Infrared spectroscopy of serum as a potential diagnostic screening approach for naturally occurring canine osteoarthritis associated with cranial cruciate ligament rupture.

OBJECTIVE: To evaluate infrared (IR) spectroscopy of serum as a screening tool to differentiate dogs affected by naturally occurring osteoarthritis (OA) associated with cranial cruciate ligament rupture (CrCLR) and controls. METHOD: 104 adult dogs with CrCLR (affected group) and 50 adult control dogs were recruited for this prospective observational study. Serum samples were collected preoperatively from CrCLR dogs and from a subset of these dogs at 4-, and 12-week post-surgical intervention to stabilize the affected stifles. Serum was collected once from control dogs. Dry films were made from serum samples, and IR absorbance spectra acquired. Data preprocessing, principal component analysis and multivariate analysis of covariance were performed to separate samples from the two groups, and to evaluate temporal differences. Weighted logistic regression with L1 regularization method was used to develop a predictive model. Model performance based on an independent test set was evaluated. RESULTS: Spectral data analysis revealed significant separation between the sera of CrCLR and control dogs (P < 0.0001), but not amongst different time points in the OA group. The sensitivity, specificity, AUC and accuracy of the test set were 84.62%, 96.15%, 93.20% and 92.31% respectively. CONCLUSIONS: These findings confirm the potential of IR-spectroscopy of serum with chemometrics methods to differentiate controls from dogs with OA associated with CrCLR. This is the first step in development of an economic, and comparatively simple IR-based screening serum test for OA. Utility of this tool as a clinical screening and diagnostic test requires further investigation and validation.

Associations between the IASLC/ATS/ERS lung adenocarcinoma classification and EGFR and KRAS mutations.

We sought to investigate the frequency of mutations in epidermal growth factor receptor (EGFR) and Kirsten-RAS (KRAS) by each pathological subtype for patients with resected pulmonary adenocarcinoma as defined by the IASLC/ATS/ERS classification. Histological examination determined the predominant subtype according to the IASLC/ATS/ERS classification. EGFR and KRAS mutations were determined by high-resolution melting and Sanger sequencing. Clinical data were collected from medical records and clinicians. The 178 consecutive patients consisted of 48% males, median age 68 years (range 20-87) and smoking history 78%. The tumour stage was I in 62%, II in 18% and III in 20%. The mutation rates were: EGFR 30%; KRAS 28%. The rate of EGFR mutations in the acinar predominant reference group (n=76), was 37%. The solid predominant subtype showed significantly fewer EGFR mutations [3/33 (9%), odds ratio 0.17 (0.05-0.61), p=0.007]. No differences in mutation rate were observed in other subtypes. No association was found between KRAS mutations and predominant histological subtype. Advanced stage and solid predominant subtype were negative prognostic factors. EGFR mutations can be present in adenocarcinoma of any predominant subtype, however rarely in solid predominant tumours. No association was found between KRAS mutation and the predominant histological subtype.

Development of the microsporidian parasite, Loma salmonae, in a rainbow trout gill epithelial cell line (RTG-1): evidence of xenoma development in vitro.

Growth and propagation of fish-infecting microsporidians within cell culture has been more difficult to achieve than for insect- and human-infecting microsporidians. Fish microsporidia tend to elicit xenoma development rather than diffuse growth in vivo, and this process likely increases host specificity. We present evidence that the fish microsporidian, Loma salmonae, has the capacity to develop xenomas within a rainbow trout gill epithelial cell line (RTG-1). Spore numbers increased over a 4 weeks period within cell culture flasks. Xenoma-like structures were observed using phase contrast microscopy, and then confirmed using transmission electron microscopy. Optimization of the L. salmonae-RTG-1 cell model has important implications in elucidating the process of xenoma development induced by microsporidian parasites.

Ultrastructure of trichocysts in Hematodinium spp. infecting Atlantic snow crab, Chionoecetes opilio.

