Comprehensive high-resolution genomic profiling and cytogenetics of human chondrocyte cultures by GTG-banding, locus-specific FISH, SKY and SNP array.

In the development of cell-based medicinal products, it is crucial to guarantee that the application of such an advanced therapy medicinal product (ATMP) is safe for the patients. The consensus of the European regulatory authorities is: "In conclusion, on the basis of the state of art, conventional karyotyping can be considered a valuable and useful technique to analyse chromosomal stability during preclinical studies". 408 chondrocyte samples (84 monolayers and 324 spheroids) from six patients were analysed using trypsin-Giemsa staining, spectral karyotyping and fluorescence in situ hybridisation, to evaluate the genetic stability of chondrocyte samples from non-clinical studies. Single nucleotide polymorphism (SNP) array analysis was performed on chondrocyte spheroids from five of the six donors. Applying this combination of techniques, the genetic analyses performed revealed no significant genetic instability until passage 3 in monolayer cells and interphase cells from spheroid cultures at different time points. Clonal occurrence of polyploid metaphases and endoreduplications were identified associated with prolonged cultivation time. Also, gonosomal losses were observed in chondrocyte spheroids, with increasing passage and duration of the differentiation phase. Interestingly, in one of the donors, chromosomal aberrations that are also described in extraskeletal myxoid chondrosarcoma were identified. The SNP array analysis exhibited chromosomal aberrations in two donors and copy neutral losses of heterozygosity regions in four donors. This study showed the necessity of combined genetic analyses at defined cultivation time points in quality studies within the field of cell therapy.

Ultrasound palpation for fast in-situ quantification of articular cartilage stiffness, thickness and relaxation capacity.

Most current cartilage testing devices require the preparation of excised samples and therefore do not allow intra-operative application for diagnostic purposes. The gold standard during open or arthroscopic surgery is still the subjective perception of manual palpation. This work presents a new diagnostic method of ultrasound palpation (USP) to acquire applied stress and strain data during manual palpation of articular cartilage. With the proposed method, we obtain cartilage thickness and stiffness. Moreover, repeated palpations allow the quantification of relaxation effects. USP measurements on elastomer phantoms demonstrated very good repeatability for both, stage-guided (97.2%) and handheld (96.0%) applications. The USP measurements were compared with conventional indentation experiments and revealed very good agreement on elastomer phantoms ([Formula: see text]) and good agreement on porcine cartilage samples ([Formula: see text]). Artificially degenerated cartilage samples showed reduced stiffness, weak capacity to relax after palpation and an increase of stiffness of approximately 50% with each single palpation. Intact cartilage was measured by USP directly at the patella (in situ) and after excision and removal of the subchondral bone (ex situ), leading to stiffness values of [Formula: see text] and [Formula: see text] ([Formula: see text]), respectively. The results demonstrate the potential of the USP system for cartilage testing, its sensitivity to degenerative changes and as a method for quantifying relaxation processes by means of repeated palpations. Furthermore, the differences in the results of in-situ and ex-situ measurements are of general interest, since such comparison has not been reported previously. We point out the limited comparability of ex-situ cartilage with its in-situ biomechanical behavior.

3D ultrasound biomicroscopy for assessment of cartilage repair tissue: volumetric characterisation and correlation to established classification systems.

Objective and sensitive assessment of cartilage repair outcomes lacks suitable methods. This study investigated the feasibility of 3D ultrasound biomicroscopy (UBM) to quantify cartilage repair outcomes volumetrically and their correlation with established classification systems. 32 sheep underwent bilateral treatment of a focal cartilage defect. One or two years post-operatively the repair outcomes were assessed and scored macroscopically (Outerbridge, ICRS-CRA), by magnetic resonance imaging (MRI, MOCART), and histopathology (O'Driscoll, ICRS-I and ICRS-II). The UBM data were acquired after MRI and used to reconstruct the shape of the initial cartilage layer, enabling the estimation of the initial cartilage thickness and defect volume as well as volumetric parameters for defect filling, repair tissue, bone loss and bone overgrowth. The quantification of the repair outcomes revealed high variations in the initial thickness of the cartilage layer, indicating the need for cartilage thickness estimation before creating a defect. Furthermore, highly significant correlations were found for the defect filling estimated from UBM to the established classification systems. 3D visualisation of the repair regions showed highly variable morphology within single samples. This raises the question as to whether macroscopic, MRI and histopathological scoring provide sufficient reliability. The biases of the individual methods will be discussed within this context. UBM was shown to be a feasible tool to evaluate cartilage repair outcomes, whereby the most important objective parameter is the defect filling. Translation of UBM into arthroscopic or transcutaneous ultrasound examinations would allow non-destructive and objective follow-up of individual patients and better comparison between the results of clinical trials.

