In this study, we analyzed TL-3 efficacy using a highly pathogenic FIV-C isolate, which causes a severe acute phase immunodeficiency syndrome, with high early mortality rates.The protease inhibitor, TL-3, demonstrated broad efficacy p ≤ 0.05) higher viral RNA loads as compared to asymptomatic animals. A marked and progressive increase in CD8+ T lymphocytes in animals surviving acute phase infection was noted, which was not evident in symptomatic animals (p ≤ 0.05). Average viral loads were lower in TL-3 treated animals and of the 6 animals requiring euthanasia, four were from the untreated cohort. At eight weeks post infection, half of the TL-3 treated animals and only one of six untreated animals had viral loads below detection limits. Analysis of protease genes in TL-3 treated animals with higher than average viral loads revealed sequence variations relative to wild type protease. In particular, one mutant, D105G, imparted 5-fold resistance against TL-3 relative to wild type protease.Twenty cats were infected with uncloned FIV-C and half were treated with TL-3 while the other half were left untreated. Two uninfected cats were used as controls. The general health and the immunological and virological status of the animals was monitored for eight weeks following infection. All infected animals became viremic independent of TL-3 treatment and seven of 20 FIV-C infected animals developed severe immunodepletive disease in conjunction with significantly (The findings indicate that the protease inhibitor, TL-3, when administered orally as a monotherapy, did not prevent viremia in cats infected with high dose FIV-C. However, the modest lowering of viral loads with TL-3 treatment, the greater survival rate in symptomatic animals of the treated cohort, and the lower average viral load in TL-3 treated animals at eight weeks post infection is indicative of a therapeutic effect of the compound on virus infection. Infected cats exhibit similar disease patterns as human immunodeficiency virus (HIV) infected patients by developing multiple immuno-depletive symptoms collectively referred to as acquired immunodeficiency syndrome (AIDS). As with HIV, differences in virulence among the different FIV subgroups are evident -4. Thus,in vitro with similar effectiveness [in vivo studies using the predominantly neurotropic FIV-PPR strain, showed that TL-3 treatment lowered plasma viral loads and resulted in a significant protective effect against neurologic alterations in the CNS in FIV infected cats [One of the major breakthroughs in HIV-1 treatment has been the use of specific inhibitors of the viral aspartic protease family as part of a drug cocktail, called highly active anti-retroviral therapy (HAART), with the ultimate goal of suppressing HIV-1 replication in patients to low or undetectable levels -8. Effectiveness  led us tted cats . In the 5 RNA copies/ml) of FIV-C-infected plasma I.V. with TL-3 drug treatment. Group 2 (n = 5) received 0.1 ml FIV-C-infected plasma without TL-3 treatment. Group 3 (n = 5) received 0.5 ml FIV-C-infected plasma with TL-3 and Group 4 (n = 5) received 0.5 ml FIV-C-infected plasma without TL-3 treatment. Blood (1 ml) was drawn from all cats prior to the start of the experiment, at weekly intervals for the first four weeks after infection, and at bi-monthly intervals from week 4 until the end of the study. Complete blood counts were assessed as a function of infection and TL-3 treatment. In addition, quantitative reverse transcription PCR (QRT-PCR) analyses were performed to assess plasma viral load. All animals were continuously observed for any changes in general health. No significant differences were noted between viral load or disease phenotype between the two plasma dosages used in infection and subsequent discussion will not distinguish between these two groups.Twenty-two female specific pathogen-free (SPF) cats were randomly divided into five groups. Group 0 consisted of two cats, which received TL-3 treatment without viral infection and were considered controls. Group 1 (n = 5) received 0.1 ml  were showing clinical signs of debilitating acute phase disease. Four of the seven affected animals (215, 219, 220, 234) were from the untreated groups, and three animals (222, 221, 229) were from the TL-3 treated groups. Symptoms in all seven symptomatic cats varied from conjunctivitis, anorexia, corneal ulcerations, and gingivitis to increases in temperature, dermatitis and marked lethargy. Despite intensive antibiotic treatment, the general state of health of 6 of the cats did not improve (221, 220, 234, 229, 215, 219) and they received mandated euthanasia between six to seven weeks post-infection. Cat 222 (TL-3 treated) responded to antibiotic therapy and recovered from acute phase symptoms.Control and infected cats gained weight at approximately the same rate during the first 4 weeks post infection, regardless of drug treatment status . However, analysis of CD4+ and CD8+ T cells counts of symptomatic cats as compared to treatment-matched asymptomatic animals showed interesting differences . Uninfected control animals maintained their T cell values during the same time frame. The CD8+ population of T cells showed an even more drastic decrease in the symptomatic animals compared to the asymptomatic infected animals  from week 2 onward, consistent with a strong CD8+ T cell response in the protected cats.Peripheral blood mononuclear cells (PBMC) were isolated from whole EDTA-treated blood from each animal and CD4s Figure , resp.. d Figure . CD4+ T s Figure . BetweenConsistent with previous reports -16, we ai of 47.3 nM as compared to 9.9 nM for wild-type, consistent with a 5-fold increase in resistance to TL-3.Drug pressure induced viral resistance to protease inhibitors represents one of the major hurdles of HIV HAART treatment regimens ,18. HIV in vivo in the context of the highly pathogenic, uncloned CABCpady00C species (FIV-C) [The protease inhibitor, TL-3, demonstrated broad efficacy against FIV, SIV and HIV in tissue culture , as well (FIV-C) , which c8 RNA copies/ml, p ≤ 0.05) than asymptomatic infected animals independent of drug treatment. This finding suggests that the intense viral infection severely compromised the immune system leading to immunodeficiency and the development of concomitant AIDS, as evidenced by the rapid loss of CD4+ T cells as well as neutrophils in the affected cats during the first few weeks  developed severe disease syndromes, which suggests that TL-3 efficacy was limited to a specific viral threshold in this study. Once the threshold has been crossed, TL-3 may not be able to overcome the full-blown, acute, viral infection, resulting in rapid onset of immune suppression.+ T cells in animals surviving the acute phase infection and a lack of such responses in animals that required euthanasia within the first 8 weeks following infection. Although not formally tested, the findings imply a strong cell-mediated response in the surviving animals contributed to controlling the viral infection.We were unable to show any correlation between humoral antibody responses and clinical outcome (data not shown). However, a consistent observation was a marked and progressive increase of CD8The above analyses underscore the natural variation in the response to FIV challenge in outbred cats, similar to the observed variation in responses to HIV infection in humans. However, more consistent responses, both in peak viral loads Figure  and in lin vitro, the findings here suggest that in vivo, drug resistance to the compound may develop. The results were not unexpected in that we had been able to develop TL-3 resistant HIV variants [Viral protease inhibitors are of paramount importance for HIV treatment and successful tempering of viral infection. However, drug resistant escape variants are an important consideration in treatment protocols ,18. Althvariants  and it sThe findings indicate that the protease inhibitor TL-3, when given orally as a monotherapy, did not prevent viremia in cats infected with a high dose challenge with FIV-C and substantial virus loads were evident in circulation throughout the acute phase (between 2–6 weeks post infection) in all infected animals, regardless of drug regimen. Average peak viral loads in the acute phase were lower in TL-3 treated animals, but variability was such that the numbers did not reach statistical significance. However, of six animals that required euthanasia, four were from the untreated cohort and two were from the TL-3 treated group. Additionally, at eight weeks post infection, half the surviving TL-3 treated animals had viral loads below the detection limits, whereas only one of six untreated animals had markedly reduced viral loads. Thus, therapeutic benefit was noted with TL-3 treatment, even in the face of an aggressive FIV infection.