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In this article, we report on an effort to study the development and usefulness of a large, broad-use, opt-in biorepository for genomic research, focusing on three ethical issues: providing appropriate understanding, recruiting in ways that do not comprise autonomous decisions, and assessing costs versus benefits. We conclude the following: (a) Understanding can be improved by separating the task of informing subjects from documenting informed consent (Common Rule) and permission to use personal health information and samples for research (Health Insurance Portability and Accountability Act [HIPAA]); however, regulations might have to be changed to accommodate this approach. (b) Changing recruiting methods increases efficiency but can interfere with subject autonomy. (c) Finally, we propose a framework for the objective evaluation of the utility of biorepositories and suggest that more attention needs to be paid to use and sustainability.

A novel, nonresorbable, monolithic composite structure ceramic, developed using a partially stabilized zirconia ceramic common to implantable devices, was used in a cementless weight-bearing articular implant to test the feasibility of replacing a region of degenerated or damaged articular cartilage in the knee as part of a preclinical study using male mongrel dogs lasting up to 24 weeks. Gross/histological cartilage observations showed no differences among control, 12-week and 24-week groups, while pull-out tests showed an increase in maximum pull-out load over time relative to controls. Hence, the use of a novel ceramic implant as a replacement for a focal cartilage defect leads to effective implant fixation within 12 weeks and does not cause significant degradation in opposing articular cartilage in the time frame evaluated.

In response to a request for proposal from the American College of Rheumatology (ACR), our group was charged with developing non-pharmacologic and pharmacologic guidelines for treatments in gout that are safe and effective, i.e., with acceptable risk-benefit ratio. These guidelines for the management and anti-inflammatory prophylaxis of acute attacks of gouty arthritis complements our manuscript on guidelines to treat hyperuricemia in patients with evidence of gout (or gouty arthritis) (1). Gout is the most common cause of inflammatory arthritis in adults in the USA. Clinical manifestations in joints and bursa are superimposed on top of local deposition of monosodium urate crystals. Acute gout characteristically presents as self-limited, attack of synovitis (also called “gout flares”). Acute gout attacks account for a major component of the reported decreased health-related quality of life in patients with gout (2, 3). Acute gout attacks can be debilitating and are associated with decreased work productivity (4, 5). Urate lowering therapy (ULT) is a cornerstone in the management of gout, and, when effective in lowering serum urate (SUA), is associated with decreased risk of acute gouty attacks (6). However, during the initial phase of ULT, there is an early increase in acute gout attacks, which has been hypothesized due to remodeling of articular urate crystal deposits as a result of rapid and substantial lowering of ambient urate concentrations (7). Acute gout attacks attributable to the initiation of ULT may contribute to non-adherence in long-term gout treatment, as reported in recent studies (8). In order to systematically evaluate a broad spectrum of acute gouty arthritis, we generated multifaceted case scenarios to elucidate decision making based primarily on clinical and laboratory test-based data that can be obtained in a gout patient by both non-specialist and specialist health care providers in an office practice setting. This effort was not intended to create a novel classification system of gout, or new gout diagnostic criteria, as such endeavors are beyond the scope of this work. Prior gout recommendations and guidelines, at the independent (i.e, non pharmaceutical industry-sponsored) national or multinational rheumatology society level, have been published by EULAR (9, 10), the Dutch College of General Practitioners (11), and the British Society for Rheumatology (BSR)(12). The ACR requested new guidelines, in view of the increasing prevalence of gout (13), the clinical complexity of management of gouty arthritis imposed by co-morbidities common in gout patients (14), and increasing numbers of treatment options via clinical development of agents(15–17). The ACR charged us to develop these guidelines to be useful for both rheumatologists and other health care providers on an international level. As such, this process and resultant recommendations, involved a diverse and international panel of experts. In this manuscript, we concentrate on 2 of the 4 gout domains that the ACR requested for evaluation of pharmacologic and non-pharmacologic management approaches: (i) analgesic and anti-inflammatory management of acute attacks of gouty arthritis, and (ii) pharmacologic anti-inflammatory prophylaxis of acute attacks of gouty arthritis. Part I of the guidelines focused on systematic non-pharmacologic measures (patient education, diet and lifestyle choices, identification and management of co-morbidities) that impact on hyperuricemia, and made recommendations on pharmacologic ULT in a broad range of case scenarios of patients with disease activity manifested by acute and chronic forms of gouty arthritis, including chronic tophaceous gouty arthropathy(1). Each individual and specific statement is designated as a “recommendation”, in order to reflect the non-prescriptive nature of decision making for the hypothetical clinical scenarios. So that the voting panel could focus on gout treatment decisions, a number of key assumptions were made, as described in Part I of the guidelines (1). Importantly, each proposed recommendation assumed that correct diagnoses of gout and acute gouty arthritis attacks had been made for the voting scenario in question. For treatment purposes, it was also assumed that treating clinicians were competent, and considered underlying medical comorbidities (including diabetes, gastrointestinal disease, hypertension, and hepatic, cardiac, and renal disease), and potential drug toxicities and drug-drug interactions, when making both treatment choicesand dosing decisions on chosen pharmacologic interventions. The RAND/UCLA methodology used here emphasizes level of evidence, safety, and quality of therapy, and excludes analyses of societal cost of health care. As such, the ACR gout guidelines are designed to reflect best practice, supported either by level of evidence or consensus-based decision-making. These guidelines cannot substitute for individualized, direct assessment of the patient, coupled with clinical decision making by a competent health care practitioner. The motivation, financial circumstances, and preferences of the gout patient also need to be considered in clinical practice, and it is incumbent on the treating clinician to weigh the issues not addressed by this methodology, such as treatment costs, when making management decisions. Last, the guidelines for gout management presented herein were not designed to determine eligibility for health care cost coverage by third party payers.

DINESH KHANNA, JOHN D. FITZGERALD, PUJA P. KHANNA, SANGMEE BAE, MANJIT K. SINGH, TUHINA NEOGI, MICHAEL H. PILLINGER, JOAN MERILL, SUSAN LEE, SHRADDHA PRAKASH, MARIAN KALDAS, MANEESH GOGIA, FERNANDO PEREZ-RUIZ, WILL TAYLOR, FREDERIC LIOTE, HYON CHOI, JASVINDER A. SINGH, NICOLA DALBETH, SANFORD KAPLAN, VANDANA NIYYAR, DANIELLE JONES, STEVEN A. YAROWS, BLAKE ROESSLER, GAIL KERR, CHARLES KING, GERALD LEVY, DANIEL E. FURST, N. LAWRENCE EDWARDS, BRIAN MANDELL, H. RALPH SCHUMACHER, MARK ROBBINS, NEIL WENGER, AND ROBERT TERKELTAUB

