Influences of alignment and obesity on knee joint loading in osteoarthritic gait.

OBJECTIVE: To determine the influences of frontal plane knee alignment and obesity on knee joint loads in older, overweight and obese adults with knee osteoarthritis (OA). METHODS: Cross-sectional investigation of alignment and obesity on knee joint loads using community dwelling older adults (age ≥ 55 years; 27 kg m(-2) ≥ body mass or body mass index (BMI) ≤ 41 kg m(-2); 69% female) with radiographic knee OA that were a subset of participants (157 out of 454) enrolled in the Intensive Diet and Exercise for Arthritis (IDEA) clinical trial. RESULTS: A higher BMI was associated with greater (P = 0.0006) peak knee compressive forces [overweight, 2411 N (2182, 2639), class 1 obesity, 2772 N (2602, 2943), class 2+ obesity, 2993 N (2796, 3190)] and greater (P = 0.004) shear forces [overweight, 369 N (322, 415), class 1 obesity, 418 N (384, 453), class 2+ obesity, 472 N (432, 513)], independent of alignment, and varus alignment was associated (P < 0.0001) with greater peak external knee adduction moments, independent of BMI [valgus, 18.7 Nm (15.1, 22.4), neutral, 27.7 Nm (24.0, 31.4), varus, 37.0 Nm (34.4, 39.7)]. CONCLUSION: BMI and alignment were associated with different joint loading measures; alignment was more closely associated with the asymmetry or imbalance of loads across the medial and lateral knee compartments as reflected by the frontal plane external adduction moment, while BMI was associated with the magnitude of total tibiofemoral force. These data may be useful in selecting treatment options for knee OA patients (e.g., diet to reduce compressive loads or bracing to change alignment).

The seroprevalence of Lawsonia intracellularis in horses in The Netherlands.

Equine proliferative enteropathy caused by Lawsonia intracellularis is an emerging disease of weanling foals and affects their growth and development. The prevalence of Lawsonia intracellularis in The Netherlands is not known. The aim of the study was to investigate the seroprevalence of Lawsonia intracellularis in horses in The Netherlands. Blood samples were taken from healthy foals before and after weaning and from healthy yearlings and mature horses on farms throughout The Netherlands. These samples were analysed for the presence of Lawsonia intracellularis-specific antibodies with a blocking ELISA. White blood cell count, packed cell volume, and total protein concentration were also measured in all foals. Information regarding housing, pasture access, and contact with pig manure on the premises was obtained for all animals. The prevalence of Lawsonia intracellularis antibodies in foals increased significantly from 15% before weaning to 23% after weaning (p = 0.019); it was 89% in yearlings and 99% in horses older than 2 years. There was no significant difference in seroprevalence between the pasture-kept and stable-confined adult horses (97% and 100%, respectively), and there was no significant influence of contact with pig manure. None of the sampled animals showed clinical disease. In conclusion, the results suggest that Lawsonia intracellularis is widespread in The Netherlands and that seropositivity is not necessarily associated with clinical problems. The high seroprevalence in adult horses suggests long-term persistence of antibodies against Lawsonia intracellularis or constant exposure to the bacterium.

Longitudinal sampling is required to maximize detection of intrahost A/H3N2 virus variants.

Seasonal human influenza viruses continually change antigenically to escape from neutralizing antibodies. It remains unclear how genetic variation in the intrahost virus population and selection at the level of individual hosts translates to the fast-paced evolution observed at the global level because emerging intrahost antigenic variants are rarely detected. We tracked intrahost variants in the hemagglutinin and neuraminidase surface proteins using longitudinally collected samples from 52 patients infected by A/H3N2 influenza virus, mostly young children, who received oseltamivir treatment. We identified emerging putative antigenic variants and oseltamivir-resistant variants, most of which remained detectable in samples collected at subsequent days, and identified variants that emerged intrahost immediately prior to increases in global rates. In contrast to most putative antigenic variants, oseltamivir-resistant variants rapidly increased to high frequencies in the virus population. Importantly, the majority of putative antigenic variants and oseltamivir-resistant variants were first detectable four or more days after onset of symptoms or start of treatment, respectively. Our observations demonstrate that de novo variants emerge, and may be positively selected, during the course of infection. Additionally, based on the 4-7 days post-treatment delay in emergence of oseltamivir-resistant variants in six out of the eight individuals with such variants, we find that limiting sample collection for routine surveillance and diagnostic testing to early timepoints after onset of symptoms can potentially preclude detection of emerging, positively selected variants.

