Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation.     

Mn2+ activates skinned smooth muscle cells in the absence of myosin light chain phosphorylation

Two effects of Mn²⁺ on skinned fibers from chicken gizzard smooth muscle were observed, dependent on the presence of absence of dithiothreitol (DTT) reducing agent. One involves protein oxidation (in the absence of DTT) with production of a latch-like state, and the other involves direct Mn²⁺ activation of contractile proteins. Cells activated by Mn²⁺ in the presence of ATP and the absence of Ca²⁺, Mg²⁺ and DTT did not relax when transferred to normal relaxing solutions. In contrast, when 5 mM DTT was included in the Mn²⁺ contracting solution to prevent protein oxidation by Mn²⁺, the cells still contracted when exposed to Mn²⁺, but relaxed rapidly when the Mn²⁺ was removed. In the presence of DTT both the Mn²⁺ activation and the relaxation following removal of Mn²⁺ were more rapid than normal Ca²⁺-activated contractions and relaxations. The skinned fibers activated by Mn²⁺ in the absence of DTT showed little active shortening unless DTT was added. This rigor-like state is probably due to oxidation of contractile proteins since the cells relaxed when exposed to a relaxing solution containing DTT (50mM) and then contracted again in response to Ca²⁺ and relaxed normally. The Mn²⁺ activation was not associated with myosin light chain phosphorylation, in contrast to Ca²⁺-activated contractions.A preliminary report of this work was given at the Biophysical Society Meeting, February 1987: Hoar PE, Kerrick WGL (1987) Mn²⁺ activates skinned smooth muscle cells directly without myosin light chain phosphorylation and by reversible oxidation. Biophys J 51:332a     

A comparison of two data analyses from two observer performance studies using Jackknife ROC and JAFROC

The authors compared two methodological approaches, Jackknife ROC and JAFROC, in analyzing data ascertained during FROC (free-response receiver operating characteristics) type studies. Observer rating data obtained from two observer performance studies were analyzed. During the first study, seven radiologists interpreted 120 mammography examinations depicting 57 masses under five different conditions with and without the results of computer-aided detection (CAD). In the second study, eight radiologists interpreted 110 examinations depicting 51 masses under six different display conditions with and without CAD results. Readers rated the detection task in a FROC type response. Jackknife ROC (using the software of LABMRMC with the highest rating per case) and JAFROC were used to compute differences, if any, in summary performance levels among all reading modes in each study as well as for all paired data sets. The results of the different analytical approaches are compared. The overall results for all modes were significantly different for the first study (p < 0.05) and not significant (p > 0.05) for the second study using either analytical approach. In the first study, the performance levels represented by three paired data sets were significantly different (p < 0.05) when computed using LABMRMC and four pairs were significantly different (p < 0.05) using JAFROC. In eight of ten pairs, JAFROC produced lower p values than LABMRMC. In the second study, LABMRMC showed no significant differences for any paired data sets and JAFROC showed a significant difference for one pair. In 15 of 16 pairs, p values computed by JAFROC were lower than those computed by LABMRMC.     

Factors associated with the response of the cell to herpes simplex virus infection

Summary Four factors were shown to participate in the outcome of herpes simplex infection in tissue cultures: (a) the innate susceptibility of the cell (b) temperature (c) antibody and (d) interferon. Antibody appeared to function only to localize infection in KB cells (it failed to protect rabbit kidney cells) when added to the cultures following adsorption of virus. The critical factor in long-term protection of cells was a substance with several properties of interferon. The substance was produced when the entire infectious process was allowed to proceed at 35° C but was not produced under conditions where infected cells were first maintained at 25° C and later shifted to 35° C. Under the latter conditions antibody failed to restrain the progress of cellular destruction. Death of the total population occurred whenever interferon was not produced or was deliberately removed. Survival of the majority of cells was possible in the absence of antibody only as long as interferon was present in the culture. Certain findings also imply that although at 25° C there was neither virus multiplication nor interferon production, an early event(g) leading to the former and apparently blocking the latter takes place at this suboptimal temperature.This investigation was supported by Public Health Service Training Grant No. CA 5075 from the National Cancer Institute, Public Health Service General Research Support Grant No. FR 5516 and Office of Naval Research Contract No. 3310(00).     

