Analysis of change in gait in the ovine stifle: normal, injured, and anterior cruciate ligament reconstructed.

BACKGROUND: Many patients who undergo anterior cruciate ligament (ACL) reconstructive surgery develop post-traumatic osteoarthritis (PTOA). ACL reconstructive surgery may not fully restore pre-injury joint biomechanics, thereby resulting in further joint damage and contributing to the development of PTOA. In an ovine model of idealized ACL reconstruction (ACL-R), it has been shown that signs of PTOA develop within surgical joints by 20 weeks post-surgery. The aim of the present study was to investigate whether altered kinematics contribute to early PTOA development within ACL-R joints of the ovine injury model by comparing the gait of these surgical animals to the gait of a stable normal control group, and an unstable injury group in which the ACL and medial collateral ligament (MCL) had been transected. METHODS: Fifteen skeletally mature female sheep were allocated evenly into 3 treatment groups: normal control, ACL-R, and ACL/MCL Tx (each group n = 5). Each animal's gait was recorded at baseline, 4 weeks post injury, and 20 weeks post injury. Principal component analysis (PCA) was used to identify the kinematic patterns that may be discriminant between treatment groups. Results from previous studies were referenced to present the amount of gross PTOA-like changes that occurred in the joints. RESULTS: ACL-R and ACL/MCL transected (Tx) animals developed a similar amount of early PTOA-like changes within the surgical joints, but differed significantly in the amount of kinematic change present at 20 weeks post-surgery. We showed that the stifle joint kinematics of ACL/MCL Tx differed significantly from those of CTRL and the majority of ACL-R animals, while no significant differences in joint kinematic changes were found between ACL-R and CTRL animals. CONCLUSIONS: These results suggest that the early PTOA-like changes reported in the ACL-R model cannot be attributed exclusively to post-surgical kinematic changes, and therefore biologic components in the post-injury environment must be contributing significantly to PTOA development.

Cartilage boundary lubrication of ovine synovial fluid following anterior cruciate ligament transection: a longitudinal study.

OBJECTIVE: To assess ovine synovial fluid (oSF) from different post-injury time points for (1) proteoglycan-4 (PRG4) and hyaluronan (HA) concentration, (2) HA molecular weight (MW) distribution, (3) cartilage boundary lubrication function, and (4) lubricant composition-function relationships. The association between cartilage boundary lubrication and gross cartilage changes after injury was also examined. METHODS: oSF was collected 2, 4, 10, and 20 weeks post anterior cruciate ligament (ACL) transection in five skeletally mature sheep. PRG4 and HA concentrations were measured using sandwich enzyme-linked immunosorbent assay, and HA MW distribution by agarose gel electrophoresis. Cartilage boundary lubrication of oSF was assessed using a cartilage-cartilage friction test. Gross damage to articular cartilage was also quantified at 20 weeks using modified Drez scoring protocol. RESULTS: Early (2-4 weeks) after ACL injury, PRG4 concentrations were significantly higher (P = 0.045, P = 0.037), and HA concentrations were substantially lower (P = 0.005, P = 0.005) compared to 20 weeks. The HA MW distribution also shifted towards lower ranges in the early post-injury stage. The kinetic friction coefficients were significantly higher 2-4 weeks post injury (P = 0.008 and P = 0.049) compared to 20 weeks. Poor cartilage boundary lubricating ability early after injury was associated with cartilage damage at 20 weeks. CONCLUSION: Altered composition and diminished boundary lubrication of oSF early after ACL transection may pre-dispose the articular cartilage to degenerative changes and initiate osteoarthritis (OA). These observations also provide potential motivation for biotherapeutic interventions at earlier time points post injury.

Immunohistochemical expression of melanocytic antigen PNL2, Melan A, S100, and PGP 9.5 in equine melanocytic neoplasms.

The immunoreactivity of PNL2, Melan A, and protein gene product (PGP) 9.5 was compared with that of S100 protein in 50 formalin-fixed, paraffin-embedded equine melanocytic neoplasms. PNL2, PGP 9.5, and S100 protein were detected in all 50 neoplasms; none expressed Melan A. PNL2 was not expressed in 62 nonmelanocytic tumors (equine sarcoids, schwannomas, carcinomas, sarcomas, endocrine tumors, sex-cord stromal tumors, germ cell tumors, and leukocytic tumors) or in normal tissues other than epidermis. In summary, antibody PNL2 is a sensitive marker of equine melanocytic neoplasms and is more specific than S100 protein or PGP 9.5. In contrast, the monoclonal antibody to Melan A did not react with any of the equine melanomas.

