Prospective clinical and serological follow-up in early childhood reveals a high rate of subclinical RSV infection and a relatively high reinfection rate within the first 3 years of life.

Children encounter repeated respiratory tract infections during their early life. We conducted a prospective clinical and serological follow-up study to estimate the respiratory syncytial virus (RSV) primary infection and reinfection rates in early childhood. Sera were collected from 291 healthy children at the ages of 13, 24 and 36 months and antibody levels against RSV antigens were determined by enzyme immunoassay. The RT-PCR method was also used for identifying the possible presence of RSV in symptomatic patients. At ages 1, 2 and 3 years, 37%, 68% and 86%, respectively, of studied children were seropositive for RSV. In children seropositive at age 1 year, RSV reinfection rate was at least 37%. Only one of reinfected children showed evidence for a third reinfection by age 3 years. Of children who turned RSV seropositive between ages 1 and 2 years, the reinfection rate was 32% during the third year of life. The mean antibody levels at primary infection were very similar in all age groups. The average decrease of antibody levels was 25-30% within a year. In 66 cases RSV infection was identified by RT-PCR. RSV infection rate in early childhood is 86% and reinfection rate is around 35%. This prospective serological follow-up study also provided evidence for the presence of RSV infections in children that did not show clinical signs warranting RSV RNA detection.

Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice.

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.

Replication of and protein synthesis by TT viruses.

The host cells and the events in the cells during Torque teno (TT) virus infection are at present unknown. Replicating TT virus DNA has been detected in liver, in peripheral blood mononuclear cells (PBMC), and in bone marrow. By alternative splicing this small virus generates three mRNA species, from which by alternative translation initiation at least six proteins are produced. The functions of the proteins are not yet fully understood. However, functions associated with, e.g., DNA replication, immunomodulation, and apoptosis have been suggested to reside in the multifunctional proteins of anelloviruses.

Genoprevalence in human tissues of TT-virus genotype 6.

TT virus (TTV) is a newly discovered human virus of high genotypic diversity. TTV is widely distributed among humans, but the possible genotype-related differences in TTV biology are not well known. The prevalence and amount of TTV-DNA, especially of genotype 6, was determined by nested-PCR in various human tissues, and human parvovirus B19, another ssDNA virus, was used as a reference. TTV DNA was detected simultaneously in bile, peripheral blood mononuclear cells (PBMC) and plasma of 77% subjects, in 38% skin samples, in 38% synovial samples and in all (100%) adenoids, tonsils and liver samples. The relative concentrations of TTV-DNA did not vary significantly among the different samples. Genotype 6 TTV-DNA was detected in bile and plasma of one subject (3%), in skin and serum of one subject (8%) and in one liver (5%). The overall prevalence of TTV genotype 6 was 4% in subjects and 4% in sera. TTV genotype 6 was shown to occur in human tissues with no obvious tissue-type or symptom specificity. Parvovirus B19 DNA was detected overall in 38% subjects, and bile was the only sample type tested that did not persistently harbour B19 DNA.

Predictors of biological aggressiveness of prostate specific antigen screening detected prostate cancer.