Trichocyst morphology and development were explored using transmission electron microscopy in Hematodinium spp. isolated directly from Atlantic snow crab (Chionoecetes opilio) hemolymph and from in vitro cultures. Appearance of trichocysts defines the initiation of a morphological transition in the parasites life cycle from vegetative stage to the transmission stage. Trichocysts within sporonts were found in distinct clusters near the nucleus in close apposition to the Golgi. As cells transitioned to more mature dinospores however, trichocysts were found randomly distributed throughout the cytoplasm. Clusters contained both primordial and maturing trichocysts at various stages indicating an asynchronous development. The random distribution of mature trichocysts suggests deployment to the cell membrane for future extrusion. Mature trichocysts of Hematodinium spp. appeared structurally similar to trichocysts from photosynthetic dinoflagellates. Hematodinium spp. trichocysts differed by the presence of peripheral tubules associated with novel cuboidal appendages in the apical region rather than a network of central electron dense fibres as found in photosynthetic dinoflagellates. Additionally, the trichocyst membrane of Hematodinium spp. was in close apposition to the square crystalline core. Trichocyst expulsion was not observed during our study which along with features of development and maturation within Hematodinium life stages should provide insight into proposed roles in host attachment or defense that could further our understanding of the mechanisms of pathogenesis and transmission of the parasite.

An experiment on the dynamics of ion implantation and sputtering of surfaces.

A major impediment towards a better understanding of the complex plasma-surface interaction is the limited diagnostic access to the material surface while it is undergoing plasma exposure. The Dynamics of ION Implantation and Sputtering Of Surfaces (DIONISOS) experiment overcomes this limitation by uniquely combining powerful, non-perturbing ion beam analysis techniques with a steady-state helicon plasma exposure chamber, allowing for real-time, depth-resolved in situ measurements of material compositions during plasma exposure. Design solutions are described that provide compatibility between the ion beam analysis requirements in the presence of a high-intensity helicon plasma. The three primary ion beam analysis techniques, Rutherford backscattering spectroscopy, elastic recoil detection, and nuclear reaction analysis, are successfully implemented on targets during plasma exposure in DIONISOS. These techniques measure parameters of interest for plasma-material interactions such as erosion/deposition rates of materials and the concentration of plasma fuel species in the material surface.

Histochemical and ultrastructural analysis of pathology and cell responses in gills of channel catfish affected with proliferative gill disease.

Pond-reared channel catfish Ictalurus punctatus with proliferative gill disease (PGD), caused by the myxozoan parasite Henneguya spp., were examined with light and transmission electron microscopy to better characterize the inflammatory response during infection. The early stages of disease are characterized by the destruction of collagen in the matrix of the gill filament cartilage causing weakness and breaks within the gill filaments. These early lesions lacked a notable inflammatory response around the disrupted cartilage, a chondrocyte response was not apparent, and the parasite was not present, suggesting that the cartilage breaks occur prior to inflammation and arrival of the parasite in the gill. In later lesions, a significant inflammatory response was generated in areas of disrupted cartilage, and the inflammatory infiltrate was composed of a mixed population of granulocytes including neutrophils and cells that resembled eosinophils. The majority of eosinophil-like cells demonstrated evidence of degranulation. Trophozoites of Henneguya spp. were surrounded by a uniform population of cells believed to be neutrophils. The granulocytes were infiltrated within the dense collagen layer of the gill filament cartilage and often appeared within chondrocyte lacunae in place of the chondrocyte. The gill lamellae adjacent to the lesions were fused and contained an inflammatory infiltrate containing granulocytes and cells with pericentriolar granules that resembled previous descriptions of Langerhans-like cells. These cells were abundant within damaged lamellar epithelium, but were only rarely found within the gill filament. Lesions that appeared to be recovering lacked the dense collagenous layer around the cartilage and contained hyperplastic and hypertrophic chondrocytes that formed a callus. Other chondrocytes in the lesions had ultrastructural features indicative of cell death.

Phylogeny and morphology of Glugea hertwigi from rainbow smelt Osmerus mordax found in Prince Edward Island, Canada.

Infection of rainbow smelt Osmerus mordax with the microsporidian Glugea hertwigi was diagnosed for the first time in Prince Edward Island, Canada. The prevalence of infection was 24%, 45 infected out of 187 examined fish captured in February and March 2009. Both large and small xenomas of G. hertwigi observed within the submucosa of the gastrointestinal tract and along the mesentery of the host contained only mature spores. Advanced and degraded xenomas associated with host reaction were described using light and transmission electron microscopy. The first rDNA sequence of G. hertwigi prepared in the present study completed the set of sequences of Glugea spp. available for comparison. The high level of rDNA sequence identity between Glugea spp. suggests that these may be variants of a single species.

High sensitivity imaging Thomson scattering for low temperature plasma.