Severe, late-onset graft-versus-host disease in a liver transplant recipient documented by chimerism analysis

A 52-year-old liver transplant recipient presented 8 months after transplantation with oral thrush, then 3 days later with oral ulcers and a diffuse rash, and 5 days later with an acutely reduced white blood cell count, rash, fever, and diarrhea. Bone marrow biopsy revealed severe aplasia. Although graft-versus-host disease (GVHD) was considered, the late onset of these symptoms was felt to render this etiology unlikely because GVHD usually occurs 2 to 6 weeks after transplantation. All potentially myelosuppressive medications were discontinued, and the patient was treated with high doses of hematopoietic growth factors. Because his symptoms continued, chimerism analysis was performed, which indicated that 96% of the peripheral blood mononuclear cells were of liver-donor origin. Ultimately, the patient underwent an allogeneic peripheral blood hematopoietic progenitor cell transplant from a human leukocyte antigen-identical brother, but he died 5 days after transplantation of overwhelming Candida kruseii infection. To our knowledge, this is the first chimerism-analysis-documented case of severe acute GVHD presenting so late after liver transplantation. It is of note that the patient had no known risks for GVHD in that he was relatively young and shared only one major human leukocyte antigen with his donor. Consideration should be given to GVHD as a cause of bone marrow aplasia at any time after organ transplantation. Storage of cell pellets from all transplant recipients and donors is highly recommended to facilitate the diagnostic evaluation.     

Radiology information systems,picture archiving and communication systems,teleradiology— Overview and design criteria

Information technology (IT), long taken for granted in commercial settings, is now being utilized for healthcare applications. Medical imaging has lagged comparatively due to the extremely vast data content of each frame; thus, the requirement for expensive high-end components. Further, IT in radiology has evolved from two distinctly separate camps—information systems, known as RIS (radiology information systems) and PACS (picture archiving and communications systems). Both RIS and PACS applications have migrated to the PC environment, enabling cost-effective implementation, but from two backgrounds: RIS from vendors using conventional information systems platforms and products, and PACS from radiographic film and modality vendors. The radiology department at Texas Tech University has assembled a seamlessly integrated, enterprise-wide RIS/PACS/teleradiology intranet. The design criteria include user-friendliness, flexibility to respond to changing needs, and open modular architecture to assure interoperability, cost-effectiveness, and future-proofing of investment. Since no single venor could provide an integrated system meeting our specifications, we decided to assume the burden of constructing our own system. As the system integrator, we embrace open architecture, thus enabling the incorporation of industry-standard-compliant, COTS (commercially off the shelf) products as modules. Microsoft Windows NT operating system, Visual C++ programming language, TCP/IP (transmission control protocol/internetworking protocol), relational SQL (structured query language) database, ODBC (open database connectivity), HL-7 (health level seven) and DICOM (digital imaging and communications in medicine) interfaces are utilized. The usage of COTS components reduces the cost to very affordable levels. With this approach, any module in our system can be replaced when outmoded, without affecting other modules in our system, making it truly future-proof. Construction and evolution of our system (TECHRAD) is reviewed.     

Relative carriage rates of nuclear dehydrogenating clostridia in two populations of different colorectal cancer risk

Carriage of nuclear dehydrogenating clostridia has been associated with colon cancer and implicated in its aetiology. This study has compared the carriage of these organisms in a British population at high risk for the development of colon cancer with a low risk Nigerian population. Clostridia were found in all of the stools from both populations. Nuclear dehydrogenating clostridia were only found in the stools of the British subjects (32%). These results support the suggestion that the carriage rate of nuclear dehydrogenating clostridia in a population is related to the risk of colon cancer.     

Autoimmune associated recurrent abortions

A possible relationship between recurrent spontaneous abortionsand autoimmune abnormalities was studied. Eight serologicalautoimmune or autoimmune-correlated parameters were investigatedin 91 women with unexplained recurrent abortions (3 consecutive,spontaneous abortions) and 89 fertile control women. Five parameterswere seen significantly more frequently in 19 women with atleast one second trimester miscarriage which had been associatedwith severe intrauterine growth regardation (IUGR), than incontrols. Seventeen of these 19 patients (89%) had at leastone positive autoimmune parameter, compared to 15 of 72 patients(21%) with no second trimester abortions with IUGR (P < 0.0001)and 14 (16%) of the controls (P < 0.0001). No single autoantibodycharacterized patients who exhibited a significant accumulationof autoimmune parameters. These findings may suggest that womenwith recurrent abortions, in whom autoimmunity is thought toplay a role, cannot be identified merely by one laboratory assay,such as that for cardiolipin antibodies, but must be definedby positivity of several criteria. Using our own test panel,preliminary clinical and serological criteria have been setup for the definition of an autoimmune-associated recurrentabortion condition. Twenty-three per cent of the patients inour material fulfilled these criteria, and seven out of nineof these women (78%) have to date been treated successfullywith heparin/aspirin during pregnancy.     

Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis - acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.