Short-, mid-, and long-term effects of a polymer-free tacrolimus-eluting stent in a porcine coronary model

Stent-based delivery of tacrolimus has shown neointimal hyperplasia and restenosis reduction; FK506 is a water insoluble macrolide immunosuppressant. The purpose of this study was to evaluate acute and chronic tissue response to a polymer-free FK506 drug-eluting stent implantation in a porcine coronary artery model. Seventy-eight nonatherosclerotic minipigs underwent successful placement of 134 stents (control n = 56; FK506 (1.5 microg/mm(2)) n = 44; FK506 (2.6 microg/mm(2)) n = 34) at 7, 15, 30, 90, or 180 days. Endothelialisation was almost complete at 7 days, complete at 15 days. At 30 and 90 days, mean neointimal thickness, neointimal area, and % stenosis was significantly less for drug-eluting stents compared with controls. At 180 days, histomorphometric values were similar for eluting and control stents. The FK506-eluting stent allows for a complete re-endothelialisation at 15 days and favorably moderate neointimal hyperplasia at 30 and 90 days in the porcine coronary model. Because of a possible limited bioavailability of FK506, long-term inhibition of neointimal formation was not sustained at the considered follow-up.     

Comparison of stent graft, sirolimus stent, and bare metal stent implanted in patients with acute coronary syndrome: clinical and angiographic follow-up

Strozzi and Anić     

Polymerized degradable hyaluronan--a platform for stent coating with inherent inhibitory effects on neointimal formation in a porcine coronary model

Biodegradable hyaluronan (hyaluronic acid, HA) made insoluble by self-cross-linking in the presence of N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide (EDC) has been used to cover stents. The maximum polymer-mass on a 16-mm stainless steel stent is approximately 2 mg. During manual crimping and simulated application, the loss of polymerized HA is negligible. The insoluble HA coating has an advantageous inherent antiproliferative effect regarding neointimal formation after local vessel wall injury (overstretch model) and leads to a reduced inflammatory response compared to uncoated stainless-steel stents, used as control, in undiseased pig coronary arteries, over a follow-up period of four weeks. Thus, cross-linked HA stent coating warrants further research as an interactive degradable biomaterial with an inherent inhibitory effect on neointimal formation as a possible biomatrix for local drug delivery to reduce restenosis rate.     

Effect of p27 deficiency and rapamycin on intimal hyperplasia: in vivo and in vitro studies using a p27 knockout mouse model.

SUMMARY: Rapamycin, an immunosuppressant and antiproliferative agent, reduces intimal hyperplasia after arterial injury in animal models and in a preliminary study in humans. Rapamycin treatment reportedly increases expression of p27, a cyclin-dependent kinase inhibitor. This mechanism was tested using a p27-deficient (p27 -/-) murine model. Aortic smooth muscle cells from wild-type (WT) and p27 -/- mice were isolated and cultured. Cell proliferation, assessed by cell count and (3)H-thymidine incorporation, was inhibited significantly by rapamycin in WT and p27 -/- cells at concentrations of 1 ng/ml, 10 ng/ml, and 100 ng/ml (p < 0.05, versus control). The in vivo effect on intimal hyperplasia was studied in p27 -/- and WT mice after femoral artery transluminal injury. Rapamycin treatment was started 2 days before injury and maintained for 2 weeks (1 mg/kg per 48 hours, ip). No significant differences in intima-to-media ratio were found between WT (1.1 +/- 0.1) and p27 -/- mice (1.0 +/- 0.1) 4 weeks after injury. Rapamycin significantly (p < 0.05) reduced intima-to-media ratios in both WT (0.7 +/- 0.1) and p27 -/- mice (0.5 +/- 0.1), compared with untreated mice. p27 deficiency did not alter the arterial wall proliferative response to injury. The inhibitory effect of rapamycin on intimal hyperplasia occurred via a p27-independent mechanism. The in vitro data showed that this effect was mediated through decreased proliferation and enhanced apoptosis.     