Because of its semi-solid character in dissemination and growth, advanced ovarian cancer with its hundreds of peritoneal tumor nodules and plaques appears to be an excellent in vivo model for studying the cancer stem cell hypothesis. The most important obstacle, however, is to adequately define and isolate these tumor-initiating cells endowed with the properties of anoikis-resistance and unlimited self-renewal. Until now, no universal single marker or marker constellation has been found to faithfully isolate (ovarian) cancer stem cells. As these multipotent cells are known to possess highly elaborated efflux systems for cytotoxic agents, these pump systems have been exploited to outline putative stem cells as a side-population (SP) via dye exclusion analysis. Furthermore, the cells in question have been isolated via flow cytometry on the basis of cell surface markers thought to be characteristic for stem cells.In the Vienna variant of the ovarian cancer cell line A2780 a proof-of-principle model with both a stable SP and a stable ALDH1A1+ cell population was established. Double staining clearly revealed that both cell fractions were not identical. Of note, A2780V cells were negative for expression of surface markers CD44 and CD117 (c-kit). When cultured on monolayers of healthy human mesothelial cells, green-fluorescence-protein (GFP)-transfected SP of A2780V exhibited spheroid-formation, whereas non-side-population (NSP) developed a spare monolayer growing over the healthy mesothelium. Furthermore, A2780V SP was found to be partially resistant to platinum. However, this resistance could not be explained by over-expression of the "excision repair cross-complementation group 1" (ERCC1) gene, which is essentially involved in the repair of platinated DNA damage. ERCC1 was, nonetheless, over-expressed in A2780V cells grown as spheres under stem cell-selective conditions as compared to adherent monolayers cultured under differentiating conditions. The same was true for the primary ovarian cancer cells B-57.In summary our investigations indicate that even in multi-passaged cancer cell lines hierarchic government of growth and differentiation is conserved and that the key cancer stem cell population may be composed of small overlapping cell fractions defined by various arbitrary markers.

Background Common marmoset monkeys (Callithrix jacchus) are readily used in biomedical research. However, superovulation for embryonic stem cell production and developmental research still remain difficult. Inexplicably, follicle-stimulating hormone (FSH) as key player in superovulation has to be administered in extremely high dosages in this non-human primate compared to human. Methods To evaluate whether marmoset FSH (cjFSH) is functionally more competent than its human homologue on the marmoset FSH receptor (cjFSHR), we established in vitro a homologous system characterizing homologous and recombinantly produced cjFSH. Results Upon stimulation of two cell lines stably expressing either the marmoset or the human FSH receptor (cj/hFSHR), respectively, the second messenger signaling of the activated receptor displayed no significant difference in ED50 values. Thermostability of cjFSH was significantly prolonged by roughly 20% on both FSHRs. Conclusion High FSH dosage in marmoset superovulation cannot be explained by enhanced biopotency of the natural animal’s gonadotropin.

Twenty-four subjects with metastatic melanoma were treated on a randomized Phase Ib trial evaluating an autologous tumor lysate-pulsed dendritic cell (DC) vaccine with or without interleukin (IL)-2. The vaccine consisted of autologous DCs obtained from peripheral blood mononuclear cells (PBMCs) cultured in granulocyte macrophage-colony stimulating factor and IL-4 then pulsed with autologous tumor cell lysate and keyhole limpet hemocyanin (KLH). The primary end points of the trial were safety and immune response to vaccine. Subjects were randomized to vaccine administered every other week times 3, vaccine x 3 followed by low-dose IL-2, or vaccine x 3 followed by high-dose IL-2. Immune response was monitored pretreatment and at 2 and 4 weeks after the third vaccine administration. Disease evaluation was performed at 4 weeks after the third vaccination. Therapy was well tolerated with no local vaccine toxicity greater than grade 1 in any arm. IL-2 toxicity was as expected without additional toxicity from the addition of IL-2 to vaccine. Immune response defined as delayed-type hypersensitivity, PBMC interferon-gamma enzyme-linked immunosorbent spot, and PBMC proliferation, to both autologous tumor and KLH were detected in all arms. Interferon-gamma enzyme-linked immunosorbent spot response to KLH (7 of 10 patients) and autologous tumor (4 of 10 patients) were also detected in subjects with available vaccine draining lymph node cells. There were no differences in immune response between treatment arms. No clinical responses were seen. Autologous tumor lysate-pulsed DC vaccine with or without IL-2 was well tolerated and immunogenic but failed to induce clinical response in patients with advanced melanoma.

Alternative treatment strategies for claudication are needed and cell-based therapies designed to induce angiogenesis are promising. The purpose of this report was to conduct a Phase I safety, dose-escalating, non-randomized, open-label study of autologous, fully differentiated venous endothelial and smooth muscle cells called MultiGeneAngio (MGA) for claudication due to peripheral artery disease. Twelve subjects, at two centers, received a single intra-arterial infusion of a suspension of equal amounts of transduced autologous venous smooth muscle cells expressing vascular endothelial growth factor (VEGF165) and endothelial cells expressing angiopoietin-1 (Ang-1) (Cohort 1: 1 × 107, Cohort 2: 2 × 107, Cohort 3: 5 × 107, Cohort 4: 7 × 107). The treatment was given unblinded and in the more symptomatic lower extremity. Transduced cells were tested for in vitro doubling time, telomerase activity, and gene expression. The main outcomes were clinical safety and tolerability. Other safety measures included ankle–brachial index (ABI) and walking time on a treadmill. All subjects were male (mean age 60 ± 5 years) including 25% with diabetes mellitus. At 1-year follow-up, there was one serious adverse event possibly related to MGA. Safety endpoints including VEGF and Ang-1 plasma protein levels were within normal ranges in all subjects. The mean maximal walking time increased from baseline to 1 year and the index limb ABI was unchanged, indicating no safety concerns. MGA, an autologous, transduced, cell-based therapy was well tolerated and safe in this Phase I study. Further evaluation is warranted in randomized human studies. Clinical Trial Registration: ClinicalTrials.gov Identifier: NCT00390767.

Application of Raman spectroscopy to analysis of subchondral bone is described. The effect of cartilage health on subchondral bone has been widely studied using radiological and histological methods; however, there is no method to directly assay mineral components. We present Raman spectra of femur condyles and observe mineral bands that arise from the subchondral bone. In two separate experiments, transgenic mouse models of early-onset osteoarthritis (OA) and lipoatrophy were compared to tissue from wild-type mice. Raman spectroscopy was used to identify chemical changes in the mineral of subchondral bone that may accompany or precede morphological changes that can be observed by histology. The transgenic mice were compared to age-matched wild-type mice. Subtle alterations in the mineral or collagen matrix were observed by Raman spectroscopy using established Raman markers such as the carbonate-to-phosphate ratio, mineral-to-matrix ratio (MTMR), and amide I ratio. The Raman microscope configuration enabled rapid collection of Raman spectra from the mineralized layer that lies under an intact layer of non-mineralized articular cartilage. The effect of the cartilage layer on collection of spectra is discussed. The technique proposed is capable of providing insight into the chemical changes that occur in subchondral bone on a molecular level.