A novel MSC-seeded triphasic construct for the repair of osteochondral defects.

Mesenchymal stem cells (MSC) are increasingly replacing chondrocytes in tissue engineering based research for treatment of osteochondral defects. The aim of this work was to determine whether repair of critical-size chronic osteochondral defects in an ovine model using MSC-seeded triphasic constructs would show results comparable to osteochondral autografting (OATS). Triphasic implants were engineered using a beta-tricalcium phosphate osseous phase, an intermediate activated plasma phase, and a collagen I hydrogel chondral phase. Autologous MSCs were used to seed the implants, with chondrogenic predifferentiation of the cells used in the cartilage phase. Osteochondral defects of 4.0 mm diameter were created bilaterally in ovine knees (n = 10). Six weeks later, half of the lesions were treated with OATS and half with triphasic constructs. The knees were dissected at 6 or 12 months. With the chosen study design we were not able to demonstrate significant differences between the histological scores of both groups. Subcategory analysis of O'Driscoll scores showed superior cartilage bonding in the 6-month triphasic group compared to the autograft group. The 12-month autograft group showed superior cartilage matrix morphology compared to the 12-month triphasic group. Macroscopic and biomechanical analysis showed no significant differences at 12 months. Autologous MSC-seeded triphasic implants showed comparable repair quality to osteochondral autografts in terms of histology and biomechanical testing.

Mesenchymal stem cells in cartilage repair: state of the art and methods to monitor cell growth, differentiation and cartilage regeneration.

Degenerative joint diseases caused by rheumatism, joint dysplasia or traumata are particularly widespread in countries with high life expectation. Although there is no absolutely convincing cure available so far, hyaline cartilage and bone defects resulting from joint destruction can be treated today by appropriate transplantations. Recently, procedures were developed based on autologous chondrocytes from intact joint areas. The chondrocytes are expanded in cell culture and subsequently transplanted into the defect areas of the affected joints. However, these autologous chondrocytes are characterized by low expansion capacity and the synthesis of extracellular matrix of poor functionality and quality. An alternative approach is the use of adult mesenchymal stem cells (MSCs). These cells effectively expand in 2D culture and have the potential to differentiate into various cell types, including chondrocytes. Furthermore, they have the ability to synthesize extracellular matrix with properties mimicking closely the healthy hyaline joint cartilage. Beside a more general survey of the architecture of hyaline cartilage, its composition and the pathological processes of joint diseases, we will describe here which advances were achieved recently regarding the development of closed, aseptic bioreactors for the production of autologous grafts for cartilage regeneration based on MSCs. Additionally, a novel mathematical model will be presented that supports the understanding of the growth and differentiation of MSCs. It will be particularly emphasized that such models are helpful to explain the well-known fact that MSCs exhibit improved growth properties under reduced oxygen pressure and limited supply with nutrients. Finally, it will be comprehensively shown how different analytical methods can be used to characterize MSCs on different levels. Besides discussing methods for non-invasive monitoring and tracking of the cells and the determination of their elastic properties, mass spectrometric methods to evaluate the lipid compositions of cells will be highlighted.

Cartilage tissue engineering by collagen matrix associated bone marrow derived mesenchymal stem cells.

In this study we compared the in vitro formation of cartilaginous grafts composed of collagen type I hydrogel with both ovine primary articular chondrocytes (AC) and bone marrow derived mesenchymal stem cells (MSC). During 4 weeks of culture, aggregate properties were quantitatively verified, cell viability and the expression of cartilage markers were assayed. Different microscopic techniques indicated a subdivision of MSC based scaffolds into a central construct region with uniformly distributed stem cells with low levels of apoptosis, and peripheral layers of proliferative cells, which undergo differentiation. Immunohistochemical staining and quantitative measurements of sulfated glycosaminoglycans (s-GAG) of MSC hydrogels showed a significant increase in matrix deposition, mainly in outer areas. Furthermore, semi-quantitative RT-PCR of MSC specimens reflected a constant collagen type I activity with enhanced collagen type II, aggrecan and Sox9 expression which would suggest hyaline-like cartilage formation. We propose the application of MSC seeded grafts for the repair of focal cartilage lesions.