A novel Staphylococcus aureus vaccine: iron surface determinant B induces rapid antibody responses in rhesus macaques and specific increased survival in a murine S. aureus sepsis model

Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.     

A novel frameshift mutation 840delA and a novel polymorphism D203A in the steroidogenic acute regulatory protein gene in a Japanese patient with congenital lipoid adrenal hyperplasia

Congenital lipoid adrenal hyperplasia (CLAH) is an autosomalrecessive disorder characterized by impaired production of allsteroids including glucocorticoids, mineralocorticoids and sexsteroids. It has recently been reported that mutations in thesteroidogenic acute regulatory protein (StAR) gene cause CLAH. We analyzed the StAR gene in a Japanese patient with CLAH. The patient was revealed to be a compound heterozygote bearing a nonsense mutation Q258X, changing codon 258 (CAG) encoding Gln to the stop codon TAG, and a novel frameshift mutation 840delA resulting from deletion of one of the three adenosines normally present in codon 238 (AAA), thus leading to a frameshift after codon 237 (Thr) in the StAR gene. The patient was also revealed to be homozygous for a novel missense point mutation D203A, changing codon 203 (GAC) encoding Asp to GCC encoding Ala in the StAR gene. To elucidate the significance of the D203A mutation, we analyzed the StAR gene sequence in twenty normal subjects, and found that all of them were homozygous for the D203A mutation, indicating that the D203A mutation is an innocent polymorphism. In conclusion, we have identified a novel frameshift mutation 840delA which seems to cause 840delA and the first polymorphism D203A in the human StAR gene. Hum Mutat 11:331, 1998. © 1998 Wiley-Liss, Inc.     

Polycarbonate membranes: a novel surface for solid-phase determinations with utility in field format serological assays

A dipstick suitable for immunoassay procedures has been developed, and its utility demonstrated by the development of tests for the determination of immunoglobulin levels in newborn bovine blood, and for the detection of bovine antibodies against Brucella abortus. The dipstick is of simple design, consisting of three components: a polycarbonate membrane, an adhesive, and a support material. The polycarbonate membrane is a porous filter material upon which the immunoassay is undertaken. Biomolecules bind to the membrane with sufficient affinity to permit the development of useful immunoassay procedures; however, this membrane exhibits low non-specific binding of reagents. Collectively, these properties make polycarbonate a useful material for solid-phase immunoassay procedures.     

A novel and simple cell-based detection system with a collagen-encapsulated B-lymphocyte cell line as a biosensor for rapid detection of pathogens and toxins

Cell-based biosensors (CBBs) are becoming important tools for biosecurity applications and rapid diagnostics in food microbiology for their unique capability of detecting physiologically hazardous materials. A multi-well plate-based biosensor containing B-cell hybridoma, Ped-2E9, encapsulated in type I collagen matrix, was developed for rapid detection of viable cells of pathogenic Listeria, the toxin listeriolysin O, and the enterotoxin from Bacillus species. This sensor measures the alkaline phosphatase release from infected Ped-2E9 cells colorimetrically. Pathogenic L. monocytogenes cells and toxin preparations from L. monocytogenes or B. cereus showed cytotoxicity ranging from 24 to 98% at 3-6 h postinfection. In contrast, nonpathogenic L. innocua (F4247) and B. subtilis induced minimal cytotoxicity, ranging only 0.4-7.6%. Laser scanning cytometry and cryo-nano scanning electron microscopy confirmed the live or dead status of the infected Ped-2E9 cells in gel matrix. This paper presents the first example of a cell-based sensing system using collagen-encapsulated mammalian cells for rapid detection of pathogenic bacteria or toxin, and demonstrates a potential for onsite use as a portable detection system.     

