Nfasc155H and MAG are specifically susceptible to detergent extraction in the absence of the myelin sphingolipid sulfatide.

Mice incapable of synthesizing the myelin lipid sulfatide form paranodes that deteriorate with age. Similar instability also occurs in mice that lack contactin, contactin-associated protein or neurofascin155 (Nfasc155), the proteins that cluster in the paranode and form the junctional complex that mediates myelin-axon adhesion. In contrast to these proteins, sulfatide has not been shown to be enriched in the paranode nor has a sulfatide paranodal binding partner been identified; thus, it remains unclear how the absence of sulfatide results in compromised paranode integrity. Using an in situ extraction procedure, it has been reported that the absence of the myelin sphingolipids, galactocerebroside and sulfatide, increased the susceptibility of Nfasc155 to detergent extraction. Here, employing a similar approach, we demonstrate that in the presence of galactocerebroside but in the absence of sulfatide Nfasc155 is susceptible to detergent extraction. Furthermore, we use this in situ approach to show that stable association of myelin-associated glycoprotein (MAG) with the myelin membrane is sulfatide dependent while the membrane associations of myelin/oligodendrocyte glycoprotein, myelin basic protein and cyclic nucleotide phosphodiesterase are sulfatide independent. These findings indicate that myelin proteins maintain their membrane associations by different mechanisms. Moreover, the myelin proteins that cluster in the paranode and require sulfatide mediate myelin-axon adhesion. Additionally, the apparent dependency on sulfatide for maintaining Nfasc155 and MAG associations is intriguing since the fatty acid composition of sulfatide is altered and paranodal ultrastructure is compromised in multiple sclerosis. Thus, our findings present a potential link between sulfatide perturbation and myelin deterioration in multiple sclerosis.

Adult CST-null mice maintain an increased number of oligodendrocytes.

The galactolipids galactocerebroside and sulfatide have been implicated in oligodendrocyte (OL) development and myelin formation. Much of the early evidence for myelin galactolipid function has been derived from antibody and chemical perturbation of OLs in vitro. To determine the role of these lipids in vivo, we previously characterized mice lacking galactocerebroside and sulfatide and observed abundant, unstable myelin and an increased number of OLs. We have also reported that mice incapable of synthesizing sulfatide (CST-null) while maintaining normal levels of galactocerebroside generate relatively stable myelin with unstable paranodes. Additionally, Hirahara et al. (2004; Glia 45:269-277) reported that these CST-null mice also contain an increased number of OLs in the forebrain, medulla, and cerebellum at 7 days of age. Here, we further the findings of Hirahara et al. by demonstrating that the number of OLs in the CST-null mice is also increased in the spinal cord and that this elevated OL population is maintained through, at least, 7 months of age. Moreover, we show that the enhanced OL population is accompanied by increased proliferation and decreased apoptosis of oligodendrocytic-lineage cells. Finally, through ultrastructural analysis, we show that the CST-null OLs exhibit decreased morphological complexity, a feature that may result in decreased OL competition and increased OL survival.

Heterologous expression of the invertebrate FMRFamide-gated sodium channel as a mechanism to selectively activate mammalian neurons.

Considerable effort has been directed toward the development of methods to selectively activate specific subtypes of neurons. Focus has been placed on the heterologous expression of proteins that are capable of exciting neurons in which they are expressed. Here we describe the heterologous expression of the invertebrate FMRFamide (H-phenylalanine-methionine-arginine-phenylalanine-NH2) -gated sodium channel from Helix aspersa (HaFaNaC) in hippocampal slice cultures. HaFaNaC was co-expressed with a fluorescent protein (green fluorescent protein (GFP), red fluorescent protein from Discosoma sp (dsRed) or mutated form of red fluorescent protein from Discosoma sp (tdTomato)) in CA3 pyramidal neurons of rat hippocampal slice cultures using single cell electroporation. Pressure application of the agonist FMRFamide to HaFaNaC-expressing neuronal somata produced large prolonged depolarizations and bursts of action potentials (APs). FMRFamide responses were inhibited by amiloride (100 microM). In contrast, pressure application of FMRFamide to the axons of neurons expressing HaFaNaC produced no response. Fusion of GFP to the N-terminus of HaFaNaC showed that GFP-HaFaNaC was absent from axons. Bath application of FMRFamide produced persistent AP firing in HaFaNaC-expressing neurons. This FMRFamide-induced increase in the frequency of APs was dose-dependent. The concentrations of FMRFamide required to activate HaFaNaC-expressing neurons were below that required to activate the homologous acid sensing ion channel normally found in mammalian neurons. Furthermore, the mammalian neuropeptides neuropeptide FF and RFamide-related peptide-1, which have amidated RF C-termini, did not affect HaFaNaC-expressing neurons. Antagonists of NPFF receptors (BIBP3226) also had no effect on HaFaNaC. Therefore, we suggest that heterologous-expression of HaFaNaC in mammalian neurons could be a useful method to selectively and persistently excite specific subtypes of neurons in intact nervous tissue.

