The patient's view on rare disease trial design - a qualitative study.

BACKGROUND: Clinical trials in rare diseases are more challenging than trials in frequent diseases. Small numbers of eligible trial participants, often complicated by heterogeneity among rare disease patients, hamper the design and conduct of a 'classical' Randomized Controlled Trial. Therefore, novel designs are developed by statisticians. However, it is important to be aware of possible design aspects that may jeopardize the feasibility of trial conduct. If the burden of participation is considered out of proportion by patients or parents, recruitment may fail or participants may drop out before trial completion. In order to maximize the chance of success of trials in small populations, it is important to know which aspects of trial design are considered important by patients. RESULTS: We have interviewed all ten members of the Patient Think Tank (PTT) of the ASTERIX project, a European research consortium on methodology for clinical trials in small populations. The PTT members are rare disease patient representatives who have completed extensive training in clinical trial methodology. We have analyzed the interviews qualitatively according to Grounded Theory using a thematic analysis, and we structured the topics in four chronologically ordered themes: 1. Involvement in trial design; 2. Opinions on trial design; 3. Trial participation; 4. Phase after the trial. Our main findings are that the PTT-members recommend that patients are involved in trial design from an early stage on, and have influence on the outcomes and measurement instruments that are chosen in the trial, the length of the study, the choice of participants, and the information that is sent to potential participants. Also, according to the PTT-members, patient groups should consider setting up disease registries, placebo groups should be minimized, and more education on clinical trials is advised. CONCLUSIONS: Rare disease patient representatives who have been educated about clinical trial methodology think it is important to involve patient representatives in research at an early stage. They can be of advice in trial design in such a way that the ratio of potential benefit and burden of trial participation as well as the chosen outcome measures and in- and exclusion criteria are optimized.

Long-Term Cognitive and Functional Outcomes in Children with Mucopolysaccharidosis (MPS)-IH (Hurler Syndrome) Treated with Hematopoietic Cell Transplantation.

The long-term cognitive and functional outcomes of children with mucopolysaccharidosis type I (MPS-IH) post-hematopoietic cell transplant (HCT) are not well documented, and the role of genetic and treatment factors in these outcomes has yet to be defined. In this multi-site, international study, we (1) characterize the cognitive and functional status of 47 individuals (ages 2-25, mean of 10.6 years) with MPS-IH who are 1-24 years post HCT (mean = 9 years) and (2) examine contributions of genotype, transplant characteristics, and sociodemographic factors to cognitive ability, adaptive behavior, and quality of life. The overall cognitive ability of our sample was mildly impaired, more than two standard deviations below general population norms. Parent reported adaptive behaviors (i.e., communication, daily living, and motor skills) were similarly impaired with a relative strength in socialization. Quality of life, as reported by parents, fell more than two standard deviations below population norms for physical functioning; however, psychosocial quality of life (emotional well-being) approximated population norms. In linear regression analysis, adjusted for demographic and treatment factors, mutation severity was associated with lower cognitive ability (p = 0.005) and adaptive functioning (p = 0.004), but not parent ratings of children's quality of life. Older age at HCT was associated with poorer physical quality of life (p = 0.002); lower socioeconomic status (p = 0.028) and unrelated bone marrow HCT (p = 0.010) were associated with poorer psychosocial quality of life. Implications for screening and early intervention for children at risk for poorer cognitive and functional outcomes are described.

Pseudotyping the adenovirus serotype 5 capsid with both the fibre and penton of serotype 35 enhances vascular smooth muscle cell transduction.

Ex vivo gene therapy during coronary artery bypass grafting (CABG) holds great potential to prevent excessive smooth muscle cell (SMC) proliferation, neointima formation and graft failure. The most successful preclinical strategies to date have utilised vectors based on the species C adenovirus, Ad5, which engages the Coxsackie and Adenovirus receptor (CAR) as its primary attachment receptor. Profiling receptors on human SMCs demonstrated the absence of CAR but substantial expression of the species B receptor CD46. We performed transduction experiments using Ad5 and the CD46-utilising adenovirus Ad35, and found Ad35 significantly more efficient at transducing SMCs. To evaluate whether transduction could be further augmented, we evaluated chimeric CD46-utilising Ad5/Ad35 vectors comprising the Ad5 capsid pseudotyped with the Ad35 fibre alone (Ad5/F35) or in combination with the Ad35 penton (Ad5/F35/P35). In human smooth muscle cells (hSMCs), Ad5/F35/P35 mediated significantly higher levels of transduction than either parental vector or Ad5/F35. Ex vivo transduction experiments using mouse aortas from CD46 transgenics demonstrated that Ad5/F35/P35 was significantly more efficient at transducing SMCs than the other vectors tested. Finally, ex vivo transduction and immunofluorescent colocalisation experiments using human tissue from CABG procedures confirmed the preclinical potential of Ad5/F35/P35 as an efficient vector for vascular transduction during CABG.

