting these periodontal pathogens.

Aggregatibacter actinomycetemcomitans↗
PubMed1994

Nucleotide sequence of mouse spermidine synthase cDNA.

The nucleotide sequence of mouse cDNA for spermidine synthase appeared to contain 75 nucleotides of 5' untranslated region, an open reading frame of 909 nucleotides and 297 nucleotides of 3' untranslated region. The open reading frame encoded a polypeptide of 302 amino acids, displaying 95% similarity to human and 33% similarity to E. coli spermidine synthase. The 3' flanking region contained an unusual polyadenylation signal AATACA.

Amino Acid Sequence↗
PubMed1994

Automated fluorescent genomic sequencing as applied to the methylation analysis of the human ornithine decarboxylase gene.

A genomic sequencing method for an automated DNA sequencer was developed. The method described here is an improved version of the previously published protocol, which utilizes bisulfite-induced modification of genomic DNA. In our method, the modified DNA is purified without a time-consuming dialysis, and the subsequent 2-step DNA amplification is carried out with one biotinylated primer in order to separate and isolate the strands of the product with the aid of streptavidin-coated magnetic beads. The strands are then sequenced with fluorescent primers and automated DNA sequencer. This provides means to determine reliably the methylation status of cytosines as well as the degree of methylation in a given CpG, site of the target sequence. The method was successfully applied to analyze the promoter region and the 11th exon of the human ornithine decarboxylase ODC gene in various human myeloma cell lines. The study revealed a totally unmethylated promoter region in every cell line studied, whereas the protein coding region appeared to be extensively methylated, although a dexamethasone resistant cell line displayed demethylation in certain CpG sequences. Also, a previously unknown ODC allele was detected.

Automation↗
PubMed1992

Genomic hypomethylation in human chronic lymphocytic leukemia.

DNA samples from patients with chronic lymphocytic (CLL), chronic myelocytic (CML), acute myelocytic (AML), and acute lymphocytic leukemia (ALL), as well as samples from patients with multiple myeloma (MM) and healthy volunteers, were analyzed for their genomic methylation status using Hpa II and Msp I digestions followed by a simple gel electrophoresis and ethidium bromide staining. A densitometric method was developed to measure more accurately the extent of methylation in genomic DNA samples and the results were confirmed by high-performance liquid chromatography (HPLC) analysis of hydrolyzed DNA. Southern analysis with ornithine decarboxylase (ODC) gene probe was also employed, and the levels of ODC mRNA were determined with the aid of polymerase chain reaction (PCR). The results indicated that a general genomic hypomethylation was present in almost all of the samples obtained from patients with B-cell CLL. This hypomethylation was most striking among the patients who were freshly diagnosed and among untreated chronic patients. CML appeared to be a heterogenous group, comprising patients with normal methylation status in addition to patients with slight hypomethylation. Patients with ALL, AML, or MM did not show any signs of DNA methylation changes in comparison to healthy volunteers. Although all analyzed samples from patients with B-CLL showed hypomethylation of ODC sequences, little correlation existed between the mRNA levels and the extent of hypomethylation of ODC gene.

Chromatography, High Pressure Liquid↗