Imaging of musculoskeletal tissues is an important aspect of diagnostic and therapeutic rheumatology. The past 20 years have witnessed the development and refinement of multiple technical platforms for imaging of musculoskeletal tissues. In addition to conventional radiographs, these include computed tomography, magnetic resonance imaging, and ultrasound [1]. Each of these platforms provide important and often complementary diagnostic and disease progression information to the clinical rheumatologist. However, every one of these platforms fails to provide critical information about the structural composition of extracellular matrix (ECM) components at the molecular level. There is increasing experimental evidence that molecular changes in ECM components may determine macroscopic changes in bone, tendon, synovium and cartilage, which are recognized by available imaging techniques and associated with clinical disease. Therefore, an unmet medical need remains for imaging technologies that can identify early molecular changes in ECM components that may predict disease risk or progression. Over the same period of time, there has been extensive development of visible and infrared light-based vibrational spectroscopy techniques for the study of complex organic polymers, including ECM of musculoskeletal tissues. These studies owe much to extensive research on the spectroscopy of synthetic polymers of industrial interest, including the ultrahigh-molecularweight polyethylene used in artificial joint implants. Much of the effort has been directed at the analysis of bone ECM, but in the last few years, several groups have also begun to investigate the spectroscopic properties of articular cartilage ECM. The term ‘vibrational spectroscopy’ includes several very different techniques that share the ability to detect and analyze molecular vibrations: the bending and stretching motions of the chemical bonds that hold organic molecules together. The strength of these methods is that molecular vibrations occur as specific and well-known optical frequencies. The vibrational spectrum represents an optical fingerprint for a given complex organic molecule. For example, collagen proteins are a major ECM component in bone, tendon and articular cartilage, and, at a molecular level, are derivatized organic polymers composed of repetitive amide linkages. Since structural collagens are characterized by long half lives, aging or disease can cause alterations in the secondary structure of the collagen fibrils that will be characterized by small changes in the vibrational frequencies and intensities of the amide backbone spectra. Because vibrational spectroscopy is unfamiliar to most rheumatologists, we will briefly review the basis for several available methods, discuss the current research using osteoarthritis (OA) as a disease model, and assess prospects for clinical application in OA. Introductory texts on both infrared [2] and Raman [3] spectroscopy are available, and although these are aimed at advanced undergradutate chemistry students, they should be easily understood by anyone who has completed approximately 2 years of college chemistry.

Photoacoustic tomography (PAT), a nonionizing, noninvasive, laser-based technology was adapted to joint imaging for the first time. Pulsed laser light in the near-infrared region was directed toward a joint with resultant ultrasonic signals recorded and used to reconstruct images that present the optical properties in subsurface joint tissues. The feasibility of this joint imaging system was validated on a Sprague Dawley rat tail model and verified through comparison with histology. With sufficient penetration depth, PAT realized tomographic imaging of a joint as a whole organ noninvasively. Based on the optical contrast, various intra- and extra-articular tissues, including skin, fat, muscle, blood vessels, synovium and bone, were presented successfully in images with satisfactory spatial resolution that was primarily limited by the bandwidth of detected photoacoustic signals rather than optical diffusion as occurs in traditional optical imaging. PAT, with its intrinsic advantages, may provide a unique opportunity to enable the early diagnosis of inflammatory joint disorders, e.g., rheumatoid arthritis, and to monitor therapeutic outcomes with high sensitivity and accuracy.

We are developing retroviral-mediated gene transfer to human fibroblast-like synovial cells (FLS) as one approach to characterizing genetic pathways involved in synoviocyte pathophysiology. Prior work has suggested that FLS are relatively refractory to infection by Moloney murine leukemia virus based vectors. To determine if viral titer influenced the transduction efficiency of FLS, we optimized a rapid, efficient, and inexpensive centrifugation method to concentrate recombinant retroviral supernatant. The technique was evaluated by measurement of the expression of a viral enhanced green fluorescent protein transgene in transduced cells, and by analysis of viral RNA in retroviral supernatant. Concentration (100-fold) was achieved by centrifugation of viral supernatant for four hours, with 100% recovery of viral particles. The transduction of FLS increased from approximately 15% with unconcentrated supernatant, to nearly 50% using concentrated supernatant. This protocol will be useful for investigators with applications that require efficient, stable, high level transgene expression in primary FLS.

Sessile drop formation, also called drop deposition, has been studied as a potential medical diagnostic, but the effects of complex biofluid rheology on the final deposition pattern are not well understood. We studied two model biofluids, blood plasma and synovial fluid, when deposited onto slightly hydrophilic substrates forming a contact angle of 50–90°. Drops were imaged during the evaporation process and geometric properties of the drop, such as contact angle and drop height, were calculated from the images. The resulting dried biofluid drops were then examined using light microscopy and Raman spectroscopy to assess morphological and chemical composition of the dried drop. The effect of substrate contact angle (surface wetting) and fluid concentration was examined. We found that when biofluids are deposited onto slightly hydrophilic surfaces, with a contact angle of 50–90°, a ring-shaped deposit was formed. Analysis of the drying drop's geometric properties indicates that biofluid dynamics follow the piling model of drop formation, as proposed by Deegan et al. The final deposition pattern varied with substrate surface and concentration, as shown by light microscopy photos of dried drops. The chemical composition of the outer ring was minimally affected by substrate surface, but the spatial heterogeneity of protein distribution within the ring varied with concentration. These results indicate that biofluid drop deposition produces ring-shaped deposits which can be examined by multiple analytical techniques.

The synovial membrane that lines diarthrodial joints is composed of a loose layer of cells (1-4 cells in depth) that overlie the surface of articular and periarticular subsynovial tissues including connective tissue, adipose tissue, tendon, bone, and articular cartilage. Using light microscopy, the synovial lining tissue appears discontinuous and lacks a clearly defined basement membrane, although the cells appear to be retained by a reticulum. Cells present below this membrane include mast cells and endothelial cells (up to 10% of the total cell population; refs. 1,2). Ultrastructural studies of the synovium have indicated that at least two major types of cells are present within the synovial membrane. Type A cells are monocytoid in appearance and contain abundant endoplasmic reticulum with fewer vesicles, vacuoles, and mitochondria. Type B cells are fibroblastoid in appearance and contain large granules, numerous filopodia, mitochondria, and intracellular vesicles. The percentage of Type A and B cells present within adult synovium varies between species (3-5). In addition, cells with an intermediate ultrastructural appearance and morphology may exist within adult synovium (6).

REPRO, a 3D computer-based simulation program for reef and carbonate platform growth, was tested using published data from the late Miocene Llucmajor carbonate platform in Mallorca. The device central to the program is a depth-dependent logistic growth function for carbonate growth combined with linear diffusion, so accounting for the lateral spreading of reefs. Evolution of the carbonate platform was simulated using a composite sea level curve consisting of superposed sine functions with different frequencies and amplitudes. Overall carbonate platform shape and internal geometries as produced by the program match fairly well with the outcrop data. Thus we demonstrate that REPRO has the potential to model lateral facies changes and lateral variations in the depositional geometries of carbonate platforms in three dimensions.