New techniques for imaging, digitization and analysis of three-dimensional neural morphology on multiple scales.

Cognitive impairment in normal aging and neurodegenerative diseases is accompanied by altered morphologies on multiple scales. Understanding of the role of these structural changes in producing functional deficits in brain aging and neuropsychiatric disorders requires accurate three-dimensional representations of neuronal morphology, and realistic biophysical modeling that can directly relate structural changes to altered neuronal firing patterns. To date however, tools capable of resolving, digitizing and analyzing neuronal morphology on both local and global scales, and with sufficient throughput and automation, have been lacking. The precision of existing image analysis-based morphometric tools is restricted at the finest scales, where resolution of fine dendritic features and spine geometry is limited by the skeletonization methods used, and by quantization errors arising from insufficient imaging resolution. We are developing techniques for imaging, reconstruction and analysis of neuronal morphology that capture both local and global structural variation. To minimize quantization error and evaluate more precisely the fine geometry of dendrites and spines, we introduce a new shape analysis technique, the Rayburst sampling algorithm that uses the original grayscale data rather than the segmented images for precise, continuous radius estimation, and multidirectional radius sampling to represent non-circular branch cross-sections and anisotropic structures such as dendritic spine heads, with greater accuracy. We apply the Rayburst technique to 3D neuronal shape analysis at different scales. We reconstruct and digitize entire neurons from stacks of laser-scanning microscopy images, as well as globally complex structures such as multineuron networks and microvascular networks. We also introduce imaging techniques necessary to recover detailed information on three-dimensional mass distribution and surface roughness of amyloid beta plaques from human Alzheimer's disease patients and from the Tg2576 mouse that expresses the "Swedish" mutation of the amyloid precursor protein. By providing true three-dimensional morphometry of complex histologic structures on multiple scales, the tools described in this report will enable multiscale biophysical modeling studies capable of testing potential mechanisms by which altered dendritic structure, spine geometry and network branching patterns that occur in normal aging and in many brain disorders, determine deficits of functions such as working memory and cognition.

Preimplantation diagnosis of a lysosomal storage disorder by in situ enzymatic activity: 'proof of principle' in acid sphingomyelinase-deficient mice.

Genetic diagnosis of preimplantation embryos (PGD) can substantially reduce the chance that at-risk couples have children afflicted with inherited diseases. However, PGD requires DNA,which is usually obtained from single cells following embryo biopsy. In addition, PGD requires that the genetic defect(s) causing the disorder be known. We have therefore developed an alternative to PGD, which we term preimplantation enzymatic diagnosis (PED). PED has several advantages over PGD, including the facts that it does not require embryo biopsy and that the gene defect(s) causing the disorder need not be known. We have demonstrated 'proof of principle' for this approach using embryos obtained from a mouse model (ASMKO mice) of acid sphingomyelinase (ASM)-deficient Niemann-Pick disease, an inherited lysosomal storage disorder. For this technique, fluorescently (BODIPY)-conjugated sphingomyelin was used to detect ASM activity in situ. Wild-type, preimplantation embryos degraded the substrate following a short 'pulse-chase' period, resulting in markedly reduced fluorescence compared to ASMKO embryos, which retained the fluorescent substrate. Thus, the two embryo types could be easily distinguished by fluorescent microscopy. The fluorescent sphingomyelin was not toxic to the embryos, and the entire procedure could be accomplished within 48 h without embryo biopsy. We suggest that PED may be useful for the preimplantation diagnosis of lysosomal storage disorders, and perhaps other enzymatic defects where similar in situ assay methods are available.

