Aminoglycoside-mediated rescue of a disease-causing nonsense mutation in the V2 vasopressin receptor gene in vitro and in vivo

Many human diseases are caused by inactivating mutations in specific G-protein-coupled receptors (GPCRs). In about 10% of these cases, a premature stop codon leads to the generation of a truncated, functionally inactive receptor protein. In this study, we tested the hypothesis that such GPCR mutations can be functionally rescued in vitro and in vivo by treatment with aminoglycoside antibiotics, which are known for their ability to suppress premature termination codons. As a model system, we studied a mutant V2 vasopressin receptor (AVPR2) containing the inactivating E242X nonsense mutation which mimics human X-linked nephrogenic diabetes insipidus (XNDI) when introduced into mice via gene targeting techniques. Studies with cultured mammalian cells expressing the E242X mutant receptor showed that G418 (geneticin) was by far the most potent aminoglycoside antibiotic capable of suppressing the E242X nonsense codon. Strikingly, G418 treatment increased AVP-mediated cAMP responses in cultured kidney collecting duct cells prepared from E242X mutant mice in vitro, and significantly improved the urine-concentrating ability of E242X mutant mice in vivo. This is the first study demonstrating that G418 (aminoglycosides) can ameliorate the clinical symptoms of a disease-causing premature stop codon in a member of the GPCR superfamily.     

Molecular epidemiologic evaluation of endocarditis due to Oerskovia turbata and CDC group A-3 associated with contaminated homograft valves

Oerskovia turbata is an unusual bacterial cause of endocarditis and septicemia in immunocompromised patients. In this study, we compared 12 isolates from a 1975 medical center cluster, 11 originally identified as O. turbata (four from the blood of a homograft aortic valve-associated endocarditis patient and seven from contaminated homograft valves) and one CDC group A-3 strain from the blood of a second endocarditis patient with fatal outcome, with eight control strains from unrelated locations. The control strains included type and reference strains of O. turbata, Cellulomonas hominis, and CDC group A-3. The four blood isolates from the first patient and six of the valve isolates shared identical biochemical, antimicrobial susceptibility, and BglI ribotype patterns that differed from the second patient's isolate and control strains. The blood isolate from the second patient and the remaining valve isolate shared a phenotypic and genotypic profile and were phenotypically identical to, but epidemiologically different from, the CDC group A-3 reference strain with the strain-specific enzyme. Also, these isolates differed from the type strain and the other reference strains of C. hominis and O. turbata. Our results indicate that the four blood isolates from the first patient and six of the homograft valve isolates represent a single clone of O. turbata associated with endocarditis. Additionally, our results indicate that the blood isolate from the second patient and one of the homograft valve isolates differ from O. turbata and C. hominis and represent a unique clone of CDC group A-3 associated with fatal endocarditis.     

Rapid identification of Nocardia farcinica clinical isolates by a PCR assay targeting a 314-base-pair species-specific DNA fragment

Nocardia farcinica is the most clinically significant species within the Nocardia asteroides complex. Differentiation of N. farcinica from other members of N. asteroides complex is important because this species characteristically demonstrates resistance to several extended-spectrum antimicrobial agents. Traditional phenotypic characterization of this species is time- and labor-intensive and often leads to misidentification in the clinical microbiology laboratory. We previously observed a 409-bp product for all strains of N. farcinica by using randomly amplified polymorphic DNA analysis with the primer DKU49. In this investigation, the 409-bp fragment was sequenced and then used to design a specific primer pair, Nf1 (16-mer) and Nf2 (16-mer), complementary to the 409-bp fragment. PCR amplification of genomic DNA from 28 N. farcinica isolates with Nf1 and Nf2 generated a single intense 314-bp fragment. The specificity of the assay with these primers was verified, since there were no PCR amplification products observed from heterologous nocardial species (n = 59) or other related bacterial genera (n = 41). Restriction enzyme digestion using CfoI and direct sequencing of the 314-bp fragment further confirmed the specificity of the assay for N. farcinica. This highly sensitive and specific PCR assay provides a rapid (within 1 day of obtaining DNA) method for identification of this medically important emerging pathogen. Rapid diagnosis of N. farcinica infection may allow for earlier initiation of effective therapy, thus improving patient outcome.     