PURPOSE: It is not known whether screening of asymptomatic men with prostate specific antigen (PSA) decreases the mortality of prostate cancer. We evaluated the extent to which PSA screening identifies clinically significant prostate cancer by analyzing markers of biological aggressiveness. MATERIALS AND METHODS: We compared prostate cancer in 56 patients in the screening and 21 in the randomized control arm of a population based cohort of 8,975 men 55 to 67 years old participating in the Finnish arm of the European Randomized Study of Screening for Prostate Cancer to 47 clinically detected organ confined, 30 clinically detected metastatic and 16 latent prostate tumors identified at autopsy in 46 consecutive subjects. Biological aggressiveness was determined by histological grading using the Gleason and Mostofi scales, tumor proliferation rate by Ki-67 immunostaining, p53 over expression by immunostaining and aneuploidy by fluorescence in situ hybridization using formalin fixed, paraffin embedded tumor specimens. RESULTS: A total of 56 neoplasms were detected in 2,781 men (2%) who participated in PSA screening and 21 were detected in 5,975 nonscreened controls (0.35%) during the study period. Disease in nonscreened controls more often involved a high tumor proliferation rate (p = 0.004) and p53 over expression (p = 0.015) than screening detected disease. At least 1 feature of biological aggressiveness was present in 19% of latent, 34% of screening detected, 51% of clinically detected and organ confined, 62% of randomized control and 87% of metastasis cases. Of the screening detected tumors defined as biologically aggressive 74% were identified at organ confined stages pT1-2N0M0. CONCLUSIONS: PSA screening detects a significant number of biologically aggressive cancers at an early clinical stage, implying that screening may decrease mortality.

Androgen receptor gene amplification at primary progression predicts response to combined androgen blockade as second line therapy for advanced prostate cancer.

PURPOSE: Amplification of the androgen receptor gene has been found in a third of hormone refractory prostate carcinomas. It is possible that amplification facilitates cell growth ability in low concentrations of androgens remaining in the serum after androgen deprivation therapy. We evaluate whether androgen receptor gene amplification at primary progression is associated with response to second line combined androgen blockade for prostate cancer. MATERIALS AND METHODS: A total of 77 patients with prostate cancer were treated initially with androgen deprivation monotherapy followed by combined androgen blockade after the first progression. After initiation of second line combined androgen blockade patients were followed every 3 months to evaluate treatment responses. Biopsies were taken from the prostate at the first progression under endocrine monotherapy. Androgen receptor gene copy number was determined by fluorescence in situ hybridization. RESULTS: Androgen receptor gene amplification was found in 10 of the 77 cases (13%) at the primary disease progression, and was associated with a favorable response to second line combined androgen blockade. Only 1 of 34 (3%) patients classified as nonresponders had androgen receptor gene amplification, whereas 9 of 41 (21%) classified as having either stable disease or response had amplification (p = 0.016). Patients with androgen receptor gene amplification also had a decrease in prostate specific antigen more often after combined androgen blockade than those with no amplification (p = 0.079). However, androgen receptor gene amplification was not associated with patient survival after the first progression. CONCLUSIONS: Androgen receptor gene amplification detected in tumors progressing during androgen deprivation monotherapy is associated with favorable treatment response to second line combined androgen blockade. This finding suggests that at least some androgen receptor amplified tumors retain a high degree of dependency on residual androgens remaining in serum after monotherapy.

Amplification and overexpression of p40 subunit of eukaryotic translation initiation factor 3 in breast and prostate cancer.

Amplification at the long arm of chromosome 8 occurs in a large fraction of breast and prostate cancers. To clone the target genes for this amplification, we used suppression subtraction hybridization to identify overexpressed genes in the breast cancer cell line SK-Br-3, which harbors amplification at 8q (8q21 and 8q23-q24). A differentially expressed gene identified by SSH, the p40 subunit of eukaryotic translation initiation factor 3 (eIF3), was localized to 8q23 and found to be highly amplified and overexpressed in the breast and prostate cancer cell lines studied. High-level amplification of eIF3-p40 was found in 30% of hormone-refractory prostate tumors and in 18% of untreated primary breast tumors. In the vast majority of the cases, p40 and c-myc were amplified with equal copy numbers. Tumors with higher copy numbers of p40 than c-myc were also found. Expression of p40 mRNA was analyzed with in situ hybridization. The amplification of eIF3-p40 gene was associated with overexpression of its mRNA, as expected for a functional target gene of the amplification. These results imply that genomic aberrations of translation initiation factors, such as eIF3-p40, may contribute to the pathogenesis of breast and prostate cancer.

Genetic alterations in hormone-refractory recurrent prostate carcinomas.