Psychosocial outcomes of bone marrow transplant for individuals affected by Mucopolysaccharidosis I Hurler Disease: patient social competency.

AIM: To explore the frequency with which children and young people participate in social activities with peers, when they are affected by Mucopolysaccharidosis I Hurler Disease (MPS IH) post bone marrow transplant (BMT). This was investigated in relation to patient age, and in comparison with a normative sample. Patient withdrawal, adaptive and social skills are also described in terms of patient age and in comparison with a normative sample. METHOD: Forty-four individuals affected by MPS IH post BMT participated in this investigation. Their ages ranged from 16 months to 25 years. Semi-structured interviews with patients' mothers were utilized, which included the Behaviour Assessment System for Children and a socialization questionnaire. Normative data for the socialization questionnaire were collected from 46 mothers of children not affected by chronic illness or disability. RESULTS: A one-way ANOVA revealed that children not affected by disability or chronic illness (mean = 20.63) participated overall in social activities more frequently than children aged under 12 years (mean = 14.87) and over 12 years (mean = 13.25) who were affected by MPS IH post BMT (F = 21.01, P < 0.001). Young people aged 12 years and over affected by this condition were found to participate in social activities the least. A pattern also emerged, which indicated greater withdrawal and less well-developed adaptive and social skills for MPS IH patients aged 2.5-5 years and 12 years and over, but scores were within the normal range for those aged 6-11 years. CONCLUSION: These data illustrate a lack of social competency and a tendency towards inhibition and withdrawal in this patient group, particularly among the very young children and the adolescents and young adults. Further research is needed to explore these issues longitudinally and to examine the role played by the family, and indeed disability, in the quality and quantity of social interaction experienced by these individuals.

Evidence that herpes simplex virus VP16 is required for viral egress downstream of the initial envelopment event.

During infection with herpes simplex virus type 1 (HSV-1), VP16 serves multiple functions, including transcriptional activation of viral immediate early genes and downregulation of the virion host shutoff protein vhs. Furthermore, VP16 has been shown to be involved in some aspect of virus assembly and/or maturation. Experiments with a VP16 null virus, 8MA, suggested that VP16 plays a direct role in virion assembly, since removal of VP16 from the HSV-1 genome results in reduced levels of encapsidated DNA and a failure to produce extracellular enveloped particles. However, VP16 null mutants display a severe translational arrest due to unrestrained vhs activity, thus complicating interpretation of these data. We examine here the role of VP16 in virion assembly and egress in the context of a vhs null background, using the virus 8MA/DeltaSma (VP16(-) vhs(-)). Comparison of 8MA and 8MA/DeltaSma with respect to viral DNA accumulation and encapsidation and accumulation of the major capsid protein, VP5, revealed that the 8MA lethal phenotype is only partially due to uncontrolled vhs activity, indicating that VP16 is required in HSV-1 virion formation. Electron microscopy confirmed these results and further showed that VP16 is required for HSV-1 egress beyond the perinuclear space. In addition, we describe the isolation and characterization of an 8MA derivative capable of propagation on Vero cells, due to second site mutations in the vhs and UL53 (gK) genes. Taken together, these results show that VP16 is required for viral egress downstream of the initial envelopment step and further underscore the importance of VP16 in controlling vhs activity within an infected cell.

Up-regulation of CIR1/CROC1 expression upon cell immortalization and in tumor-derived human cell lines.