BK virus-transformed inbred hamster brain cells: status of viral DNA in subclones.

We have recently reported that viral DNA sequences in inbred LSH hamster brain cells transformed by the GS variant of BK virus (LSH-BR-BK) are present predominantly in a free form (Beth et al., J. Virol. 40:276-284, 1981). In this report, we confirm that the presence of viral DNA sequences in these cells is not due to virus production, since viral capsid proteins were not detected by immunoprecipitation. Furthermore, we examined the status of viral DNA in 15 subclones of this cell line and detected free and integrated viral DNA sequences in only 5 of the subclones. The other 10 subclones contained exclusively integrated viral DNA sequences, as shown by the blot hybridization of high-molecular-weight cell DNA which was uncleaved or digested with HincII, for which there are no sites in viral DNA. The arrangement of viral DNA in these clones was further analyzed by cleavage of cellular DNA with HpaII and HindIII. Mitomycin (0.03 microgram/ml) treatment of subclones containing only integrated sequences resulted in the appearance of free viral DNA sequences in some of these cells. This result supports the postulation that free viral DNA in LSH-BR-BK cells is made up of excision products of observed tandemly repeated integrated sequences. In addition to the large T- and small t-antigens, LSH-BR-BK and all of its 15 subclones contained two antigen species which were larger than large T and one species which was smaller than small t. The number of tumor antigens in the LSH- BR-BK cell line and its subclones with a large copy number in a free form was not more than in the subclones with low copy number and integrated DNA. This suggests that free viral DNA is not a template for tumor antigen production in transformed cells.

Glucocorticoid requirement for growth of human papillomavirus 16-transformed primary rat kidney epithelial cells: correlation of development of hormone resistance with viral RNA expression and processing.

The establishment of transformation of primary baby rat kidney epithelial cells by human papillomavirus type 16 DNA requires glucocorticoid hormones (Pater et al., Nature (Lond.), 335: 832-835, 1988). In this report we provide evidence that growth of transformed baby rat kidney cells in culture also requires glucocorticoids. However, transformed cells for which growth does not require hormone readily arise after a brief period of crisis, if cultured without added hormone. No reduction of glucocorticoid receptor was evident in non-hormone-requiring cells. The expression of human papillomavirus 16 RNA in these cells was analyzed by Northern blot, primer extension, and RNase protection analysis. Cells that do not require hormone had greatly reduced levels of transcripts initiated from the viral P97 promoter. However, there is evidence for compensating alterations to allow more efficient expression of E7 mRNA, since the growth of these cells is correlated with altered patterns of viral RNA expression and processing.

Squamous metaplasia of normal and carcinoma in situ of HPV 16-immortalized human endocervical cells.

The importance of cervical squamous metaplasia and human papillomavirus 16 (HPV 16) infection for cervical carcinoma has been well established. Nearly 87% of the intraepithelial neoplasia of the cervix occur in the transformation zone, which is composed of squamous metaplastic cells with unclear origin. HPV DNA, mostly HPV 16, has been found in 90% of cervical carcinomas, but only limited experimental data are available to discern the role of HPV 16 in this tissue specific oncogenesis. We have initiated in vivo studies of cultured endocervical cells as an experimental model system for development of cervical neoplasia. Using a modified in vivo implantation system, cultured normal endocervical epithelial cells formed epithelium resembling squamous metaplasia, whereas those immortalized by HPV 16 developed into lesions resembling carcinoma in situ. In contrast, their ectocervical counterparts formed well differentiated stratified squamous epithelium and a lesion with mild dysplastic change, respectively. The HPV 16-immortalized cells showed in vivo cytokeratin expression patterns similar to their respective normal counterparts, confirming their different origins. Thus, this study provides direct experimental evidence for the transformation of simple epithelial cells of endocervical origin into stratified squamous metaplasia and indicates the differential susceptibility of endo- and ectocervical epithelial cells for conversion to cancer by HPV 16.

Different HPV16 E6/E7 oncogene expression patterns in epithelia reconstructed from HPV16-immortalized human endocervical cells and genital keratinocytes.