Expression plasmids encoding chloramphenicol acetyltransferase (CAT) or human interferon-alpha2 cDNA were formulated in water-in-oil nanoemulsions and applied to murine skin. The histological location of transfected cells was assessed by in situ DNA PCR and showed that the deposition of plasmid DNA was primarily in follicular keratinocytes. Transgene expression in the skin was monitored for 24-72 h, following topical application of either single or multiple daily doses by quantitative RT-PCR and ELISA. It was found that transgene expression was optimal at 24 h following topical application of a single dose of water-in-oil nanoemulsion containing plasmid DNA. Dose-response studies using a total dose of 3, 10 or 30 microg of plasmid DNA suggested that topical transfection using nanoemulsions is subject to both threshold and saturation effects. None of the cationic liposome formulations tested as controls mediated transgenic protein expression at levels higher than background values of the ELISAs used to assay transgenic protein. Single and multiple dose experiments using human interferon-alpha2 as a transgene indicated that the efficiency of nanoemulsion mediated transfection was most effective in the context of normal versus atrophic hair follicles. In addition, the total amount of human interferon-alpha2 present in skin appeared to accumulate as a consequence of multiple dosing. Histologic evaluation of treated skin showed no overt signs of toxicity or irritation associated with the short-term application of the nanoemulsions. The results suggest that water-in-oil nanoemulsions can be used to facilitate transfection of follicular keratinocytes in vivo.

Background and Purpose —Chemokines have been shown to play an important role in leukocyte and monocyte/macrophage infiltration into ischemic regions. The purpose of this study is to identify whether overexpression of the active human transforming growth factor-β1 (ahTGF-β1) can downregulate expression of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and intercellular adhesion molecule-1 (ICAM-1) and reduce ischemic brain injury. Methods —Overexpression of transforming growth factor-β1 (TGF-β1) was achieved through adenoviral gene transfer. Five days after adenoviral transduction, the mouse underwent 30 minutes of middle cerebral artery occlusion followed by 1 to 7 days of reperfusion. TGF-β1, MCP-1, MIP-1α, and ICAM-1 were detected by enzyme-linked immunosorbent assay and immunohistochemistry. Infarct areas and volumes were measured by cresyl violet staining. Results —MCP-1 and MIP-1α expression is increased after middle cerebral artery occlusion, and double-labeled immunostaining revealed that MCP-1 is colocalized with neurons and astrocytes. Viral-mediated TGF-β1 overexpression was significantly greater at measured time points, with a peak at 7 to 9 days. The expression of MCP-1 and MIP-1α, but not ICAM-1, was reduced in the mice overexpressing ahTGF-β1 ( P P Conclusions —This study demonstrates that MCP-1 and MIP-1α expressed in the ischemic region may play an important role in attracting inflammatory cells. The reduction of MCP-1 and MIP-1α, but not ICAM-1, in the mice overexpressing ahTGF-β1 suggests that the neuroprotective effect of TGF-β1 may result from the inhibition of chemokines during cerebral ischemia and reperfusion.

We have developed a model of transforming growth factor (TGF)beta1 gene transfer into mouse vascularized cardiac allografts to study the use of gene transfer as an immunosuppressive therapy in transplantation. Donor hearts were perfused with either DNA-liposome complexes or adenoviral vectors that encode the active form of human TGFbeta1. DNA-liposome mediated transfection prolonged allograft survival in approximately two-thirds of transplant recipients, while adenoviral delivery of TGFbeta1 was not protective. Protective TGFbeta1 gene transfer was associated with reduced Th1 responses and an inhibition of the alloantibody isotype switch. The protective effects of TGFbeta1 gene transfer were overridden by exogenous interleukin-12 administration. Interestingly, alloreactive CD4+ and CD8+ cells exhibited distinct sensitivities to TGFbeta1 gene transfer: CD4+ Th1 function was abrogated by this modality, although CD8+ Th1 function was not. Transient depletion of recipient CD8+ cells markedly prolonged the survival of grafts transfected with either DNA-liposome complexes or adenoviral vectors. Transgene expression persisted for at least 60 days, and Th1 responses were not detectable until CD8+ T cells repopulated the periphery. However, long-term transfected allografts appeared to exhibit exacerbated fibrosis and neointimal development. These manifestations of chronic rejection were absent in long-term transfected isografts, suggesting that long-term expression of active TGFbeta1 alone is not sufficient to induce fibrosis of the grafts. Collectively, these data illustrate the utility of immunosuppressive gene therapy as a treatment for transplantation when combined with additional conditioning regimens. Further, they illustrate that alloreactive CD4+ and CD8+ cells may be differentially influenced by cytokine manipulation strategies.

Recombinant adenoviruses are useful vectors for basic research. When the vectors are used for delineating protein function, several viruses, each containing a mutated version of the transgene are compared at the same time. However, methods to generate multiple vectors simultaneously within a short time period are cumbersome. In this report, we show that a novel backbone plasmid, when cotransfected with routinely used shuttle vectors into HEK293 cells allowed for production of recombinant viruses in an average of 14 days. The recombinant viruses had no detectable wild-type virus contamination by A549 plaque assay and only three to 300 E1a copies per 109 adenovirus genomes by a sensitive PCR-based assay. Further culturing or serial amplification did not result in wild-type revertants nor did cultures show increased levels of E1a copy number by quantitative PCR. Thus, recombinant adenovirus vectors can be produced very simply, rapidly and with little to no contaminating wild-type particles. This system should facilitate the generation of multiple genetic variants by eliminating the need for time-consuming plaque purification and the need to manipulate and screen very large plasmids. We call this the RAPAd.I system.

Gene transfer has been performed in a variety of organs. In the mammalian inner ear, viral vectors have been used to introduce exogenous reporter genes via the scala tympani into the cochlea. While scala tympani inoculation is clinically feasible, it is not without risks. Moreover, transgene expression has so far been restricted to the cochlear tissues in the perilymphatic spaces that are contiguous with the scala tympani. To achieve gene transfer of vestibular organs and cells surrounding the endolymphatic space, and to extend the clinical utility of inner ear gene therapy, we developed a new surgical approach for vector inoculation. A replication-deficient adenoviral vector, Ad.RSVntlacZ, was injected into the guinea pig endolymphatic sac. A large number of blue (LacZ-positive) cells was observed in the endolymphatic sac and duct, the vestibule, and the ampulla. Blue cells were also detected in the cochlea, mainly in cells bordering the endolymphatic space: marginal cells in the stria vascularis and supporting cells in the organ of Corti. These findings indicate that inoculation of viral vectors into the endolymphatic sac can provide efficient gene transfer into a variety of cell types that are not accessible via scala tympani inoculation.

