Biomedical subjects

V Hukkanen

Publications and source records attributed to V Hukkanen.

At least 37 records · Page 2Linked to original sources

PubMed1994

Semliki Forest virus infects mouse brain endothelial cells and causes blood-brain barrier damage.

Induction of experimental allergic encephalomyelitis is facilitated in a genetically resistant BALB/c mouse strain by a nonpathogenic strain of a neurotropic alphavirus, Semliki Forest virus (SFV-A7). One possible explanation for this enhancement is virus infection of endothelial cells (EC), causing increased permeability of the blood-brain barrier. We have now sought evidence for virus infection of EC in vivo by immunocytochemistry and in situ hybridization. SFV-A7 antigens and RNA were detected in vascular EC and perivascular neurons in cerebellar and spinal cord white matter. Expression of viral antigens was followed by fibrinogen leakage from the blood vessels into brain parenchyma. This was shown by immunoperoxidase staining detecting fibrinogen extravascularly in central nervous system sections of infected mice. Simultaneously, expression of ICAM-1 (intercellular adhesion molecule 1) was induced on brain EC. SFV-A7 replicated in mouse brain microvascular EC in vitro and caused lysis of the cells. SFV-A7 did not induce ICAM-1 expression of mouse brain microvascular EC in vitro, while ICAM-1 was readily induced by gamma interferon and interleukin 1 beta. The observed increase of ICAM-1 expression on EC is immune mediated and not a direct effect of the virus infection. We conclude that SFV-A7 infection causes cerebral microvascular damage which contributes to the facilitation of experimental allergic encephalomyelitis in BALB/c mice.

Animals↗

PubMed1993

Cell proteins bind to sites within the 3' noncoding region and the positive-strand leader sequence of measles virus RNA.

The genomic 3' noncoding region (NCR) of nonsegmented negative-strand RNA viruses contains recognition site(s) for the polymerase complex, while the RNA plus-strand leader sequence (LS) is probably involved in RNA encapsidation. It is known that host-encoded factors play a role in transcription and replication of some of this group of viruses. Here we report that cellular proteins interact with the genomic 3' NCR and with the plus-strand LS RNA of an important human pathogen, measles virus (MV), a member of the family Paramyxoviridae. Using gel retardation assay and RNA footprinting analysis, we demonstrated that in Vero cells, host-encoded proteins bind specifically to domains within these two sequences. A polypeptide of about 20 kDa binding to the 3' NCR and two polypeptides of about 22 and 30 kDa interacting with plus-strand LS were detected by RNA-protein UV cross-linking. Different RNA-binding activities were found in cells differing in permissiveness to MV replication. The results suggest a role for host-encoded proteins in MV replication.

Animals↗

PubMed1993

L-myc and N-myc in hematopoietic malignancies.

The myc proto-oncogenes encode nuclear DNA-binding phosphoproteins which regulate cell proliferation and differentiation. The c-myc gene is implicated in hematopoietic malignancies on the basis of its frequent deregulation in naturally occurring leukemias and lymphomas. Recent evidence suggests that also the N-myc and L-myc genes may have a role in normal and malignant hematopoiesis. N-myc and to a certain degree L-myc can substitute for c-myc in transformation assays in vitro, and their overexpression can block the differentiation of leukemia cell lines. Immunoglobulin heavy chain enhancer (IgH) -driven overexpression of N-myc or L-myc genes cause lymphatic and myeloid tumors, respectively, in transgenic mice. Furthermore, the L-myc and N-myc genes are expressed in several human leukemias and leukemia cell lines, L-myc predominantly in myeloid and N-myc both in myeloid and in some lymphoid leukemias. All N/L-myc positive leukemias and leukemia cell lines coexpress the c-myc gene, thus exemplifying a lack of negative cross-regulation between the different myc genes in leukemia cells. Taken together, these data suggest that L-myc and N-myc may participate in the growth regulation of hematopoietic cells.

Animals↗

PubMed1992

Expression of the L2 and E7 genes of the human papillomavirus type 16 in female genital dysplasias.