Loss of Ly-6A.2 expression on immature developing T cells in the thymus is necessary for their normal growth and generation of the Vbeta T-cell repertoire.

Stage-specific expression of a number of cell-surface and signaling proteins is critical for normal development of T cells in the thymus. Equally important may be the loss of expression/signaling of developmentally regulated proteins for proper transitioning of developing T cells into thymic subsets. Ly-6A.2 exhibits a regulated pattern of expression on T cells maturing in the thymus, and dysregulating its expression results in arrest of developing T cells within the CD3-CD4-CD8- triple negative (TN) stage where the normal expression of Ly-6A.2 is extinguished. To further characterize the mechanisms underlying this block, we examined whether cell signaling and/or cell adhesion properties of the Ly-6A.2 molecule influenced the block in T-cell development. Analysis of bone marrow chimeras generated by injecting CFSE-labeled Ly-6A.2 transgenic bone marrow cells into irradiated syngeneic non-transgenic mice revealed normal trafficking of developing T cells from the cortex into the medulla. Production of LAT but not p56lck was diminished in CD4-CD8- DN cells from Ly-6A.2 dysregulated mice when compared with control littermates. Dysregulated expression of Ly-6A.2 did not suppress endogenous TCR-Vbeta expression. Finally, dysregulated expression of Ly-6A.2 enhanced apoptosis of an immature CD4+CD8+ (DP) subset of developing cells and altered the selected TCR-Vbeta repertoire. Taken together, these observations indicate that the termination of Ly-6A.2 expression and signaling within the CD4-CD8-CD3- subset of developing T cells is an important checkpoint during normal thymic development.

Thyroid organoid formation in simulated microgravity: influence of keratinocyte growth factor.

The generation of artificial human thyroid tissues in suspension (low-shear environment, present in simulated microgravity [MG] and generated by a rotary cell culture system [RCCS]), was enhanced by increasing medium kinematic viscosity with a (3% v/v) suspension of extracellular matrix (basement membrane extract [BME]) in serum-free medium to generate artificial human thyroid organoids. Recombinant human keratinocyte growth factor (KGF, 7 ng/mL) facilitated human thyrocyte aggregation and three-dimensional (3-D) differentiation. There was an MG-associated decrease in extractable DNA that was reversed after addition of keratinocyte growth factor (KGF). In simulated MG, the increase in extractable DNA after KGF addition was up to 170% over non-KGF control cultures. In contrast, monolayer cultures in unit gravity showed a maximum DNA increase of 39% after KGF addition. Morphologically, differentiated thyroid neofollicles displayed polarization and were located in close proximity after 2 weeks of culture. Immunogold labeling with antibody to human thyroglobulin (Tg) revealed staining of follicular lumina and secretory vesicles, and a time-dependent increase in human Tg was detected in the culture media. Culture under simulated MG thus allowed direct visualization of KGF-facilitated thyrocyte/extracellular matrix interaction. Such artificial human thyroid organoids-generated in MG and in the presence of KGF-structurally resembled natural thyroid tissue. The above findings may have implications for autoimmune thyroid disease where KGF (if, for example, secreted locally by intraepithelial gammadelta T cells among other cells) may contribute to thyroid cell growth.

Peroxisomal ghosts are intracellular structures distinct from lysosomal compartments in Zellweger syndrome: a confocal laser scanning microscopy study.

Peroxisome ghosts are aberrant peroxisomal structures found in cultured skin fibroblasts from patients affected by Zellweger Syndrome (ZS), a genetic disorder of peroxisomal assembly. They contain peroxisomal integral membrane proteins (PxIMPs) and they lack most of the matrix enzymes that should be inside the organelle (Santos et al., Science 239 (1988) 1536-1538). Considerable evidence indicates that these ghosts result from genetic defects in the cellular machinery for importing newly-synthesized peroxisomal proteins into the organelle. In contrast to these observations, (Heikoop et al., Eur. J. Cell Biol. 57 (1992) 165-171) report that in Zellweger Syndrome, peroxisomal membranes are located within lysosomes and/or contain lysosomal enzymes. We have undertaken a more detailed and systematic investigation of this matter, employing confocal laser scanning microscopy (CLSM). In fibroblasts derived from ZS patients belonging to different complementation groups, peroxisomes were labeled with antibodies against PxIMPs and lysosomes were labeled with an antibody against a lysosome associated membrane protein (LAMP-2) or with LysoTracker. The results unambiguously demonstrated no appreciable colocalization of PxIMPs and LAMPs (or LysoTracker), indicating that peroxisomal ghosts are distinct subcellular structures, occupying separate subcellular locations.

