A homozygous ADAMTS2 nonsense mutation in a Doberman Pinscher dog with Ehlers Danlos syndrome and extreme skin fragility.

An eight-week old Doberman Pinscher was diagnosed with Ehlers Danlos syndrome based on the dog's hyper-mobile carpal, tarsal and stifle joints and abnormal skin. The skin was loose and hyper-elastic with several wounds and large atrophic scars. The dog was euthanized after a severe degloving injury from minimal trauma. A whole-genome sequence, generated with DNA from the dog's blood, contained a rare, homozygous C-to-T transition at position 2408978 on chromosome 11. This transition is predicted to alter the ADAMTS2 transcript (ADAMTS2:c.769C>T) and encode a nonsense mutation (p.Arg257Ter). Biallelic ADAMTS2 mutations have caused a type of Ehlers Danlos syndrome known as dermatosparaxis in other species.

Long-term evaluation of mesenchymal stem cell therapy in a feline model of chronic allergic asthma.

BACKGROUND: Mesenchymal stem cells (MSCs) decrease airway eosinophilia, airway hyperresponsiveness (AHR), and remodelling in murine models of acutely induced asthma. We hypothesized that MSCs would diminish these hallmark features in a chronic feline asthma model. OBJECTIVE: To document effects of allogeneic, adipose-derived MSCs on airway inflammation, AHR, and remodelling over time and investigate mechanisms by which MSCs alter local and systemic immunologic responses in chronic experimental feline allergic asthma. METHODS: Cats with chronic, experimentally induced asthma received six intravenous infusions of MSCs (0.36-2.5 × 10E7 MSCs/infusion) or placebo bimonthly at the time of study enrollment. Cats were evaluated at baseline and longitudinally for 1 year. Outcome measures included: bronchoalveolar lavage fluid cytology to assess airway eosinophilia, pulmonary mechanics and clinical scoring to assess AHR, and thoracic computed tomographic (CT) scans to assess structural changes (airway remodelling). CT scans were evaluated using a scoring system for lung attenuation (LA) and bronchial wall thickening (BWT). To assess mechanisms of MSC action, immunologic assays including allergen-specific IgE, cellular IL-10 production, and allergen-specific lymphocyte proliferation were performed. RESULTS: There were no differences between treatment groups or over time with respect to airway eosinophilia or AHR. However, significantly lower LA and BWT scores were noted in CT images of MSC-treated animals compared to placebo-treated cats at month 8 of the study (LA P = 0.0311; BWT P = 0.0489). No differences were noted between groups in the immunologic assays. CONCLUSIONS AND CLINICAL RELEVANCE: When administered after development of chronic allergic feline asthma, MSCs failed to reduce airway inflammation and AHR. However, repeated administration of MSCs at the start of study did reduce computed tomographic measures of airway remodelling by month 8, although the effect was not sustained at month 12. Further study of MSC therapy including repeated MSC administration is warranted to assess impact on remodelling in chronic asthma.

Induction of airway mucus production By T helper 2 (Th2) cells: a critical role for interleukin 4 in cell recruitment but not mucus production.

Airway inflammation is believed to stimulate mucus production in asthmatic patients. Increased mucus secretion is an important clinical symptom and contributes to airway obstruction in asthma. Activated CD4 Th1 and Th2 cells have both been identified in airway biopsies of asthmatics but their role in mucus production is not clear. Using CD4 T cells from mice transgenic for the OVA-specific TCR, we studied the role of Th1 and Th2 cells in airway inflammation and mucus production. Airway inflammation induced by Th2 cells was comprised of eosinophils and lymphocytes; features found in asthmatic patients. Additionally, there was a marked increase in mucus production in mice that received Th2 cells and inhaled OVA, but not in mice that received Th1 cells. However, OVA-specific Th2 cells from IL-4-deficient mice were not recruited to the lung and did not induce mucus production. When this defect in homing was overcome by administration of TNF-alpha, IL-4 -/- Th2 cells induced mucus as effectively as IL-4 +/+ Th2 cells. These studies establish a role for Th2 cells in mucus production and dissect the effector functions of IL-4 in these processes. These data suggest that IL-4 is crucial for Th2 cell recruitment to the lung and for induction of inflammation, but has no direct role in mucus production.

T helper 1 cells and interferon gamma regulate allergic airway inflammation and mucus production.