Herpes simples virus thymidine kinase (HSV-TK) expression plasmid DNA was injected into the joint space of rabbits with antigen-induced arthritis (AIA). Purified plasmid DNA was able to mediate transfection of synovial lining cells and transient overexpression of HSV-TK in the context of active synovial inflammation. The pharmacodynamic distribution of intraarticular expression plasmid DNA was confined to the joint space. Arthritic rabbits treated with intraarticular expression plasmid DNA followed by intravenous ganciclovir (GCV, 5 mg/kg) twice daily for 3 days showed histologic evidence of synovial lining layer cytolysis when articular tissues were examined 21 days posttreatment. There was also a reduction in joint swelling in the TK-treated knees. No untoward clinical effects were observed in the rabbits and no evidence of cytolytic damage specific to the TK-GCV gene therapy was observed either in the articular cartilage or bone. The application of TK-GCV intraarticular gene therapy using purified expression plasmid DNA for the induction of synovial cytolysis may be applicable to the treatment of human inflammatory arthritis.

Adenoviral vectors are being developed for oral delivery of therapeutic genes to the intestine. Initial studies in the rat using mucolytics and direct application of adenovirus encoded with the interleukin-1 receptor antagonist gene to the jejunum produced limited gene expression. The goal of this study was to determine the role of integrins in adenovirus-mediated gene delivery to the intestinal epithelium. Integrins are involved in cellular differentiation and tight junction formation and mediate adenoviral internalization. Results from Caco-2 and IEC-18 cells suggest that, as enterocytes differentiate, cell-surface integrin expression decreases. Pretreatment of Caco-2 cells with RGD peptides reduced adenoviral transduction efficiency by 80% in undifferentiated cells and 20% in differentiated cells. Both differentiated and undifferentiated IEC-18 cells showed a 70% drop in transduction when pretreated with the peptide. Infection inhibition studies with monoclonal antibodies further suggest that alpha(v)beta3 and alpha6beta1 integrins play significant roles in adenoviral internalization in the intestine. Expression of integrins in cell culture models of the intestine correlated with in vivo expression in intestinal segments. These results indicate that the ileum is a prime target for efficient adenovirus-mediated gene transfer in the rat. To enhance transduction in differentiated enterocytes (probable targets for oral gene delivery), Caco-2 cells were treated with interleukin-1beta (a cytokine known to increase integrin expression) prior to administration of the virus. Transduction efficiency increased four-fold.

Purpose. Our goals are to establish an in vitro screening system and to evaluate a new approach in improving oral absorption of peptides and peptide-like drugs by overexpression of the human intestinal oligo-peptide transporter (hPepTl). This study characterizes the expression of hPepTl in human intestinal Caco-2 cells, rat intestinal epithelial cells (IEC-18), and human cervix epithelial cells (Hela) after adenoviral transduction.

This report identifies formulation and processing factors that influence stability of viral preparations such as selection of appropriate buffer systems, cryoprotectants, and cooling rates. Adenovirus type 5 containing the lacZ marker gene was suspended in combinations of trehalose, sorbitol, sucrose, mannitol, glycine, CaCl2, and gelatin. X-gal stains of 293 cells were used to determine the lac-forming units (lfu)/ml of each preparation before and after treatments. Phosphate-buffered solutions (except those containing sucrose or trehalose) demonstrated a drop of 3 pH units upon freezing regardless of cryoprotectant used. Tris-buffered solutions demonstrated a variation in pH which was dependent upon chosen cryoprotectant, with 1 M trehalose exhibiting no change and a 5% mannitol/10 mM CaCl2 combination showing a 3-unit drop in pH. 4-[2-Hydroxyethyl]-1-piperazine ethanesulfonic acid (HEPES)-buffered solutions showed little change in initial pH when frozen regardless of cryoprotectant chosen. In solution, adenovirus was not affected by incubation for 24 hr in buffers ranging from pH 4 to 8. However, when the solutions were frozen, the number of remaining infectious virions was dependent upon the final pH of the suspending medium. Cryoprotectant solutions that significantly maintained viral stability during a single freeze--thaw cycle were 0.5 M sucrose, 0.5 M trehalose, and 10% sorbitol/0.4% gelatin. Long-term stability studies were performed at 4 degrees C with lyophilized sorbital/gelatin and sucrose preparations. Both formulations provided adequate stability for the adenovirus, with 2.6 and 5.6 x 10(11) lfu/ml detected 150 days after drying, respectively.

Purpose. In general, the intestinal epithelium is quite refractory to viral and non-viral methods of gene transfer. In this report, various cyclodextrin formulations were tested for their ability to enhance adenoviral transduction efficiency in two models of the intestinal epithelium: differentiated Caco-2 cells and rat jejunum.

Recently, replication-deficient adenoviruses have received increasing attention as vector for gene delivery and as potential vaccine carriers. With the increased use of the vector in vivo and in clinical trails, the demand for a safe, rapid, and cost effective purification process has been heightened. In this report, a simple and efficient method for the purification of large quantities of live adenoviral vectors was developed. The process involved the replacement of cesium chloride (CsCl) gradients with sucrose gradients. Ultracentrifugation times were reduced and the desalting step eliminated, decreasing total preparation time by 15 hr. A 20-80% linear sucrose gradient provided optimal recovery of infectious viral particles and positioning of the viral band in the gradient. Purification with this gradient system produced a preparation containing 1.39 x 10(14) lac-forming units (lfu)/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the process also removed all associated cellular proteins from the preparation. Studies have shown that direct lyophilization of the vector in sucrose after purification produces a product containing 1.4 x 10(12) lfu/ml. Minimal degradation was seen in the lyophilized preparation. A viral concentration of 6 x 10(11) lfu/ml was detected in the product after 150 days in storage at -20 degrees C. This approach will not only simplify the preparation of adenoviral vectors for in vivo studies and clinical trials, but will facilitate production of stable adenoviral formulations for oral gene delivery.

Gene transfer is an exciting new tool in medical therapy and scientific investigation, but only very recently has it begun to be developed in the auditory system. This paper describes in vivo and ex vivo experiments using an adenoviral vector (Ad. RSVntlacZ), which is a replication-deficient virus based on a human adenoviral (serotype 5[ genomic backbone. The in vivo experiments demonstrate successful gene transfer into multiple types of cochlear cells. We observed a relatively efficient transduction, several weeks of sustained transgene expression and an absence of major lethal cytotoxicity in spiral ganglion and epithelial cells of the cochlea in adult animals. The ex vivo experiments were performed using fibroblasts transduced in vitro with Ad. RSVntlacZ. Two weeks after inoculation of the fibroblasts into the perilymph, we observed transplanted fibroblasts, which were adherent to the lining of the perilymphatic spaces, and were expressing the lacZ transgene. We speculate that, as the genetic basis of degenerative cochlear diseases is characterized on a mutational level, transgene expression will allow us to test hypotheses regarding the effects of specific genes on cochlear cell biology. Gene transfer will not only increase our understanding of the pathophysiology of hearing loss, but also may provide gene therapy for disease.

