Detection of human papillomavirus in the oral cavities of persons with Fanconi anemia.

OBJECTIVE: We conducted a cross-sectional study to describe the prevalence and correlates of type-specific human papillomavirus (HPV) DNA in the oral cavities of persons with Fanconi anemia. MATERIALS AND METHODS: Oral swabs were collected from 67 participants with Fanconi anemia and tested for 27 HPV genotypes using polymerase chain reaction-based methods. RESULTS: Participants were a mean of 18.6 (standard deviation, 10.0) years of age (range 4-47 years). The prevalence of oral HPV infection was 7.5%, and the prevalence of high-risk HPV infection was 6.0%. HPV type 16 was not detected in any samples. Prevalence was higher in adults than in children (13.3% vs 2.7% in those ≥18 vs <18 years of age). Among adults, prevalence was higher in males than in females (25.0% vs 9.1%, respectively). CONCLUSIONS: Prevalence of oral HPV infection in persons with Fanconi anemia was comparable to estimates from other studies in the general population. However, in contrast to previous studies, we did not identify HPV type 16 (the type found in most HPV-related head and neck cancers) in any participants.

Gene therapy progress and prospects: development of improved lentiviral and retroviral vectors--design, biosafety, and production.

Replication defective vectors derived from simple retroviruses or the more complex genomes of lentiviruses continue to offer the advantages of long-term expression, cell and tissue specific tropism, and large packaging capacity for the delivery of therapeutic genes. The occurrence of adverse events caused by insertional mutagenesis in three patients in a gene therapy trial for X-linked SCID emphasizes the potential for problems in translating this approach to the clinic. Several genome-wide studies of retroviral integration are now providing novel insights into the integration site preferences of different vector classes. We review recent developments in vector design, integration, biosafety, and production.

In vivo treatment of hemophilia A and mucopolysaccharidosis type VII using nonprimate lentiviral vectors.

Gene therapy holds great promise for the treatment of a variety of inherited diseases, including hemophilia A and mucopolysaccharidosis type VII (MPS VII). In both these disorders, subnormal levels of replacement protein have therapeutic effects. Thus we hypothesized that transduction of a small proportion of cells by feline immunodeficiency virus (FIV)-based lentiviral vectors might provide sufficient levels of transgene expression for phenotypic correction. We intravenously injected replication-deficient FIV-based vectors encoding either human factor VIII or human beta-glucuronidase into factor VIII-deficient or beta-glucuronidase-deficient mice, respectively. This route of delivery targeted multiple organs, with the liver as the primary transduction site. In the hemophilia A mice, factor VIII expression persisted for the duration of the experiments (approximately 5 months), and recipient mice survived an otherwise lethal bleeding episode (tail-clipping). In mucopolysaccharidosis type VII mice, substantial beta-glucuronidase activity was detected in several tissues and corresponded with marked reduction of lysosomal storage in liver and spleen. These findings indicate that gene transfer with FIV-based lentiviral vectors can permanently introduce transgenes into a sufficient number of hepatocytes for long-term therapeutic effect and suggest potential clinical value of FIV-based lentiviral vectors for treatment of hemophilia A and MPS VII.

FIV vector systems.

Why is feline immunodeficiency virus (FIV) such an appealing candidate for gene therapy vector development? Phylogenetic analysis suggests FIV is only distantly related to the primate lentiviruses, and despite repeated exposure, neither seroconversion nor other detectable evidence of human infection occurs. FIV naturally infects diverse Felidae worldwide, including the domestic cat. Here, the disease progression parallels the immunodeficiency caused by HIV, and for that reason, FIV and the cat provide an excellent model for anti-virals and AIDS vaccine research. Simple genome organization also facilitates vector development and analysis: FIV has only three accessory/regulatory proteins. To overcome FIV's cat-specific tropism, feline vectors are equipped with hybrid LTRs, since the FIV LTR shows low activity in human cells. Recombinant FIV vectors generate titers comparable to other lentiviral systems, are capable of incorporating heterologous envelopes and efficiently transduce dividing and nondividing cells in the presence and absence of the accessory proteins in vitro. Compared to HIV vectors, FIV vector development is still in its infancy, but initial in vivo data in various species and tissues indicate long-term gene expression at therapeutic levels, and thus FIV vectors hold great promise. Future efficacy studies in animal models and primates will determine the FIV vectors' suitability for gene therapy. The design of recombinant FIV vectors incorporates safety features described for primate lentiviral vectors with the benefit that biosafety testing of FIV vectors can occur in the natural host. Currently, FIV vectors are generated in a transient fashion, but the availability of a stable producer system amenable to better characterization and scale-up will considerably increase the potential for use of FIV vectors in the clinic.