Acquisition of the immortal phenotype by tumor cells represents an essential and potentially rate-limiting step in tumorigenesis. To identify changes in gene expression that are associated with the early stages of cell immortalization, we compared genetically matched pairs of pre-immortal and immortal human cell clones by mRNA differential display. Two transcripts, denoted CIR1 and CIR2, were identified which were up-regulated in immortal cells. Sequence analysis revealed CIR1 to be identical to the recently cloned CROC1/UEV-1 gene, whereas CIR2 corresponds to an as yet uncharacterized 1.2 kb mRNA. A 5-6-fold elevation in CIR1/CROC1 expression and a 2-3-fold elevation in CIR2 expression were observed in SV40-transformed human embryonic kidney cells immediately following proliferative crisis, suggesting a potential role for these genes in immortalization. Expression of CIR1/CROC1 was found to be elevated also in a variety of immortal human tumor-derived cell lines, as compared to their normal tissue counterparts. These results are compatible with induction of CIR1/CROC1 being an early event in the acquisition of immortality and with a role for this gene in the immortal phenotype of tumor cells.

Nonuniform nighttime distribution of acute cardiac events: a possible effect of sleep states.

BACKGROUND: Although 250,000 myocardial infarctions and 38,000 sudden cardiac deaths occur at night annually, this public health problem is underappreciated and poorly understood. We examined whether the incidence of myocardial infarction, sudden cardiac death, and automatic implantable cardioverter-defibrillator (AICD) discharge was nonuniform, a result that may implicate physiological triggers such as sleep-state dependent changes in autonomic nervous system activity. METHODS AND RESULTS: We conducted a review of the circadian pattern of the onset of myocardial infarction (n=19), sudden cardiac death (n=12), and AICD discharge (n=7). The nighttime period was chosen a priori as midnight to 5:59 AM. These reports documented 11,633 nocturnal myocardial infarctions (20% of the total myocardial infarctions), 1981 nocturnal sudden cardiac deaths (14.6% of the total sudden cardiac deaths), and 1200 nocturnal AICD discharges (15.0% of the total discharges). The distributions of myocardial infarction, sudden cardiac death, and AICD discharge were each significantly nonuniform (P<.001). The peak incidence of myocardial infarction and AICD discharge occurred between midnight and 0:59 AM, whereas the peak incidence of sudden cardiac death was between 1:00 and 1:59 AM. The trough in incidence occurred between 4:00 and 4:59 AM for sudden cardiac death and between 3:00 and 3:59 AM for myocardial infarction and AICD discharge. CONCLUSIONS: Nocturnal myocardial infarction, sudden cardiac death, and AICD discharge exhibit nonuniform distributions. This finding is consistent with the hypothesis that sleep-state dependent fluctuations in autonomic nervous system activity may trigger the onset of major cardiovascular events and provides further impetus for more directly testing this hypothesis at population, individual, and mechanistic levels. A better understanding of nocturnal triggers may make it possible to reduce the incidence of myocardial infarction, ventricular tachyarrhythmias, and sudden cardiac death during the nocturnal period.

A 3- to 6-year follow-up of former long-stay psychiatric patients in Northern Ireland.

Little is known about the first patients who left hospital before and during the official implementation of the hospital discharge policy in Northern Ireland. This study describes patterns of residential provision for former long-stay patients (approximately two-thirds of whom had an ICD-9 diagnosis of schizophrenia) discharged from the six major psychiatric hospitals in Northern Ireland between 1987 and 1990 (n = 321). It also employs several instruments within a retrospective survey design to examine outcomes for a 35% sample of people (112/321) discharged between 1997 and 1990 and followed up in 1993. Almost two-thirds (61%) had been discharged to independent living or low-staffed statutory settings. None of the group was homeless, one person was in prison and three people had committed suicide during the first 2 years after discharged. Almost one-third had to be re-admitted at some stage during the 6-year period and 13% had died. 'Moderate' to 'major problems' with most daily living skills were reported for less than 25% of people, while 15% or less had problem behaviour. Approximately 90% or more were satisfied with most aspects of their new homes and most also reported feeling happier (77%), healthier (63%) and more independent (78%) since discharge. However, social, recreational and occupational opportunities were limited. Purchasers, providers and practitioners need to review ways in which former long-stay patients might be empowered to live more meaningful and integrated lives in the community, particularly as the current government strategy for health and social well-being (1997-2002) in Northern Ireland points to the closure of existing psychiatric hospitals.