Screw oversizing for anterior cruciate ligament graft fixation in primary and enlarged tibial tunnels: A biomechanical study in a porcine model

Background

Ideal diameter for tibial interference screw fixation of the anterior cruciate ligament (ACL) graft remains controversial. Tibial graft fixation with screws matching the tunnel diameter vs. one-millimetre oversized screws were compared.

A model for the rapid vegetative segregation of multiple chloroplast genomes in Chlamydomonas: Assumptions and predictions of the model

Summary Physical evidence indicates that the chloroplast DNA of Chlamydomonas reinhardtii is composed of approximately 75 copies of a small unique sequence. Genetic analysis of zygotes biparental for chloroplast genes shows rapid vegetative segregation of parental chloroplast alleles. Zygote clones composed entirely of homoplasmic progeny cells predominate within 10–20 post-mating generations. A model is proposed here which reconciles the high multiplicity of chloroplast genes with their rapid vegetative segregation rates. Clustering of genomes into a small number of discrete areas (nucleoids) within the chloroplast reduces the effective number of segregating units. A non-random distribution of nucleoids to daughter cells, dictated solely by the spatial arrangement of parental nucleoids with respect to the plane of chloroplast division, further increases the rate of segregation from heteroplasmic cells. Recombination between parental chloroplast genomes is viewed as an indication of nucleoid fusion, and can account for differences in the patterns and rates of segregation at different gene loci. Within such fused nucleoids, clustering of parental genomes and a non-random distribution, again based solely on physical positioning of the genomes, to daughter nucleoids, could act to promote rapid genetic purification of heteroplasmic nucleoids. The effects of biased parental nucleoid ratios, and of potentially unequal nucleoid distributions to daughter chloroplasts are also discussed with respect to observed rates and patterns of chloroplast gene segregation.     

Comparison of commercial latex agglutination and sandwich enzyme immunoassays with a competitive binding inhibition enzyme immunoassay for detection of antigenemia and antigenuria in a rabbit model of invasive aspergillosis

A commercial latex agglutination assay (LA) and a sandwich enzyme immunoassay (SEIA) (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) were compared with a competitive binding inhibition assay (enzyme immunoassay [EIA]) to determine the potential uses and limitations of these antigen detection tests for the sensitive, specific, and rapid diagnosis of invasive aspergillosis (IA). Toward this end, well-characterized serum and urine specimens were obtained by using a rabbit model of IA. Serially collected serum or urine specimens were obtained daily from control rabbits or from rabbits immunosuppressed and infected systemically with Aspergillus fumigatus. By 4 days after infection, EIA, LA, and SEIA detected antigen in the sera of 93, 93, and 100% of A. fumigatus-infected rabbits, respectively, whereas antigen was detected in the urine of 93, 100, and 100% of the rabbits, respectively. False-positive results for non-A. fumigatus-infected rabbits for EIA, LA, and SEIA were as follows: for serum, 14, 11, and 23%, respectively; for urine, 14, 84, and 90%, respectively. Therefore, although the sensitivities of all three tests were similar, the specificity was generally greater for EIA than for LA or SEIA. Infection was also detected earlier by EIA, by which the serum of 53% of A. fumigatus-infected rabbits was positive as early as 1 day after infection, whereas the serum of only 27% of the rabbits tested by LA was positive. Although the serum of 92% of A. fumigatus-infected rabbits was positive by SEIA as early as 1 day after infection, the serum of a high percentage (50%) was false positive before infection. The urine of 21% of A. fumigatus-infected rabbits was positive by EIA as early as 1 day after infection, and the urine of none of the rabbits was false positive before infection. When EIA results for urine specimens were combined with those for serum, sensitivity was improved (i.e., 67% of rabbits were positive by 1 day after infection and only one rabbit gave a false-positive result). A total of 93% of A. fumigatus-infected rabbits were positive for antigen in urine as early as 1 day after infection and the urine of 100% of the rabbits was positive by SEIA. However, before infection, 79% of A. fumigatus-infected rabbits were false positive for antigen in urine by LA and 90% were false positive for antigen in urine by SEIA. These data indicate that the EIA has the potential to be used to diagnose IA with both serum and urine specimens and to detect a greater number of infections earlier with greater specificity than the specificities achieved with the commercial tests.     