Changes of early post-traumatic osteoarthritis in an ovine model of simulated ACL reconstruction are associated with transient acute post-injury synovial inflammation and tissue catabolism.

The study described here tested the hypothesis that early intra-articular inflammation is associated with the development of post-traumatic osteoarthritis (PTOA) in a sheep model. We extended previously published work in which we investigated joint gross morphology and synovial mRNA expression of inflammatory and catabolic molecules 2 weeks after anatomic Anterior cruciate ligament (ACL) autograft reconstructive surgery (ACL-R). The same variables have been analyzed at 20 weeks post surgery together with new experimental variables at both time points. Animals were sacrificed at 20 weeks post ACL-R surgery and their joints graded for signs of PTOA. Synovial samples were harvested for histological grading plus mRNA and protein analysis for a panel of inflammatory and catabolic molecules. The mRNA expression levels for this panel plus connective tissue matrix turnover molecules were also investigated in cartilage samples. Results of gross morphological assessments at 20 weeks post surgery showed some changes consistent with early OA, but indicated little progression of damage from the 2 week time point. While significant alterations in mRNA levels for synovial inflammatory and catabolic molecules were detected at 2 weeks, values had normalized by 20 weeks. Similarly, all mRNA expression levels for inflammatory and catabolic molecules in articular cartilage had returned to normal levels by 20 weeks post ACL-R surgery. We conclude that synovial inflammatory processes are initiated very early after ACL-R surgery and may instigate events that lead to the gross cartilage and joint abnormalities observed as early as 2 weeks. However, the absence of sustained inflammation and joint instability may prevent OA progression.

[Digital X-ray: interhospital data transfer by CD-ROM. Prospective data evaluation during the import process]. / Digitales Röntgen: interhospitaler Datentransfer auf CD-ROM. Prospektive Datenevaluation während des Arbeitsprozesses.

BACKGROUND: Increasing numbers of radiological imaging diagnostics are archived in digital form. In addition to the results of diagnostics performed in hospital a growing number of patients present with digital results of outpatient radiological investigations. These digitized images represent a challenge for the internal hospital work flow. The aim of the study was to determine the expenditure for the hospital when dealing with digital outpatient diagnostic results. METHOD: Several parameters were observed and analyzed within the import process of nearly 400 CD-ROMs over a time period of 5 months. Only a negligible number of data on CD-ROMs could not be transferred into the hospital archive (1.5%). The duration of the process depended on the amount of data and the time period. RESULTS: During regular hours the import process took on average 13 min per CD and 19 min per patient while the time increased significantly during on-call duties. This study demonstrates the significance of the import of digital outpatient radiological diagnostic results into the hospital archive which can in particular influence patient treatment.

[PHTLS team course: a pilot project. Structured student education in prehospital care of severely injured patients]. / Der PHTLS-TEAM-Kurs ­ ein Pilotprojekt. Strukturierte studentische Ausbildung in der präklinischen Versorgung schwerverletzter Patienten.

BACKGROUND: The training of medical school students at the University of Heidelberg seems to be improvable regarding prehospital trauma treatment compared to an established anaesthesiology-based training for medical emergencies. This study addresses the current situation and possibilities for advancing this training. MATERIAL AND METHODS: A baseline was set by interviews of the medical school students. Based on this the hypothesis was postulated that there is a deficit in the education of the medical school students concerning the training in prehospital trauma treatment. This was proved by questionnaires given to the students in the 7th and 8th semesters at the University of Heidelberg Medical School. The results were evaluated and a possible approach for improvement was developed. RESULTS: A total of 111 questionnaires could be evaluated. It could be shown that the existing education was not effectual and that there is a need for a praxis-orientated 1-day course in prehospital trauma treatment. CONCLUSION: Especially the treatment of multiply injured patients is a challenge for young medical professionals. However, there is a high motivation to learn and train in emergency medicine. The students long for a practical trauma course compared to the advanced medical CPR course provided by the Department of Anaesthesiology of the University of Heidelberg. Those algorithm-based trauma courses do exist with PHTLS® and ATLS®. Based on these courses we developed the PHTLS® TEAM course.

New understanding of the complex structure of knee menisci: implications for injury risk and repair potential for athletes.