A highly sensitive imaging Thomson scattering system was developed for low temperature (0.1-10 eV) plasma applications at the Pilot-PSI linear plasma generator. The essential parts of the diagnostic are a neodymium doped yttrium aluminum garnet laser operating at the second harmonic (532 nm), a laser beam line with a unique stray light suppression system and a detection branch consisting of a Littrow spectrometer equipped with an efficient detector based on a "Generation III" image intensifier combined with an intensified charged coupled device camera. The system is capable of measuring electron density and temperature profiles of a plasma column of 30 mm in diameter with a spatial resolution of 0.6 mm and an observational error of 3% in the electron density (n(e)) and 6% in the electron temperature (T(e)) at n(e) = 4 x 10(19) m(-3). This is achievable at an accumulated laser input energy of 11 J (from 30 laser pulses at 10 Hz repetition frequency). The stray light contribution is below 9 x 10(17) m(-3) in electron density equivalents by the application of a unique stray light suppression system. The amount of laser energy that is required for a n(e) and T(e) measurement is 7 x 10(20)n(e) J, which means that single shot measurements are possible for n(e)>2 x 10(21) m(-3).

Endoscopic ultrasound-guided fine-needle aspiration when combined with positron emission tomography improves specificity and overall diagnostic accuracy in unexplained mediastinal lymphadenopathy and staging of non-small-cell lung cancer.

BACKGROUND: The aim of this study was to assess the incremental value of endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) to positron emission tomography (PET) in the diagnosis of unexplained mediastinal lymphadenopathy and staging of non-small-cell lung cancer (NSCLC). METHODS: Patients who had both EUS-guided FNA and PET were retrospectively identified from an EUS database at a tertiary hospital. All EUS-guided FNA were carried out by one endoscopist between August 2002 and April 2005, either for the diagnosis of unexplained mediastinal lymphadenopathy or for the staging of NSCLC. Results of PET and EUS were compared with histology. A true histological positive result was defined as histological involvement in either surgery (mediastinoscopy or resection) or EUS-guided FNA. A true histological negative result was defined as negative involvement at surgery (mediastinoscopy or resection). RESULTS: Forty-nine patients who had both PET scanning and EUS-guided FNA for diagnosis of unexplained mediastinal lymphadenopathy or staging of NSCLC were identified. Of these, 33 (73% males, n = 24, age range = 44-78 years, mean = 62 years) had surgical confirmation of mediastinal lymph node pathology. In these patients, PET alone showed sensitivity, 95%; specificity, 90%; positive predictive value, 87%; negative predictive value, 90% and accuracy, 88%; whereas the addition of EUS-guided FNA increased the overall specificity and positive predictive value to 100%, with an overall accuracy of 97%. CONCLUSIONS: This study suggests that EUS-guided FNA complements PET by improving the overall specificity and thereby the accuracy for diagnosis of unexplained mediastinal lymphadenopathy. It provides a minimally invasive technique to assess the mediastinum in patients with NSCLC and is particularly valuable in cases in which PET findings are equivocal.

Identification and localization of a putative ATP-binding cassette transporter in sea lice (Lepeophtheirus salmonis) and host Atlantic salmon (Salmo salar).

Some members of the ABC-transporter superfamily, such as P-glycoprotein and the multidrug resistance associated protein, may confer resistance to the avermectin subclass of macrocyclic lactones. The aim of this study was to examine the presence of ABC transporters in both sea lice (Lepeophtheirus salmonis) and its Atlantic salmon host (Salmo salar) using monoclonal antibodies (C219 and JSB-1, with high selectivity for P-gp) and a new polyclonal antibody (SL0525) generated against a putative sea louse ABC transporter. The antibody raised to SL0525 did not react with rat P-gp, suggesting that an ABC transporter, not necessarily P-gp, was isolated. C219 was the only antibody to localize P-gp in all 3 salmon tissues (intestine, kidney and liver). American lobster (Homarus americanus) was used as a reference crustacean for L. salmonis immunostaining reactions and showed positive staining in the hepatopancreatic and intestinal tissues with all 3 antibodies. The L. salmonis showed positive staining in the intestinal epithelial lining with all antibodies. This report represents the first documented evidence for the expression of ABC transporters in L. salmonis, its Atlantic salmon host, and the American lobster.

Ultrastructural examination of the host cellular response in the gills of Atlantic salmon, Salmo salar, with amoebic gill disease.