A percutaneous implant using a porous metal surface coating for adhesion to bone and a velour covering for soft tissue attachment: results of trials in pigs.

A percutaneous implant for the attachment of an artificial limb has been designed and tested in 14 pigs. Firm fixation to bone was achieved with the porous-surface layered metal intramedullary stem design in some cases. Dacron velour was used at the soft tissue interface. Evidence of soft tissue ingrowth was seen. However, the velour was unable to maintain adequate epithelial adhesion to form an anatomical seal and a barrier to bacteria.     

A novel frameshift mutation 840delA and a novel polymorphism D203A in the steroidogenic acute regulatory protein gene in a Japanese patient with congenital lipoid adrenal hyperplasia. Mutations in brief no. 117. Online

Congenital lipoid adrenal hyperplasia (CLAH) is an autosomalrecessive disorder characterized by impaired production of all steroids including glucocorticoids, mineralocorticoids and sexsteriods. It has recently been reported that mutations in the steriodogenic acute regulatory protein (StAR) gene cause CLAH. We analyzed the StAR gene in a Japanese patient with CLAH. The patient was revealed to be a compound heterozygote bearing a nonsense mutation Q258X, changing codon 258 (CAG) encoding Gln to the stop codon TAG, and a novel framshift mutation 840delA resulting from deletion of one of the three adenosines normally present in codon 238 (AAA), thus leading to a frameshift after codon 237 (Thr) in the StAR gene. The patient was also revealed to be homozygous for a novel missense point mutation D203A, changing codon 203 (GAC) encoding Asp to GCC encoding Ala in the StAR gene. To elucidate the significance of the D203A mutation, we analyzed the StAR gene sequence in twenty normal subjects, and found that all of them were homozygous for the D203A mutation, indicating that the D203A mutation is an innocent polymorphism. In conclusion, we have identified a novel frameshift mutation 840delA which seems to cause 840delA and the first polymorphism D203A in the human StAR gene.     

Comparison of the Gen-Probe Group A streptococcus Direct Test with culture and a rapid streptococcal antigen detection assay for diagnosis of streptococcal pharyngitis.

The Gen-Probe Group A Streptococcus Direct Test (GP-ST) is a new assay which utilizes a nucleic acid probe to detect group A streptococci directly from pharyngeal swabs. In this study, 1,103 specimens were cultured and tested by GP-ST. The sensitivities and specificities were as follows: culture, 98.8 and 100%; GP-ST, 92.4 and 99.6%. Of the 1,103 specimens, 808 were also tested with the TestPack Strep A assay. For the specimens tested by all three methods, the sensitivities and specificities were as follows: culture, 99.5 and 100%; TestPack Strep A assay, 76.3 and 99.7%; GP-ST, 93.5 and 99.7%. The GP-ST is a very user-friendly assay which has the potential to replace culture for the diagnosis of streptococcal pharyngitis.     

A novel mouse model of Graves' disease: implications for a role of aberrant MHC class II expression in its pathogenesis

Mice immunized with fibroblasts expressing an MHC class II molecule and human thyrotropin receptor (TSHR), but not either alone, develop major features characteristic of Graves' disease (GD), such as thyroid-stimulating autoantibodies directed against TSHR, increased serum thyroid hormone levels, and enlarged thyroid glands. The results indicate the need for the simultaneous expression of a class II molecule and the TSHR on the surface of the fibroblasts to develop stimulating anti-TSHR antibodies and full-blown GD in our model. A T cell line established from a mouse with hyperthyroidism proliferates in response to fibroblasts expressing a class II molecule and TSHR, but not to the fibroblasts expressing only TSHR, indicating that the class II molecules on the fibroblasts present TSHR-derived peptide(s) to T cells. These results strongly suggest that the acquisition of antigen-presenting ability by thyrocytes can lead to the induction or progression of GD. We identified a T cell epitope of TSHR by the proliferative response of spleen cells from mice immunized with fibroblasts expressing a class II molecule and TSHR to 80 overlapping peptides spanning the extracellular domain of human TSHR. The identification of a major T cell epitope provides an important clue to a novel therapy of GD.     