Menisci help maintain the structural integrity of the knee. However, the poor healing potential of the meniscus following a knee injury can not only end a career in sports but lead to osteoarthritis later in life. Complete understanding of meniscal structure is essential for evaluating its risk for injury and subsequent successful repair. This study used novel approaches to elucidate meniscal architecture. The radial and circumferential collagen fibrils in the meniscus were investigated using novel tissue-preparative techniques for light and electron microscopic studies. The results demonstrate a unique architecture based on differences in the packaging of the fundamental collagen fibrils. For radial arrays, the collagen fibrils are arranged in parallel into ∼10 µm bundles, which associate laterally to form flat sheets of varying dimensions that bifurcate and come together to form a honeycomb network within the body of the meniscus. In contrast, the circumferential arrays display a complex network of collagen fibrils arranged into ∼5 µm bundles. Interestingly, both types of architectural organization of collagen fibrils in meniscus are conserved across mammalian species and are age and sex independent. These findings imply that disruptions in meniscal architecture following an injury contribute to poor prognosis for functional repair.

Technical issues in using robots to reproduce joint specific gait.

Reproduction of the in vivo motions of joints has become possible with improvements in robot technology and in vivo measuring techniques. A motion analysis system has been used to measure the motions of the tibia and femur of the ovine stifle joint during normal gait. These in vivo motions are then reproduced with a parallel robot. To ensure that the motion of the joint is accurately reproduced and that the resulting data are reliable, the testing frame, the data acquisition system, and the effects of limitations of the testing platform need to be considered. Of the latter, the stiffness of the robot and the ability of the control system to process sequential points on the path of motion in a timely fashion for repeatable path accuracy are of particular importance. Use of the system developed will lead to a better understanding of the mechanical environment of joints and ligaments in vivo.

Cystic pseudotumours in both upper ankle joints in gouty arthritis.

This case demonstrates a yet unreported clinical entity of bilateral tophaceous cystic lesions of the tibiotalar joints of an 37-year-old white male who presents with moderate painful swelling in both upper ankle joints for 2 years. Radiodiagnostic studies show cystic lesions in both upper ankle joints and the right talus considering neoplastic processes in the differential diagnosis. The incisional biopsy reveals chalk-like material of the intraosseous lesions which was pathognomonic for tophaceous gout. Antihyperuricemic medication led to a stagnation of growth of the lesions without a sign of osseous remodeling. However, due to limitation of discomfort, personal preferences and a lack of surgical options conservative treatment was maintained.

Primary Meningeal Rhabdomyosarcoma of the Spinal Cord of a Young Dog with Neuromelanocytosis and Multiple Cutaneous Neurofibromas.

A 7-week-old male black Labrador retriever puppy was presented for post-mortem examination following progressive hindlimb paralysis and multiple masses within the skin. A highly compressive and infiltrative intradural mass was found within the T9-T11 spinal cord. Microscopical and immunohistochemical analysis revealed features compatible with spindle cell rhabdomyosarcoma (RMS). The adjacent spinal cord had numerous melanin-containing cells, arranged in small nodules, predominantly within the grey matter (proposed term of 'micronodular neuromelanocytosis') and the left lateral thorax had multifocal dermal neurofibromas. In this case, the constellation of proliferative/neoplastic lesions represents a unique case presentation with unclear aetiology. Primary canine meningeal RMS of the spinal cord has not been reported previously and represents a novel differential diagnosis for spinal tumours of young dogs. Moreover, such cases should be assessed for the presence of additional congenital abnormalities.

Malignant anal sac melanoma in dogs: eleven cases (2000 to 2015).

OBJECTIVES: To report the signalment, clinical presentation, treatments pursued and outcomes of dogs with malignant anal sac melanoma. METHODS: Medical records from five institutions from January 2000 through December 2015 were reviewed and dogs with cytologically- or histologically-confirmed malignant anal sac melanoma were identified. Signalment, clinical signs, staging, cytology, histopathologic analysis, surgical and non-surgical treatments were extracted from the medical records. The referring veterinarians and owners were contacted for follow-up data. RESULTS: Eleven dogs were included and survival data was available for all. The most common clinical signs were bloody anal sac discharge and perianal licking. Initial treatments pursued included surgery (n=8), chemotherapy (n=1), and palliative treatment with pain medications and stool softeners (n=2). In an adjuvant setting, melanoma vaccine was pursued following surgery in three dogs and chemotherapy in one dog. Regardless of treatment, progression-free survival (mean 92 · 5 days) and overall survival times (median 107 days) were short. CLINICAL SIGNIFICANCE: Dogs in this case series had a guarded to poor prognosis regardless of treatment. Ten of 11 dogs were euthanased due to local or distant disease progression. Only 1 of 11 dogs was alive one year after diagnosis. An understanding of tumour behaviour in this location could lead to improved survival times with earlier diagnosis and treatment.