CD4 T helper (Th) type 1 and Th2 cells have been identified in the airways of asthmatic patients. Th2 cells are believed to contribute to pathogenesis of the disease, but the role of Th1 cells is not well defined. In a mouse model, we previously reported that transferred T cell receptor-transgenic Th2 cells activated in the respiratory tract led to airway inflammation with many of the pathologic features of asthma, including airway eosinophilia and mucus production. Th1 cells caused inflammation with none of the pathology associated with asthma. In this report, we investigate the role of Th1 cells in regulating airway inflammation. When Th1 and Th2 cells are transferred together into recipient mice, there is a marked reduction in airway eosinophilia and mucus staining. To address the precise role of Th1 cells, we asked (i), Are Th2-induced responses inhibited by interferon (IFN)-gamma? and (ii) Can Th1 cells induce eosinophilia and mucus in the absence of IFN-gamma? In IFN-gamma receptor(-/-) recipient mice exposed to inhaled antigen, the inhibitory effects of Th1 cells on both airway eosinophilia and mucus production were abolished. In the absence of IFN-gamma receptor signaling, Th1 cells induced mucus but not eosinophilia. Thus, we have identified new regulatory pathways for mucus production; mucus can be induced by Th2 and non-Th2 inflammatory responses in the lung, both of which are inhibited by IFN-gamma. The blockade of eosinophilia and mucus production by IFN-gamma likely occurs through different inhibitory pathways that are activated downstream of Th2 cytokine secretion and require IFN-gamma signaling in tissue of recipient mice.

Essential role of nuclear factor kappaB in the induction of eosinophilia in allergic airway inflammation.

The molecular mechanisms that contribute to an eosinophil-rich airway inflammation in asthma are unclear. A predominantly T helper 2 (Th2)-type cell response has been documented in allergic asthma. Here we show that mice deficient in the p50 subunit of nuclear factor (NF)- kappaB are incapable of mounting eosinophilic airway inflammation compared with wild-type mice. This deficiency was not due to a block in T cell priming or proliferation in the p50(-/-) mice, nor was it due to a defect in the expression of the cell adhesion molecules VCAM-1 and ICAM-1 that are required for the extravasation of eosinophils into the airways. The major defects in the p50(-/-) mice were the lack of production of the Th2 cytokine interleukin 5 and the chemokine eotaxin, which are crucial for proliferation and for differentiation and recruitment, respectively, of eosinophils into the asthmatic airway. Additionally, the p50(-/-) mice were deficient in the production of the chemokines macrophage inflammatory protein (MIP)-1alpha and MIP-1beta that have been implicated in T cell recruitment to sites of inflammation. These results demonstrate a crucial role for NF-kappaB in vivo in the expression of important molecules that have been implicated in the pathogenesis of asthma.

Prevalence of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus carriage in three populations.

BACKGROUND: A higher prevalence of methicillin-resistant Staphylococcus aureus (MRSA) colonization is reported in healthcare workers compared with nonhealthcare workers. HYPOTHESIS: The prevalence of MRSA colonization differed in people and pets in households with healthcare workers as compared with households without healthcare workers. SUBJECTS: A person and 1 dog or cat from 586 households defined as either a nonhealthcare (n = 213), veterinary healthcare (n = 211), or human healthcare (n = 162) worker household. METHODS: Prospective cross-sectional study. Samples from humans and pets were cultured in vitro. Staphylococcus aureus was identified as methicillin sensitive (MSSA) or MRSA with mecA polymerase chain reaction. Pulsed-field gel electrophoresis and spa-typing were used to characterize relatedness of S. aureus and MRSA and assign USA types. RESULTS: The prevalence of MSSA and MRSA in humans was 21.5% (126/586) and 5.63% (33/586), respectively, and 7.85% (46/586) and 3.41% (20/586), respectively, in pets. There were no differences in prevalences of either MSSA or MRSA between household types. The proportion of MRSA among all S. aureus isolates in humans and pets was 20.8% (33/159) and 30.3% (20/66), respectively. In < 1.0% (4/586) of households, the same strain of MRSA was found in both a person and a pet. CONCLUSIONS AND CLINICAL IMPORTANCE: There were no differences in the prevalences of MSSA or MRSA between healthcare worker and nonhealthcare worker households. Pets and people colonized with S. aureus were as likely to be colonized with MRSA. Colonization of a person and their pet with the same strain of MRSA was rare.

Clinical, metabolic, and molecular genetic characterization of hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency in 30 dogs.