Human papillomavirus type 16 (HPV16) E6/E7 oncogenes immortalize two types of human genital epithelial cells in vitro, endocervical cells and ectocervical or foreskin keratinocytes. Epithelia reconstructed in in vivo nude mouse implants or in vitro organotypic raft cultures from immortalized endocervical cells form higher grade dysplasia than those from keratinocytes. Here, we compared viral E6/E7 mRNA expression in immortalized cell lines of the three cell types using implants, rafts and in situ hybridization assays. Endocervical cells expressed E6/E7 throughout their reconstructed epithelia. In contrast, oncogenes were limited to basal cells for keratinocyte lower grade dysplasias. To study the role of the HPV16 promoter/enhancer in this repression in the upper layers of keratinocyte epithelia, new cell lines were established by immortalization with E6/E7 controlled by the SV40 promoter. The oncogenes were shown to be controlled from the SV40 elements after immortalization. Nevertheless, E6/E7 in the two cell types had the same cell-specific expression pattern as that controlled from the homologous HPV16 promoter. In addition, naturally occurring premalignant lesions having integrated HPV16 DNA expressed E6/E7 extensively in the high-grade dysplastic region of undifferentiated metaplasia. On the other hand, oncogene expression was restricted to lower layers in the lower grade dysplastic region of more mature differentiation. Our data suggest that keratinocytes have an inherent HPV16 promoter-nonspecific mechanism of repression. Apparently this mechanism, which can be acquired during maturation, is initially nonfunctional in in vitro and in vivo epithelia derived from metaplastic endocervical cells.

Human BAG-1/RAP46 protein is generated as four isoforms by alternative translation initiation and overexpressed in cancer cells.

Previously, a Bcl-2-interacting protein, BAG-1, was cloned from mouse cells and was shown to interact with several other proteins and to be important for inhibition of apoptosis. Human BAG-1 (hBAG-1) cDNA, recently isolated by us and two other groups, has been shown to be identical to a hormone receptor-binding protein, RAP46. However, different molecular masses of hBAG-1 protein products were noted by these three groups. Here we demonstrated that hBAG-1 protein was expressed as four isoforms, designated p50, p46, p33 and p29, with apparent molecular masses of 50 kDa, 46 kDa, 33 kDa and 29 kDa, respectively. Deletion, site-directed mutagenesis and in vitro transcription/translation analysis showed that the four protein products of hBAG-1 were expressed by alternative initiation from four different start codons through a leaky scanning mechanism. Furthermore, we demonstrated that the distinct forms of hBAG-1 have different subcellular localizations, suggesting that they may have distinct functions in the cells. Characterization of hBAG-1 RNA and protein also showed that hBAG-1 was overexpressed in human cervical, breast and lung cancer cell lines. Taken together, these data clarify the conflicting observations reported in the literature and suggest that hBAG-1 is expressed as four forms of protein products, which may play a differential role in apoptosis and oncogenesis of human cells.

Role of steroid hormones in potentiating transformation of cervical cells by human papillomaviruses.

Human papillomaviruses (HPVs) are etiologically involved in cervical neoplasia, and epidemiological evidence suggests that steroid hormones can increase the risk of this cancer in HPV-infected women. Steroids can interact with hormone-response elements in the viral long control region, enhancing HPV transcription and resulting in transformation of cervical cells. Subsequent malignant progression may involve virus-induced chromosomal instability, facilitating viral DNA integration and deregulation of gene expression.

Differential regulation by c-jun and c-fos protooncogenes of hormone response from composite glucocorticoid response element in human papilloma virus type 16 regulatory region.