BCL2 and MYC are expressed at high levels in canine diffuse large B-cell lymphoma but are not predictive for outcome in dogs treated with CHOP chemotherapy.

Diffuse large B-cell lymphoma (DLBCL) is the most common haematopoietic malignancy in dogs. Recently, MYC and BCL2 expression levels determined with immunohistochemistry (IHC) were found to be prognostic in people with DLBCL. We hypothesized that canine DLBCL can be similarly subdivided into prognostic subtypes based on expression of MYC and BCL2. Cases of canine DLBCL treated with CHOP chemotherapy were retrospectively collected and 43 dogs had available histologic tissue and complete clinical follow-up. Median values of percent immunoreactive versus immunonegative cells were used to determine positive or negative expression status. Completion of CHOP was significantly associated with a positive outcome. Compared with human patients, our canine DLBCL patients had high IHC expression of both MYC and BCL2, and relative expression levels of one or both markers were not associated with clinical outcome.

Cerebral Candidal Abscess and Bovine Viral Diarrhoea Virus Infection in an Aborted Bovine Fetus.

Candida species are opportunistic fungi associated with immunosuppression and are the most commonly isolated fungal pathogens from the human central nervous system. Invasive candidiasis is reported uncommonly in animals and there have only been two reports of candidal infection of the brain. This report presents a case of a cerebral candidal abscess in an aborted late-term calf co-infected with bovine viral diarrhoea virus. Candida etchellsii, a species not previously identified as pathogenic, was identified as the causative agent by polymerase chain reaction.

Immunohistochemical Detection of Pax8 and Napsin A in Canine Thyroid Tumours: Comparison with Thyroglobulin, Calcitonin and Thyroid Transcription Factor 1.

Expression of thyroid transcription factor (TTF)-1 corroborates a thyroid origin of neoplasms. Thyroglobulin and calcitonin immunohistochemistry (IHC) can distinguish between a follicular and C-cell origin of thyroid tumours, respectively. Pax8 (expressed by normal canine thyroid follicular cells) and napsin A (expressed mainly by C-cells) labelling was compared with labelling for TTF-1, thyroglobulin and calcitonin in 114 canine proliferative thyroid lesions. All 81 follicular tumours expressed thyroglobulin and were negative for calcitonin; 79/81 (98%) of these tumours expressed TTF-1 and Pax8 and 60/81 (74%) expressed napsin A. All 25 C-cell lesions expressed calcitonin and were negative for expression of thyroglobulin; 22 (88%) were positive for TTF-1, 13 (57%) for Pax8 and 24/24 for napsin A. Six mixed follicular-medullary carcinomas expressed all five markers. Both carcinosarcomas expressed TTF-1 and napsin A, and one each of these tumours expressed thyroglobulin, calcitonin or Pax8. Pax8 expression was also detected in epididymal cells, endometrial cells and vas deferens epithelium, in Sertoli-like ovarian cells, and in some cases of ovarian adenoma, pancreatic carcinoma, renal cell carcinoma and Sertoli cell tumour. Napsin A was also detected in adrenocortical cells, ovarian granulosa cells, epididymal and endometrial cells, as well as in some renal cell carcinomas, pulmonary adenocarcinomas and Sertoli cell tumours. In summary, Pax8 was as sensitive as TTF-1 and slightly less sensitive than thyroglobulin for identification of follicular tumours, but had low sensitivity for C-cell tumours. Napsin A was as sensitive as calcitonin for C-cell neoplasms, but was less sensitive than thyroglobulin for follicular neoplasms. Thus, these markers are sensitive and, except for renal cell carcinoma (for Pax8, napsin A) and pulmonary adenocarcinoma (for napsin A), are specific thyroid tumour markers.

Postmortem stability of total RNA isolated from rabbit ligament, tendon and cartilage.