Expression plasmid DNA for the human interleukin-1 receptor antagonist (IL-1ra) protein was formulated with nonionic:cationic (NC) liposomes or phosphatidylcholine:cationic (PC) liposomes and applied to the auricular skin of hamsters in single- and multiple-dose protocols. Confocal microscopy identified delivery of plasmid DNA proximal to perifollicular cells, and successful transfection of perifollicular cells was identified by immunohistochemistry and ELISA. Skin treated for 3 days with the NC liposomes had statistically significant levels of transgenic IL-1ra present for 5 days post-treatment. Expression of transgenic IL-1ra was specific to areas of skin treated with NC liposomes but not PC liposomes. The results indicate that the NC liposomes can deliver expression plasmid DNA to perifollicular cells and mediate transient transfection in vivo.

The recent developments in human gene therapy have created a great deal of interest in using oral gene transfection approaches to induce the production and secretion of natural peptides and proteins by intestinal cells. In this study the receptor antagonist protein for interleukin-1 (IL-1ra), which shows potential therapeutic effects in inflammatory bowel disease (IBD), serves as a model protein for GI delivery. The target delivery of recombinant adenovirus encoding the IL-1ra protein to intestinal epithelium is suggested as a strategy for utilizing gene therapy for treatment of IBD. The human intestinal cell line Caco-2 was used as an in vitro model for gene transfer to the gastrointestinal epithelium. Adenovirus containing the β-galactosidase reporter gene (Ad.RSVntlacZ) infected Caco-2 monolayers with a marked preference for non-confluent and undifferentiated cells. In order to simulate a gastrointestinal environment, we showed that exposure of Ad.RSVntlacZ to gastrointestinal proteases and bile acids only slightly affected the infectivity of the virus over the testing period. Furthermore, variations of the luminal pH in the range of 5.5 to 7.4 did not lead to changes in infectivity. Infection of Caco-2 cells grown on permeable supports with the adenoviral vector containing the IL-1ra gene (Ad.RSVIL-1ra) resulted in an average secretion of 10 ng/day to the basolateral compartment, the target site for IBD therapy using this approach. Additionally, the cell viability and integrity of the monolayers were not affected by infection with adenoviral vectors. In summary, we have shown in vivo synthesis and secretion of a therapeutic protein using intestinal epithelial cells, indicating that this gene therapeutic approach not only may be valuable in treating IBD, but also has considerable potential for oral therapeutic protein delivery.

In Fisher's equation, the mechanism of logistic growth and linear diffusion are combined in order to model the spreading and proliferation of population, e.g., in ecological contexts. A Galerkin Finite Element method in two space dimensions is presented, which discretises a 1 + 2 dimensional version of this partial differential equation, and thus, providing a system of ordinary differential equations (ODEs) whose numerical solutions approximate those of the Fisher equation. By using a particular type of form functions, the off-diagonal elements of the matrix on the left-hand side of the ODE system become negligibly small, which makes a multiplication with the inverse of this matrix unnecessary, and therefore, leads to a simpler and faster computer program with less memory and storage requirements. It can, therefore, be considered a borderline method between finite elements and finite differences. A simple growth model for coral reefs demonstrates the program's adaptability to practical applications.

Purpose. Adenoviral (Ad) vectors have been used as efficient tools for gene therapy in various tissues, whereas in some differentiated epithelium transduction efficiency is almost abolished.

OBJECTIVE Osteomyelitis in the diabetic foot is a major risk factor for amputation, but there is a limited understanding of early-stage infection, impeding limb-preserving diagnoses. We hypothesized that bone composition measurements provide insight into the early pathophysiology of diabetic osteomyelitis. RESEARCH DESIGN AND METHODS Compositional analysis by Raman spectroscopy was performed on bone specimens from patients with a clinical diagnosis of osteomyelitis in the foot requiring surgical intervention as either a biopsy (n = 6) or an amputation (n = 11). RESULTS An unexpected result was the discovery of pathological calcium phosphate minerals in addition to normal bone mineral. Dicalcium phosphate dihydrate, also called brushite, and uncarbonated apatite were found to be exclusively associated with infected bone. CONCLUSIONS Compositional measurements provided a unique insight into the pathophysiology of osteomyelitis in diabetic foot ulcers. At-patient identification of pathological minerals by Raman spectroscopy may serve as an early-stage diagnostic approach.

In seven-day-old rats, intracerebral injection of N-methyl-D-aspartate transiently stimulates expression of Interleukin-1 beta messenger RNA. To evaluate the role of Interleukin-1 beta in the pathogenesis of excitotoxic injury, we sought to determine if Interleukin-1 receptor antagonist, an endogenous competitive inhibitor of Interleukin-1 beta, could attenuate N-methyl-D-aspartate-induced injury. To induce sustained over-expression of Interleukin-1 receptor antagonist in the brain, a recombinant adenovirus encoding Interleukin-1 receptor antagonist was administered by intracerebroventricular injection into three-day-old rats. Increased brain concentrations of Interleukin-1 receptor antagonist two to six days later were documented by assays of tissue homogenates and by immunocytochemistry. To evaluate the impact of Interleukin-1 receptor antagonist on N-methyl-D-aspartate neurotoxicity, three-day-old animals received intracerebroventricular injections of either adenovirus encoding Interleukin-1 receptor antagonist or a control adenovirus encoding beta-galactosidase, followed four days later by right intrastriatal injections of N-methyl-D-aspartate (10 nmol/0.5 microliter), a dose that typically elicits excitotoxic injury in the ipsilateral striatum and adjacent hippocampus, or saline. Animals were killed five days later, and brain damage was quantitated by measurement of bilateral cross-sectional areas of the striatum and anterior hippocampus. In three independent experiments, in N-methyl-D-aspartate-lesioned animals, both striatal and hippocampal injuries were reduced in animals that had been infected with adenovirus that encoded Interleukin-1 receptor antagonist, in comparison with littermates infected with the control adenovirus (right striatal volume loss ranged from 16 to 24%, compared with 54-65% volume loss in control). There was no striatal atrophy in adenovirus-infected saline-injected animals. These results provide strong support for the hypothesis that Interleukin-1 beta is a mediator of excitotoxic brain injury in perinatal rats.