The expression of the E7 and L2 genes of HPV 16 was studied in benign and precancerous female genital lesions to evaluate their role in the development of dysplasias. Ninety biopsy specimens from 70 patients, selected on basis of dot blot DNA hybridization, were included in immunohistochemical and in situ hybridization analyses. In the HPV 16 DNA positive cases, L2 mRNA and E7 mRNA were detected in biopsies from 24 and 21 patients, respectively. L2 mRNA was found in eight of 16 cases of condyloma and mild dysplasia, and in 13 of 14 cases of moderate to severe dysplasia. The figures for E7 mRNA were 6/16 and 13/14, respectively. We found L2 mRNA in four of 12 normal or condylomatous specimens and E7 mRNA in only one of these. The detection rates for L2 and E7 mRNAs increased along with the severity of the lesions (P = 0.0064 and P = 0.0001, respectively). The L2 protein was found in one condyloma and in 12 dysplasias, eight of which were moderate or severe. The L2-antibody-reactive cells were localized in superficial layers of the epithelium. The detection rate for L2 mRNA and especially for E7 mRNA increased along with the histopathologic grade of the lesion.

Biopsy↗

PubMed1991

Expression of L-myc and N-myc proto-oncogenes in human leukemias and leukemia cell lines.

The myc proto-oncogenes encode nuclear phosphoproteins, which are believed to participate in the control of cell proliferation and differentiation. Deregulated expression of c-myc has been implicated in several human hematopoietic malignancies. We have studied the expression and mRNA processing of human L-myc, N-myc, and c-myc genes in a panel of human leukemias, leukemia cell lines, and normal hematopoietic cells. L-myc mRNA was expressed in three acute myeloid leukemias (AML) studied and in several myeloid leukemia cell lines. Only low expression levels were observed in adult bone marrow and in fetal spleen and thymus. The K562 and Dami leukemia cell lines showed a unique pattern of L-myc mRNA processing, with approximately 40% of L-myc mRNA lacking exon III and intron I. N-myc was expressed in five of six AML cases studied, in one of nine acute lymphocytic leukemia (ALL) cases, and in several leukemia cell lines, while c-myc mRNA was detected in all leukemias and leukemia cell lines studied. Coexpression of all three myc genes was observed in Dami and MOLT-4 cell lines and in two AMLs, and either L-myc or N-myc was coexpressed with c-myc in several other cases. These results show that in addition to c-myc, the L-myc and N-myc genes are expressed in some human leukemias and leukemia cell lines, and suggest a lack of mutually exclusive cross-regulation of the myc genes in human leukemia cells.

Blotting, Northern↗

PubMed1991

Expression of the herpes simplex virus 1 alpha transinducing factor (VP16) does not induce reactivation of latent virus or prevent the establishment of latency in mice.

A feature of the cascade regulation of herpes simplex virus 1 gene expression in productive infection is that the first genes to be expressed, the alpha genes, are transactivated by a structural component of the virion designated as the alpha transinducing factor (alpha TIF). In this study, we have tested the hypothesis that latent infection of sensory neurons results from the failure of alpha TIF, a tegument protein, to be transported from the nerve endings to the nucleus of the sensory neuron. Two viruses were constructed. The first recombinant virus (R6003) contained a second copy of the alpha TIF gene placed under the control of a metallothionein promoter. The second recombinant virus (R6004) is identical to R6003 except for the presence of a stop codon inserted at amino acid 70 of the second alpha TIF gene. The metallothionein promoter inserted into the viral genome was shown to be expressed, and alpha TIF mRNA was detected by in situ hybridization of sections of trigeminal ganglia of mice infected with R6003, both untreated and those given cadmium injections. In all experiments, there were no significant differences in the recovery of latent virus from mice infected with R6003 or R6004, whether injected with cadmium or not. Cadmium administration at the time of infection and at intervals thereafter did not preclude establishment of latency. In another series of experiments, transgenic mice expressing the metallothionein-driven alpha TIF did not differ from nontransgenic siblings with respect to the incidence of latent virus in trigeminal ganglia. We conclude that the absence of alpha TIF cannot alone account for the establishment of latency.

Animals↗

PubMed1990

Genomic variation of herpes simplex virus type 2 isolates analysed by hybridization after electroblotting from polyacrylamide gels.