+ cell numbers starting around three weeks post infection in animals that eventually required humane euthanasia versus a pronounced and significant increase in CD8+ T cell numbers in animals that survived the acute phase.The findings also show clear differences in the lymphocyte responses of animals that succumb to acute phase illness versus those that survive to the asymptomatic phase. The most pronounced difference was in the lack of an increase in CD8Certain animals that received TL-3 had higher than average viral loads after the acute phase. Analyses of the protease genes of FIV quasi-species prevalent in these animals revealed sequence variations relative to protease of wild type FIV-C. One particular protease, cloned and expressed from two TL-3-treated animals, contained the mutation D105G, which imparted 5-fold resistance against TL-3 relative to wild type protease. This may represent the initial stages of drug resistance development and preparation of this mutation in the context of isogenic virus will address this issue. The findings suggest that the cat model will serve as a valuable animal model for study of resistance development against lentivirus infections.22 female purpose-bred 8–9 week old kittens purchased from Liberty Laboratories were inspected upon arrival for signs of illness, examined by a veterinarian and weighed. Animals were maintained in a 2-week quarantine and observed for any signs of illness prior to the beginning of the study. IACUC number ARC 61 JAN 3.5 RNA copies/ml) from a cat, that had died from an acute infection with CABCpady00C (FIV-C), were kindly provided by Dr. E. Hoover, of Colorado State University. Cats were injected I.V. with either 0.1 ml (105 RNA copies/ml) or 0.5 ml of plasma.Plasma samples carbonyl]-L-alanyl-L-valyl]amino])  treatmenUninfected and FIV-infected animals were intermittently scheduled for analyses of evoked potentials in conjunction with the blood sampling, including testing for both auditory and visual evoked potentials as previously described . Once reAnimals were examined daily and in case of health concerns further therapy/diagnostics were initiated. Animals with abnormal weight, or on antibiotics were placed on supplemental feeding with moist food and Nutrical. Dehydrated animals received subcutaneous fluid therapy. More affected animals received supportive care and medications, consisting of BID administration of antibiotics (Baytril), BID subcutaneous fluid therapy, BID temperature evaluation, BID application of antibiotic ophthalmic ointment supportive care. Any animals that required extensive supportive care (TID fluid therapy, TID force feeding) were euthanized. Euthanasia of research animals was conducted with strict adherence to NIH Office of Laboratory Animal Welfare protocols.Blood samples  were collected weekly during the first month of the study and then every two weeks thereafter, as described . SamplesPlasma was separated from blood by centrifugation at 3000 rpm for 5 minutes at room temperature. Blood cells were resuspended in 3 ml PBS and PBMC were separated from buffy coats by density gradient centrifugation using Ficoll-Hypaque Plus (Amersham Biosciences, Sweden). PBMC were washed once in PBS and twice in PBS/2% FBS for flow cytometry analyses.Statistical p values for the FIV-C viral load were determined by the Student's two-tailed t-test (paired, two-tailed distribution between the treated and non-treated group and the symptomatic vs asymptomatic group). Statistical p values for the weekly total CD4 and CD8 cell counts were also determined by the Student's two-tailed t-test (paired, two-tailed distribution compared to base line levels at week 0).1κ FITC and PE (BD PharMingen, San Diego, CA) were used as isotype controls. Cells were fixed with 2% PFA prior to analysis performed on a FACScan flow cytometer (Beckton Dickenson Immunocytometry Systems) using the Cell Quest Software program.Two-color flow cytometry analysis was performed on cells stained with mouse α-feline CD4 FITC and mouse α-feline CD8 PE (Southern Biotech, Birmingham, AL). Anti-mouse IgG9 copies RNA /280 μl plasma), corresponding to the 1.2 kb KAN gene (Promega, Madison, WI), was used to normalize plasma volumes between samples and to correct for sample loss from viral RNA extraction and cDNA synthesis. An on-column DNase/ RNase free (Qiagen) incubation step for 10 minutes at room temperature was added to remove residual cellular DNA. Complimentary DNA (cDNA) was generated in a 20 μl total reaction using 13 μl of sample RNA, 0.5 μl (2 μM stock) each of KAN and FIV specific reverse primers (sequences below) and StrataScript reverse transcriptase, following the manufacturer's protocol (Stratagene, La Jolla, CA). After incubation, each cDNA sample was diluted in water to 30 μl and stored at -80°C for use in real-time PCR.Plasma for weeks 0, 2, 4, 6 , 6.5 , and 8, were isolated from whole blood by centrifugation and stored at -20°C. Viral RNA was extracted using the QiaAmp Viral RNA Isolation Kit  according to manufacturer's instructions with slight modifications: Plasma samples (280 μl) were lysed in buffer AVL (1,120 μl) (Qiagen) for 10 minutes at room temperature in the presence of carrier RNA (10 μg/ml) and an external Kanamycin (KAN) RNA spike. Equal amounts of the external RNA spike  (Invitrogen, Carlsbad, CA), forward, reverse primers and probe mix (7.5 μl), and cDNA target (5 μl). The mixture was incubated at 50°C for 2 minutes, 95°C for 10 minutes, then cycled at 95°C for 15 seconds and 60°C for 60 seconds 55 times, using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Data were analyzed using the ABI 7700 Sequence Detection Software. Forward and reverse primers  used in real-time PCR were made at IDT, , while the fluorescein-dabsyl Amplifluor UniPrimer  was purchased from Serologicals (Norcross, GA). The primer sequences used for real-time PCR are as follows:FIV reverse-transcriptase forward: 5'-ACTGAACCTGACCGTACAGATAAATTACAGGAA GAACCCCCATA-3'FIV reverse-transcriptase reverse: 5'-TGTTAATGGATGTAATTCA TAACCCATC-3'KAN forward: 5'-ACTGAACCTGACCGTACACGCTCAGGCGCAATCAC-3'KAN reverse: 5'-CCAGCCATTACGCTCGTCAT-3'8 to 5 × 102 copies per well) of dsDNA plasmids of known copy number , against the cycle threshold (Ct) determined for that value. The pET28 vector (Novagen) was used as the target plasmid for the KAN gene, while a plasmid containing the molecular clone of FIV-C was used for the FIV reverse-transcriptase gene. Calculated values for each plasma sample represent relative copy numbers for the purposes of evaluation between individual samples.To determine the relative copy numbers of KAN and FIV from plasma samples, a linear standard curve was generated by plotting 10-fold dilutions (5 × 10Complementary DNA (cDNA) was synthesized from isolated plasma viral RNA of infected cats. The cDNA pool was used as a template for PCR reactions using 5' primer MFIVCPL5' (5'-GATTTATAAATCATATG GCATATAATAAAGTGGGTACCACTACAACATTAG-3'), which adds an NdeI restriction site, methionine, and alkaline to the N-terminal of the protease and 3' primer MFIVCPL33' (5'-CTGAGATCTGAGCAAGCTTTTACATTACTAATCT AATATTAAATTTAACCATG TTATC-3'), which adds a stop codon and a Hind III restriction site to the C-terminus of the protease. The amplified PCR product was gel purified and cloned into pCR-TOPOII vector (Invitrogen, Carlsbad, CA) for sequencing. Selected DNA of mutants, N55D, H72R, D105G, and M107R were digested with Nde I/Hind III and ligated into pET21a expression vector (Novagen). The mutant FIV-C proteases were expressed in Rosetta pLysS cells (Novagen) and purified as previously described .2)-Val-Gln-Ser-Lys-Gly-Arg-NH2. The concentration was determined by active-site titration with inhibitor TL-3. Inhibitor constant (Ki) of TL-3 against mutant FIV-C protease was analyzed as described [Enzyme kinetics of FIV-C protease were assayed on the flourogenic substrate Arg-Ala-Leu-Thr-Lys(Abz)-Val-Gln/Phe(NOescribed .None of the authors have commercial interests or direct association with a company that is marketing TL-3. J.H.E. is a co-author of a patent application regarding use of protease inhibitors like TL-3 reported here, containing small P3 residues, as inhibitors of HIV and FIV.S.R. and C.H.S contributed equally to this work and carried out all tissue culture analyses, purification and characterization of cell populations isolated from PBMC, statistical analyses, and preparation of results for publication. Quantitative PCR analyses was carried out by D.A.S. K.J.C. was responsible for all in vivo animal work, including TL-3 administration and oversight of veterinary care for the animals. S.H-R. carried out measurements of brainstem auditory evoked potential changes. S.H., B.E.T, and J.H.E. acted as mentors for the various facets of the project, oversaw the writing of the manuscript, and provided space and funding to carry out the work.