A review of functional status measures for workers with upper extremity disorders.

In order to identify functional status measures for epidemiological studies among workers with mild to moderate disorders of the neck and upper extremity, a literature search was conducted for the years 1966 to 2001. Inclusion criteria were: (1) relevance to neck and upper extremity; (2) assessment among workers; and (3) relevance to mild to moderate disorders. Of 13 instruments reviewed, six measures were tested among workers. The three best measures, depending on the purpose of research, included the standardised Nordic Musculoskeletal Questionnaire, the Upper Extremity Questionnaire, and the Neck and Upper Limb Instrument. Development of a functional protocol is regarded as a realistic enhancement for research of neck and upper extremity disorders in the workplace. For research and clinical practice, measures of functional status, sensitive enough to measure the subtle conditions in mild to moderate disorders, may provide prognostic information about the risk of developing musculoskeletal disorders in apparently healthy patients. Appropriate use of functional status questionnaires is imperative for a meaningful portrayal of health.

Proliferative responses to selected peptides of IA-2 in identical twins discordant for Type 1 diabetes.

BACKGROUND: The aim of the study was to define T lymphocyte reactivity to selected peptides of an islet antigen IA-2, associated with Type 1 diabetes. METHODS: We used 10 peptides selected from the IA-2 molecule due to their predicted ability to bind to HLA-DRB1*0401, a Type 1 diabetes-associated allele. We tested 21 identical twin pairs discordant for the disease and 15 control subjects and then followed them prospectively; seven non-diabetic twins developed diabetes. RESULTS: Twins of identical pairs tended to respond to different peptides suggesting that the T cell response is, to a degree, shaped by non-genetically determined factors (p<0. 0001). However, there was no difference in the T cell responses between diabetic twins and either their non-diabetic identical twins or control subjects and the response was heterogenous. T cell responses did not differ in those seven non-diabetic twins who developed diabetes from those twins who did not. T cell responses to peptide 11 (amino acids 502-514) was immunodominant in diabetic twins as well as their non-diabetic twins and controls; responses were not correlated with HLA, IA-2 antibodies, age or duration of disease. CONCLUSION: We conclude that T cell responses to selected IA-2 peptides are not genetically determined, heterogeneous, not strictly HLA controlled and did not distinguish diabetic or prediabetic twins from non-diabetic twins or controls. The identification of an immunodominant T cell response to IA-2 peptide 502-514 raises the possibility that this, or similar, epitopes may be of therapeutic value in disease prevention.

Elders with dementia living in the community with and without caregivers: an epidemiological study.

BACKGROUND: Previous studies of dementia and family caregiving have focused on individuals seeking diagnosis and treatment, and have rarely been conducted in representative community samples. Identifying demented individuals participating in a community survey, we determined (a) the factors associated with demented elderly living alone; (b) the factors associated with the demented elderly having caregivers; (c) the factors associated with increased levels of burden among caregivers of persons with dementia. POPULATION AND METHODS: During an epidemiological survey of a mostly rural U.S. community, the authors identified 116 noninstitutionalized elderly individuals with dementia. These individuals were classified into those living alone and those living with others; both groups were further classified into those with and without identifiable family caregivers. Characteristics of both caregivers and care recipients were examined. RESULTS: Approximately a third of the subjects with dementia lived alone, and only half of them had caregivers. The average age of the caregivers was 67.4 years, and 73% of them were women. Almost half of the caregivers were spouses, whereas almost a third were offspring, of the demented individuals. Over two thirds of caregivers lived with the subjects. Female caregivers were significantly younger than male caregivers. Multivariate analyses revealed that subjects with dementia who were living alone were independently and significantly more likely to be women and to have dementias of shorter duration, lesser severity, and lesser functional impairment than those living with others. Demented subjects with caregivers were more likely to have greater dementia severity, functional impairment, and cognitive impairment and more current cognitive and behavioral symptoms than those without caregivers. Demented subjects whose caregivers reported higher levels of burden were more likely to be women and to have greater dementia severity, functional impairment, and cognitive impairment and more current symptoms than those whose caregivers had no/minimal burden. CONCLUSIONS: These results draw attention to the problems of persons with dementia living alone, particularly those without caregivers. Our data also provide epidemiological confirmation of previous clinical/volunteer studies of dementia caregiving, as well as a preliminary assessment of need in the community at large. Living arrangements and caregiver issues should be taken into account when planning services for the elderly.