Gills from Atlantic salmon with experimentally induced amoebic gill disease (Neoparamoeba spp.) were examined with transmission electron microscopy to assess pathology and host-cell responses. Amoebae were found either on the surface epithelium or with pseudopodia extending deeply into invaginations of epithelial cells. The amoebae had various densities along the plasma membrane and contained electron-dense deposits within their cytoplasm. Surface epithelial cells sloughed from the gills and had features consistent with apoptosis, including rounded shape, loss of surface microridges, and hypercondensation of nuclear chromatin. Affected areas of gills had fusion of secondary lamellae with interlamellar spaces occupied by mitotic epithelial cells and eosinophils. Eosinophils contained abundant fusiform-shaped granules that measured approximately 1 microm long and 360 nm wide. The granule consisted of an electron-dense matrix with a central inclusion that was less electron-dense, consisting of particulate and fibrillar material. In many instances, the central inclusion appeared empty and 90% of the eosinophils had morphology suggestive of piecemeal degranulation. Also observed within affected areas were a few neutrophils, mucous cells releasing mucus, and a small number of dendritic-like cells.

Correlation of virus replication in tissues with histologic lesions in Atlantic salmon experimentally infected with infectious salmon anemia virus.

We have studied the replication of virus in tissues and development of lesions associated with infectious salmon anemia virus (ISAV) infection in Atlantic salmon using in situ hybridization (ISH) with a riboprobe targeting ISAV RNA segment 7 messenger RNA. Fish were infected with three ISAV isolates (U5575-1, RPC-01-0593-1, Norway 810/9/99) and then euthanatized sequentially at 3, 6, 10, and 13 days postinoculation (dpi) and thereafter once a week for 8 weeks. Severe histopathologic lesions were observed in tissues from all groups beginning at the onset of mortality. The severe histopathologic lesions correlated with maximum intensity and frequency of ISH signals (P < 0.001). There was a strong association between the hybridization signals and severity of lesions in the liver, kidney, and heart (R = 0.81, 0.70, and 0.78, respectively; P < 0.001). The distribution of ISH signals indicated the presence of a viremia because signals were observed predominantly in individual blood cells and endothelial cells, and possibly hematopoietic cells of head kidney, but not in the necrotic hepatocytes and renal epithelium. Of the organs sampled, the heart was the first and last to show ISH signals, possibly because of increased activity of the endocardial endothelial cells and the underlining macrophages, which continuously trap and remove circulating virus, and therefore represents the best tissue sample for screening of suspected infected fish. On the basis of mortality, severity of lesions, and intensity and frequency of ISH signals, ISAV isolate Norway 810/9/99 was the most virulent and U5575-1 the least virulent isolate studied.

Morphological characterization and notes on the life cycle of a newly discovered variant of Loma salmonae (Putz, Hoffman & Dunbar) from a natural infection of chinook salmon, Oncorhynchus tshawytscha (Walbaum).

Two variants of Loma salmonae occur in net-pen reared chinook salmon, Oncorhynchus tshawytscha. The typical variant (OA) has a host specificity for salmonids of the genus Oncorhynchus whereas the atypical variant (SV) has a host specificity for brook trout, Salvelinus fontinalis, and in this study, the ultrastructure of the two are compared. In fish at 8 weeks post-exposure xenomas of the SV variant have a very high proportion of mature spores compared with other developmental stages, while in xenomas of the OA variant there are fewer spores and many other developmental stages. Spores of the SV variant had up to 20 turns of their polar tube whereas those of the OA variant only had 17. Furthermore, the spores of the SV variant were significantly larger than those of the OA variant. The sporophorous vesicle for both variants appears to form around a sporogonial plasmodia, which results in many spores developing within the vesicle.

Ultrastructural changes in the lungs of neonatal rats intratracheally inoculated with meconium.

Meconium aspiration syndrome has been for many years an important cause of neonatal respiratory distress in newborn babies and sporadically reported in animals. This investigation was designed to study the ultrastructural and morphometric changes in the lungs of neonatal rats following the intratracheal inoculation of meconium. Seven-day-old Fischer-344 rats (n = 24) were randomly allocated in two groups. One group was intratracheally inoculated with saline solution and the second group received homologous meconium. Neonates were euthanatized at 1, 3 and 7 postinoculation days (PID) and lungs were examined by light and electron microscopy. Saline solution did not induce any ultrastructural changes in the lung. In contrast, meconium induced deciliation, recruitment of neutrophils and pulmonary alveolar macrophages to the bronchoalveolar space, intravascular sequestration of neutrophils and aggregation of platelets at PID 1 and 3. Other ultrastructural changes at PID 1 and 3 included interstitial edema and escape of red cells and fibrin into the alveolar space and interstitium. Interstitial edema and sequestration of neutrophils were responsible for the significant increase in thickness of alveolar septa. At PID 7 there was hyperplasia and enlargement of type II pneumocytes as well as interstitial proliferation of mesenchymal cells with intra-alveolar fibrosis. It was concluded that intratracheal inoculation of meconium in neonatal rats induces acute ultrastructural changes followed by a reparative response.