Genotype-phenotype correlations of humans and dogs with hereditary methemoglobinemia are not yet well characterized. We determined total hemoglobin and methemoglobin (MetHb) concentrations, cytochrome b5 reductase (CYB5R) enzyme activities, genotypes, and clinical signs in 30 dogs with persistent cyanosis without cardiopulmonary disease. Erythrocytic CYB5R enzyme activities were low in all dogs assayed. Owner-reported quality of life ranged from subclinical to occasional exertional syncope. Two previously reported and two novel CYB5R3 missense variants were identified among the methemoglobinemic cohort and were predicted to impair enzyme function. Two variants were recurrent: a homozygous Ile194Leu substitution was found in Pomeranians and other small dogs, and a homozygous Arg219Pro change occurred predominately in pit bull terriers. The other two variants were Thr202Ala and Gly76Ser substitutions in single dogs. Of the two common CYB5R3 genotypes, Arg219Pro was associated with a more severe metabolic phenotype. We conclude that CYB5R3 deficiency is the predominate cause of canine hereditary methemoglobinemia. Although this finding is unlikely to alter the clinical approach to hereditary methemoglobinemia in dogs, it demonstrates the possibility of how genotype-phenotype cohort analysis might facilitate precision medicine in the future in veterinary medicine.

A dominant negative form of transcription activator mTFE3 created by differential splicing.

Transcription factor E3 (mTFE3) is a murine transcription activator that binds to the intronic enhancer of the immunoglobulin heavy chain gene. A naturally occurring splice product of mTFE3 messenger RNA (mRNA) lacked 105 nucleotides that encode an activation domain; both absolute and relative amounts of long and truncated mRNAs varied in different tissues. Cells were cotransfected with complementary DNAs that encoded the two mRNA forms in amounts that corresponded to the amounts of each mRNA found in different cells. Small changes in substoichiometric amounts of the truncated form of mRNA effected trans-dominant negative modulation of mTFE3 activity. These findings identify a function for differential splicing in the regulation of transcription factor activity.

Strategies for endovascular mitral valve repair.

Technological advances have recently enabled mitral valve repair to be performed using endovascular techniques and thus open the possibility of nonsurgical treatment of mitral valve disease. While balloon valvotomy has been applied to mitral stenosis for over 20 years, a number of devices aimed at correcting mitral regurgitation are currently in preclinical and clinical development. While some of these, such as edge-to-edge repair, are catheter adaptations of established surgical techniques, others represent true departures from the current surgical paradigms of correcting mitral regurgitation. This review will summarize the current status of percutaneous transcatheter techniques for mitral valve repair. Included are balloon mitral valvotomy, indirect annuloplasty, direct annuloplasty, ventricular shape change, and edge-to-edge repair. These techniques certainly represent a new interdisciplinary paradigm between cardiac surgery and interventional cardiology and may be the next frontier in minimally-invasive cardiac surgery.

Local photodynamic therapy for equine squamous cell carcinoma: evaluation of a novel treatment method in a murine model.

The objective of this study was to evaluate the effect of local photodynamic therapy (PDT) with verteporfin on tumor growth inhibition of squamous cell carcinoma (SCC) in a murine model. SCC was implanted in 85 nude mice by subcutaneous injection of A-431 SCC cells. Treatment groups (10 mice/group) received an intra-tumoral injection of verteporfin dissolved in dimethyl sulfoxide (DMSO) or 5% dextrose solution at a dose of 0.01 or 0.1mg/cm3. Controls received only solvent, or no injectate. All groups received identical light illumination (100J/cm2). Relative change in tumor volume (RCTV) at day 30 was compared between groups using the Wilcoxon rank sum test (P< 0.05). Local PDT with verteporfin at a dose of 0.1mg/cm3 resulted in significantly lower RCTV at day 30 compared to controls. Choice of solvent (DMSO versus D5W) did not affect the results. Local PDT may be an effective adjunctive therapy for the treatment of periocular equine SCC.

Endocrine and immunologic effects of inhaled fluticasone propionate in healthy dogs.

BACKGROUND: Inhaled glucocorticoids reduce airway inflammation while minimizing systemic effects in several species. HYPOTHESIS: Inhaled fluticasone suppresses the hypothalamic-pituitary-adrenal axis (HPAA), modifies immune function, and induces clinical signs to a lesser extent than PO-administered prednisone in dogs. ANIMALS: Seven healthy adult pet dogs. METHODS: Dogs were randomized to 1 of 3 treatment groups in a crossover design: fluticasone propionate (220 mug actuation of a metered dose inhaler delivered via a spacer and mask, q12h), placebo (spacer and mask alone, q12h), or prednisone (1 mg/kg PO q24h). Each treatment was administered for 3 weeks followed by a 4-week washout. Appetite, attitude, and water consumption were recorded during the last week of each treatment period. Urine cortisol : creatinine ratios, ACTH stimulation tests, white blood cell counts, lymphocyte phenotype, and serum IgM and IgA concentrations were recorded at each baseline and after the last day of each treatment. Clinical observations were expressed descriptively. Friedman's test was applied to all data comparisons. Pairwise comparisons were made with a mixed model analysis when data were normally distributed, whereas signed rank tests were used otherwise (significance P-value <.01). RESULTS: Appetite and water consumption increased during prednisone treatment. Peak serum cortisol concentrations post-ACTH were significantly decreased in prednisone- and fluticasone-treated dogs compared with placebo (prednisone > fluticasone). Serum IgM concentrations were significantly decreased in dogs treated with prednisone. CONCLUSIONS AND CLINICAL IMPORTANCE: As used, fluticasone suppresses the HPAA to a lesser extent than prednisone and may avert systemic signs associated with PO-administered glucocorticoids in dogs.