Genomic variation of herpes simplex virus type 2 (HSV-2) strains was analysed by polyacrylamide gradient gel electrophoresis and subsequent hybridization to cloned HSV-2 sequences. Two probes were used, one from the L-segment and one from the S-segment of the HSV-2 genome. The probes did not contain a-repeat sequences. Hybridization to the specific sequences in individual DNA fragments obtained by use of the frequently cleaving restriction endonuclease Alu I revealed variations in the genome not detectable by analysing the fragment size only. The use of 35S-labelled deoxynucleotide in the radioactive labelling of the probe further improved the resolution of the method.

DNA, Viral↗

PubMed1989

Detection of human papilloma virus (HPV) DNA in genital biopsy specimens by in situ hybridization with digoxigenin-labeled probes.

The presence of human papillomavirus (HPV) nucleotide sequences in paraffin sections of genital biopsies was examined by in situ hybridization using non-isotopic, digoxigenin-labeled probes representing HPV types 11, 16 and 18. Digoxigenin-labeling of the probes was performed using DNA labeling and a commercially provided detection kit. Hybridization was performed under stringent conditions. The hybrids were detected by using anti-digoxigenin alkaline phosphatase conjugate and visualized with enzyme catalyzed color reaction. In situ hybridization with digoxigenin-labeled probes was a useful technique for identification of HPV infection. The results were compared with the results obtained with radiolabeled DNA probes. The sensitivity of the digoxigenin-labeled probes was equal to the sensitivity of the radiolabeled probes. The background with digoxigenin-labeled probes was very low. Using nonradioactive probes the localization of hybrids at the cellular level was better than 35S-labeled probes.

Autoradiography↗

PubMed1989

Polyethylene glycol increases specificity of hybridization typing of HPV specimens.

Different hybridization conditions for the typing of human papillomaviruses (HPV) in clinical biopsy specimens were tested. The stringency of the hybridization reaction was varied by altering the formamide concentration in the solution. Two polymers, dextran sulphate and polyethylene glycol (PEG), were compared as accelerators of the hybridization reaction. The PEG-containing hybridization solution was found to be suitable for typing clinical HPV specimens. Single-stranded RNA probes proved to be more specific than DNA probes in typing clinical specimens. Specimens that were positive both for HPV6 and HPV11 in spot hybridization were confirmed by Southern hybridization. In total, 467 biopsy specimens from genital, anal, oral, aural and nasal lesions were examined for HPV6, HPV11, HPV16 and HPV18 DNA by spot hybridization. Approximately one-third of the specimens were positive for these types.

DNA Probes↗

PubMed1989

The N-myc proto-oncogene and IGF-II growth factor mRNAs are expressed by distinct cells in human fetal kidney and brain.

We studied the expression of the N-myc proto-oncogene and the insulin-like growth factor-II (IGF-II) gene in human fetuses of 16-19 gestational wk. Both genes have specific roles in the growth and differentiation of embryonic tissues, such as the kidney and neural tissue. Since continued expression of N-myc and IGF-II mRNAs is also a characteristic feature of Wilms' tumor, a childhood neoplasm of probable fetal kidney origin, we were particularly interested in the possibility that their expression might be linked or coordinately regulated in the developing kidney. Expression of N-myc mRNA was observed in the brain and in the kidney by Northern hybridization analysis. In in situ hybridization of the kidney, N-myc autoradiographic grains were primarily located over epithelially differentiating mesenchyme while most of the mesenchymal stromal cells showed only a background signal with the N-myc probe. N-myc mRNA was detectable throughout the developing brain with a slight accentuation in the intermediate zone cells in between the subependymal and cortical layers. Thus, even postmitotic neuroepithelial cells of the fetal cerebrum expressed N-myc mRNA. In Northern hybridization, IGF-II mRNA signal was abundant in the kidney but much weaker, though definite, in the brain. The regional distribution of IGF-II mRNA in the kidney was largely complementary to that of N-myc. IGF-II autoradiographic grains were located predominantly over the stromal and blastemal cells with a relative lack of hybridization over the epithelial structures. In the brain, IGF-II mRNA was about two- to threefold more abundant in the subependymal and intermediate layers than in the cortical plate and ependymal zone, respectively. The fetal expression patterns of the N-myc and IGF-II mRNAs are reflected by the types of tumors known to express the corresponding genes during postnatal life such as Wilms' tumor. However, the apparent coexpression of the IGF-II and N-myc genes in immature kidneys occurs largely in distinct cell types.