The knowledge of factors accurately predicting the long lasting sick leaves is sparse, but information on medical condition is believed to be necessary to identify persons at risk. Based on the current practice, with identifying sick-listed individuals at risk of long-lasting sick leaves, the objectives of this study were to inquire the diagnostic accuracy of length of sick leaves predicted in the Norwegian National Insurance Offices, and to compare their predictions with the self-predictions of the sick-listed.Based on medical certificates, two National Insurance medical consultants and two National Insurance officers predicted, at day 14, the length of sick leave in 993 consecutive cases of sick leave, resulting from musculoskeletal or mental disorders, in this 1-year follow-up study. Two months later they reassessed 322 cases based on extended medical certificates. Self-predictions were obtained in 152 sick-listed subjects when their sick leave passed 14 days. Diagnostic accuracy of the predictions was analysed by ROC area, sensitivity, specificity, likelihood ratio, and positive predictive value was included in the analyses of predictive validity.The sick-listed identified sick leave lasting 12 weeks or longer with an ROC area of 80.9% (95% CI 73.7–86.8), while the corresponding estimates for medical consultants and officers had ROC areas of 55.6% (95% CI 45.6–65.6%) and 56.0% (95% CI 46.6–65.4%), respectively. The predictions of sick-listed males were significantly better than those of female subjects, and older subjects predicted somewhat better than younger subjects. Neither formal medical competence, nor additional medical information, noticeably improved the diagnostic accuracy based on medical certificates.This study demonstrates that the accuracy of a prognosis based on medical documentation in sickness absence forms, is lower than that of one based on direct communication with the sick-listed themselves. The increasing rate of sick leave experienced in most Western countries challenges insurance companies, employers, and public authorities to identify measures to reduce burdens at the individual, workplace and societal levels.To reduce the expenses of sick leave and the risk of expulsion from work, the Norwegian government introduced legislation in 1993 that anticipated early and more vigorous interventions of the Norwegian National Insurance Scheme , 2000, uThe selection process is currently based on information in medical sickness certificates supplied by access to the register of previous sickness benefits. A medical sickness certificate  is required if sick leave exceeds 3 days, and after 8 weeks an extended medical certificate is mandatory  .Based on the current practice with identifying sick-listed individuals at risk of long-lasting sick leaves, the objectives of this study were to inquire diagnostic accuracy of predictions within the NIOs, and to compare their predictions with the self-predictions of the sick-listed.In October and November 1997 and March and April 1998, newly sick-listed persons with musculoskeletal or mental disorders (ICPC, L- and P- diagnoses)  were incA total of 495 randomly selected persons received a questionnaire on the expected length of their ongoing sick leave period. The answer categories were: less than 4 weeks, 4 to 7 weeks, 8 to 11 weeks, 12 to 15 weeks, 16 to 25 weeks, 26 to 51 weeks, and at least 1 year. Some 152 persons (30.7%), called the responder group, returned the questionnaire with this question filled in.Based on SC1s available after 14 days of sick leave, two NIO officers without formal medical competence, but experienced in working with sick-listed persons, and two experienced physicians working part time as insurance medical officers , assessed the expected length in each of the 993 ongoing sick leave cases. In 496 randomly chosen cases, the NIO assessors had additional access to information on sick leave periods during the previous 3 years. Of potentially 1986 assessments in each profession, the officers and medical consultants had 18 and 25 missing assessments, respectively.SC2s became available in 322 of the 459 cases where sick leave exceeded 8 weeks, and the NIO assessors reassessed these cases.Reproducibility of assessments by medical consultants were analysed in 20 cases reassessed by the two NIO medical consultants, and assessed by another eight of their colleagues.The reference standard lengths of individual sick leaves within 1 year were collected from the National Sickness Benefit Register. Sick leaves interrupted by only 1–2 days without sickness benefits, typically on weekends, were registered as a single period. The observed length of sick leave thus comprised the total period of continuous full-time or part-time absence due to sickness within 1 year.The diagnostic accuracy of predicted lengths was compared on the basis of sensitivity, specificity, likelihood ratio and the area under the receiver operating characteristics curves (ROC area) ,7. The nThe predictive validity is presented as sensitivity, specificity, positive predictive value (PPV) and likelihood ratio at different thresholds, cut-offs, in predicted length . ReliabiThe Regional Ethical Committee approved the protocol, and the Norwegian Data Inspectorate licensed the necessary register of sick-listed subjects.The mean observed continuous sickness absence was 100.8 days (median 48 days). Sick leaves in females lasted a mean of 105.1 days, compared to 94.6 days in men (medians 55 and 43 days, respectively). The mean length among persons with musculoskeletal disorders was 90.2 days in 335 males and 108.6 days in 489 females. The mean length among persons with mental disorders was 120.6 days in 56 males and 90.0 days in 113 females.The mean length of the sick leave in the responder group was 107.4 days , compared to 92.4 days in the 343 non-responders. Stratified analysis revealed longer mean sick leaves among responders 40 years and younger, of 109.3 days (95% CI 81.4–134.5 days), compared to the 79.3 days (95% CI 65.6–93.1 days) in non-responders. Stratification on gender or musculoskeletal or mental disorders did not reveal any significant differences in the length of sick leave between responders and non-responders.All assessors, including the sick-listed themselves, systematically overestimated the length of short sick leaves (lasting 4–11 weeks) and underestimated the length of long sick leaves , and sick leaves lasting 26 weeks or longer with a ROC area of 75.5% (95% CI 67.9–82.1%). Sick-listed persons with mental disorders or with neck, or shoulder and arm disorders, were most accurate in their assessment Figure . This waThe sick-listed subjects predicted their sick leaves with higher sensitivity and PPV than the NIO assessors Tables , 4. MaleDuration of at least 8 weeks was the preferable cut-off in predicted length, to identify sick leaves lasting at least 12 weeks Table . A prediThe sensitivity of identifying sick leaves lasting at least 26 weeks was generally low when medical consultants and officers predicted on the basis of SC1s. Table . The senAccording to the results, the effects of the different predictive strategies can be illustrated by considering a program designed to intervene in all cases where the subject is expected to be sick-listed for more than 12 weeks at 14 days of sick leave. Out of every 1000 sick-listed persons, 333 will be sick-listed for more than 12 weeks according to the prevalence in this study. The random selection of 333 persons will include 111 true positives, while 333 persons selected by officers will include 133 of the 333 persons that will be sick-listed at least 12 weeks. The evaluation of 1000 sick-listed individuals thus increases the number of true positives by 22 in a selection of 333 sick-listed persons. The alternative strategy of asking the sick-listed themselves will include 210 true positives in a selection of 333 persons.Agreement between medical consultants in their initial prediction of sick leaves lasting at least 12 weeks, was fair, with a kappa of 0.31 (95% CI 0.20–0.43). The corresponding kappa value between officers was 0.05 (95% CI -0.05–0.14).In the prediction of sick leaves lasting at least 12 weeks based on the SC2, agreement was moderate between medical consultants (kappa = 0.42, 95% CI 0.29–0.54) and fair between officers (kappa = 0.26, 95% CI 0.10–0.42). The corresponding agreements in the prediction of sick leaves lasting at least 26 weeks were moderate between medical consultants (kappa = 0.55, 95% CI 0.40–0.70) and fair between insurance officers (kappa = 0.31, 95% CI 0.17–0.47).The differences in diagnostic accuracy, between the two participating medical consultants and their eight colleagues in the reproducibility group, were not significant.The results of the present study question any practical value of using information in medical sickness certificates in predicting the length of sick leave, as is the current practice in Norwegian NIOs. Instead, the sick-listed themselves predicted their length of sick leaves far more accurately, but this information is not routinely sought.The officers in the present study were selected from experienced officers who had shown an interest in the field of sick leave. This might introduce a bias of overestimating the officers' general ability to predict the length of sick leaves. The performances of the two medical consultants were representative of eight of their colleagues who participated in the reproducibility part of the study. We therefore consider the diagnostic accuracy of the assessors to be representative of their professional groups, or at least not underestimated due to bias. Although the diagnostic accuracy varied within each group, the main conclusion of better predictive ability among the sick-listed, was challenged neither by comparing with the mean length predicted by assessors, nor by comparing with the best-performing NIO assessor.The distributions of gender and diagnosis among the 993 persons included in the study were comparable with those in the National Sickness Benefits Register. The findings of longer sick leaves in women with musculoskeletal disorders, and longer sick leaves in men with mental disorders, are consistent with the Register and other studies -13.The low responder rate among the sick-listed introduced a possible selection bias, although we could not identify any selection bias in gender, age, diagnosis or occupation . If therIf the lengths of sick leaves were predominantly related to loss of function caused by sickness, in line with the legislation, we would expect that the medical consultants' professional competence would favour them in predictions of the lengths of sick leaves. The differences we observed between medical consultants and officers in mean ROC area, were however minor. Furthermore, we could not demonstrate any significant differences in diagnostic accuracy between medical consultants and officers when aggregate information on disease, treatment, function related to work, and prognoses were available in the SC2. The improvement in ROC area with this aggregated information was minor, with the area just reaching 70%, which is considered borderline useful for some purposes , and is The Norwegian NIO is obliged by legislation to perform early intervention on the sick-listed in an effort to reduce the length of sick leave and the risk of expulsions from work. Limited resources and the large number of sick-listed individuals make selection desirable before any intervention is initiated. An alternative to selection on the basis of medical certificates is to communicate directly with the sick-listed themselves. This selection for intervention by NIOs might be seen as screening. The aim is to reach – at an acceptable cost – as many as possible of those that might profit from intervention. The potential individual gain by intervention will be greater when longer lasting sick leaves can be anticipated, and greater the sooner individual intervention programs are established.The marginal predictive ability and modest agreement between NIO assessors questions the use of resources in selection based on information from medical certificates. The predictions of medical consultants tend to be better than those of officers, but not to an extent that makes it more meaningful to use medical consultants in the selection process, rather than officers.With limited resources for intervention, it might be more cost effective to identify those whose sick listing will last longer than 26 weeks instead of 12 weeks. Based on self-reporting, eight out of ten would be true positives, and one fourth of the individuals would be reached. To reach the same number of true positives at 14 days of sick leave, the ratio of true positives would be reversed from eight out of ten, to two or three out of ten, if the selection were based on medical certificates.In the search for tests predicting long-lasting sick leaves, such as The Örebro Musculoskeletal Pain Questionnaire , the preSick-listed individuals predicted their length of sick leave far more accurately than did NIO medical consultants and officers based on information from sickness certificates and the history of past sick leaves. The predictions of sick-listed males were better than those of females, and older persons predicted better than younger persons. The availability of more information, as through the SC2, had only a minor effect on the predictive ability of the medical consultants and officers. Neither reliability nor validity of their predictions was satisfactory.This study demonstrates the need to re-consider the diagnostic usefulness of documentation on sickness absences, and supports a change in strategy from collecting more medical information to more direct communication with the sick-listed themselves, for effective and early interventions to prevent long sick leaves and expulsions from work.The author N.F. is part-time employed as National Insurance medical consultant.NF was in charge of designing and running the study, and performed most of the analyses and the writing of this manuscript. RJ actively supervised all parts of the study, and OHF contributed to planning and writing. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