Xenotransplantation of cryopreserved equine squamous cell carcinoma to athymic nude and SCID mice.

Cryopreserved equine ocular squamous cell carcinoma (SCC) was inoculated subcutaneously into 15 athymic nude and 15 SCID mice. Xenotransplantation resulted in tumor growth in two athymic nude mice and 1 SCID mouse. Histological appearance and immunohistochemical characterization using cytokeratin 5/6 markers and p53 markers of the tumor grown in mice was in full accord with the original equine tumors. No evidence of metastasis was noted in any mouse. This model may serve as a relevant in vivo model for studying the biology of equine ocular SCC and for the testing of new therapeutic modalities.

The effects of an acetate-sensitive anion binding site on NADPH binding in glutamate dehydrogenase.

The nature of a general anion binding site that regulates NADPH binding to L-glutamate dehydrogenase has been explored. Dissociation constants for the enzyme-NADPH complex were measured by difference spectroscopy in the presence of phosphate, pyrophosphate, ADP and acetate ions. Whereas two molecules of phosphate, binding in a cooperative fashion, raise the Kd of the enzyme-NADPH complex 50-fold from 2.3 microM, a single pyrophosphate raises the Kd only 23-fold, disproving the notion that the anion binding site is simply the pyrophosphate binding site of NADPH. ADP raises the Kd of the enzyme-NADPH complex 2-fold for a given phosphate concentration, and formation of the enzyme-ADP complex is itself interfered with by phosphate and pyrophosphate, indicating that these anions interact with the same anion binding site. Acetate ion acts in a manner opposite to that of phosphate, pyrophosphate and ADP and reverses the weakening effect that these ions exert on NADPH binding, returning the Kd of the enzyme-NADPH complex to 2.3 microM. In the absence of these anions, however, acetate exerts no measurable effect on the Kd, suggesting an allosteric mechanism.

Does coronary artery bypass grafting alone correct moderate ischemic mitral regurgitation?

BACKGROUND: The optimal management of moderate (3+ on a scale of 0 to 4+) ischemic mitral regurgitation (MR) remains controversial. Some advocate CABG alone, whereas others favor concomitant mitral annuloplasty. To clarify the optimal management of these patients, we evaluated the early impact of isolated CABG on moderate ischemic MR. METHODS AND RESULTS: Between January 1992 and August 1999, 136 patients (54% male, mean age 70.5 years, mean New York Heart Association class 2.7, mean ejection fraction 38.1%) with a preoperative diagnosis of moderate ischemic MR, without leaflet prolapse or pathology, underwent isolated CABG. Thirty-eight (28%) of 136 patients had intraoperative transesophageal echocardiography (TEE) before CABG, and 68 (50%) had postoperative transthoracic echocardiography (TTE) within 6 weeks of surgery. The subgroups of patients undergoing intraoperative TEE and postoperative TTE had preoperative characteristics similar to the overall group. The 30-day operative mortality was 2.9% (). Intraoperative TEE downgraded the severity of MR to mild or less (0 to 2+) in 89% (). On postoperative TTE, 40% () continued to have at least moderate MR (3 to 4+), 51% () improved somewhat to mild (2+) MR, and only 9% () had resolution of their MR (0 to 1+). The mean preoperative, intraoperative, and postoperative MR grades were 3.0+/-0.0, 1.4+/-1.0, and 2.3+/-0.8, respectively (P<0.001). CONCLUSIONS: CABG alone for moderate ischemic MR leaves many patients with significant residual MR and may not be the optimal therapy for most patients. Intraoperative TEE may significantly underestimate the severity of ischemic MR. A preoperative diagnosis of moderate MR may warrant concomitant mitral annuloplasty.

Sustained angina relief 5 years after transmyocardial laser revascularization with a CO(2) laser.