To study the genetic basis of tumor progression, we have screened 37 hormone-refractory prostate carcinomas for genetic changes by comparative genomic hybridization (CGH). All recurrent tumors showed genetic aberrations, with a mean total number of changes per tumor of 11.4 (range, 3 to 23). The most common genetic aberrations were losses of 8p (72.5%), 13q (50%), 1p (50%), 22 (45%), 19 (45%), 10q (42.5%), and 16q (42.5%) and gains of 8q (72.5%), 7q (40%), Xq (32.5%), and 18q (32.5%). The CGH results were further validated with fluorescence in situ hybridization (FISH) using probes for pericentromeric regions of chromosomes 7, 8, and 18 as well as probes for caveolin (7q31), c-myc (8q24), and bcl-2 (18q21.3). In addition, the samples had previously been analyzed for androgen receptor gene copy number. CGH and FISH results were concordant in 78% of cases. Seventeen of twenty-two tumors showed an increased copy number of c-myc by FISH. However, only 5 of 17 (29%) of the cases showed high-level (more than threefold) amplification. Both CGH and FISH findings suggested that in most of the cases 8q gain involves the whole q-arm of the chromosome. Four of seventeen (24%) cases showed increased copy number of bcl-2 by FISH; however, no high-level amplifications were found. To evaluate the clonal relationship of the primary and recurrent tumors, six primary-recurrent tumor pairs from the same patients were studied by CGH. In three of six cases (50%), the recurrent tumor had more than one-half of the aberrations found in the corresponding primary tumor, indicating a close clonal relationship. In the rest of the cases, such a linear clonal relationship was less evident. Altogether, these results suggest that recurrent prostate carcinomas are genetically unstable. The resulting heterogeneity may well underlie the poor responsiveness of hormone-refractory tumors to treatment.

Intracellular route of canine parvovirus entry.

The present study was designed to investigate the endocytic pathway involved in canine parvovirus (CPV) infection. Reduced temperature (18 degrees C) or the microtubule-depolymerizing drug nocodazole was found to inhibit productive infection of canine A72 cells by CPV and caused CPV to be retained in cytoplasmic vesicles as indicated by immunofluorescence microscopy. Consistent with previously published results, these data indicate that CPV enters a host cell via an endocytic route and further suggest that microtubule-dependent delivery of CPV to late endosomes is required for productive infection. Cytoplasmic microinjection of CPV particles was used to circumvent the endocytosis and membrane fusion steps in the entry process. Microinjection experiments showed that CPV particles which were injected directly into the cytoplasm, thus avoiding the endocytic pathway, were unable to initiate progeny virus production. CPV treated at pH 5.0 prior to microinjection was unable to initiate virus production, showing that factors of the endocytic route other than low pH are necessary for the initiation of infection by CPV.

Characterization of a nuclear localization signal of canine parvovirus capsid proteins.

We investigated the abilities of synthetic peptides mimicking the potential nuclear localization signal of canine parvovirus (CPV) capsid proteins to translocate a carrier protein to the nucleus following microinjection into the cytoplasm of A72 cells. Possible nuclear localization sequences were chosen for synthesis from CPV capsid protein sequences (VP1, VP2) on the basis of the presence of clustered basic residues, which is a common theme in most of the previously identified targeting peptides. Nuclear targeting activity was found within the N-terminal residues 4-13 (PAKRARRGYK) of the VP1 capsid protein. While replacement of Arg10 with glycine did not affect the activity, replacement of Lys6, Arg7, or Arg9 with glycine abolished it. The targeting activity was found to residue in a cluster of basic residues, Lys5, Arg7, and Arg9. Nuclear import was saturated by excess of unlabelled peptide conjugates (showing that it was a receptor-mediated process). Transport into the nucleus was an energy-dependent and temperature-dependent process actively mediated by the nuclear pores and inhibited by wheat germ agglutinin.