The Internet provides a new meeting ground, especially for gay men, that did not exist in the early 1990s. Several studies have found increased levels of high risk sexual behaviour and sexually transmissible infections (STI) among gay men who seek sex on the Internet, although the underlying processes are not fully understood. Research funded by the UK Medical Research Council (2002–2004) provided the opportunity to consider whether the Internet represents a new sexual risk environment for gay and bisexual men living in London.The objectives of the Internet and HIV study are to: (i) measure the extent to which gay men living in London seek sexual partners on the Internet; (ii) compare the characteristics of London gay men who do and do not seek sex on the Internet; (iii) examine whether sex with Internet-partners is less safe than with other sexual partners; (iv) compare use of the Internet with other venues where men meet sexual partners; (v) establish whether gay men use the Internet to actively seek partners for unprotected anal intercourse; (vi) determine the potential for using the Internet for HIV prevention. These objectives have been explored using quantitative and qualitative research methods in four samples of London gay men recruited and interviewed both online and offline. The four samples were: (i) gay men recruited through Internet chat rooms and profiles; (ii) HIV positive gay men attending an NHS hospital outpatients clinic; (iii) gay men seeking an HIV test in an NHS HIV testing or sexual health clinic; (iv) gay men recruited in the community.Quantitative data were collected by means of confidential, anonymous self-administered questionnaires (n>4000) completed on-line by the Internet sample. Qualitative data were collected by means of one-to-one interviews (n = 128) conducted either face-to-face or on-line.The strength of the Internet and HIV study is its methodological plurality, drawing on both qualitative and quantitative research among online and offline samples, as well as taking advantage of recent advances in web survey design. The study's findings will help us better understand the role of the Internet in relation to gay men's sexual practice Several studies have found elevated levels of high risk sexual behaviour among people who seek – and meet – sexual partners through the Internet -7 In a sA San Francisco study also found that gay men were more likely than heterosexual men and women to use the Internet to meet sexual partners . Around Similar reports have also emerged from European studies. An investigation of gay men in London gyms found that in the year 2000 over one-third of those with Internet access had used it to look for sex . When suThe association between seeking sex on the Internet and high risk sexual behaviour raises a number of important, as yet unanswered questions. Does the excess risk for HIV and STI occur with sexual partners whom men actually meet through the Internet? Does the association reflect the fact that high risk men are selectively using the Internet to look for sex? Or does the Internet in some way facilitate high risk behaviour? Is the Internet attracting a constituency of men who have little contact with the established gay scene or health promotion agencies ? For exaTo address these questions we have undertaken research funded for two years (2002–2004) by the UK Medical Research Council and the Department of Health. This has been conducted by researchers at City University London in collaboration with colleagues at the MRC Social and Public Health Sciences Unit Glasgow and University College London (see appendix 1).The core research question is: Does the Internet represent a new sexual risk environment for gay/bisexual men living in London?" (referred to as "gay men" throughout the rest of this paper). And if so, what are the underlying processes? The research focuses on gay men living in London since the incidence and prevalence of HIV infection among gay men in London is higher than elsewhere in the UK -13.• measure the extent to which London gay men seek sexual partners on the Internet• compare the characteristics of gay men who do and do not seek sex on the Internet• examine whether sex with Internet-partners is less safe than with other sexual partners• compare use of the Internet with other venues such as saunas and backrooms• establish whether gay men use the Internet to actively seek partners for unprotected anal intercourse• determine the potential for using the Internet for HIV preventionThese objectives have been explored using both quantitative and qualitative research methods. While quantitative research methods can provide data on a range of outcomes they can rarely offer insight into underlying processes. Qualitative research, on the other hand, illuminates our understanding of human behaviour but is limited in the extent to which findings can be generalized. By adopting methodological plurality and employing both quantitative and qualitative methods, we will be able to build on the strengths of both approaches within an integrated research programmeIn desribing the design and methods of the Internet and HIV study we focus on sampling strategy, recruitment into the study, data collection and data analysis.Research among hard-to-reach groups such as gay men is usually based on convenience rather than probability samples . For exaConsequently, we recruited gay men from four different sources – one online, three offline. Each sample has specific features which are pertinent to the research question.HIV positive men are central to any research concerning HIV risk, transmission and prevention -21. FurtMost people seeking an HIV test have been at risk of HIV infection. Furthermore, HIV negative gay men with a history of multiple repeat testing report elevated levels of high risk sexual behaviour . This grSurveying gay men in the community allows us to examine the extent to which they use the Internet for seeking sexual partners and the associated risks. Previous research has shown that gay men surveyed in central London gyms are broadly representative of men "on the scene" in London ie men who go to gay bars, clubs and other venues . HoweverMen who use Internet chat rooms and profiles to seek sex with other men are clearly of central importance to this research project. The Internet may attract a constituency of men who would not otherwise be included in behavioural surveys among gay men ie men who do not go to gay bars, clubs or other venues and men who have not been tested for HIV . CompariBased on previous research conducted among gay men in both clinic and community settings ,8,22,23 For the qualitative arm of the study, we recruited at least 20 men from each of the four samples to allow us to derive accounts of Internet dating and sexual practice from a diverse range of men, selected purposively according to age, education, employment, HIV status and use of the Internet for seeking sex.Men diagnosed with HIV infection attending an outpatient treatments clinic at the Royal Free Hampstead NHS Trust hospital, London over an 8 month period (October 2002–May 2003) were invited to participate in the research. Patients with a limited command of English were ineligible for the study as were those who were too ill to complete a questionnaire. Eligible patients were approached in the clinic's waiting area by a trained member of the research team who discussed the project with them. Patients were provided with written information about the research, contact details of the research team as well as helpline numbers. Once they had provided written consent, respondents were asked to complete a pen-and-paper questionnaire in the clinic and return it in a sealed envelope to the team member (further information about the questionnaire in Research Methods below). Some patients were only in the waiting area for a short time so there wasn't an opportunity to invite them to take part in the study.Over the eight month survey period (October 2002–May 2003), 1001 individual male patients attended the clinic of whom 939 were deemed eligible for the study. Of those who were eligible, 864 were asked to complete a questionnaire and 620 did so. The response rate was 72% of men who were offered a questionnaire and 66% of all eligible men who attended the clinic. Of the 620 men who completed a questionnaire, 542 described their sexual orientation as gay or bisexual or had had sex with another man in the previous year. Of these, 523 men provided sufficient information to be included in the quantitative sample  (further information about the one-to-one interviews in Research Methods below). If they agreed, the researcher contacted them to arrange a time for the interview. This could be in the researcher's office at City University London, in the hospital or at the respondent's home. In this way 20 HIV positive gay men were recruited for one-to-one interviews as part of the qualitative arm of the study table .People seeking an HIV test at the same-day HIV testing clinic, Royal Free Hampstead NHS Trust Hospital over a 13 month period (October 2002–November 2003) were invited to take part in the study. Based on previous research in the HIV testing clinic, we estimated that this would generate a sample of approximately 500 gay men, 500 heterosexual men and 500 heterosexual women . AlthougUsing a similar strategy to the outpatient HIV treatments clinic, everyone seeking an HIV test was asked to participate in the research. Those who agreed completed a detailed, self-administered pen-and-paper questionnaire, after providing written consent, while they were waiting for their pre-test counselling. People with limited command of English were deemed ineligible for the study as were those who were too ill, too young (under 18 years) or too anxious to complete a questionnaire. Those attending the testing clinic on more than one occasion during the survey period were asked to only complete the questionnaire once.Over the 13 month survey period (October 2002–November 2003), 1889 individuals came to the Royal Free clinic for an HIV test of whom 1753 were eligible for the study. Of those who were eligible, 1640 were asked to complete a questionnaire and 1230 did so. The response rate was 75% of people who were offered a questionnaire and 70% of all eligible persons who attended the clinic. Of the 1230 people who completed a questionnaire, 345 described their sexual orientation as gay or bisexual, 435 as heterosexual male and 450 as heterosexual female; 334 gay/bisexual men provided sufficient information to be included in the quantitative sample.Gay men who completed a questionnaire were asked if they would be willing to have an in-depth, face-to-face interview with the qualitative researcher (MD). If they