Current gene therapy strategies using adenoviral vectors to target the lung or liver have been complicated by an acute inflammatory response that can result in loss of transgene expression as well as tissue injury and necrosis. Skeletal muscle comprises 40% of total body weight; it possesses a high density, accessible capillary network that is resistant to injury and thus may be a logical target for adenoviral vectors. We hypothesized that adenoviral transduction of the rat skeletal muscle capillary bed during vascular isolation would achieve efficient gene transfer sufficient to achieve systemic serum levels of a recombinant protein without significant tissue injury. During vascular isolation of the hindleg, a replication-incompetent adenovirus (Ad) encoding for either the marker gene, human placental alkaline phosphatase (hpAP), or interleukin-1 receptor antagonist (IL-1ra) was infused and subsequently flushed from the circulation after a 30-min dwell period. Gene transfer over a 10(9)-10(12) particle/ml range to the gastrocnemius capillary endothelium and muscle fibers was highly efficient and titer-dependent, reaching maximum transduction rates of 71 +/- 7% and 25 +/- 5%, respectively, 5 days after gene transfer (n = 3-8 rats/group, p0.05). hpAP transgene expression was barely detectable at 14 days. No significant tissue injury or necrosis of the skeletal muscle was observed at 5 and 14 days, and distant organ gene transfer was minimal or absent. Gastrocnemius muscle from rats (n = 4) given Ad-IL-1ra had 241 +/- 66 pg IL-1ra/mg protein at 5 days, while those given Ad-hpAP, negative control (n = 3) had 35 +/- 14 pg IL-1ra/mg protein (p0.05). Ad-IL-1ra rats (n = 4) had serum levels of 185 +/- 20 pg/ml IL-1ra at 5 days whereas Ad-hpAP control rats (n = 5) had no IL-1ra detectable (p0.0001). Athymic rats given Ad-IL-1ra (n = 6) had serum levels of 493 +/- 62 pg/ml IL-1ra 14 days after transduction, and IL-1ra was detected for up to 98 days. Sera from Ad-IL-1ra athymic rats significantly inhibited IL-1 beta-induced (1 ng/ml) prostaglandin E2 (PGE2) production from cultured endothelial cells by 82 +/- 2% (p0.001). Thus, this gene transfer strategy is the first to result in substantial transduction of both skeletal muscle capillary endothelium and fibers, sufficient to achieve pharmacologic levels of IL-1ra. Although no acute tissue injury or necrosis was observed, persistence of transgene expression in athymic rats suggests that loss of expression in normal rats was by an immune-mediated mechanism.

The diverse secretory and absorptive functions of the intestinal epithelium are conducted by a mixed population of absorptive cells and mucus-producing goblet cells as the major cell types. In order to approach the main characteristics in an in vitro model, a coculture system of absorptive Caco-2 cells and mucus-secreting HT29-MTX cells was developed and the permeability of a range of different drugs was tested. Variable goblet cell frequency can be achieved, preserving a significant barrier to drug transport and maintaining the differentiated features of both cell types. Absorption rates for actively transported drugs are rather underestimated in the cell culture model when compared to in vivo data. However, a good correlation with fraction absorbed in humans was attained separating the range of passively transported drugs into two groups of well-absorbable compounds with Peffor = 10 x 10(-6) cm/s and drugs that are absorbed 40-70% with Peff = 0.1-1 x 10(-5) cm/s. A permeability of Peff0.1 x 10(-5) cm/s is suggested for low absorbable drugs.

In this study we show that recombinant adenovirus can augment hypoxanthine-guanine phosphoribosyltransferase (HPRT) levels in the central nervous system (CNS) of HPRT-deficient mice. Recombinant adenovirus containing the cDNA for rat HPRT (rHPRT) expressed from the Rous sarcoma virus LTR (RSV LTR) was constructed (AdRSVrHPRT). AdRSVrHPRT was injected into the right caudate nucleus of 7-week-old HPRT-deficient mice. Brains were analyzed for gene transfer, transgene expression and function by DNA PCR, in situ RNA hybridization, and enzyme bioactivity. The results show that rHPRT cDNA delivered by an adenoviral vector can augment HPRT levels in brain tissue and documents the utility of gene transfer to restore HPRT activity in an HPRT-deficient CNS.

Degradable matrices containing expression plasmid DNA [gene-activated matrices (GAMs)] were implanted into segmental gaps created in the adult rat femur. Implantation of GAMs containing beta-galactosidase or luciferase plasmids led to DNA uptake and functional enzyme expression by repair cells (granulation tissue) growing into the gap. Implantation of a GAM containing either a bone morphogenetic protein-4 plasmid or a plasmid coding for a fragment of parathyroid hormone (amino acids 1-34) resulted in a biological response of new bone filling the gap. Finally, implantation of a two-plasmid GAM encoding bone morphogenetic protein-4 and the parathyroid hormone fragment, which act synergistically in vitro, caused new bone to form faster than with either factor alone. These studies demonstrate for the first time that repair cells (fibroblasts) in bone can be genetically manipulated in vivo. While serving as a useful tool to study the biology of repair fibroblasts and the wound healing response, the GAM technology may also have wide therapeutic utility.

Loss of ganglion cells is a common and irreversible complication of hair cell loss in the cochlea. Gene transfer could potentially be used to prevent this neuronal degeneration and other pathologies in the cochlea. Human adenoviruses should provide a feasible gene transfer vehicle for transducing the quiescent cochlear neurons and organ of Corti epithelium. We now describe in vivo experiments in which a replication-deficient adenoviral vector, Ad.RSVntlacZ was injected into the perilymphatic fluid of six normal guinea pigs. Postoperative recovery of animals was complete. Inner ear tissues were assessed for histology and for presence of lacZ-positive cells 1 or 2 weeks after the injection. A large number of blue (lacZ-positive) cells were observed in the neural, epithelial and connective tissues of the cochlea. In four ears spiral ganglion cell infection exceeded 50%, throughout the length of the cochlear spiral. No major pathology was detected in the organ of Corti and other cochlear tissues, and no infection was present in the vestibular tissues or the contralateral cochlea. Immunocytochemical assessment of T cells revealed an increased in the number of lymphocytes in the connective tissue lining the perilymphatic spaces. We conclude that efficient gene transfer into multiple types of cochlear cells in vivo can be achieved without major morphological signs of pathology or toxicity.

Recombinant adenovirus has recently become a promising gene delivery vehicle that may be used therapeutically for various medical disorders. However, in vivo expression of transgenes delivered by E1 region-deleted adenoviral vectors is transient in immunocompetent animals. It has been proposed that destruction of adenovirally transduced cells by the host immune mechanisms, particularly cytotoxic T-lymphocytes, may play a major role in limiting the duration of transgene expression in vivo. In the present study, Southern blot analysis of genomic DNA prepared from transduced liver tissues showed the persistent presence of the viral genome in both immunocompetent and immunodeficient animals, indicating the survival of the adenovirally transduced liver cells. Furthermore, active expression of the surviving factor IX transgenes was shown by the presence of recombinant human factor IX as well as specific human factor IX mRNA and protein in the transduced liver tissues. The transient appearance of human factor IX in the circulation of normal as well as partially immunodeficient mice is primarily due to the generation of mouse antihuman factor IX antibodies in these mice rather than host immune destruction of transduced cells. These results suggest that liver cells transduced with recombinant adenoviral vectors can escape from being destroyed by the host immune mechanism in normal animals. The present study thus provides a new rationale for further engineering of adenoviral vectors into a durable expression system for gene therapy of various diseases including congenital disorders such as hemophilia B.