A three- to six-year follow-up of former long-stay residents of mental handicap hospitals in Northern Ireland.

OBJECTIVES: Little is known about the first cohorts of long-stay hospital residents with learning disabilities who moved to the community. This study describes the pattern of residential reprovision for all former long-stay residents discharged from the three mental handicap hospitals in Northern Ireland between 1987 and 1990 (N = 283) as well as describing aspects of quality of life for a smaller sample of people. METHOD: The study employs a retrospective survey design and the method and findings are discussed within a quality of life framework. Information about destinational outcomes between 1987 and 1993 was collected for each former resident. Several instruments were also used to assess material, emotional and social well-being and development and activity for a 40 per cent sample of people (114/283) discharged from hospital during 1987-1990 and followed up in 1993. RESULTS: Approximately 70 per cent of residents were discharged to, and subsequently remained in, highly supported settings such as residential and nursing homes. Only 3 per cent were discharged to 'independent living' with their own families or foster families. Few of the sample had 'major' problems with daily living skills and serious behavioural problems were uncommon. Former patients were also more satisfied with their new homes and reported feeling happier, healthier and more independent since discharge. However, social networks were poor and there was no evidence to suggest that people were undertaking new or 'ordinary' daytime activities. CONCLUSION: Although the material needs of former hospital residents (many of whom may have been 'cream skimmed' from the long-stay population) appeared to be met and they were content with their new homes in the community, they had a limited choice of mainly private sector accommodation and few opportunities for personal and social development.

Meta-analysis of the morning excess of acute myocardial infarction and sudden cardiac death.

To evaluate the impact of elimination of the morning peak of cardiovascular events, we performed a meta-analysis of studies of circadian variation of myocardial infarction and sudden cardiac death. The impact would be significant because approximately 1 of every 11 acute myocardial infarctions and 1 of every 15 sudden cardiac deaths are attributable to the morning excess incidence.

Effect of resection margins on the recurrence of Crohn's disease in the small bowel. A randomized controlled trial.

OBJECTIVE: The authors assess the effect of surgical margin width on recurrence rates after intestinal resection of Crohn's Disease (CD). BACKGROUND: The optimal width of margins when resecting DC of the small bowel is controversial. Most studies have been retrospective and have had conflicting results. METHODS: Patients undergoing ileocolic resection for CD (N = 152) were randomly assigned to two groups in which the proximal line of resection was 2 cm (limited resection) or 12 cm (extended resection) from the macroscopically involved area. Patients also were classified by whether the margin of resection was microscopically normal (category 1), contained nonspecific changes (category 2), were suggestive but not diagnostic for CD (category 3), or were diagnostic for CD (category 4). Recurrence was defined as reoperation for recurrent preanastomotic disease. RESULTS: Data were collected on 131 patients. Median follow-up time was 55.7 months. Disease recurred in 29 patients: 25% of patients in the limited resection group and 18% of patients in the extended resection group. In the 90 patients in category 1 with normal tissue, recurrence occurred in 16, whereas in the 41 patients with some degree of microscopic involvement, recurrence occurred in 13. Recurrence rates were 36% in category 2, 39% in category 3, and 21% in category 4. No group differences were statistically at the 0.01 level. CONCLUSION: Recurrence of CD is unaffected by the width of the margin of resection from macroscopically involved bowel. Recurrence rates also do not increase when microscopic CD is present at the resection margins. Therefore, extensive resection margins are unnecessary.

Herpes simplex virus VP16 rescues viral mRNA from destruction by the virion host shutoff function.

Herpes simplex virus (HSV) virions contain two regulatory proteins that facilitate the onset of the lytic cycle: VP16 activates transcription of the viral immediate-early genes, and vhs triggers shutoff of host protein synthesis and accelerated turnover of cellular and viral mRNAs. VP16 and vhs form a complex in infected cells, raising the possibility of a regulatory link between them. Here we show that viral protein synthesis and mRNA levels undergo a severe decline at intermediate times after infection with a VP16 null mutant, culminating in virtually complete translational arrest. This phenotype was rescued by a transcriptionally incompetent derivative of VP16 that retains vhs binding activity, and was eliminated by inactivating the vhs gene. These results indicate that VP16 dampens vhs activity, allowing HSV mRNAs to persist in infected cells. Further evidence supporting this hypothesis came from the demonstration that a stably transfected cell line expressing VP16 was resistant to host shutoff induced by superinfecting HSV virions. Thus, in addition to its well known function as a transcriptional activator, VP16 stimulates viral gene expression at a post-transcriptional level, by sparing viral mRNAs from degradation by one of the virus-induced host shutoff mechanisms.