Transduction of murine cerebellar neurons with recombinant FIV and AAV5 vectors.

Our data demonstrate that vectors derived from recombinant feline immunodeficiency virus (rFIV) and adeno-associated virus type 5 (rAAV5) transduce cerebellar cells following direct injection into the cerebellar lobules of mice. Both recombinant viruses mediated gene transfer predominantly to neurons, with up to 2500 and 1500 Purkinje cells transduced for rAAV5 or rFIV-based vectors, respectively. The vectors also transduced stellate, basket and Golgi neurons, with occasional transduction of granule cells and deep cerebellar nuclei. rAAV5 also spread outside the cerebellum to the inferior colliculus and ventricular epithelium, while rFIV demonstrated the ability to undergo retrograde transport to the physically close lateral vestibular nuclei. Thus, AAV5 and FIV-based vectors show promise for targeting neurons affected in the hereditary spinocerebellar ataxias. These vectors could be important tools for unraveling the pathophysiology of these disorders, or in testing factors which may promote neuronal survival.

VSV-G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum.

Lentiviral vectors transduce dividing and postmitotic cells and thus are being developed toward therapies for many diseases affecting diverse tissues. One essential requirement for efficacy will be that vector particles are resistant to inactivation by human serum complement. Most animal studies with lentiviral vectors have utilized VSV-G pseudotyped envelopes. Here we demonstrate that VSV-G pseudotyped HIV and FIV vectors produced in human cells are inactivated by human serum complement, suggesting that alternative envelopes may be required for therapeutic efficacy for many clinical applications of lentiviral vectors.

Generation of retroviral packaging and producer cell lines for large-scale vector production and clinical application: improved safety and high titer.

For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.

A field study of supplementary rest breaks for data-entry operators.

This study examined the effects of supplementary rest breaks on musculoskeletal discomfort, eyestrain, mood, and performance in data-entry workers. Two rest break schedules were compared in a within-subjects design. Workers alternated between a 'conventional' and a 'supplementary' schedule in 4-week intervals. The conventional schedule contained a 15-min break during the first half of the work shift and a 15-min break during the second half of the shift. The supplementary schedule contained the same two 15-min breaks, and a 5-min break during each hour which otherwise did not contain a break, for a total of 20 extra minutes of break time. Results are based on data from 42 workers. They indicated that discomfort in several areas of the body, and eyestrain, were significantly lower under the supplementary than under the conventional schedule. While symptoms increased from pre- to post-work periods under both schedules, the magnitude of the increases was significantly less under the supplementary schedule. In addition, increases in discomfort of the right forearm, wrist and hand over the course of the work week under the conventional schedule were eliminated under the supplementary schedule. These beneficial effects were obtained without reductions in data-entry performance.

The use and development of retroviral vectors to deliver cytokine genes for cancer therapy.

In this review, we describe technical advancements of retroviral vectors to address issues of safety, titer, and clinical scale manufacturing to produce high-quality retroviral vector preparations that have made direct intratumoral administration of cytokine encoding recombinant vectors a feasible cancer therapy in the clinic. We also review possible further advances in retroviral vector design, which may prove important in expanding these clinical applications.

Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect.