Autoradiography↗

PubMed1989

Comparison of smear specimens with biopsy specimens in a nucleic acid hybridization test for human papilloma virus (HPV) infection.

A total of 323 pairs of specimens from women with Papanicolaou class II or III cytology were examined for human papillomavirus (HPV) types 6, 11, 16 and 18 by spot hybridization. Each pair consisted of a representative biopsy specimen and a smear specimen from cervical, vaginal or, more rarely, vulvar lesions. We found a close correlation between HPV findings in biopsies and smears. In 83.9% (271) of the cases, both specimens were either positive for a given HPV type or negative. No discordant HPV-types in the two types of specimens were found. In 15.8% (51) of the cases, one specimen proved positive for a given HPV-type while the other specimen from the same patient was negative. In 8.0% (26) of the cases, the biopsy specimen proved positive and the corresponding smear specimen was negative. On the other hand, in 7.7% (25) of the cases we were able to detect HPV DNA in the smear specimen, whereas the corresponding biopsy specimen was negative. We suggest that a smear specimen would be advantageous for screening large groups of patients for the presence of a HPV infection in the genital tract. By using smear specimens together with biopsy specimens it is possible to maximize the number of HPV infection diagnoses.

Condylomata Acuminata↗

PubMed1989

Hepatitis B virus DNA in blood specimens of anti HBc IgM positive patients.

This study compares the presence of hepatitis B virus (HBV) DNA and HBV e antigen in sera positive for IgM antibody to HBV core antigen. Of the 31 sera included in the study, 14 were HBV DNA positive. Eight sera were positive for HBV DNA but negative for HBeAg and 3 sera were negative for HBV DNA but positive for HBeAg. The HBV DNA positive specimens included also 3 HBsAg negative sera. The importance of the level of the anti HBc IgM antibody is discussed.

DNA, Viral↗

PubMed1988

Polyamine depletion of cells reduces the infectivity of herpes simplex virus but not the infectivity of Sindbis virus.

The effect of polyamines on the viral growth was examined using cell strains that could be effectively depleted of polyamines. In order to avoid the polyamines present in serum we used a polyamine auxotrophic Chinese hamster ovary cell line P22 growing in serum-free medium and Vero cells growing in low serum medium. The final yield of an enveloped RNA virus, Sindbis, in P22 cells was not decreased by depletion of cellular polyamines although the onset of the viral replication was delayed. In contrast the final yield of an enveloped DNA virus, Herpes simplex virus (HSV), was considerably reduced in Vero cells, depleted of polyamines by alpha-difluoromethylornithine, an inhibitor of polyamine synthesis. However, the number of HSV particles detected by electronmicroscopy was not decreased. Southern blot analysis of HSV-DNA from the polyamine depleted and the control cells showed changes in the relative abundance of the DNA fragments suggesting that impairment in DNA synthesis may have caused the decreased infectivity of HSV.

Animals↗

PubMed1987

Autolytic changes of human white matter: an electron microscopic and electrophoretic study.

The effect of autolysis on the electrophoretic pattern of the 3H-labeled membrane glycoproteins derived from human brain white matter (WM) was investigated and correlated with electron microscopic findings. Samples were taken from the WM of outer zones of routine surgical specimens sent for pathologist's examination. The WM samples were incubated at +4 and +25 degrees C for 6 and 24 hr and analyzed by electron microscopy and by galactose oxidase labeling with subsequent SDS-polyacrylamide gradient gel electrophoresis. The effect of freezing and thawing was also studied. In electron microscopic examination myelin was found to have degenerated after an incubation of 24 hr at +4 and +25 degrees C, and it was severely disrupted after 24 hr at +25 degrees C, when a periaxonal network-like change was observed. The major labeled membrane glycoprotein band with an estimated molecular weight (MW) of 138,000 was stable during the first 24 hr of incubations at both +4 and +25 degrees C. No marked changes were noticed in the electrophoretic pattern of all of the WM membrane proteins labeled by [3H]acetic anhydride technique. In frozen and thawed specimens there was an increase in the intensity of the band with a MW of 86,500 and a new band appeared at the MW region of 25,000. The present results suggest that despite the apparent stability of the major glycoprotein band during the first 24 hr of autolysis, there are ultrastructural abnormalities in the myelin sheath, which should be taken into consideration during interpretation of changes in pathologic conditions.