Construction and preliminary characterization of a nondefective herpes simplex virus recombinant bearing the genome of human papillomavirus type 16.

Studies of the molecular biology of human papillomavirus type 16 have been limited by the lack of a tissue culture system that is fully permissive for virus replication; as a result, high-titre stocks of infectious virus are not readily available. Therefore, studies of viral gene expression have relied on analysis of transformed or tumour cell lines harbouring latent or integrated viral genomes, or on the behaviour of transfected reporter gene constructs. To provide a method of efficiently delivering papillomavirus information into the nuclei of mammalian cells, we constructed a herpes simplex virus type 1 recombinant bearing the entire human papillomavirus type 16 genome. The resulting recombinant was capable of lytic replication and induced the accumulation of papillomavirus mRNAs initiated from the p97 early promoter during infection of Vero cells. This and other herpes simplex - papillomavirus recombinants should facilitate molecular analysis of the life cycle of human papillomavirus type 16.

The herpes simplex virus type 1 (HSV-1) a sequence serves as a cleavage/packaging signal but does not drive recombinational genome isomerization when it is inserted into the HSV-2 genome.

We inserted the terminal repeat (a sequence) of herpes simplex virus type 1 (HSV-1) strain KOS into the tk gene of HSV-2 strain HG52 in order to assess the ability of the HSV-1 a sequence to provoke genome isomerization events in an HSV-2 background. We found that the HSV-1 a sequence was cleaved by the HSV-2 cleavage/packaging machinery to give rise to novel genomic termini. However, the HSV-1 a sequence did not detectably recombine with the HSV-2 a sequence. These results demonstrate that the viral DNA cleavage/packaging system contributes to a subset of genome isomerization events and indicate that the additional recombinational inversion events that occur during infection require sequence homology between the recombination partners.

Association of oral contraceptive use and human papillomaviruses in invasive cervical cancers.

In a study of 197 cases of histologically confirmed invasive cervical cancer, 61% of biopsies were positive for human papillomavirus (HPV) DNA by Southern or dot-blot hybridization. An association between detection of HPV DNA and oral contraceptive use was observed when HPV-positive and -negative cases were compared. Women reporting recent or long-term (greater than 4 yrs) oral contraceptive use were at 2.3 and 2.9-fold increased risks of HPV positivity, respectively. An increased risk of HPV positivity was also associated with formal education and with urban residence, while long-term smoking was negatively associated with HPV detection. A non-significant trend of increasing risk of HPV positivity with increasing number of sexual partners of the women and of the male partners of monogamous women was observed. Detection of HPV DNA was not associated with other cervical cancer risk factors examined, including age at first coitus, number of pregnancies, and Pap smear screening history. Our findings suggest either an interaction between HPV infection and oral contraceptive use in the genesis of cervical cancer or an increased expression of HPV genome in neoplasms of oral contraceptive users. These observations also support a multifactorial model of cervical cancer causation.

PIP: 197 cases of invasive cervical cancer were biopsied and tested for presence of human papilloma virus (HPV) DNA, and virus-positive and - negative cases were compared as to oral contraceptive use and other risk factors. These cases were all histologically confirmed invasive cervical cancers seen in the Panamanian National Oncology Institute ascertained from July 1985-June 1987. HPV DNA was identified by Southern or dot-blot hybridization, using probes for HPV-16, -18 and - 33. 61% of the cases were considered positive for at least 1 of the tests. Women reporting oral contraceptive use within the last year, or long-term (44 years) use, were 2.3 and 2.9-fold more likely to he HPV- positive than were non-users. Increased risk of HPV was also associated with urban residence and some, rather than no, formal education. Smoking was negatively associated with HPV. A non-significant trend was evident for multiple sexual partners of the women, or for monogamous women, of her partner. HPV was not linked with other cervical cancer risk factors, such as age at 1st coitus, parity, or Pap screen history. The possibility of an interaction between HPV infection and oral contraceptive use in the genesis of cervical cancer, or an increased expression of HPV genome in neoplasms of oral contraceptive users, was discussed, suggesting a multifactorial model of cervical cancer causation.