Unequal access to cadaveric kidney transplantation in California based on insurance status.

OBJECTIVE: To assess the impact of insurance status on access to kidney transplantation among California dialysis patients. STUDY SETTING: California Medicare and Medicaid dialysis populations. STUDY DESIGN: All California ESRD dialysis patients under age 65 eligible for Medicare or Medicaid in 1991 (n = 9,102) took part in this cohort analytic study. DATA COLLECTION: Medicare and California Medicaid Program data were matched to the Organ Procurement and Transplantation Network Kidney Wait List files. PRINCIPAL FINDINGS: Only 31.4 percent of California Medicaid dialysis patients were placed on the kidney transplant waiting list compared to 38.8 percent and 45.0 percent of dually eligible Medicate/Medicaid and Medicare patients, respectively. Compared to the Medicaid population, Medicare enrollees were more likely to be placed on the kidney transplant waiting list (adjusted Relative Risk [RR] = 2.10, Confidence Interval [CI] 1.68, 2.62) as were dually eligible patients (RR = 1.54, CI 1.24, 1.91). Once on the waiting list, however, Medicare enrollment did not influence the adjusted median waiting time to acquire a first cadaveric transplant (p > .05). CONCLUSIONS: California dialysis patients excluded from Medicare coverage, who are disproportionately minority, female, and poor, are much less likely to enter the U.S. transplant system. We hypothesize that patient concerns with potential subsequent loss of insurance coverage as well as cultural and educational barriers are possible explanatory factors. Once in the system, however, insurance status does not influence receipt of a cadaveric renal transplant.

Early events in the pathogenesis of avian salmonellosis.

Salmonellae are gastrointestinal pathogens of man and animals. However, strains that are host-specific avian pathogens are often avirulent in mammals, and those which are nonspecific are commensal in poultry. The objective of this study was to determine whether host specificity was exhibited by bacterial abilities to invade epithelial cells or resist leukocyte killing. In this study, leukocytes isolated from humans and chickens were used to kill Salmonella in vitro. Both Salmonella pullorum, an avian-specific serotype, and Salmonella typhimurium, a broad-host-range serotype, were sensitive to killing by polymorphonuclear leukocytes isolated from both species. Both serotypes replicated in cells of the MQ-NCSU avian-macrophage cell line. In contrast, S. pullorum was noninvasive for cultured epithelial Henle 407, chick kidney, chick ovary, and budgerigar abdominal tumor cells. In the bird challenge, however, S. typhimurium rapidly caused inflammation of the intestinal mucosa, but S. pullorum preferentially targeted the bursa of Fabricius prior to eliciting intestinal inflammation. Salmonella serotypes which cause typhoid fever in mice have been shown to target the gut-associated lymphoid tissue. Observations from this study show that S. pullorum initiated a route of infection in chicks comparable to the route it takes in cases of enteric fever.

ESI/ion trap/ion mobility/time-of-flight mass spectrometry for rapid and sensitive analysis of biomolecular mixtures.

An ion trap/ion mobility/time-of-flight mass spectrometry technique is shown to be a rapid and sensitive means of analyzing peptide/protein mixtures. In this approach, an ion trap is used to accumulate ions that have been electrosprayed from a mixture into concentrated packets. The ion packets are injected into a drift tube where components of the mixture are separated based on differences in mobility through a buffer gas. Ions that exit the drift tube are dispersed in a time-of-flight mass spectrometer for mass-to-charge (m/z) determination. The gas-phase separation strategy reduces congestion in the mass spectrum, and experimental mobilities complement m/z measurements in assigning peaks. Examples of the application of the approach to identification of peptides (from tryptic digests) and to separation of charge-state distributions from electrospray of a mixture containing ubiquitin and myoglobin are presented. Most peptides that are observed from tryptic digests of proteins such as cytochrome c and myoglobin can be identified from data that are acquired in under 1 min; studies of mixtures with known compositions indicate that detection limits are approximately 0.5-3 pmol for individual components. Factors that may influence the distributions that are observed, such as storage time in the trap, injection voltages used for the mobility experiment, and variations in ion cross section with charge state, are discussed.