Several problems limit the application of gene transfer to correct the cystic fibrosis (CF) Cl(-) transport defect in airway epithelia. These include inefficient transduction with vectors applied to the apical surface, a low rate of division by airway epithelial cells, failure of transgene expression to persist, and immune responses to vectors or vector-encoded proteins. To address these issues, we used a feline immunodeficiency virus-based (FIV-based) vector. FIV vector formulated with a calcium chelator transduced fully differentiated, nondividing human airway epithelia when applied to the apical surface. FIV-based vector encoding the cystic fibrosis transmembrane conductance regulator cDNA corrected the Cl(-) transport defect in differentiated CF airway epithelia for the life of the culture (>3 months). When this approach was applied in vivo, FIV vector expressing beta-galactosidase transduced 1-14% of adult rabbit airway epithelia. Transduced cells were present in the conducting airways, bronchioles, and alveoli. Importantly, gene expression persisted, and cells with progenitor capacity were targeted. FIV-based lentiviral vectors may be useful for the treatment of genetic lung diseases such as CF. This article may have been published online in advance of the print edition.

Occupational health psychology: an emerging discipline.

There is growing concern that rapidly changing patterns of work organization and employment pose risk for occupational illness and injury. In the present article, we assert that these changes create new needs and opportunities for research and practice by psychologists in the area of work organization and health. We begin with an historical overview of the contribution of psychologists to the occupational safety and health field, and to the study of work organization and health. We then describe new initiatives by the American Psychological Association and national health organizations in the United States and Europe to frame a new field of study--called "occupational health psychology"--that focuses on the topic of work organization and health. We conclude with a discussion of emerging research needs and trends within this field.

Minimum requirements for efficient transduction of dividing and nondividing cells by feline immunodeficiency virus vectors.

The development of gene delivery vectors based on feline immunodeficiency virus (FIV) is an attractive alternative to vectors based on primate sources for the delivery of genes into humans. To investigate the requirements for efficient transduction of dividing and nondividing cells by vector particles based on FIV, a series of packaging and vector constructs was generated for which viral gene expression was minimized and from which unnecessary cis-acting sequences were deleted. Pseudotyped vector particles produced in 293T cells were used to transduce various target cells, including contact-inhibited human skin fibroblasts and growth-arrested HT1080 cells. FIV vectors in which the U3 promoter was replaced with the cytomegalovirus promoter gave rise to over 50-fold-higher titers than FIV vectors containing the complete FIV 5' long terminal repeat (LTR). Comparison of the transduction efficiencies of vectors containing different portions of the FIV Gag coding region indicates that at least a functional part of the FIV packaging signal (Psi) is located within an area which includes the 5' LTR and the first 350 bp of gag. Transduction efficiencies of vectors prepared without FIV vif and orf2 accessory gene expression did not differ substantially from those of vectors prepared with accessory gene expression in either dividing or nondividing cells. The requirement for FIV rev-RRE was, however, demonstrated by the inefficient production of vector particles in the absence of rev expression. Together, these results demonstrate the efficient transduction of nondividing cells in vitro by a multiply attenuated FIV vector and contribute to an understanding of the minimum requirements for efficient vector production and infectivity. In addition, we describe the ability of an FIV vector to deliver genes in vivo into hamster muscle tissue.

Behavioral science activities at the Centers for Disease Control and Prevention. A selected overview of exemplary programs.

Behavioral research and surveillance activities are conducted across the Centers for Disease Control and Prevention (CDC). This article highlights activities in 4 program areas: violence against women, tuberculosis elimination, HIV prevention, and occupational health. The unique constraints and opportunities of each organization and program focus have shaped the way research has developed in each of these areas. Behavioral scientists also face many common challenges at CDC. Despite the difficulties of integrating behavioral research into an institution that historically has focused on biomedical and epidemiological research, behavioral scientists have made important contributions to public health. Many opportunities remain for psychologists to translate theory and operationalize constructs for use in solving important public health problems.

The impact of keyboard design on comfort and productivity in a text-entry task.