Cardiac damage in rodents after exposure to bis(2-chloroethoxy)methane

We report that an environmental agent, bis(2-chloroethoxy)methane (CEM), caused cardiac toxicity in male and female F344 rats and B6C3F1 mice exposed to the chemical by dermal administration at doses of 0, 50, 100, 200, 400 or 600 mg/kg 5 days a week for up to 14 weeks. Treatment-related deaths occurred in 10/10 male and 10/10 female rats at 600 mg/kg, in 2/10 female rats at 400 mg/kg, and in 3/10 female mice at 600 mg/kg. The heart lesions were more severe in rats than mice, and more severe in females than males. In rats, the no-observed-adverse-effect level (NOAEL) for the heart lesions was 200 mg/kg for males and 100 mg/kg for females; in mice, it was more than 600 mg/kg for males and 200 mg/kg for females. Multifocal, widespread vacuolization of the myocytes comprised the main morphological feature of the lesions, and only in rats was it accompanied by mononuclear cell infiltration, myocytic necrosis and atrial thrombosis. Hearts from male rats were immunohistochemically stained for troponin T (cTnT) protein. Loss of cytoplasmic cTnT correlated with histopathological damage only in the 600 mg/kg animals. CEM is metabolized to thiodiglycolic acid, a chemical that causes mitochondrial dysfunction. It is hypothesized that mitochondrial damage leads to the heart toxicity from bis(2-chloroethoxy)methane.     

Primary porcine endothelial cells express membrane-bound B7-2 (CD86) and a soluble factor that co-stimulate cyclosporin A-resistant and CD28-dependent human T cell proliferation

Increasing evidence suggests that endothelial cells can directlyactivate syngeneic, allogeneic and xenogeneic T cells. In thisstudy we demonstrate that unstimulated, paraformaldehyde-fixedprimary porcine aortic endothelial cells (PAEC) and microvascularendothelial cells (PMVEC) can provide co-stimulation for humanT cell IL-2 secretion and proliferation. EC-mediated co-stimulationhas both cyclosporin A (CsA)-sensitive and CsA-resistant components.The CsA-resistant component is completely suppressed eitherby blocking with anti-CD28 F(ab) fragments or CTLA-4-lg. Northernblot analysis of unstimulated PAEC and PMVEC with porcine-specificprobes reveals constitutive expression of B7-2 mRNA while B7-1message was not detected. hCTLA-4-lg and anti-B7-2 mAb immunoprecipitatesa single 79 kDa PMVEC surface protein. Surprisingly, PMVEC conditionedmedia also has soluble co-stimulatory activity that is blockedby anti-CD28 F(ab) fragments or anti-B7-2 mAb. These findingsdemonstrate that primary unstimulated porcine EC can co-stimulateCsA-resistant human T cell proliferation through binding ofmembrane bound, constitutively expressed EC B7-2 (CD86) to humanT cell CD28, providing one of the first demonstrations of functionalB7-2 on cells outside the immune system. In addition, PMVECsecrete or shed a soluble factor that mediates CD28-dependenthuman T cell proliferation, demonstrating the existence of solublemediators of CD28 activation.     

Comparative phenotypic assessment of cardiac pathology, physiology, and gene expression in C3H/HeJ, C57BL/6J, and B6C3F1/J mice

Human cardiomyopathies often lead to heart failure, a major cause of morbidity and mortality in industrialized nations. Described here is a phenotypic characterization of cardiac function and genome-wide expression from C3H/HeJ, C57BL/6J, and B6C3F1/J male mice. Histopathologic analysis identified a low-grade background cardiomyopathy (murine progressive cardiomyopathy) in eight of nine male C3H/HeJ mice (age nine to ten weeks), but not in male C57BL/6J and in only of ten male B6C3F1/J mice. The C3H/HeJ mouse had an increased heart rate and a shorter RR interval compared to the B6C3F1/J and C57BL/6J mice. Cardiac genomic studies indicated the B6C3F1/J mice exhibited an intermediate gene expression phenotype relative to the 2 parental strains. Disease-centric enrichment analysis indicated a number of cardiomyopathy-associated genes were induced in B6C3F1/J and C3H/HeJ mice, including Myh7, My14, and Lmna and also indicated differential expression of genes associated with metabolic (e.g., Pdk2) and hypoxic stress (e.g. Hif1a). A novel coexpression and integrated pathway network analysis indicated Prkaa2, Pdk2, Rhoj, and Sgcb are likely to play a central role in the pathophysiology of murine progressive cardiomyopathy in C3H/HeJ mice. Our studies indicate that genetically determined baseline differences in cardiac phenotype have the potential to influence the results of cardiotoxicity studies.     