The stability of RNA, particularly mRNA, in tissues is under complex regulation. Most studies to date have focused on very cellular tissues and not connective tissues such as ligaments, tendons and cartilage. As the availability of such tissues for transplantation or research purposes is frequently delayed following death, it is important to determine whether RNA stability in such tissues is influenced by time postmortem. To approach this question, skeletally mature NZW rabbits were used to investigate RNA integrity over time in dense, hypocellular connective tissues and in several hypercellular organ tissues such as brain, kidney, liver and lung. Samples were analyzed at varying intervals postmortem with respect to rRNA integrity by agarose gel electrophoresis and ethidium bromide staining and mRNA integrity by Northern blot analysis and RT-PCR. No degradation of rRNA or loss in integrity of mRNA for genes of low and high copy number was observed up to 96 h postmortem. These findings confirm that it is likely appropriate to use properly stored postmortem dense connective tissues for molecular biological investigations.

Strength of medial structures of the knee joint are decreased by isolated injury to the medial collateral ligament and subsequent joint immobilization.

Past studies of the healing of the medial collateral ligament (MCL) in animal models have been conducted over a variety of healing intervals, some as early as 1 week. One concern with testing at early healing intervals is the difficulty in identifying and isolating the tissues that carry load. The purpose of this study was to determine if isolation of the MCL and healing time are critical factors in the assessment of structural strength in this model. Furthermore, the effect of immobilization on these critical factors was investigated. Our approach was to calculate the load-sharing ratio between the MCL and the MCL plus capsule. A 4 mm gap was created in the midsubstance of both hindlimb MCLs of 52 female New Zealand White rabbits (n=104). Of these, 29 rabbits had their right hindlimb pin immobilized (immobilized group), leaving the left hindlimb non-immobilized. Testing was performed at 3 (n=12), 6 (n=22), and 14 (n=24) weeks. The remaining 23 rabbits, which had both limbs non-immobilized (non-immobilized group), were tested at 3 (n=10), 6 (n=12), 14 (n=12), and 40 (n=12) weeks. For both groups, half of the specimens at each healing interval were used to test the MCL alone and half to test the MCL plus capsule, except for 3 week immobilized joints where only the MCL plus capsule was tested. Additionally, MCL (n=12), MCL plus capsule (n=6), and capsule alone (n=5) were tested from normal animals. The load-sharing ratio at MCL failure for the normal joint was 89%, suggesting an MCL-dominated response. For the non-immobilized group, the load-sharing ratio was 24% at 3 weeks of healing, suggesting a capsule-dominated response. At and after 6 weeks of healing, an MCL-dominated response was observed, with the ratio being 68% or greater. Thus, at less than 6 weeks of healing, the structural strength capabilities of the joint may be better represented by the medial structures rather than the isolated MCL. Immobilization delayed the transition from a capsule-dominated response to an MCL-dominated response in this model.

Altered levels of extracellular matrix molecule mRNA in healing rabbit ligaments.

RT-PCR methods were used to amplify, semi-quantify, clone and sequence cDNA fragments specific for rabbit extracellular matrix molecules biglycan, collagen I, collagen III, decorin, lumican, versican, fibromodulin, and also glyceraldehyde-3-phosphate dehydrogenase (G3PDH), using RNA isolated from rabbit ligaments. Sequence analysis of two independent clones of PCR products was used to verify the identity of the cDNA. Semi-quantitative RT-PCR was used to study mRNA levels for these matrix molecules in normal and healing rabbit ligament at three, six, and fourteen weeks post-injury. The yield of RNA from the ligament scar was increased at three and six weeks post-injury, but it had returned to near normal levels by fourteen weeks. On a microgram RNA basis, it was demonstrated that biglycan, collagen I, collagen III and lumican mRNA levels are significantly elevated, versican mRNA levels significantly depressed, and decorin and fibromodulin mRNA levels showed no significant change in response to tissue injury in the ligament during the course of healing. These findings suggest that differential regulation of mRNA levels for these extracellular matrix molecules occurs during ligament healing.

The relation between hypochondriasis and age.