Concerns have arisen that the keyboard is a causal factor in the development of work-related musculoskeletal disorders (WRMDs) among video display terminal (VDT) operators. A number of alternative keyboard designs have been developed with altered geometry in an effort to improve comfort in keyboard operation. However, few data are available to substantiate whether these new keyboard designs are actually effective in reducing discomfort and musculoskeletal problems in users. The purpose of this study was to provide data on the efficacy of certain alternative keyboard design features (e.g. splitting the keyboard in half, and laterally inclining the keyboard halves) in reducing fatigue and musculoskeletal discomfort among keyboard operators. The study also explored the effects of these design features on performance. Fifty subjects performed a text-entry task for one day on a standard keyboard, then were assigned to one of five keyboard conditions for an evaluation period of two days (i.e. 10 subjects/condition). Outcome measures included performance (i.e. keystrokes/h, errors/h) and self-report measures of discomfort and fatigue. The results indicated an initial decline in productivity when subjects began typing on two of the alternative keyboards, but these productivity losses were recovered within the two-day evaluation period. The results also indicated no significant differences between keyboard conditions in discomfort and fatigue. These results suggest a minimal impact of the keyboard design features examined in this study on productivity, comfort and fatigue, at least after two days of exposure.

Psychosocial correlates of harassment, threats and fear of violence in the workplace.

OBJECTIVES: The purpose of this study was to investigate work climate factors and structural job aspects as predictors of workplace violence, with particular attention to the relative influence of both sets of factors. METHODS: Telephone survey data collected by a large midwestern insurance company were analyzed. Interviewers asked 598 full-time workers about their work climate, structural job aspects, and subject and workplace demographics, all of which were used as predictor variables in regression analyses. The participants were also asked about incidents of threats, harassment, physical attacks, and fear of becoming a victim of workplace violence, all of which were used as outcome measures. RESULTS: Separate logistic regressions were carried out for each of the outcome measures. The study identified a variety of factors which appear to place workers at risk of nonfatal occupational violence. Work climate variables, such as co-worker support and work group harmony, were predictive of threats, harassment, and fear of becoming a victim of violence. Structural aspects of the job, such as work schedule, were also significant in predicting threats and fear of becoming a victim of violence, but they were not predictive of harassment. CONCLUSIONS: This is the first study which suggests that both work climate and structural aspects of work may be important in promoting workplace violence. This finding suggests that intervention strategies should consider organizational and climate issues in addition to basic security measures.

Work-physiological synchronization as a determinant of performance in repetitive computer work.

The present study tested the hypothesis that performance would improve when the work rhythm of a highly repetitive task was synchronous with a worker's internal physiological rhythms. Experienced office workers (n = 20) used video display terminals (VDTs) to perform a repetitive, self-paced data-entry task in a simulated office environment over a 2-day period. Each work day consisted of six 40-min work periods. Work rhythm changes were induced by varying input data field lengths (3-13 characters) across eleven of the twelve work periods. The degree of synchronization between the work and breathing rhythms, and also between the work rhythm and variations in the interbeat interval, was scored using cross-spectral analysis. Synchronization scores were then used to predict keying performance using multiple regression analysis. The degree of synchronization between the work and breathing rhythms was not predictive of performance. However, increased synchronization between the work and cardiac rhythms was predictive of (a) increased keystroke output, (b) lower error rate and (c) lower correction rate. The results suggest that performance in repetitive VDT work might improve if the task is designed to promote work-physiological synchronization.

Musculoskeletal disorders among visual display terminal users in a telecommunications company.

The relationship between workplace factors and work-related upper extremity musculoskeletal disorders (UE disorders) was assessed in a cross-sectional study of 533 telecommunication employees utilizing video display terminals (VDTs). Cases of UE disorders were defined using symptom questionnaires and physical examinations. Data on demographics, individual factors (medical conditions and recreational activities), work organization and practices, and psychosocial aspects of work, including electronic performance monitoring (EPM), were obtained by questionnaire. Associations between workplace factors and UE disorders were assessed by multiple logistic models generated for each of the four UE areas (neck, shoulder, elbow, hand/wrists). One-hundred and eleven (22%) participants met our case definition for UE disorders. Probable tendon-related disorders were the most common (15% of participants). Probable nerve entrapment syndromes were found in 4% of participants. The hand/wrist was the area most affected, 12% of participants. The following variables had associations in the final models (p < 0.05) with at least one of the four UE disorders, although the strength of these associations were modest. Non-white race, a diagnosis of a thyroid condition (self-reported) use of bifocals at work, and seven psychosocial variables (fear of being replaced by computers, increasing work pressure, surges in workload, routine work lacking decision-making opportunities, high information processing demands, jobs which required a variety of tasks and lack of a production standard) were associated with UE disorders. This study indicates that work-related UE musculoskeletal disorders are relatively common among telecommunication workers who use VDTs, and adds to the evidence that the psychosocial work environment is related to the occurrence of these disorders.