Umbilical cord blood xenografts in immunodeficient mice reveal that T cells enhance hematopoietic engraftment beyond overcoming immune barriers by stimulating stem cell differentiation.

Clinical experience and animal models have shown that donor T cell depletion (TCD) adversely affects engraftment of hematopoietic stem cells (HSCs). Although it is known that donor T cells are acting to overcome residual host immune barriers, they may also exert effects independent of host resistance via direct or indirect interactions with donor stem cells, their microenvironment, or key differentiation events. To more precisely define the effect of T cells on engraftment, we have performed human umbilical cord blood (UCB) transplantation into immunodeficient mice under limiting dilution conditions. UCB mononuclear cells (MNC) or TCD UCB were transplanted into NOD/LtSz-scid/scid B2m(null) (NOD/SCID-beta(2)m(-/-)) mice. Cohorts of mice received UCB MNC or TCD UCB at 5 dose levels between 5 x 10(4) and 5 x 10(6) cells. At dose levels at or above 10(5) cells, engraftment was higher in the MNC recipients (n = 32) than the TCD recipients (n = 31) in a dose-dependent manner. Despite this difference, limiting dilution analysis to determine functional stem cell frequency revealed that SCID repopulating cells in TCD UCB was not significantly less than in CB MNCs, suggesting that T cells may facilitate engraftment at stages beyond the stem cell. Add-back of CD3/CD28 costimulated T cells restored and appeared to enhance engraftment, both in NOD/SCID-beta(2)m(-/-) as well as NOD/LtSz-scid IL2Rgamma(null) (NOG) recipients. These results, in a model where there are minimal host immune barriers to overcome, suggest T cells possess additional graft-facilitating properties. CD3/CD28 costimulation of UCB T cells represents a potential strategy for enhancing the engraftment of UCB.     

Accessory gene regulator group polymorphisms in methicillin-resistant Staphylococcus aureus: an association with clinical significance

PURPOSE: Virulent gene expression in Staphylococcus aureus is controlled by regulators such as the accessory gene regulator (agr). Strains can be divided into four major agr groups (agr I-IV) on the basis of agrD and agrC polymorphisms. The purpose of this study was to define the proportion of agr I, II, and III polymorphisms and to compare the clinical characteristics between group I and non-group I polymorphisms of methicillin-resistant Staphylococcus aureus (MRSA) strains in a Korean tertiary care teaching hospital. MATERIALS AND METHODS: A total of 158 clinical isolates were evaluated by RFLPs (restriction fragment length polymorphisms). RESULTS: The mean age of the patients was 50.2 +/- 21.9 years old. There were 74 (49.3%), 66 (44.0%), 10 (6.7%), 7 (4.4%), and 1 (0.6%) strains in agr group I, II, III, I + II, and I + III polymorphisms, respectively. Only ear infections were a statistically significant clinical parameter according to univariate (p=0.001) and multivariate analysis (OR, 4.721 (1.273-17.508), p=0.020). CONCLUSION: This study suggests that agr group I is the most prevalent in Korea, and ear infections are correlated with the group I polymorphism, which is a different clinical trend from western countries. It can also be inferred that community-acquired MRSA correlates with agr group I.     

In vivo imaging of human burn injuries with polarization-sensitive optical coherence tomography

The accurate determination of burn depth is critical in the clinical management of burn wounds. Polarization-sensitive optical coherence tomography (PS-OCT) has been proposed as a potentially non-invasive method for determining burn depth by measuring thermally induced changes in the structure and birefringence of skin, and has been investigated in pre-clinical burn studies with animal models and ex vivo human skin. In this study, we applied PS-OCT to the in-vivo imaging of two pediatric burn patients. Deep and superficial burned skins along with contralateral controls were imaged in 3D. The imaging size was 8 mm × 6 mm × 2 mm in width, length, and depth in the air respectively, and the imaging time was approximately 6 s per volume. Superficially burned skins exhibited the same layered structure as the contralateral controls, but more visible vasculature and reduced birefringence compared to the contralateral controls. In contrast, a deeply burned skin showed loss of the layered structure, almost absent vasculature, and smaller birefringence compared to superficial burns. This study suggested the vasculature and birefringence as parameters for characterizing burn wounds.