Autolysis↗

PubMed1987

Improved sensitivity of restriction endonuclease analysis of herpes simplex virus type 2 DNA with polyacrylamide gradient gel electrophoresis.

Polyacrylamide gradient gel electrophoresis was used to resolve fragments of herpes simplex virus type 2 (HSV-2) DNA, produced by the restriction endonucleases Alu I, Bam HI, Pst I, and Sma I, which cleave the HSV-2 DNA into more than 30 fragments each. HSV-2 strains isolated from different individual patients could be easily distinguished from each other by the endonucleases Bam HI and Sma I. Successive virus isolates from a single person, analyzed using Alu I and Sma I, showed variability of fragment patterns. The effect of passaging the virus in cell cultures for several cycles was evaluated with the restriction endonuclease Alu I. No differences were found after 29 successive passages in VERO cells. Polyacrylamide gradient gel analysis of restriction endonuclease digests of HSV-2 DNA enables the use of enzymes that cleave the DNA into a great number of fragments, thus improving the sensitivity of analysis.

Animals↗

PubMed1987

Use of enzyme immunoassay and nucleic acid hybridization for detecting Sindbis virus in infected mosquitoes.

Aedes aegypti mosquitoes were inoculated intrathoracically with prototype Sindbis virus, held at 26.7 degrees C for from 0-95 h and placed at -70 degrees C. Individual mosquitoes were tested for virus by plaque assay in Vero cells, for viral RNA by nucleic acid hybridization using a cloned cDNA probe, and for viral protein by enzyme-linked immunosorbent assay. Virus was detected by plaque assay as early as 8 h after infection. Sindbis virus RNA was detected by nucleic acid hybridization 18 h after infection and by enzyme-linked immunosorbent assay 10 h after infection. The results of these comparisons suggest that both nucleic acid hybridization and enzyme-linked immunosorbent assay are applicable to direct detection of Sindbis virus in mosquitoes containing virus at levels usually found during arbovirus epidemics.

Animals↗

PubMed1986

Rapid effect on endemic measles, mumps, and rubella of nationwide vaccination programme in Finland.

An immunisation programme to eliminate measles, mumps, and rubella from Finland within 10 years was started in November, 1982. A combined live vaccine is being given twice, at the ages of 14-18 months and 6 years, but at the beginning of the project children between these age limits are also being immunised. Because vaccinations are traditionally done by the public health nurses, special attention was paid to their motivation. 2.5 years after the launch of the project, 80.9% of the target children had been vaccinated. The incidence of measles has fallen by 93% and that of mumps by 87% compared with a normal prevaccination year (1982). A similar fall in incidence was seen for rubella, but only in the vaccinated age groups. Although elimination of measles, mumps, and rubella is not likely to be achieved with the present vaccination coverage, a drastic fall in the incidence of all three target diseases has occurred already. Every effort is being made to improve the coverage and thus to achieve the ultimate goal of the project.

Adolescent↗

PubMed1985

Typing of herpes simplex virus isolates with monoclonal antibodies and by nucleic acid spot hybridization.

Fifty-one clinical isolates of herpes simplex virus (HSV) were typed by an enzyme immunoassay (EIA) using mouse monoclonal antibodies, by DNA spot hybridization, and by restriction enzyme analysis using restriction endonuclease Eco RI. Extracts of VERO cells infected with the isolates were used for coating microtitre plates or denatured and spotted onto nitrocellulose filters. Viral antigens passively adsorbed to microtitre plates were detected by an indirect EIA using mouse monoclonal antibodies specific for HSV type 1 (HSV-1) or HSV type 2 (HSV-2). Spotted DNA was hybridized with 32P-labeled probes containing Hind III/Sal I-fragments of either HSV-1 or HSV-2 DNA and bound radioactivity was detected by autoradiography and counted in a liquid scintillation counter. All the three methods gave identical results for the 51 isolates studied. Twenty-six isolates were identified as HSV-1 and 25 as HSV-2. An additional 30 specimens were tested only by EIA and hybridization. Results by both techniques were in complete agreement.

Animals↗