Regional distribution of human papillomavirus DNA and other risk factors for invasive cervical cancer in Panama.

A population-based national cancer registry has documented strikingly different regional incidence rates of cervical cancer in the Republic of Panama. Such regional differences in disease rates could represent regional differences in the occurrence of risk factors, in particular, human genital papillomaviruses (HPV). This study enrolled newly diagnosed invasive cancer patients in the Republic of Panama over an 18-mo period. Behavioral risk factors were measured by interviewing cases and matched controls. In addition, DNA extracted from biopsies of the cancers was tested for HPV sequences. Early age at first coitus, multiple pregnancies, and nonparticipation in Pap smear screening programs were significant risk factors for cervical cancer in this population. These factors and low levels of education occurred more frequently among women residing in regions with higher cancer rates than women residing in the region with lower cancer rates. HPV DNA was detected most frequently (70%) among cases from the region with the lowest cancer rate (30 of 100,000) and least frequent (54%) among cases where the cancer rate was the highest (51 of 100,000). The observations suggest that risk factors other than HPV contribute to the differences in cervical cancer rates among women residing in various regions of Panama.

Comparison of risk factors for cervical cancer in different populations.

The incidence of cervical cancer has been found to vary between populations. Risk factors of cervical cancer include early age at first marriage, multiple marriages and antibodies to herpes simplex virus type 2 (HSV-2). The interrelatedness of these risk factors was examined by comparing data collected from 428 cancer cases and 947 control women selected from 6 populations having standardized cervical cancer incidence rates varying from 9.3 to 85.1 per 100,000. Logistic regression analysis revealed that multiple marriages, early age at first marriage or pregnancy and HSV-2 antibodies were all associated with significant risk when all 3 factors were entered into the model. Cervical cancer incidence rates were best predicted by the occurrence of HSV-2 antibodies among control women. To further assess the relation between cervical cancer rates and HSV-2 antibody, 2,306 additional sera representing an 0.8% random sample of females over 9 years of age residing in the Republic of Panama were assayed for antibodies to the virus, and the occurrence of antibodies was correlated with invasive cervical cancer rates specific to each Province. Data from both the random sample and the other study populations yielded a linear relation between the occurrence of HSV-2 antibodies and the incidence of cervical cancer. An exception was found for women living in Herrera Province, Republic of Panama, who had a higher cancer rate than predicted by HSV-2 antibody occurrence. The data suggested that infection with HSV-2 is a co-variable of venereal factors, although a role for the virus in the genesis of a certain proportion of cervical cancers is not excluded.

Competitive inhibition by human sera of mouse monoclonal antibody binding to glycoproteins C and D of herpes simplex virus types 1 and 2.

A competitive enzyme-linked immunosorbent assay was used to test for human antibodies to antigenic sites on herpes simplex virus (HSV) glycoproteins C and D, which are recognized by mouse monoclonal antibodies. Antibodies capable of blocking the monoclonal antibodies were detected in the human sera, and the inhibition of binding correlated with the histories of herpetic infections. The binding of monoclonal antibody to glycoprotein C of HSV type 2 was inhibited primarily by sera from patients with recurrent herpes genitalis; however, the binding of the monoclonal antibodies to gC of HSV type 1 was inhibited by sera from patients previously infected with either HSV type 1 or HSV type 2. The observations suggest that the antigenic sites defined by the mouse monoclonal antibodies are recognized by the human host.

Identification and typing of herpes simplex viruses with monoclonal antibodies.

Monoclonal antibodies which reacted with type-specific antigens of herpes simplex virus type 2 or with antigens shared by herpes simplex virus types 1 and 2 were used in an indirect immunofluorescence assay to type virus isolates and to detect viral antigens in cells obtained from herpetic lesions. Complete concordance was obtained for 42 isolates typed by endonuclease restriction analysis of viral DNA and by indirect immunofluorescence with monoclonal antibodies. Examination of a limited number of ulcerative lesions revealed that indirect immunofluorescence and virus isolation were comparable in detecting herpes simplex virus. The results indicate that monoclonal antibodies can be used to accurately identify and type isolates of herpes simplex virus.