Reduction in health care resource utilization associated with extended-release theophylline.

BACKGROUND: People with airway disease are high utilizers of health care resources. Few studies document the value of alternative therapies in reducing utilization. Studies examining theophylline, which demonstrate reduction in resource utilization, have been primarily of short duration in hospitalized settings with small samples. OBJECTIVE: The purpose of this study was to examine the role of oral extended-release theophylline in reducing health care utilization over an extended period of time when added to existing inhaler therapy for ambulatory patients with airway disease. METHODS: We used a retrospective, pretest/posttest design in examining the 1990-1993 South Carolina Medicaid database to compare health care utilization of 455 ambulatory patients for 4 months before and 6 months after extended-release theophylline was added to their treatment regimen. We assessed the following three outcomes: inhaler use, physician office visits, and emergency department visits, all measured in units/person/month. RESULTS: Our sample consisted of patients taking beta2-agonist only (n = 393), steroid only (n = 25), and beta2-agonist plus steroid (n = 37). Inhaler use and physician office visits declined significantly among beta2-agonist users, as well as within the entire sample. Initiation of extended-release theophylline therapy was associated with a 30% decline in utilization of inhaler and physician office visits, influenced mostly by the decline with the beta2-agonist group. CONCLUSION: The results of this effectiveness study using an administrative claims database are consistent with the published randomized clinical trials that document the value of extended-release theophylline when added to existing inhaler therapy.

A novel murine P-450 gene, Cyp4a14, is part of a cluster of Cyp4a and Cyp4b, but not of CYP4F, genes in mouse and humans.

Genomic clones for Cyp4a12 and a novel member of the murine Cyp4a gene family were isolated. The novel gene, designated Cyp4a14, has a GC rich sequence immediately 5' of the transcription start site, and is similar to the rat CYP4A2 and CYP4A3 genes. The Cyp4a14 gene spans approximately 13 kb, and contains 12 exons; sequence similarity to the rat CYP4A2 gene sequence falls off 300 bp upstream from the start site. In view of the known sex-specific expression of the rat CYP4A2 gene, the expression and inducibility of Cyp4a14 was examined. The gene was highly inducible in the liver when mice were treated with the peroxisome proliferator, methylclofenapate; induction levels were low in control animals and no sex differences in expression were observed. By contrast, the Cyp4a12 RNA was highly expressed in liver and kidney of control male mice but was expressed at very low levels in liver and kidney of female mice. Testosterone treatment increased the level of this RNA in female liver slightly, and to a greater extent in the kidney of female mice. In agreement with studies on the cognate RNA, expression of Cyp4a12 protein was male-specific in the liver of control mice and extremely high inducibility of Cyp4a10 protein, with no sex differences, was also demonstrated. In view of the overlapping patterns of inducibility of the three Cyp4a genes, we investigated whether the three genes were co-localized in the genome. Two overlapping yeast artificial chromosome (YAC) clones were isolated, and the three Cyp4a genes were shown to be present on a single YAC of 220 kb. The Cyp4a genes are adjacent to the Cyp4b1 gene, with Cyp4a12 most distant from Cyp4b1. The clustering of these two gene subfamilies in the mouse was replicated in the human, where the CYPA411 and CYP4B1 genes were present in a single YAC clone of 440 kb. However, the human CYP4F2 gene was mapped to chromosome 19. Phylogenetic analysis of the CYP4 gene families demonstrated that CYP4A and CYP4B are more closely related than CYP4F.

Quantifying the clinical significance of drug-drug interactions: scaling pharmacists' perceptions of a common interaction classification scheme.