agreed, the researcher contacted them to arrange a time for the interview as described above. In this way, 16 gay men seeking an HIV test at the Royal Free were recruited for one-to-one interviews for the qualitative arm. Heterosexual men and women who completed the questionnaire were not asked to have a one-to-one interview.Previous research suggested that the majority of clinic attenders would test HIV negative while approximately 6% of gay men were expected to test HIV positive. It was considered inappropriate to ask men who had just been diagnosed HIV positive if they would be willing to have a face-to-face interview. Consequently, the qualitative sample from the HIV testing clinic solely comprised men who had tested negative. Anyone who volunteered for an interview who subsequently received a positive test result was not included in the qualitative sample.After 6 months it became apparent that while the response rate at the Royal Free HIV testing clinic was high the number of gay men seeking a test was not as large as had been expected. We decided therefore to extend recruitment to men seeking an HIV test at a sexual health clinic specifically for gay men at Barts and the London NHS Trust hospital, London. All men attending this clinic are gay.Using a similar strategy to the one developed at the Royal Free, everyone attending the gay men's sexual health clinic at Barts and the London hospital between June and November 2003 was invited to participate in the research. Those who agreed completed a self administered questionnaire while they were waiting in the clinic for their appointment. Although all men were asked to complete a questionnaire, only men seeking an HIV test were eligible for inclusion in the Internet and HIV quantitative sample.Over the 6 month survey period at the Barts and the London sexual health clinic (June – November 2003), 211 gay men came for an appointment of whom 209 were eligible for the study. Of those who were eligible, 198 were asked to complete a questionnaire and 156 did so. The response rate was 79% of men who were offered a questionnaire and 75% of all eligible men attending the clinic.Of the 156 gay men who completed a questionnaire at Barts and the London, 70 were seeking an HIV test and provided sufficient information to included in the quantitative sample for the study. These men were also asked if they were willing to have a one-to-one interview with the qualitative researcher. Four men were recruited in this way for the qualitative arm of the study.Combining the data from the two clinics, 404 gay men seeking an HIV test were recruited for the quantitative arm of the study between October 2002–November 2003, of whom 20 agreed to have a one-to-one interview for the qualitative arm table . In addiPrevious research has shown that gyms in central London provide a suitable environment for undertaking detailed behavioural research among gay men at risk of HIV infection ,17,24,25In the mixed gyms the number of questionnaires handed out to gay men was estimated by multiplying the total number of questionnaires distributed in the gym by the proportion of male members who were gay (according to the managers' estimates). To calculate the response rates we divided the number of questionnaires returned by gay men (as indicated on the questionnaire) by the estimated number of questionnaires handed out to gay men, as described above. In 2002, 921 gay men completed the questionnaire while in 2003, 550 men did so. Those men who provided information on their HIV status  were included in the quantitative sample . If they agreed, the researcher contacted them to arrange a time for the interview as described above. Some interviewees were recruited through snowballing. In this way, 23 men were recruited from the gyms for the qualitative arm of the study table .)or gay.com  were invited to take part in the study. Gaydar and gay.com are two of the UK's most popular websites for gay men . Over a four week period in May-June each year, a series of pop-ups and banners advertised the research project in UK chatrooms and on profiles pages. Clicking on a popup or banner took men to the homepage of the online questionnaire. Men who agreed to complete the questionnaire then did so after providing informed consent – all online.In both 2002 and 2003, men using UK chat rooms or personal profiles on gaydar  . Those who agreed were asked to send an email to the researcher who then contacted them, also by email, to provide further information about the study and to arrange a time for the interview as described above. Interviews were either conducted online or face-to-face (see Research methods below). Of the London men who completed a questionnaire online in 2002 or 2003, 65 went on to have a one-to-one interview as part of the qualitative arm table ; 30 men The questionnaires sought detailed information on the men's socio-demographic characteristics (age, ethnicity, employment, education), sexual orientation, HIV test history (date and result of last test), history of STIs, access to and use of the Internet, seeking sex on the Internet, use of other venues (eg back rooms, saunas), as well as sexual risk behaviour in the previous 3 months, differentiating between Internet- and other sex partners. Unprotected anal intercourse (UAI) in the previous 3 months was categorized according to type  and HIV status of partner. Detailed information was collected about sexual behaviour with partners met through the Internet and with partners met elsewhere in order to compare the level and nature of risk with Internet and other sexual partners. Data were also collected on potential confounding factors such as recreational drug use, alcohol consumption, relationships, attitudes towards new treatments for HIV and mental health. Standard and validated questionnaire items were used extensively (copies of the questionnaires are available from JE).Core questions, included in the questionnaires for all four samples, were worded identically to ensure direct comparability between the different groups. In addition, some questions specific to each group were included eg, detailed questions on HIV test history and reasons for testing for people seeking an HIV test; questions on HIV medication, CD4 and viral load for HV positive men. The questionnaires were piloted both online and offline among gay men at the developmental stage of the study and revised in the light of any feedback and comments.People recruited offline, in clinics or gyms, completed a pen-and-paper questionnaire. All questionnaires were confidential and anonymous. They contained no information that would allow an individual respondent to be identified. For the HIV positive men, information on their most recent viral load and CD4 count was abstracted from hospital records and linked to their questionnaire without breaching confidentiality. The HIV test result of those seeking an HIV test was linked to their questionnaire, again without breaching confidentiality. Once the questionnaire and clinic data had been linked in the database individual identifiers  were removed and destroyed to ensure anonymity.) working in close collaboration with the research team. Because of their technical expertise and capacity, gaydar hosted the questionnaire on their server. All questionnaires were confidential and anonymous. Identifiers such as IP addresses were removed from the questionnaires completed online before the data were downloaded to a database.Men recruited through Internet chatrooms and profiles completed the questionnaire online. The online questionnaire was constructed by a computer programmer at gaydar (Within each sample men were asked (a) to complete only one questionnaire and (b) whether they had been in any of the other samples. For example, men who completed the questionnaire online were asked whether they had also completed a questionnaire in the gyms or clinics, etc.Qualitative data were collected by means of one-to-one, in-depth interviews conducted face-to-face (n = 93) or on-line (n = 35)The interviewees were volunteers from the quantitative samples recruited in the HIV treatments clinic, HIV testing clinic, gyms or online as described above. They provided written consent for the one-to-one interview which lasted between 50 and 90 minutes and was audio-taped for transcription. The interviews were generally conducted in the research office at City University London but occasionally at the interviewee's home or in the clinic. Confidentiality was provided in two ways; first, the interviewee's contact details were not linked to the interview transcript and secondly personal identifiers such as the person's name, where he was born, lived or worked were removed from the transcripts. Once all the data had been transcribed and entered into the database, individual identifiers (eg interviewees' contact details) were destroyed.online quantitative samples. They provided informed consent by email. Interviews were prearranged and conducted in a private room on  or  which only the interviewer and interviewee could enter. The interviews were synchronous and conducted entirely through text generated as online chat. Each interview lasted between 50 and 90 minutes at the end of which the interview text was copied and pasted into a Word document. Confidentiality was provided in the same manner as the face-to-face interviews..The interviewees were volunteers from the Interviews were conducted in two phases. Phase one (n = 24 interviews) focused on how gay men used the Internet for sexual partnering while phase two (n = 104) focused on risk behaviour related to the Internet.phase one were based on a topic guide comprising questions about age, residence, schooling, employment, relationship status and HIV testing. Social and sexual lifestyles were explored in depth paying particular attention to the role of the Internet. The topic guide included seeking sexual partners, preferences for different ways of meeting partners (including the Internet), Internet experience and skills, learning how to use the Internet for sexual partnering, online communication skills, other uses of the Internet and related media, a recent sexual episode and its relationship with the Internet.The interviews for The topic guide for phase two comprised social background; sexual lifestyle and role of the Internet; risk episodes including a description of anal sex with a condom and without a condom with Internet and non-Internet partners; HIV testing (where relevant); general discussion of Internet experience and web profiles; safer sex ; and sources of knowledge and skills about risk reduction . For HIV positive men the topic guide also covered their experience of HIV treatment and care  and the relationship between treatments and risk ;The research protocol was approved by the following ethics committees: Royal Free Hospital and Medical School Local Research Ethics Committee, the East London and The City Research Ethics Committee and City University London Research Ethics Committee.Over 4000 London gay men were recruited for the quantitative arm of the study in 2002 and 2003 from the HIV treatments and testing clinics, gyms and through the Internet. Of these, a subset of 128 men were interviewed one-to-one for the qualitative arm . On the other hand, men surveyed online were more likely to have used the Internet to look for sex (approximately 90%) than men who surveyed in the clinics or gyms (40–50%). The differences between the samples will be explored further in future analyses and subject to formal statistical testing. For the most part the four samples were independent; only a minority of men said they had completed a questionnaire in more than one recruitment site (eg in a clinic and in a gym); range 2–10%.Data analysis, using standard statistical packages, will allow us to examine; whether sex with Internet partners is of higher risk than with other men  and the extent to which Internet sex seekers were specifically looking for unprotected anal intercourse either online or offline. The characteristics of those who have and have not used the Internet to find a sexual partner will be compared to explore whether selected groups of men  seek sex on the Internet. The importance of the Internet in relation to other venues will be examined. The samples, ranging in size from 404 to 1218, are sufficiently large to allow us to detect statistically significant differences between and within the different groups at the 5% level of significance.surveyed