Glucocorticoid hormones positively regulate human papilloma virus (HPV) type 16 gene expression, and we have previously shown that this regulation is through three glucocorticoid response elements (GREs). The GRE at nucleotide 7640 is a composite GRE (cGRE) containing an overlapping activator protein-1 (AP-1) motif for the c-jun homodimer and c-jun/c-fos heterodimer. This report examined the effects of c-jun and/or c-fos AP-1 protooncogenes and the glucocorticoid hormone dexamethasone on expression of the HPV 16 cGRE in AP-1-deficient P19 embryonal carcinoma cells. The activity of the full-length HPV 16 enhancer was progressively increased with increasing levels of c-jun. The hormone induced an additional response. For the c-jun/c-fos heterodimer, the response to hormone was progressively diminished. Site-specific mutations of the cGRE revealed that the regulation by AP-1 and hormone required both GRE and the AP-1 motif. An enhancer fragment containing the cGRE and excluding the two simple GREs gave similar results. Two disruption mutations of the AP-1 site confirmed the requirement of this site for hormone response. A cGRE oligonucleotide construct substantiated the effect of c-jun for response to hormone. For heterodimer, activity and hormone response were both also progressively increased. The results reveal a unique cross-talk between the distinct AP-1- and hormone-signaling pathways, suggesting the involvement of a complex interaction of c-jun and c-fos and glucocorticoid hormone receptor with the HPV 16 cGRE, resulting in novel control patterns for regulating viral expression.

Identification and characterization of a JC virus pentanucleotide repeat element binding protein: cellular nucleic acid binding protein.

The JC virus (JCV) control region contains AGGGAAGGGA, the tandem pentanucleotide repeat element (Pnt2). Several proteins specifically interacted via Pnt2 to regulate the expression of JCV early promoter-enhancer (JCV(E)) or late promoter-enhancer (JCV(L)). In this study, a JCV Pnt2 oligonucleotide probe was used to screen a cDNA expression library from glial P19 mouse embryonal carcinoma cells. A cDNA clone was isolated by Southwestern blot assay and it produced a protein that reproducibly and specifically bound to Pnt2. This cDNA had 100% homology to one of three previously identified mouse cDNAs called cellular nucleic acid binding proteins (Cnbps). Cnbps are a highly homologous family of eukaryotic genes implicated in functional interactions with cytoplasmic RNA and regulatory DNA elements. An mRNA of 2.2 kb of Pnt2-interacting Cnbp (PCnbp) was seen in undifferentiated, muscle or glial P19 cells. When expressed from a cDNA expression vector as a fusion protein that also contained 115 kDa from beta-galactosidase, a Pnt2 binding protein (PCNBP) specifically bound to Pnt2 in Southwestern blots as a 30 kDa component of the 145 kDa fusion protein. Furthermore, JCV(E) expression was negatively regulated by PCnbp produced in vivo from the cDNA expression vector. Regulation of JCV(L) was unaffected. We suggest a novel role for CNBP as a PCNBP that interacts with Pnt2 in the negative transcriptional regulation of JCV(E).

Alterations in physical state and expression of human papillomavirus type 18 DNA following crisis and establishment of immortalized ectocervical cells.

Integration of episomal human papillomavirus (HPV) DNA in infected cervical lesions during malignant progression is frequently observed, but the importance of integration is poorly understood. We have studied immortalization by HPV-18 of human cervical cells as an in vitro model system. Here, the status and expression of HPV-18 DNA in precrisis ectocervical keratinocytes was compared with that in the same cells after crisis and establishment of immortalization. Southern blots revealed, and two-dimensional gel analysis confirmed, that the precrisis culture contained more than 100 copies/cell of episomal HPV-18 DNA and no detectable integrated viral DNA. In contrast, the postcrisis cells contained a low copy number of only integrated viral genome. The Northern blot patterns of E6-E7 and E2/E4 RNA expression were also different. Analysis of RNA by RT-PCR indicated that neither culture expressed the unspliced HPV-18 E6 oncogene present in tumor cell lines and that the precrisis, but not postcrisis, culture expressed the full-length E2 repressor. The two cultures displayed a similar keratinocyte morphology in vitro and a similar low grade dysplasia in vivo and both were non-tumorigenic. These results suggest that, although insufficient for complete malignant conversion, viral DNA integration during crisis is associated with the establishment of an immortalized phenotype in which HPV-18 DNA is integrated and HPV-18 RNA expression is altered.

Human papillomavirus type 16 expression in cervical keratinocytes: role of progesterone and glucocorticoid hormones.