OBJECTIVE: To develop a precise, interval level scale of the clinical significance of drug-drug interactions that reflects the professional judgments of practicing pharmacists. PARTICIPANTS: A convenience sample of 63 practicing pharmacists representing hospital (clinical and staff) and retail (chain and independent) practice settings. METHOD: Pharmacists judged the similarity among 15 interaction categories that have been commonly used to classify drug-drug interactions. A multidimensional scaling technique produced a spatial representation (i.e., a psychological map) of the structure inherent in those similarity judgments. Pharmacists' ratings of clinical significance were projected onto that same spatial representation using a multiple regression procedure, and the resulting information was used to develop a scale of clinical significance. RESULTS: The clinical significance scale developed from pharmacists' judgments was substantially different from a comparison scale published in a popular reference. The new scale was more precise than the comparison scale, and it also approximated an interval level of measurement. The judgments used to produce the new clinical significance scale were not reliably influenced by pertinent demographic characteristics of the sample. CONCLUSIONS: Inconsistencies between published clinical significance scales and the professional judgments of practitioners could affect patient care to the degree that a summary measure of clinical significance affects a practitioner's response to a potential drug-drug interaction. The clinical significance scale developed in this study has good measurement characteristics and reflects the professional judgments of practicing pharmacists. Use of the new scale is recommended on these grounds, although further assessment of its generality is warranted.

A comparison of costs and efficacy of intranasal fluticasone propionate and terfenadine tablets for seasonal allergic rhinitis.

This paper compares cost-efficacy ratios for intranasal fluticasone propionate and terfenadine tablets within a sample of patients with seasonal allergic rhinitis symptoms due to mountain cedar allergy. Efficacy was assessed using secondary data analysis of patient ratings of symptoms and their overall assessment of response to treatment within a previously conducted clinical trial. Costs include direct costs of the drugs used for therapy. Patients with documented mountain cedar allergy who were 12 years of age or older (N = 232) had been randomized to either receive intranasal fluticasone propionate, terfenadine, or placebo. The cost-efficacy ratios for intranasal fluticasone propionate 200 micrograms once daily were more favorable than the ratios for terfenadine 60 mg twice daily. This relationship remained throughout the sensitivity analysis. Because intranasal fluticasone propionate is only available in a fixed package size, the number of efficacy-adjusted days of terfenadine therapy that could be purchased to reach break-even costs for a 30-day supply of fluticasone was calculated. Cost efficacy-adjusted days ranged from 11 to 18 days. If cost-efficacy adjustments are not conducted, the upper end of the range increases from 18 to 22 days, since 22 days of terfenadine could be purchased for the price of a 30-day supply of intranasal fluticasone propionate. Depending on which of the efficacy measures the reader believes, if patients use terfenadine for longer than 11 to 22 days, fluticasone propionate is the more cost-efficacious choice. Because most allergies are seasonal and allergy seasons typically last longer than 11 to 22 days, it is likely that fluticasone propionate will frequently be the more cost-efficacious choice in the patient population represented in this study.

Drug use behavior under the constraints of a Medicaid prescription cap.

Limiting the number of prescriptions reimbursed per month is a cost-containment measure used by state Medicaid programs. The purpose of this study was to identify and examine the strategies used by patients who exceed the limits of the cap. Nineteen patients identified by community pharmacists as meeting this criterion were interviewed in their homes by a member of the project team. The sample consisted predominantly of women. Seven of the 19 individuals reported that they had altered the way they took their medication, primarily by reducing the frequency of dosing, to make the medication last longer. Eight individuals reported that, at some time during the past year, they did not obtain a prescribed medication because of the prescription cap. These medications were prescribed for a variety of conditions, including diabetes, asthma, and congestive heart failure. Patients made the purchase/nonpurchase decision based primarily on importance of the condition for which the medication was prescribed. What was deemed important, however, could change over time as a result of changing symptoms. Patients obtained some help from friends, family, and health care professionals in coping with the constraints of the prescription cap. However, the cap forced a significant group of patients into noncompliance, and thus placed them at risk for poor health outcomes.

Too many medications, too little money: how do patients cope?