Identification of a ligand-dependent switch within a muscarinic receptor.

G-protein-coupled receptors spontaneously switch between active and inactive conformations. Agonists stabilize the active conformation, whereas antagonists stabilize the inactive conformation. In a systematic search for residues that participate in receptor function, several regions of the m5 muscarinic receptor were randomly mutated and tested for their functional properties. Mutations spanning one face of transmembrane 6 (TM6) were found to induce high levels of receptor activity in the absence of agonists (constitutive activity). The same face of TM6 contained several residues crucial for receptor activation by agonists and one residue identified as a contact site for both agonists and antagonists. In addition, one mutation induced agonist-like responses from the receptor when exposed to classical antagonists. These results suggest that TM6 is a switch that defines the activation state of the receptor, and that ligand interactions with TM6 stabilize the receptor in either an active or an inactive conformation.

High-order structure and dissociation of gaseous peptide aggregates that are hidden in mass spectra.

Injected-ion mobility and high-pressure ion mobility techniques have been used to examine the conformations of bradykinin, insulin chain A, and several other peptide ions in the gas phase. Under the experimental conditions employed, evidence for multimer formation in the mass spectra of peptides is minimal or absent altogether. However, ion mobility distributions show that aggregates of peptides (containing a single charge per monomer unit) are observed at the same mass-to-charge ratios as the singly charged parent ions. Collision cross sections for these clusters show that they have tightly packed roughly spherical conformations. We have bracketed the average density as 0.87 < p < 1.00 g cm-3. In some cases, specific stable aggregate forms within a cluster size can be distinguished indicating that some high order structures are favored in the gas phase. Multimer formation between different sizes of polyalanine peptides shows no evidence for size specificity in aggregate formation. Collisional and thermal excitation studies have been used to examine structural transitions and dissociation of the multimers. Aggregates appear to dissociate via loss of singly charged monomers. The observation that peptide multimers can be concealed in mass spectral data requires that fragmentation patterns and reactivity studies of singly charged monomers be undertaken with care.

CD4+ T cells mature in the absence of MHC class I and class II expression in Ly-6A.2 transgenic mice.

The TCRs expressed on T lymphocytes recognize foreign peptides bound to MHC molecules. This reactivity is the basis of specific immune response to the foreign Ag. How such specificities are generated in the thymus is still being debated. Signals generated through TCR upon interaction with self MHC-peptide complexes are critical for maturation of the CD4+ helper and CD8+ cytotoxic subsets. We have observed maturation of CD4+ but not CD8+ T cells in Ly-6A.2 transgenic MHC null mice. Since there can be no interactions with MHC molecules in these mice, these CD4+ cells must express the T cell repertoire that exists before positive and negative selection. Interestingly, despite an absence of selection by MHC molecules, the CD4+ cells that mature recognize MHC molecules at a frequency as high as in CD4+ cells in normal mice. These results demonstrate that: 1) the germline sequences encoding TCRs are biased toward reactivity to MHC molecules; and 2) CD4+ cells as opposed to CD8+ cells have distinct lineage commitment signals. These results also suggest that signals originating from Ly-6 can promote or substitute for signals generated from TCR that are required for positive selection. Moreover, this animal model offers a system to study T cell development in the thymus that can provide insights into mechanisms of lineage commitment in developing T cells.

Influence of the NIH Consensus Conference on Helicobacter pylori on physician prescribing among a Medicaid population.