OBJECTIVE: To determine the role of the steroid hormones, progesterone and glucocorticoids, and the viral hormone response elements, in the episomal expression of human papillomavirus (HPV) type 16 in primary human ectocervical cells. METHODS: In situ hybridization and mutagenesis were used to assess the requirements of these hormones and the HPV 16 glucocorticoid/progesterone response elements in the induction of HPV 16 expression in ectocervical cells. RESULTS: The assays detected a marked increase in viral messenger RNA only after treatment of the cells with either of the steroid hormones. This response was inhibited by the anti-progestin RU 486 in a concentration-dependent manner. Mutagenesis of the previously identified hormone response element in the regulatory region of the HPV 16 genome had no effect on hormone-induced HPV gene expression. We have now identified two additional hormone response elements. Different combinations of mutations in the three hormone response elements showed that all three were independently sufficient for the hormone-mediated induction of viral transcription. CONCLUSIONS: Steroid hormones induce HPV 16 gene expression in cervical keratinocytes directly through three hormone response elements in the regulatory region of the viral genome. The anti-progestin RU 486 inhibits this induction. Because the physical state of HPV DNA in this in vitro system and in premalignant cervical lesions is extrachromosomal, steroid hormones may have a critical role in modulating HPV expression in such lesions.

Human papillomavirus 18-immortalized endocervical cells with in vitro cytokeratin expression characteristics of adenocarcinoma.

OBJECTIVE: To determine whether human papillomavirus (HPV) 18 has a role in the development of adenocarcinoma from human endo- or ectocervical cells. METHODS: Secondary cultures of human endo- and ectocervical cells were assayed for immortalization by HPV 18 DNA using lipofection. The effects of immortalization on the patterns of cytokeratin expression were determined by indirect immunofluorescence using monoclonal antibodies. The differentiation phenotype of the immortalized cells was investigated by a modified in vivo implantation system. RESULTS: Both endo- and ectocervical cells were immortalized by HPV 18. The immortalized cells contained integrated HPV 18 DNA and expressed E6-E7 RNA. The immortalized endocervical cells had a cytokeratin phenotype characteristic of adenocarcinoma, whereas the immortalized ectocervical cells retained a distinct cytokeratin expression pattern of normal parental cells. In an in vivo implantation system, endocervical cells formed a lesion resembling severe dysplasia or carcinoma in situ, whereas ectocervical cells developed into a lesion resembling mild dysplasia. Both cell lines were nontumorigenic in nude mice. CONCLUSION: Both endo- and ectocervical cells are targets for immortalization by HPV 18. Based on cytokeratin expression patterns, immortalized endocervical cells, but not ectocervical cells, may be useful as a model for premalignant lesions that progress into adenocarcinoma.

Prevention of carcinoma in situ of human papillomavirus type 16-immortalized human endocervical cells by retinoic acid in organotypic raft culture.

OBJECTIVE: To determine the effect of retinoic acid on the development of severe dysplasia or carcinoma in situ from endocervical cells containing human papillomavirus (HPV) type 16. METHODS: Two independent lines of HPV 16-immortalized endocervical cells were reconstructed into two squamous epithelial tissues using the organotypic raft culture system to examine the differentiated phenotype. The effect of retinoic acid on dysplastic morphology of differentiation of the epithelia was examined by light microscopy of stained sections and electron microscopy. The endocervical cell type cytokeratin expression pattern was determined by indirect immunofluorescence using specific monoclonal antibodies. Ribonucleic acid expression of the HPV 16 E7 oncogene was examined by in situ hybridization. RESULTS: Untreated HPV 16-immortalized endocervical cells were reconstructed into squamous dysplastic lesions resembling carcinoma in situ observed in women. Retinoic acid-treated rafts formed epithelia composed of two to three cell layers of columnar-like cells resembling simple epithelium of the endocervix. Electron microscopy and cytokeratin expression patterns confirmed the histology of a differentiated endocervical phenotype after treatment with retinoic acid. Expression of HPV 16 E7 was modestly lower in treated epithelia, preferentially in basal cells. CONCLUSION: Retinoic acid prevents the histology and cytokeratin differentiation markers of carcinoma in situ of HPV 16-immortalized endocervical cells. Because the epithelia closely mimic HPV 16-containing severe dysplasias and native endocervical epithelium in women, this immortalized endocervical cell-raft system may be useful as a model to assess the efficacy of agents such as retinoic acid for preventing progression of these lesions to malignant cervical carcinoma.

An improved experimental model for the study of in vitro release of nitric oxide by murine peritoneal macrophages.