in the community (eg in Scotland). The data collected online will also allow us to examine patterns of Internet use in a sample.that covers the whole of the UK.All analyses will be conducted for HIV positive, negative and never-tested men separately and comparisons will be made within and between the four samples. Comparison of those recruited through the Internet with the community and clinic samples will indicate whether the Internet attracts men who would otherwise be hard to reach for health promotion and HIV prevention. The data collected online from men living in the UK but outside London will provide opportunities for further analysis. For example their characteristics and behaviours can be compared with those of the London men surveyed online as well as with other men living outside London A PhD studentship, funded separately by the UK Economic and Social Research Council (ESRC), will allow us to examine methodological issues around using the Internet for data collection and research , such asIn addition, data collected in the HIV testing clinic will also allow us to explore Internet sex-seeking and risk behaviours among heterosexual men and women.The interview transcripts from both the face-to-face and online interviews were coded and analysed using Nvivo. The background characteristics of the men interviewed for the qualitative arm of the study are summarized in table Interviewees were recruited for qualitative interviews once they had completed a behavioural questionnaire for the quantitative arm of the study. Additional purposive criteria were adopted to ensure the sample included men from a range of age groups; of different HIV status (HIV positive, HIV negative and never tested); of varying educational attainment; both employed and unemployed; and who reported differential use of the Internet for sexual purposes. Online, as well as face-to-face (FTF) interviews were conducted to encourage the participation of gay men who used the Internet to look for sex. This combination of matching the quantitative arm in terms of recruitment sites, together with additional purposive criteria and two interviewing methods resulted in 128 qualitative interviews with a diverse range of gay/bisexual men. All interviewees lived in the greater London area.For the qualitative arm of the study, we addressed analytic bias using guidance provided by Barbour , Kvale  and Popa• Iteration• Team analysis• TransparencyWe conducted a mapping phase to provide an empirical context for our thinking about the Internet-related sexuality of gay men and to orient further research in terms of the sampling and topic guides. We also adopted the practice of constant comparison derived from grounded theory as another form of iteration.The research team was involved in a cycle of reflection on the data as they were generated and analysed. In the first meeting the team reviewed a transcript to identify possible themes for analysis. Notes and the transcription of the meeting contributed to the formulation of themes. At an intermediate stage the team was asked to apply the thematic framework to other transcripts. This helped to assess the utility of the framework and elaborate possible new themes. A second meeting was then held where the team reviewed the coding scheme for the qualitative research and its application to the entire data set. This process was conducted twice – first for data generated during phase 1, then for data collected in phase 2.We documented how the data were generated, catalogued and analysed to make it open to observation.The one-to-one interviews will be analysed for recurring and contradictory themes relating to the core research questions. Phase one will help orientate the qualitative research around the Internet and risk. Phase two will provide information about the range of sexual experiences linked to the Internet as well as how people communicate about sex and risk through the Internet. The qualitative interviews will allow for greater interrogation than is permitted by self-completed questionnaire alone. A number of issues will be examined including personal experiences of using the Internet for sex, partner selection, the context in which unsafe sex occurs, the emergence of social and sexual networks via the Internet and the value or meaning placed on unsafe sex with casual partners met in different settings including the Internet.Does the Internet represent a new sexual risk environment for gay men? To answer this question, we have employed a range of quantitative and qualitative research methods in online and offline samples of London gay men. The data we collect will allow us to explore in depth the association between seeking sex on the Internet and high risk behaviour and also consider the underlying processes.One of the strengths of the Internet and HIV study is its methodological plurality. Detailed behavioural data have been collected in the quantitative arm of the study from a large number of men recruited in community and clinic settings as well as through the Internet. Questionnaires were completed online by the Internet sample. On the other hand, qualitative interviews will allow for a greater understanding of the context in which unsafe sex occurs and the processes that underlie the behavioural patterns seen in the quantitative analysis. By using both quantitative and qualitative methods, we hope to garner the best that both approaches can offer within an integrated research programmeSampling men online has allowed us to develop innovative research methods for both the quantitative and qualitative arms of the study by taking advantage of recent advances in web-based data collection. The online questionnaire provided opportunities for innovation with respect to its design, format of the questions and data entry. Conducting one-to-one interviews online has opened up new ways of undertaking qualitative research. Ours is one of a small number of studies which have explored this approach to qualitative interviewing . In addiThe Internet undoubtedly offers enormous potential for HIV prevention and sexual health promotion ,35. Our None declared.JE, GH and LS conceived the study; all authors participated in its design; JE was responsible for overall project management; GB was responsible for conducting the quantitative arm of study; MD was responsible for the qualitative arm; JE drafted the manuscript with input from GB and MD. All authors read, revised and approved the final manuscript.The Internet and HIV study: MRC-funded researchCity University London, Institute of Health Sciences, St Bartholomew School of Nursing and MidwiferyProfessor Jonathan Elford, principal investigatorGraham Bolding, research fellow (quantitative arm)Mark Davis, research fellow MRC Social and Public Health Sciences Research Unit, GlasgowProfessor Graham Hart, co-investigatorRoyal Free and University College Medical School, LondonProfessor Lorraine Sherr, co-investigatorCollaborators, , Royal Free Hampstead NHS Trust Hospital London, Barts and the London NHS Trust Hospital, Camden Primary Care Trust, London, Central London gyms.The research was funded from 2002–2004 by the UK Medical Research Council and the Department of Health as part of its AIDS Epidemiological Research Programme Further information about the project can be found on our website at The Internet and research methodology: ESRC-funded PhD studentshipCity University London, School of Social Sciences and Institute of Health Sciences, St Bartholomew School of Nursing and MidwiferyAlison Evans PhD candidateSchool of Social Sciences and Institute of Health Sciences, St Bartholomew School of Nursing and MidwiferyProfessor Dick Wiggins first supervisor,School of Social Sciences, Department of SociologyProfessor Jonathan Elford second supervisor,Institute of Health Sciences, St Bartholomew School of Nursing and MidwiferyAdvisory BoardCity University LondonGraham Bolding, Mark Davis, Professor Jonathan Elford, Professor Roger Jowell, Professor Dick WigginsMRC Social and Public Health Sciences Research Unit, GlasgowProfessor Graham HartRoyal Free and University College Medical School, LondonProfessor Lorraine SherrFurther information can be found at The pre-publication history for this paper can be accessed here:


Arabidopsis.Microarray analysis and the 'InterAct class' method were used to study interactions between carbon and nitrogen signaling in  Carbon and nitrogen are two signals that influence plant growth and development. It is known that carbon- and nitrogen-signaling pathways influence one another to affect gene expression, but little is known about which genes are regulated by interactions between carbon and nitrogen signaling or the mechanisms by which the different pathways interact.Arabidopsis thaliana. An informatic analysis using InterAct Class enabled us to classify genes on the basis of their responses to carbon or nitrogen treatments. This analysis provides in vivo evidence supporting the hypothesis that plants have a carbon/nitrogen (CN)-sensing/regulatory mechanism, as we have identified over 300 genes whose response to combined CN treatment is different from that expected from expression values due to carbon and nitrogen treatments separately. Metabolism, energy and protein synthesis were found to be significantly affected by interactions between carbon and nitrogen signaling. Identified putative cis-acting regulatory elements involved in mediating CN-responsive gene expression suggest multiple mechanisms for CN responsiveness. One mechanism invokes the existence of a single CN-responsive cis element, while another invokes the existence of cis elements that promote nitrogen-responsive gene expression only when present in combination with a carbon-responsive cis element.Microarray analysis was used to study global changes in mRNA levels due to carbon and nitrogen in This study has allowed us to identify genes and processes regulated by interactions between carbon and nitrogen signaling and take a first step in uncovering how carbon- and nitrogen-signaling pathways interact to regulate transcription. Carbon and nitrogen are two major macronutrients required for plant growth and development. Specific carbon and nitrogen metabolites act as signals to regulate the transcription of genes encoding enzymes involved in many essential processes, including photosynthesis, carbon metabolism, nitrogen metabolism, and resource allocation -5. For ecis elements that have been shown to be carbon-responsive cis elements (C-elements) and none has been identified from studies in Arabidopsis thaliana. Although much less is known concerning the mechanisms controlling nitrogen signaling, microarray analysis has been used to identify nitrogen-responsive genes [A. thaliana [In plants, there are multiple carbon-responsive signaling pathways -13, and ve genes ,18. It hthaliana , but a gA. thaliana, including the identification of a putative cis regulatory element that is responsive to either light or carbon signals [cis elements that may be responsible for coordinating a gene's responses to both these signaling pathways.Previously, microarrays were used to identify genes and biological processes regulated by interactions between carbon and light signaling in  signals . In thisArabidopsis. We predicted six general models that could describe the possible modes of gene regulation due to carbon, nitrogen and CN together. Three of these models do not involve interactions between carbon and nitrogen signaling. The 'No effect' model includes genes not regulated by carbon, nitrogen and/or CN. The 'C-only' model includes genes regulated only by carbon. Finally, the 'N-only' model includes genes regulated only by nitrogen. Three additional models are needed to describe the regulation of genes affected by interactions between carbon and nitrogen signaling  depicts a gene X, for which regulation requires carbon and nitrogen, and neither carbon alone nor nitrogen alone has an effect. Model 3 (CN dependent/independent) incorporates both an independent and a dependent component to the interactions of carbon and nitrogen signaling. For gene Y, carbon alone has an independent inductive effect, while nitrogen has a carbon-dependent effect as it can enhance the effect of carbon, but has no effect on its own (Model 3 CN-enhanced). For gene Z, nitrogen alone has an independent inductive effect, while carbon has a nitrogen-dependent effect. These general models can be broken down into more descriptive sub-models. For example, Model 2 