Creation of a blood-compatible surface: a novel strategy for suppressing blood activation and coagulation using a nitroxide radical-containing polymer with reactive oxygen species scavenging activity

Various polymeric materials have been used in medical devices, including blood-contacting artificial organs. Contact between blood and foreign materials causes blood cell activation and adhesion, followed by blood coagulation. Concurrently, the activated blood cells release inflammatory cytokines together with reactive oxygen species (ROS). We have hypothesized that the suppression of ROS generation plays a crucial role in blood activation and coagulation. To confirm this hypothesis, surface-coated polymers containing nitroxide radical compounds (nitroxide radical-containing polymers (NRP)) were designed and developed. The NRP was composed of a hydrophobic poly(chloromethylstyrene) (PCMS) chain to which 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) moieties were conjugated via condensation reaction of the chloromethyl groups in PCMS with the sodium alcoholate group of 4-hydroxy-TEMPO. Blood compatibility was investigated by placing NRP-coated beads in contact with rat whole blood. The amount of ROS generated on PCMS-coated beads used as a control increased significantly with time, while NRP-coated beads suppressed ROS generation. It is interesting to note that the suppression of inflammatory cytokine generation by NRP-coated beads was shown to be significantly higher than that by PCMS-coated beads. Both platelet and leukocyte adhesion to the beads were suppressed with increasing TEMPO incorporation in the polymer. These results confirm that the suppression of ROS by NRP prevents inflammatory cytokine generation, which in turn results in the suppression of blood activation and coagulation on the beads.     

A novel role for IL-18 in corticosterone-mediated intestinal damage in a two-hit rodent model of alcohol intoxication and injury

Recent findings from our laboratory have shown that acute alcohol (EtOH) intoxication before burn injury impairs intestinal immunity and barrier functions. To further delineate the mechanism of impaired intestinal barrier function, the present study examined the role of corticosterone (CORT) and interleukin (IL)-18, as CORT and IL-18 are elevated following a combined insult of EtOH intoxication and burn injury. Male rats (approximately 250 g) were gavaged with EtOH to achieve a blood EtOH level of approximately 100 mg/dL prior to burn or sham injury (25% total body surface area). Immediately after injury, a group of rats was treated with CORT synthesis inhibitor metyrapone (25 mg/kg), with or without recombinant (r)IL-18 (50 microg/kg). Another group of rats was treated with caspase-1 inhibitor Ac-YVAD-CHO to block IL-18 production. On Day 1 after injury, there was a significant increase in blood CORT levels, intestinal levels of IL-18, neutrophil chemokines [cytokine-induced neutrophil chemoattractant 1 (CINC-1) and CINC-3], intercellular adhesion molecule-1, myeloperoxidase activity, and intestinal permeability in rats receiving a combined insult of EtOH and burn injury. Treatment of rats with CORT inhibitor or with caspase-1 inhibitor prevented the increase in all of the above parameters following a combined insult of EtOH and burn injury. Moreover, coadministration of rIL-18 in metyrapone-treated rats restored the above parameters, similar to those observed in rats receiving EtOH and burn injury. These findings suggest that a combined insult of EtOH and burn injury results in increased CORT levels, which in turn up-regulates intestinal IL-18 levels and thereby causes altered intestinal barrier function following a combined insult of EtOH intoxication and burn injury.