Many individuals with chronic illnesses have multiple medications prescribed that often are not covered by third party payers. In South Carolina, Medicaid pays for only three prescriptions per month per recipient. A qualitative pilot study was conducted to learn how Medicaid recipients with more than three prescriptions decide which ones to have filled under Medicaid and what they do about the remaining medications. Nineteen Medicaid recipients who had more than three prescriptions were interviewed in their homes. Research participants paid for medications out-of-pocket, borrowed money, were extended credit by the pharmacy, got samples from the physician, did not get prescriptions filled or refilled, took medicines less frequently or in lower doses to stretch their supply, and very infrequently took someone else's medication. These individuals decided which medications to take based on: their perceptions of the importance of the medication or the seriousness of the condition for which it was prescribed, current symptoms, and the drug's cost. Some participants had to choose monthly whether to buy medications or food.

Monoamine oxidase inhibition by the MAO-A inhibitors brofaromine and clorgyline in healthy volunteers.

The present study compared the extent and duration of MAO inhibition by the selective and reversible MAO-A inhibitor brofaromine with the selective and irreversible MAO-A inhibitor clorgyline using amine pressor tests and excretion of urinary amine metabolites (MHPG, tryptamine). The pharmacological characterization of clorgyline as an irreversible and brofaromine as a reversible MAO-A inhibitor in clinically effective doses was confirmed in humans.

Economic, clinical, and humanistic outcomes: a planning model for pharmacoeconomic research.

Medical, ethical, and societal concerns about costs, access, and quality of care are causing health care practitioners to consider a more comprehensive model for medical decision making. Consequently, interest in research to assess the outcomes of health care has been increasing. The purpose of this paper is to explicate a theoretical framework for identifying, collecting, and using outcomes data to assess the value of pharmaceutical treatment alternatives. Causal relationships between disease, health outcomes, and decisions about medical care interventions (eg, treatment with pharmaceutical products and services) are proposed to address limitations inherent in the traditional medical decision-making model. The Economic, Clinical, and Humanistic Outcomes (ECHO) model depicts the value of a pharmaceutical product or service as a combination of traditional clinical-based outcomes with more contemporary measures of economic efficiency and quality. This integrated approach provides a theoretical basis for considering potential trade-offs among economic, clinical, and humanistic variables in optimizing the allocation of health care resources. The ECHO model is a preliminary step to modeling outcomes from pharmaceutical treatments and services. Data collection instruments need to be developed, and the proposed relationships among outcomes variables should be established empirically. The ECHO model should assist health services researchers in planning, conducting, and evaluating pharmaceutical products and services from a multidimensional perspective.

Economic impact of cost-containment strategies in third party programmes in the US. Part II.

This is the second article in a 2-part series that examines the economic impact of several different strategies used to control costs in third party programmes. This article investigates 5 different methods: (a) formularies; (b) capitation; (c) drug utilisation review; (d) prior approval; and (e) drug product selection. The published literature indicates that use of formularies decreases drug expenditures, but these savings may be offset by expenditures in other areas of healthcare programmes. Capitation, though less well studied than other strategies, may show some effectiveness in reducing costs by increasing generic dispensing and promoting switching from prescription drug to over-the-counter. Drug utilisation review, as a systematic programme of claims data review, has been shown to yield positive economic return in a variety of areas, including both impersonal and face-to-face educational interventions with healthcare practitioners. Prior approval and drug product selection both result in savings when examined in isolation from other aspects of healthcare. Cost-shifting, administrative costs and costs incurred because of possible decreased access to care have yet to be fully accounted for.

Economic impact of cost-containment strategies in third party programmes in the US (part I).

The rising cost of healthcare has strained the resources of governments, private third parties and individuals with responsibility to pay for it. Various strategies have been used in an attempt to control costs. This article examines the economic impact of 4 such strategies: (a) cost sharing; (b) prescription limits; (c) rebates; and (d) cost limits. Cost sharing has been successful at reducing utilisation of prescription drugs, although the effects have not been uniform across therapeutic categories. However, the long term effect on cost and utilisation of other medical services, and the impact on overall health status, remain largely unknown. Some evidence suggests that utilisation of other services may increase. The available data regarding drug rebate programmes have been descriptive in nature. However, the designs employed in this research do not establish a direct causal relationship between rebate programmes and changes in Medicaid drug expenditure. Furthermore, still unknown is the degree of cost shifting and the effect of the rebate programme on other large public and private drug purchasers. The Maximum Allowable Cost programme led to direct savings in drug costs, but the size of these savings was variable and uncertain because of administrative costs of the programme. The Estimated Acquisition Cost programme has not resulted in significant savings.