can be broken into two sub-models for which CN results in either an inductive or repressive effect.The goal of this study was to use a genomic approach to test the hypothesis that carbon and nitrogen signaling pathways interact to regulate the expression of genes in g Figure . Model 1in vivo significance of the above models, a microarray analysis of RNA from plants treated transiently with distinct carbon and nitrogen treatments was carried out, and the results were analyzed to determine the carbon and nitrogen regulation of different genes. For this study, we analyzed RNA isolated from Arabidopsis seedlings exposed to four different transient carbon and/or nitrogen treatments (-C/-N, +C/-N, -C/+N, and +C/+N)  Figure  using AfW, X, Y and Z) were classified by InterAct Class .InterAct Class is a ranking system used to qualitatively compare gene-expression profiles across multiple treatments. For each gene, each treatment is assigned a value representing the effect of the treatment on the expression of that gene. Treatments that result in repression of a gene are assigned a negative number, treatments that do not significantly affect a gene are assigned zero, and treatments that cause induction are assigned a positive number. If more than one treatment causes induction or repression, the treatments are ranked so that the treatment that causes the most induction or repression will be assigned the number furthest from zero. The four hypothetical genes in Figure s Figure , demonstOut of 23,000 genes on the Affymetrix chip, 3,652 passed our stringent filtering criteria for reproducibility among treatment replicates and were assigned an InterAct class. Our subsequent analysis of the expression patterns of these 3,652 genes validated the existence of 60 different InterAct classes Table . For exaThe InterAct classes were assigned to one of the six general models. To identify biological processes that contain a significant number of genes regulated by carbon, nitrogen and/or CN, we determined which Munich Information Center for Protein Sequences (MIPS) functional categories (funcats) ,22 were p-value = 6.0 × 10-4). Therefore, the metabolism funcat is under-represented in the No effect model, showing that metabolism displays significant regulation by carbon and/or nitrogen. This analysis revealed several primary funcats (01 = metabolism, 02 = energy and 05 = protein synthesis) that are significantly under-represented in the No effect model (Table For example, 31.6% (1,089/3,447) of the genes assigned to an InterAct class and a funcat are assigned to the No effect model Table . This pep-value, and the funcat analysis extended to every sub-model and every funcat).For the funcats that are under-represented in the No effect model, this type of analysis was extended to examine the regulation of these funcats in all of the sub-models. This analysis enabled us to determine into which sub-models the genes from these funcats fell and to determine whether the genes in these funcats are under- and over-represented (-S and +S respectively) in these sub-models Table   (Table To begin to elucidate the mechanisms that control gene regulation in response to carbon and nitrogen treatments, we sought to identify putative ed Table . These ggulation . For thigulation , which egulation . As used2) Table . These gcis search analysis. Four nuclear genes encoding ribosomal proteins predicted to be localized to the mitochondria [cis-regulatory elements. Over-represented motifs in the promoters of the four protein synthesis genes or the three ferredoxin metabolism genes were identified using AlignAce [As we found that genes in the funcat protein synthesis are over-represented in Model 3 CN-enhanced (Table chondria  were assAlignAce ,27 (Aligcis-regulatory elements by which carbon and nitrogen can have a non-additive effect (for example, Model 3 CN-enhanced) on the transcription of a gene  that responds to carbon alone. Such carbon-responsive cis elements (C-elements) can be identified because they should also be over-represented in the promoters of genes that are induced by carbon alone (the C-only inductive model). From this analysis, a number of the AlignAce motifs identified from the ferredoxin metabolism and protein synthesis genes in the Model 3 CN-enhanced were also shown to be associated with C-only inductive model genes (Table cis element) that responds specifically to a CN-signaling pathway  exists, it would be predicted to be over-represented in the promoters of genes in Model 3 CN-enhanced, but would not be over-represented in the C-only inductive model. Two of the AlignAce motifs fit this pattern . Another prediction is that if CN1 and CN2 regulate gene expression, biologically related genes might also contain CN1 and CN2. Interestingly, ferredoxin-dependent nitrite reductase (At2g15620) contains three copies of CN1 and one copy of CN2 in its promoter. This gene is in Model 3 CN-enhanced (InterAct class 1021), its protein product is localized to the chloroplast [p-value = 0.0364), but it would not be unlikely to find one copy of CN2 . In addition, a total of four copies of CN1 and CN2 was very unlikely  in any combination (for example, three CN1 and one CN2, two CN1 and two CN2, or one CN1 and two CN2, and so on).If CN1 and CN2 regulate gene expression, they might be expected to be evolutionarily conserved. Unfortunately, oroplast  and its oroplast , suggestA. thaliana has only one copy of ferredoxin-dependent nitrite reductase, we searched the O. sativa genome sequence for ferredoxin-dependent nitrite reductase genes. Again, we found only one gene [A. thaliana ferredoxin-dependent nitrite reductase gene and the O. sativa gene for an alignment. Despite this lack of similarity, we tested for the presence of CN1 and CN2 in the promoter of this gene; three copies of CN1  and one copy of CN2  were found. Again, it was very unlikely that a total of four copies of CN1 and CN2  would occur in the promoter sequence.As one gene . BLAST [one gene  did not cis element that alone has little or no effect on gene regulation, but when present in combination with a C-element, enhances the induction caused by carbon and is dependent on a carbon-responsive transcription factor . To identify NDEs, our strategy for cis element identification was modified. NDEs would be expected to be over-represented in the promoters of Model 3 CN-enhanced genes, but only when present in combination with a separate C-element, as both elements are required to give the enhanced expression due to CN. However, some of the AlignAce motifs are potentially involved in regulating expression due to the carbon treatment in cooperation with the already identified C-elements. These cis elements would be similar to NDEs as they would be over-represented in genes induced by carbon in combination with the already identified C-elements. As these motifs are not NDEs, we sought to identify them and remove them from the analysis. AlignAce motifs were tested to determine whether they are over-represented in the promoters of genes whose promoters contain any of the C-elements and are in the C-only inductive model. Those that were found to be over-represented were eliminated from further analysis because these motifs are potentially involved in carbon regulation and are not NDEs. Next, the remaining 33 AlignAce motifs were tested to determine if any are NDEs by determining whether they are over-represented in combination with a C-element within the promoters of the Model 3 CN-enhanced genes. Seven of the potential NDEs are over-represented  with at least one C-element in the promoters of the Model 3 CN-enhanced genes, resulting in 12 significant combinations between putative NDEs and C-elements .A second mechanism by which the expression due to CN could be greater than C+N could involve a nitrogen-responsive r Figure . Other r another . In the p-value < 0.005).To determine if this approach resulted in an enrichment of NDEs, the promoter sequence of each gene was randomized, and the same test was performed. This enabled us to determine whether the remaining 33 AlignAce motifs were over-represented in combination with each C-element in the randomized promoters of the Model 3 CN-enhanced genes. Sets of the randomized promoters (200 sets) were tested, and none of them had as many significant pairs of potential nitrogen-dependent enhancers of carbon regulation and C-elements than the 12 found in the actual promoters. This randomization proves that our approach successfully enriched for NDEs in the actual promoters of the Model 3 CN-enhanced genes and that all the observed significant combinations cannot be due to false positives . Two NDEs (N1 and N2) were found to be associated with C-elements  to regulate gene expression in response to CN, then genes that contain both motifs and are in Model 3 CN-enhanced should be misrepresented in certain functional groups as these genes are truly co-regulated. This misrepresentation should occur not only with respect to the genome, but also with respect to the genes in Model 3 CN-enhanced. This result is expected because these genes are more closely related to each other than to the other genes in Model 3 CN-enhanced, and because their CN regulation is the result of the action of the same transcription factor(s). Funcat analysis was used to determine if any functional categories were misrepresented in the genes whose promoters contain N1C6, N1C7, N2C3, N2C6, N2C7 or N2C10 and are in Model 3 CN-enhanced. As the genes used to derive most of the pertinent  and C10 A. thaliana [This report contains the one of the first genome-wide investigations of carbon- and nitrogen-signaling interactions in thaliana . While tBiological processes containing genes that respond significantly to carbon, nitrogen and/or CN were initially identified by finding MIPS funcats ,22 that To gain a better understanding of how the metabolism, energy and protein synthesis funcats are regulated by carbon and/or nitrogen, the sub-models in which they are misrepresented were identified Table . This anASN1 (At3g47340), which has been proposed to be involved in producing asparagine for the transport of nitrogen when carbon levels are low and has been shown to be repressed by carbon [GDH1 (At5g18170), which has been proposed to be involved in ammonia assimilation when ammonia levels are high, is repressed by carbon, and induced by nitrogen [ASN1 is induced because it is involved in shifting the excess nitrogen to asparagine, as asparagine is an efficient way to store and transport nitrogen with respect to carbon [ASN1 is repressed. The regulation of these genes demonstrates the exquisite control of metabolic genes required to balance carbon and nitrogen availability.Surprisingly, metabolism is over-represented in Model 3 CN-suppressed, indicating that many of the genes involved in metabolism show less expression due to CN than expected. The majority of the genes (28 out of 34) were repressed by carbon, induced by nitrogen and repressed by CN, and were assigned to InterAct classes such as -21-2-1 . Several of these genes encode enzymes involved in the catabolism of complex carbohydrates, including β-fructofuranosidase (At1g12240), β-amylase (At3g23920) and β-glucosidase (At3g60130 and At3g60140). y carbon , was assnitrogen , and wasOur studies also showed that protein synthesis is one of the processes most affected by the interactions between carbon and nitrogen signaling Table . In addiTrichoderma hamatum, the gene for ribosomal protein L36 is regulated by interactions between carbon and nitrogen, as it is induced only by CN, and not by carbon or nitrogen alone [Little work has been done on the transcriptional control of protein synthesis by carbon and/or nitrogen signaling in plants. However, it has been shown in yeast that genes encoding ribosomal proteins are induced by nitrogen in the presence of carbon; whether this induction by nitrogen requires carbon to be present was not addressed in the yeast study . Furthercis-regulatory mechanisms involved in carbon and nitrogen signaling interactions. We hypothesized that there could be two general transcriptional mechanisms that would result in the expression due to CN being greater than that due to C+N . Since CN1 and CN2 , and while three copies in the promoter of the O. sativa gene did not reach the 0.05 cutoff, this might represent some small change in the specificity of the regulating factor between O. sativa and A. thaliana. The failure of BLAST to detect any similarity between the