OBJECTIVES: In February 1994, an National Institutes of Health (NIH) Consensus Development Conference panel unequivocally recommended antimicrobial therapy to eradicate Helicobacter pylori in the treatment of peptic ulcer disease. The goal of this study was to determine if these recommendations resulted in a change in physician prescribing among an underserved population. METHODS: Computerized Pennsylvania Medicaid data from January 1993 through February 1996 were used to evaluate prescribing patterns in the year before and 2 years after the NIH conference. An interrupted time series model, based on 12,737 outpatient peptic ulcer disease encounters, assessed the impact of the conference in influencing physician prescribing. RESULTS: The prescription of antimicrobial agents for the treatment of peptic ulcer disease significantly increased across the study period, from 6.5% in January 1993 to 10.2% in February 1996. Similarly, the prescription rate for the proton pump inhibitor, omeprazole, significantly increased from 9.4% in January 1993 to 25.6% in February 1996. Neither trend, however, could be attributed to the NIH Consensus Development Conference. Stratification by physician specialty, ulcer type, nonsteroidal anti-inflammatory drug use, and patient demographics did not affect these results. The traditional treatment approach, using H2-receptor antagonists, remained the preferred pharmacotherapy (72% of all prescriptions). CONCLUSIONS: Two years after the highly publicized NIH conference on the eradication of Helicobacter pylori, antimicrobial agents were not widely prescribed among the Pennsylvania Medicaid population. In treating this underserved population, physicians do not appear to be using recommendations developed by an NIH expert panel based on recent scientific advances.

Abdominal adhesiolysis: inpatient care and expenditures in the United States in 1994.

BACKGROUND: Adhesion formation represents a major complication after lower abdominal operations. It is postulated that a shift in surgical practice in recent years toward the use of less invasive techniques, such as laparoscopy, may be associated with a reduction in the incidence of intraperitoneal adhesions and in the rate of adhesiolysis procedures. Using an attributable-risk methodology, this cost-of-illness study was designed to estimate the hospitalization rate and expenditures for adhesiolysis in the United States in 1994 and to examine changes in attributable expenditures since 1988. STUDY DESIGN: A national hospital discharge data base was used to identify all abdominal adhesion procedures performed in the United States in 1994. Total hospitalization expenditures were based on Medicare payment rates for adhesiolysis hospitalizations and physician services, which were applied to the total number of inpatient days attributed to adhesiolysis. The results were compared with published rates and expenditures attributed to adhesiolysis in 1988. RESULTS: Adhesiolysis was responsible for 303,836 hospitalizations during 1994, primarily for procedures on the digestive and female reproductive systems. These procedures accounted for 846,415 days of inpatient care and $1.3 billion in hospitalization and surgeon expenditures. CONCLUSIONS: Although the adhesiolysis hospitalization rate has remained constant since 1988, inpatient expenditures have decreased by nearly 10% because of a 15% decrease in the average length of stay. The increased use of laparoscopy during this 6-year period does not appear to be associated with a concomitant reduction in the adhesiolysis hospitalization rate, suggesting that the causes of adhesion formation warrant further research.

Compound heterozygous protein C deficiency resulting in the presence of only the beta-form of protein C in plasma.

About 30% of human plasma protein C (PC) is of lower molecular weight than the predominant alpha-form. The minor beta-form arises as a consequence of the lack of glycosylation at Asn329. Although the functional role of Asn329 has been investigated by in vitro mutagenesis, until now no naturally occurring mutations have been reported at this site. We describe here the case of two identical twin sisters compound heterozygous for two novel PC mutations: Cys78-->Stop inherited from the maternal side and Asn329-->Thr inherited from the paternal side, associated with the presence of only the beta-form of PC in plasma. The Cys78-->Stop substitution is predicted to abolish PC synthesis from one allele, whereas the Asn329-->Thr substitution results in the reduced synthesis of a beta PC variant with decreased functional activity. PCN329T from the two monovular twin sisters was purified and its active form APCN329T was assessed for its ability to inactivate factor Va. Whereas no differences were observed between the activation rates of normal PC and PCN329T, APCN329T inactivated human factor Va with a rate slower than the normal APC. This is the first report of a PC defect involving glycosylation of the molecule. This defect results in the presence of only the beta-form of PC in human plasma and is responsible for the reduced anticoagulant activity observed.

Dacron stimulation of macrophage transforming growth factor-beta release.