We present a method which improves the determination of geometric parameters of unicellular organisms with prolate ellipsoid geometry, such as Dunaliella. Approximately determined axis lenghts were obtained from transmission electron micrographs of ultrathin sections of microalgae. The density functions generated by these ‘axis lengths’ have poles which can be used to determine the short half axis length and ratio of axis lengths of the ellipsoid in order to determine the true cell volume.

In this report, we present data showing that a recombinant adenoviral vector (Ad.RSVIL-1ra) containing the cDNA for human interleukin-1 receptor antagonist protein (IL-1ra) can genetically modify synoviocytes both in vitro and in vivo. Human synoviocytes infected with Ad.RSVIL-1ra in vitro expressed and secreted high levels of human IL-1ra that were detected by ELISA of tissue culture supernatants. New Zealand White rabbits that received intra-articular injections of Ad.RSVIL-1ra expressed transgenic IL-1ra in synoviocytes, and secretion was detected for at least 4 weeks post-infection. Further, biological activity of the transgenic IL-1ra was demonstrated by its ability to inhibit IL-1-induced prostaglandin E2 (PGE2) synthesis in vitro and IL-1-induced glycosaminoglycan (GAG) degradation in vivo. These data demonstrate that recombinant adenoviral vectors can mediate the intra-articular expression of anti-inflammatory proteins and may be a reasonable method to deliver therapeutically relevant proteins for the regional treatment of synovial inflammation.

Les traitements medicaux ont progressivement trouve leur place dans la prise en charge des hemangiomes et des malformations vasculaires afin, soit d’en accelerer la regression (hemangiome), soit de reduire la frequence des complications (malformations vasculaires). Les beta-bloquants ne sont plus discutes dans certaines localisations d’hemangiomes infantiles, mais leur plus large prescription en reste discutee. La cible des antithrombotiques dans les malformations veineuses est dominee par les episodes thrombotiques. Les anti-XA y ont pris une place importante dans cette indication. La Rapamycine (sirolimus) a ete proposee dans le traitement des lesions complexes symptomatiques, lymphatiques et veineuses. La cible pharmacologique de ce produit serait le controle de mutation dans les cellules endotheliales. Les experiences cliniques sont limitees, l’interet porte surtout sur la douleur, l’impact tissulaire est plus limite. Les beta-bloquants seraient interessants sur les malformations arterio-veineuses a haut debit. En l’absence de reduction du debit, l’objectif est de reduire la pression dans les ectasies veineuses en aval du nidus. Le but en est ainsi de limiter la souffrance cutanee en regard et les complications hemorragiques. C’est la premiere proposition therapeutique avant toute decision d’embolisation.

A method has been developed to measure the cell volume of the unicellular, green alga Dunaliella bioculata 19/4 during salt stress conditions, where shape change in the alga becomes problematic and the cells can no longer be recognised as 'prolate ellipsoids', by using image analysis of transmission electron micrographs. The image analysis of the micrographs employs a specialised numerical integration programme or 'variable frames analysis' for unicellular microorganisms which possess a single axis of symmetry. Basic mathematics was used to determine: (a) the functional dependence of the calculated volume on the angle of the cut to the axis of symmetry and the distance of the origin of the cut from the centre of mass; (b) errors resulting from the orientation of the longest axis off-vertical for image analysis; (c) the uppermost range of calculated volumes obtained which represent the 'true' volumes within required confidence levels. The procedure was applied to a series of experiments on the effects of salt stress on Dunaliella bioculata cells.

More than 50 mutations in the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus have been described, yet only 2 alter the AUG initiation codon. One, variant HPRT1151, results in Lesch-Nyhan syndrome (LNS), and the other, HPRTIllinois, results in partial HPRT deficiency. Although previously undetectable, we used a sensitive gel assay to demonstrate that HPRTIllinois is not only active, but has a native Mr indistinguishable from normal. Confirmatory evidence of activity and native Mr is demonstrated following transfection of HPRT cells with expression plasmids containing cDNA sequences representing HPRTIllinois. These data provide support for the hypothesis that patient RT, or variant HPRTIllinois, is spared manifestations of the LNS as a result of translation at the newly formed GUG initiation codon.

Regional alteration of brain function by directed gene transfer may allow for the investigation and treatment of both inherited and acquired diseases of brain. Examples of inherited CNS diseases that may be investigated by regional gene transfer include the mucopolysaccharidoses (MPS). a group of lysosomal storage diseases characterized by the accumulation of glycosaminoglycans within the neuroglial cells of the CNS [16]. Enzymatic correction directed to the CNS of animals with MPS would allow the analysis of the biochemical, histological and functional effects associated with transgene expression. Further, since metabolic cooperation occurs between various cell types within the brain, a regional correction of MPS deficiency may produce a more diffuse level of biochemical and/or functional improvement. On the other hand, neurodegenerative conditions such as Parkinson's or Alzheimer's are acquired diseases that exhibit a characteristic regional neuropathology, and amelioration of CNS manifestation may result from the regional expression of specific gene products [6,23]. These hypotheses could be tested by the development of methods to mediate the production of transgenic proteins in anatomically defined brain regions. Gene transfer to specific brain regions may be approached by direct somatic cell gene transfer. Since adult mammalian CNS cells do not readily undergo cell division and are long lived, they represent an ideal target for somatic cell gene transfer. Previous efforts at somatic cell

To support the translation of Raman spectroscopy into clinical applications, synthetic models are needed to accurately test, optimize and validate prototype fiber optic instrumentation. Synthetic models (also called tissue phantoms) are widely used for developing and testing optical instrumentation for diffuse reflectance, fluorescence, and Raman spectroscopies. While existing tissue phantoms accurately model tissue optical scattering and absorption, they do not typically model the anatomic shapes and chemical composition of tissue. Because Raman spectroscopy is sensitive to molecular composition, Raman tissue phantoms should also approximate the bulk tissue composition. We describe the fabrication and characterization of tissue phantoms for Raman tomography and spectroscopy. These phantoms have controlled chemical and optical properties, and also multilayer morphologies which approximate the appropriate anatomic shapes. Tissue phantoms were fabricated to support on-going Raman studies by simulating the human wrist and rat leg. Surface meshes (triangle patch models) were generated from computed tomography (CT) images of a human arm and rat leg. Rapid prototyping was used to print mold templates with complex geometric patterns. Plastic casting techniques used for movie special effects were adapted to fabricate molds from the rapid prototypes, and finally to cast multilayer gelatin tissue phantoms. The gelatin base was enriched with additives to model the approximate chemistry and optical properties of individual tissue layers. Additional studies were performed to determine optimal casting conditions, phantom stability, layer delamination and chemical diffusion between layers. Recovery of diffuse reflectance and Raman spectra in tissue phantoms varied with probe placement. These phantoms enable optimization of probe placement for human or rat studies. These multilayer tissue phantoms with complex geometries are shown to be stable, with minimal layer delamination and chemical diffusion.