OBJECTIVE: This study examined the relation between hypochondriasis and age while controlling for the possible confounding influences of medical morbidity, social isolation, and other psychiatric disorder. METHOD: Consecutive patients attending a general medical clinic on randomly selected days were screened with a hypochondriasis self-report questionnaire. Those whose scores exceeded a preestablished cutoff level and a random sample of those who scored below it completed a research battery consisting of self-report questionnaires and structured interviews for DSM-III-R diagnoses of hypochondriasis and other axis I disorders. The patients' medical records were audited, and their physicians completed questionnaires about them. The 60 patients who met the DSM-III-R criteria for hypochondriasis at interview constituted the study group, and 100 patients randomly chosen from among those who scored below the cutoff for hypochondriasis constituted the comparison group. RESULTS: The hypochondriacal group was not older than the comparison group. Hypochondriacal patients aged 65 years and over did not differ significantly from younger hypochondriacal patients in hypochondriacal attitudes, somatization, tendency to amplify bodily sensation, or global assessment of their overall health, even though their aggregate medical morbidity was greater. The elderly hypochondriacal patients had higher levels of disability, but this appeared to be attributable to their medical status rather than to any increase in hypochondriasis. Within the comparison sample, subjects aged 65 years and over were not more hypochondriacal than those under 65 years of age. CONCLUSIONS: Hypochondriasis is found to some degree in all patients and appears to be unrelated to age.

Localization and characterization of porcine patellar tendon xenograft antigens in a rabbit model of medial collateral ligament replacement.

BACKGROUND: Ligament injuries of the knee are common and, if severe, can predispose to joint pain, instability, reinjury, and, ultimately, osteoarthritis. Xenograft replacement of ligaments could have potential; however, a limited understanding of the immunology of ligament xenograft rejection has inhibited their use. The purpose of this study was to characterize the antigenic elements of a fresh porcine tendon xenograft in a rabbit model and to provide a better understanding of what would need to be done to either block or extract these antigenic elements. METHODS: Three experimental situations were evaluated in a pig to rabbit ligament transplantation model: subcutaneous implantation of fresh porcine patellar tendon (PPT), implantation of fresh PPT into a medial collateral ligament midsubstance gap, and replacement of the entire medial collateral ligament complex with either fresh or guanidinium hydrochloride-extracted PPT. Preimmune and immune sera were collected from rabbits and used to localize antigenic targets in PPT, meniscus, and cartilage with indirect immunofluorescence techniques. The reactivities of the same rabbit sera towards tissue extracts of PPT, meniscus, and cartilage by Western immunoblot analyses were used to characterize the antigenic components. RESULTS: Indirect immunofluorescence with preimmune rabbit sera on PPT showed staining of tendon fibroblasts. Immune sera from rabbits transplanted with xenografts stained regions of the extracellular matrix of PPT. Fresh PPT induced antibodies that consistently recognized six extracellular matrix components with molecular masses of >200 kDa, 180 kDa, 135 kDa, 108 kDa, 63 kDa, and 59 kDa. CONCLUSIONS: Our results suggest that naturally occurring rabbit anti-pig antibodies of the IgG isotype recognize immunogenic components on tendon fibroblasts, whereas induced rabbit anti-pig antibodies recognize a specific subset of six extracellular matrix components of PPT. PPT xenografts appeared to induce a similar humoral immune response irrespective of graft location. Finally, our results indicate that selective extraction of PPT xenograft components before implantation altered the induced rabbit anti-pig antibody response; however, such extraction did not change the ultimate fate of the transplant tissue.

Efficient transfer of intact oligonucleotides into the nucleus of ligament scar fibroblasts by HVJ-cationic liposomes is correlated with effective antisense gene inhibition.

The efficacy of two different cationic liposomes, Lipofectin and hemagglutinating virus of Japan (HVJ)-cationic liposomes, on nuclear uptake of fluorescence-labeled phosphorothioate oligodeoxyribonucleotide (S-ODN) by ligament scar fibroblasts and suppression of decorin mRNA expression when antisense decorin S-ODN was transferred was investigated. There was no significant difference in nuclear uptake of fluorescent ODN between the two methods. However, only HVJ-cationic liposomes had a significant effect on suppression of decorin mRNA expression levels. To address the discrepancy, the molecular integrity of the transferred ODN in the cells was assessed by analysis of fluorescence resonance energy transfer (FRET) within double-fluorescence-labeled S-ODN. More than 70% of the ODN transfected by HVJ-cationic liposomes remained intact within the nucleus at 20 h after transfection, while the majority of the ODN transferred by Lipofectin was degraded at this point. These results suggest a strong relationship between the nuclear integrity of transfected antisense ODN and its suppression of target mRNA expression.