In the immune system macrophages are the cells responsible for nitric oxide (NO) production. The synthesis of NO by activated macrophages correlates with their cytotoxic effect on neoplastic cells as well as killing of intracellular parasites. In the present paper we test several parameters that may influence (in vitro) NO production by murine peritoneal macrophages previously stimulated in vivo by intraperitoneal injection of thioglycollate. In our system the maximum NO/NO2- release was obtained in the culture containing 10(6) M phi/ml after 24 h incubation. For macrophage activation we used lipopolysaccharide (LPS) and several recombinant cytokines (IFN-gamma, TNF-alpha, IL-2, IL-3, IL-6). We also tested the influence of latex phagocytosis on NO production by simultaneously activated macrophages.

Isolation and characterization of papillomavirus DNA from nasal inverting (schneiderian) papillomas.

To elucidate the possible role of papillomaviruses as etiological agents in nasal inverting papillomas, DNA hybridization techniques were used. Total DNA from two nasal inverting papillomas was examined for the presence of DNA from human papillomavirus (HPV) types 6 and 11 under stringent conditions of hybridization. Both lesions contained DNA hybridizing with HPV 11. Restriction enzyme digestion and subsequent Southern blotting of the DNA samples revealed that one lesion contained viral DNA identical to HPV 11a. The DNA of the other lesion contained an extra 500 base pair insertion. These results provide definitive evidence for the first time for the association of HPV with nasal inverting papillomas.

Biomechanical responses of the back of riding horses to water treadmill exercise.

There is a lack of evidence for the presumed beneficial effects of water treadmills on the movement of the horse's back. The aim of the study was to evaluate the effects of water treadmill exercise on axial rotation (AR), lateral bending (LB) and pelvic flexion (PF) in horses. The back kinematics of a group of riding horses were studied at the walk in a water treadmill at different depths of water (hoof, fetlock, carpus, elbow and shoulder joint levels) over a period of 10 days. Skin markers were placed at anatomical locations on the back. AR, LB and PF were measured on days 1 and 10 using two high-speed video cameras. There was a significant increase in AR compared to baseline at the level of the carpus and at higher water levels, whereas LB was significantly lower than baseline values at water levels that reached the elbow and shoulder joints. PF was significantly higher than baseline values at each water depth other than hoof water depth. At increasing water depths, there were significant increases in flexion and rotation of the back. At the highest water levels, there was reduced bending of the back. After 10 days, horses exhibited more bending of the back.

RU486 inhibits glucocorticoid hormone-dependent oncogenesis by human papillomavirus type 16 DNA.

We have previously reported (Pater et al., Nature 335, 832-835, 1988) the glucocorticoid hormone-dependent oncogenic transformation of primary baby rat kidney (BRK) cells by a combination of human papillomavirus (HPV) type 16 DNA and the activated form of the human Ha-ras-1 (ras) oncogene. In this study we provide evidence for the inhibition of such hormone-dependent transformation by the hormone antagonist, RU486. The level of inhibition was dependent on the ratio of hormone to antagonist and severe inhibition was observed at a 1:5 ratio. The effect of RU486 on the growth of HPV16-transformed cells was also studied. Severe growth inhibition and cell death occurred when cells transformed in the absence of RU486 were subsequently grown in the presence of either a 1:2.5 or 1:5 ratio of hormone to hormone antagonist. RU486 did not alter the level or electrophoretic mobility pattern of HPV16 mRNA expressed in transformed cells, when antihormone was applied either continuously or subsequent to transformation by HPV. These results indicate the importance of continuous viral gene expression for initiation and maintenance of the transformed phenotype.

Transformation of primary human embryonic kidney cells to anchorage independence by a combination of BK virus DNA and the Harvey-ras oncogene.

Primary human embryonic kidney (HEK) cells were transformed by a focus assay with BK virus (BKV) DNA molecularly cloned at its unique EcoRI site. Both viral DNA sequences and viral tumor antigens were present and expressed in all the foci that we examined. However, cells isolated from foci were incapable of growth in soft agar. We then examined the transformation of HEK cells after their transfection with a combination of BKV DNA and either the normal or the activated form of the human Ha-ras oncogene (EJ c-Ha-ras-1). Only the cells transfected with a combination of BKV DNA and the activated form of Ha-ras were capable of growth in soft agar. Both BKV and Ha-ras DNAs were present in the transformed colonies. BKV tumor antigens and the Ha-ras p21 protein were also expressed.