This study evaluated the effect of Dacron on the release of macrophage transforming growth factor-beta (TGF-beta),an endothelial cell growth inhibitor. Rabbit peritoneal macrophages were grown in minimum essential medium (MEM) with 10% fetal bovine serum (FBS) in the presence or absence of Dacron (0.5 mm x 3 mm particles). Media were collected three times each week for 7 weeks. For the TGF-beta bioassay, mink lung epithelial cells (CCL64) were grown in MEM with 10% FBS. Test-conditioned media, 100 mu 1, were added (n = 4), and incubated 48 h. 3(H)-Thymidine (3(H)-TdR) uptake was determined and compared with 3(H)-TdR uptake using known pure TGF-beta standards. Media samples were additionally pre-incubated with a neutralizing anti-TGF-beta(1) antibody and the 3(H)-TdR uptake again quantitated. TGF-beta activity in the conditioned media of macrophages exposed to Dacron exceeded the control media groups in all weeks, reaching significance (P<0.05) in weeks 3, 4,5, 6 and 7. Pre-incubation of media samples with the anti-TGF-beta antibody inhibited this TGF-beta activity in all weeks with statistical significance in weeks 1, 2, 3, 5 and 7. The inhibitory effects of Dacron on endothelialization may be explained by the Dacron-induced release of TGF-beta from macrophages.

Basic fibroblast growth factor production in vitro by macrophages exposed to Dacron and polyglactin 910.

Macrophage activation by implanted blood-contacting biomaterials modulates smooth muscle cell and endothelial cell ingrowth. The present study evaluates the in vitro interactions between Dacron or polyglactin 910 with macrophages derived from rabbits fed either normal or atherogenic diets. Peritoneal macrophages were cultured in the presence or absence (negative controls) of either biomaterial for 7 weeks. Conditioned media was evaluated for mitogenic activity using a rabbit aortic smooth muscle cell bioassay with or without preincubation with neutralizing anti-basic-FGF antibody. Results demonstrated increased mitogen release from macrophages harvested from the atherosclerotic rabbits. Only macrophages harvested from normal diet fed rabbits increased their mitogen release following exposure to either polyglactin 910 (p < 0.05) or to Dacron (p < 0.005) over controls. The stimulation of mitogen release by polyglactin 910 did not significantly exceed that in response to Dacron. In rabbits fed normal diets neutralization with the anti-basic-FGF antibody inhibited 100% of the Dacron induced mitogen release as compared to 36% of the polyglactin 910 induced mitogen release (p < 0.01). These results demonstrate significant induced mitogen release from macrophages exposed to biomaterials in vitro, much of the smooth muscle cell mitogen represented by basic-FGF.

Polypropylene small-diameter vascular grafts.

Polypropylene's physical properties (e.g., high tensile strength) and relatively inert behavior suggest that fabrication into an arterial substitute may result in an efficacious prosthesis. Grafts were woven from polypropylene yarn into conduits 4 mm I.D. x 50 mm in length. Control grafts were Dacron and ePTFE. Baseline platelet aggregometry on all dogs was performed with 10(-5) M ADP. Aspirin and dipyridamole were given for three days preoperatively and maintained for 2 weeks after surgery. Fifty-four grafts were placed into the aortoiliac position, two different graft materials per dog. The grafts were explanted at intervals of 2 weeks through 16 months; photographed for thrombus-free surface area determinations; and preserved for light, scanning, and transmission electron microscopy. Late (4-16 month) patency was 81% (13/16) for polypropylene, 69% (9/13) for Dacron, and 20% (1/5) for ePTFE. These data include one year patencies of 11/12 (92%) for polypropylene and 7/10 (70%) for Dacron. Late patency for polypropylene grafts was better than for PTFE (p less than 0.05). Platelet aggregation status did not predict graft patency. Light microscopy of 2-week polypropylene explants showed inner capsules composed of myofibroblasts and macrophages, with patchy areas of endothelial cells lining the lumen. By 1 month, a confluent endothelialized surface was seen in all polypropylene explants. Progressive thickening of inner capsules with myofibroblasts and collagen continued through 4 months, reaching a mean thickness of 142 +/- 50 microns (compared to 150 +/- 30 microns for Dacron). These findings suggest potential clinical efficacy for polypropylene as an arterial substitute.