Identification of the specific oligosaccharide sites recognized by type 1 fimbriae from Escherichia coli on nonspecific cross-reacting antigen, a CD66 cluster granulocyte glycoprotein.

Nonspecific cross-reacting antigen (NCA), a CD66 cluster antigen, is a well characterized glycoprotein on granulocytes, macrophages, and lung epithelium. Structural studies at the protein and genomic levels have revealed that NCA is a member of the immunoglobulin (Ig) supergene family and contains a domain structure similar to Ig with an amino-terminal variable-like domain followed by disulfide loop-containing constant-like domains. Previous work by this laboratory and others has demonstrated that NCA is a receptor for binding of bacteria expressing type 1 fimbriae (pili). This binding is mediated by interaction between lectins on the bacteria fimbriae and carbohydrate chains on NCA. In the present work we further characterize the specificity for bacterial binding by NCA using endoglycosidases and site-directed mutagenesis. Results of these studies demonstrate that Escherichia coli expressing type 1 fimbriae binds to high mannose oligosaccharide structures on NCA and that the functionally relevant sites are located in the variable-like domain of NCA.

Expression of carcinoembryonic antigen and its predicted immunoglobulin-like domains in HeLa cells for epitope analysis.

Carcinoembryonic antigen (CEA) is a member of the immunoglobulin gene superfamily with one predicted variable domain-like region (N domain; 108 amino acids) and three sets of constant domain-like regions (A1B1, A2B2, and A3B3; 92 amino acids for A domains and 86 amino acids for B domains). In addition, CEA possesses two signal peptides, one at the amino terminus and one at the carboxyl terminus. Both are removed during posttranslational processing, with the one at the carboxyl terminus being replaced by a glycosylphosphatidylinositol (GPI) moiety. We have previously expressed the full length complementary DNA clone for CEA in Chinese hamster ovary cells and murine L cells, demonstrating proper processing of nascent polypeptide chains to mature, fully glycosylated CEA including the GPI anchor. Using the same full length CEA complementary DNA clone and the polymerase chain reaction, we have now constructed expression clones for secreted versions of the N domain, the A3B3 domain, and the A3 and B3 subdomains. The clones were expressed in HeLa cells using the beta-actin promoter. A stop codon was introduced at the end of the A3B3 and the A3 and B3 domains to allow secretion instead of retention on plasma membranes with the GPI anchor. Expressed products were purified to homogeneity by affinity chromatography using monoclonal antibodies specific for each domain and by reversed phase high pressure liquid chromatography. Purified domains were characterized by Western blotting, antibody binding and inhibition studies, amino-terminal sequence and amino acid analyses, and laser desorption/time of flight mass spectrometry. These analyses revealed that the monomeric N domain is of size 15,990, with a glycosylation mass of about 4100, in good agreement with two N-linked glycosyl units of about mass 2100. There is some evidence that the N domain forms dimers. The N domain reacted with antibodies specific for this domain with an affinity similar to that of intact CEA. The A3B3 domain had a mass of 34,462, with a glycosylation mass of 14,900, in good agreement with seven N-linked glycosylation sites of average mass 2100. The A3B3 domain reacted only with antibodies specific for this domain, with a slightly lower affinity than that of native CEA. The amino-terminal sequences of the N domain and A3B3 domain proteins demonstrated proper processing of the signal peptide.(ABSTRACT TRUNCATED AT 400 WORDS)