BACKGROUND: Although transmyocardial laser revascularization (TMR) has provided symptomatic relief of angina over the short term, the long-term efficacy of the procedure is unknown. Angina symptoms as assessed independently by angina class and the Seattle Angina Questionnaire (SAQ) were prospectively collected up to 7 years after TMR. METHODS: Seventy-eight patients with severe angina not amenable to conventional revascularization were treated with a CO(2) laser. Their mean age was 61+/-10 years at the time of treatment. Preoperatively, 66% had unstable angina, 73% had had >/=1 myocardial infarction, 93% had undergone >/=1 CABG, 42% had >/=1 PTCA, 76% were in angina class IV, and 24% were in angina class III. Their average pre-TMR angina class was 3.7+/-0.4. RESULTS: After an average of 5 years (and up to 7 years) of follow-up, the average angina class was significantly improved to 1.6+/-1 (P=0.0001). This was unchanged from the 1.5+/-1 average angina class at 1 year postoperatively (P=NS). There was a marked redistribution according to angina class, with 81% of the patients in class II or better, and 17% of the patients had no angina 5 years after TMR. A decrease of >/=2 angina classes was considered significant, and by this criterion, 68% of the patients had successful long-term angina relief. The angina class results were further confirmed with the SAQ; 5-year SAQ scores revealed an average improvement of 170% over the baseline results. CONCLUSIONS: The long-term efficacy of TMR persists for >/=5 years. TMR with CO(2) laser as sole therapy for severe disabling angina provides significant long-term angina relief.

Surgical treatment of adult atrial septal defect: early and long-term results.

OBJECTIVES: The purpose of this study is to determine the early and late results of the surgical repair of atrial septal defect in adults. BACKGROUND: Progressively limiting, untreated atrial septal defect can lead to the early death of middle-aged adults. Recently it has been suggested that the closure of atrial septal defects might be accomplished with interventional cardiac techniques. Although the long-term results of the transcatheter closure are as yet unknown, the outcome of surgical therapy has been shown to be beneficial for almost 40 years. METHODS: Between 1971 and 1991, 166 consecutive patients underwent surgical repair of a secundum or sinus venosus atrial septal defect, or both, at the Brigham and Women's Hospital, Boston. There were 120 women and 46 men in this group; the mean age was 44 years and 58 (35%) of the patients were > or = 50 years old. The average pulmonary to systemic flow ratio was 3.0, and 57 patients had a peak systolic pulmonary artery pressure > 30 mm Hg. RESULTS: There were two operative deaths (early mortality rate 1.2%), and 13% of the patients had a perioperative complication. One hundred fifty-three of the 164 survivors were followed up for a mean of 90 months (range 2 to 247). There were eight late deaths (late mortality rate 4.9%) and a late morbidity rate of 12.4% (in most cases due to arrhythmias). The 5- and 10-year survival rates are 98% and 94%, respectively, and the probability of event-free survival (with no morbidity or mortality) at 5 years is 97% and at 10 years is 92%. CONCLUSIONS: The results indicate that the surgical correction of atrial septal defect in adults is safe and efficacious as confirmed by 20 years of follow-up.

Decreased operative risk of surgical treatment of mitral regurgitation with or without coronary artery disease.

The consecutive 2 year experience with patients undergoing first-time surgery for mitral regurgitation with and without coronary artery disease was reviewed. From January 1988 to January 1990, 127 patients with pure mitral regurgitation undergoing first-time operation were surgically treated. No other valve lesion, no reoperation and no congenital defects were included. The mean patient age was 62 years with 26% of the patients greater than 70 years. Twenty-six percent of the entire group was in functional class IV. Seventy-five patients received mitral valve repair and 52 underwent mitral valve replacement with a St. Jude or Hancock valve. In patients undergoing mitral valve repair, there was a higher incidence of those greater than 70 years old and of coronary artery disease and in patients undergoing mitral valve replacement there was a higher incidence of functional class IV. The operative mortality rate was 2.3% (3 of 127 patients). No patient failed to be discontinued from cardiopulmonary bypass and all three deaths occurred after mitral valve replacement, with one from complications of chronic renal failure and dialysis. There was no significant difference in patients who either did or did not have a concomitant coronary artery bypass graft and there was no difference related to age or functional class. Postoperative complications occurred in five patients in the valve repair group, including recurrent mitral regurgitation in two necessitating reoperation, and in three patients in the valve replacement group. With newer operative and postoperative management techniques, especially preservation of the papillary muscle annular continuity, the risk of mitral valve surgery, particularly of valve repair, is considerably lower than in previous years.