promoters of these two genes suggests that their transcriptional regulators share very little sequence specificity, so a slight change in specificity is not unexpected. The same analysis suggests that CN2 is a false positive because it is not over-represented in the promoters of ferredoxin-dependent nitrite reductase genes. However, we cannot rule out the possibility that the combination of CN1 and CN2 is what is important in regulating these genes, as having a total of four copies of CN1 and CN2 is unlikely in the promoters of both genes. One possibility is that there is a positional relationship between the copies of CN1 and CN2 that is important. From a quick visual inspection, there does not appear to be a conserved relationship between the three copies of CN1 and one copy of CN2 in the A. thaliana and O. sativa promoters. These issues will have to be resolved by further experimental work; however, these results do suggest that ferredoxin, ferredoxin reductase and ferredoxin-dependent nitrite reductase are co-regulated by carbon and nitrogen due to CN1 and/or CN2. CN1 and/or CN2 therefore might act to link nitrogen reduction and energy metabolism.Further analysis suggests that CN1 is involved in regulating the expression of ferredoxin-dependent nitrite reductase. Finding three copies of CN1 in the promoter of the Our analysis found CN-elements in the promoters of the ferredoxin-related genes Table , but notcis elements that are responsive to carbon and nitrogen signaling interactions. It is noteworthy that the putative CN-elements and NDEs represent cis elements that have not been previously identified and as such may represent novel components of the CN regulatory circuit. Further study of the identified genes and cis elements is required to bring about a complete understanding of interactions between carbon and nitrogen signaling.This analysis represents a first step in understanding how carbon and nitrogen signaling interact to control gene expression and has identified genes and putative Arabidopsis thaliana seeds of the Columbia ecotype were surface-sterilized and plated on designated media and vernalized for 48 h at 8°C. Plants were grown semi-hydroponically under 16-h-light (70 E/m2/sec)/8-h-dark cycles at a constant temperature of 23°C on basal Murashige and Skoog (MS) medium (Life Technologies) supplemented with 2 mM KNO3, 2 mM NH4NO3 and 30 mM sucrose [3 and NH4NO3) or carbon (sucrose) and dark-adapted for 48 h. To perform specific metabolic treatments, 25 dark-adapted seedlings were transferred to fresh MS medium containing 0% or 1% (w/v) sucrose and/or 2 mM KNO3 and 2 mM NH4NO3 or no nitrogen, and illuminated with white light for an additional 8 h (70 E/m2/s1). Following these transient carbon and nitrogen treatments, whole seedlings were harvested, immediately frozen in liquid nitrogen, and stored at -80°C before RNA extraction. sucrose . Two-weeArabidopsis ATH1 Target (Affymetrix). Washing, staining and scanning were carried out as recommended by the Affymetrix instruction manual. Expression analysis was performed with the Affymetrix Microarray Suite software (version 5.0) set at default values with a target intensity set to 150. Three biological replicates for each treatment were carried out.RNA was isolated from whole seedlings using a phenol extraction protocol as previously described . Double-Only Affymetrix probes representing genes that were deemed to be expressed in all treatments and replicates were used for subsequent analysis by InterAct Class ,20. For p-values were calculated for the MIPS functional categories (funcats) [n; p was the number of genes assigned to the specific model being analyzed divided by the number of genes assigned to an InterAct class and funcat; k was the number of genes in the funcat being analyzed and assigned to the model being analyzed. This analysis, with the baseline being all the genes assigned an InterAct class, accounts for any biases that may have been caused by discarding all the absent genes. The one-tailed p-value was considered when the Poisson approximation of binomial probabilities was used. For the binomial-ratio and the exact binomial probability test, the p-value for k or more out of n was used.funcats) ,22 analyfuncats) . Brieflyn was the number of genes in Model 3 CN-enhanced (the child list), k was the number of genes in the pathway being analyzed and in the child list, and p was the number of genes in the pathway being analyzed and in the parent list divided by the number of genes in the parent list. We limited our search to pathways that contained more than two genes in the Model 3 CN-enhanced list. To identify cis-regulatory elements involved in regulating genes in Model 3 CN-enhanced and protein synthesis, we used genes involved in protein synthesis that were assigned Model 3 CN-enhanced, to drive the cis search: At1g07070 , At2g36620 , At5g07090 , and At5g58420 .Pathways whose genes are over-represented in Model 3 CN-enhanced were identified using the informatic tool PathExplore  functionA. thaliana promoters for every gene [n, p is the number of genes in the sub-model (for example, Model 3 CN-enhanced) divided by the total number of genes assigned an InterAct class, and k is the number of genes whose promoters contain the AlignAce motif being analyzed (in a specific copy number) and that is in the particular sub-model being tested. A p-value was only calculated if k is greater than nine. In each case, the lowest p-value is given. Cis elements over-represented in the C-only inductive model are considered to be putative C-elements, and cis elements that are over-represented in the promoters of Model 3 CN-enhanced genes and are not over-represented in the promoters of C-only inductive genes, are considered to be putative CN-elements  and that are in the C-only inductive model was used as k. In this example, the analysis will determine if the genes that contain the second motif and the C-element being analyzed are over-represented in the C-only inductive model compared to the genes that just contain the C-element. The same approach was used to identify NDEs as described below.To identify interacting elements, a similar analysis was used. For example, to identify motifs interacting with a C-element Table  in regulp-value less than 0.05 is obtained, the C-element and potential NDE are a significant combination and are likely to regulate carbon and nitrogen interactions. As each motif is tested with each of the 11 C-elements, two steps were taken to control for the multiple tests. First, single strands of the promoter sequences of the A. thaliana genes were randomized 200 times, the reverse complement of the randomized strand was determined, and the number of times the 33 remaining AlignAce motifs were found to be over-represented  with the C-elements was determined and compared to the number of significant combinations  between the 33 remaining motifs and the C-elements when the actual promoters were used. In no set of the randomized promoters were the potential NDEs found to form more significant combinations with the 11 C-elements than the actual promoter sequences . In the second control step, the number of significant combinations that each of the 33 remaining AlignAce motifs was involved in was determined and compared to the number of significant combinations found with the 200 sets of randomized promoters. For one motif, if one random set is significant with as many C-elements as the real promoters the p-value would be 0.005 (1/200).The 33 motifs ) that are not N-, CN- or C-associated elements were tested to determine whether they are potential NDEs. They were tested to see whether genes whose promoters contained these motifs plus a C-element Table  are overp-value )The promoter for At2g15620 was extracted from RSA tools ,40. The O. sativa sequence. Only one hit was found. This gene is annotated as a ferredoxin-dependent nitrate reductase [The sequence database was searched using BLAST  for a geeductase . The 1,0cis elements was performed similarly to the approach described above. Briefly, the number of genes assigned to the funcat being analyzed and Model 3 CN-enhanced was used as n; p was the number of genes assigned to Model 3 CN-enhanced and the funcat being analyzed divided by the number of genes assigned to Model 3 CN-enhanced and a funcat; k was the number of genes in the funcat being analyzed that was assigned to the Model 3 CN-enhanced category and containing the combination of C- and N-element being analyzed.Funcat analysis of the genes whose promoters contain specific Statistical significance of localization was calculated similarly. The only difference being that instead of genes assigned a funcat, genes whose protein products are predicted to be localized in the compartment being analyzed were used. Predicted protein localizations were extracted from the TAIR web page .The following additional data are available with the online version of this paper: Additional data file A table listing the Affymetrix probe ID, gene, and InterAct class for all the Affymetrix probes assigned an InterAct classClick here for additional data fileThe data from 12 Affymetrix microarray chips used in this studyClick here for additional data file


Every multicellular organism depends on the coordinated actions of a multitude of cell types, each designed to carry out specific jobs. In vertebrates, for example, the task of ferrying oxygen to organs, tissues, and cells throughout the body is shouldered by red blood cells. These cells must develop early in the embryo to nourish the body and must be maintained at the right levels throughout adulthood to keep the organism healthy.Specialized red blood cells arise from undifferentiated stem cells in a developmental process called hematopoiesis. All vertebrates have the same basic plan for hematopoiesis during their development, with one group of stem cells producing embryonic blood cells and, later, another group in a different part of the body making adult blood cells. Such developmental programs are tightly orchestrated by suites of transcription factors—proteins that turn genes on and off.But which transcription factors act—and how and where they act—is still largely unknown. Scientists and doctors alike want to better understand differentiation in order to grasp not only how it proceeds normally but also how it can go wrong and lead to diseases, such as leukemia, in which white blood cells become cancerous, and aplastic anemia, in which bone marrow stem cells make too few red blood cells.To discover the genes involved in hematopoiesis, Leonard Zon's group at Children's Hospital, Boston, and their collaborators exposed zebrafish to mutagens and then studied the individuals that developed relevant disorders. In 1996, the group produced three zebrafish lines with embryos lacking fully differentiated red blood cells; since these fish also had especially shimmery tails, the lines were named moonshine.moonshine (mon) gene, it turns out, encodes a transcription factor with wide effects on the embryo's mesoderm, the set of cells that eventually form the circulatory system, muscles, and skeleton. The researchers found that all three lines of mutant zebrafish had mutations in the mon gene.Now Zon and his colleagues have identified the mutant gene responsible for the moonshine zebrafish lines' peculiar traits. The In the mutant fish, stem cells that produce red blood cells were present initially, but the defective moonshine protein was unable to keep the blood cells alive. These blood cells underwent apoptosis, or programmed cell death, leaving the fish unable to make red blood cells, and they died at about two weeks old.mon gene is most similar to the human and mouse Tif1-gamma, one of a family of proteins known to link DNAbinding proteins with other factors that activate or repress gene activity. Using a DNA analysis technique in mouse cell cultures, the researchers found Tif1-gamma in nuclear bodies, multi-protein complexes in cell nuclei that help regulate gene expression. But the Tif1-gamma nuclear bodies didn't fit into any known class of such complexes, so it's an open question how Tif1-gamma acts and what other proteins help regulate it. Finding a transcription factor such as Tif1-gamma involved in early cell specialization opens a door to a suite of studies on the targets of the transcription factor, as well as other genes that act along with mon to affect red blood cell development.The researchers found that the protein encoded by the