Ultrastructural study of the late stages of Loma salmonae development in the gills of experimentally infected rainbow trout.

The main objective of this investigation was to examine the ultrastructural features of gills from rainbow trout experimentally infected with Loma salmonae to determine the morphological events that occur during the late stages of development of this parasite. Peripheral distribution of the mature parasites inside round xenomas was observed at weeks 5 and 6 postexposure (PE), but eventually the parasite occupied the entire xenoma. Degenerative changes were observed only in immature parasites at week 7 PE, and eventually an inflammatory reaction with a cellular infiltration was directed against mature spores. Round, flattened, and irregular shaped xenomas were observed at week 8 PE. The round xenomas showed a severe inflammatory response with disintegration of the xenoma membrane. This event was accompanied by eversion of polar tubes within the attacked xenoma and by the simultaneous presence of 2 tubular appendages, the type I and II tubules. Flattened xenomas were observed below the endothelium of gill lamella arteries. The irregular xenomas were located in the connective tissue of the gill filament and showed multiple projections occupied by spores. Both flattened and irregular xenomas showed no evidence of inflammatory reaction. An earlier proposed hypothesis is expanded to explain how L. salmonae is implanted beneath lamellar endothelium and within filament connective tissue.

Microscopic changes induced by the intratracheal inoculation of amniotic fluid and meconium in the lung of neonatal rats.

Meconium aspiration syndrome is a major contributor to neonatal respiratory distress in infants and it has been sporadically recognized in neonatal animals. This investigation was designed to study the short and long term effects of meconium and amniotic fluid in the lungs of neonatal rats. Seven-day-old rats (n = 123) divided in three groups were intratracheally inoculated with saline solution, amniotic fluid or meconium. Rats were euthanatized on 1, 3, 7, 14, 28, 56 and 112 postinoculation days (PID) and the lungs were examined by light microscopy. Saline solution did not induce any change while amniotic fluid elicited only a mild foreign body response which disappeared by PID 14. In contrast, meconium induced an exudative alveolitis characterized by recruitment of neutrophilsn in the bronchoalveolar spaces. Meconium also induced atelectasis, hyperinflation and thickening of alveolar septa all of which had disappeared by PID 14. Starting at PID 7, neutrophils were progressively replaced by macrophages, giant cells, and some fibroblasts. There were sporadic foci of mineralization starting at PID 14 and lasting up to PID 112. Some mineralized foci became lined with cuboidal epithelial cells at PID 28. Meconium was slowly degraded but still evident by PID 112. It was concluded that inoculation of meconium in neonatal rats induces acute microscopic changes typical of meconium aspiration syndrome. The long term lesions induced by meconium consisted of persistent multifocal histiocytic alveolitis and bronchiolitis reaction with occasional foci of calcification.

Video-assisted thoracoscopic thymectomy for myasthenia gravis.

BACKGROUND: Thymectomy is an effective, but radical therapy for myasthenia. Video-assisted thoracic surgery (VATS) may allow a minimally invasive alternative to the standard sternotomy approach. AIMS: To audit prospectively the outcome of VATS thymectomy for myasthenia gravis in a unit specializing in advanced VATS techniques. METHODS: Twenty-six patients underwent VATS thymectomy between 1997 and 2001. Most underwent preoperative plasma exchange therapy. Seventeen women and nine men with a median age of 36 years (range 17-71 years) had a right-sided VATS approach to remove all anterior mediastinal fat and thymic tissue. RESULTS: There was no perioperative mortality and all procedures were concluded successfully, with one patient requiring sternotomy. Twenty-five patients were extubated in theatre and one patient required 17 h of assisted ventilation. The other significant complication was a diathermy injury to the phrenic nerve, which recovered. Median postoperative stay was 4 days (range 2-6 days), with median postoperative chest drainage for 2 days (range 1-3 days). Three patients had progression of disease postoperatively. The remainder were asymptomatic (7), improved (14) or stable (2). CONCLUSION: In a dedicated unit with neurological and intensive care support, VATS thymectomy is a safe, effective method of obtaining remission or improvement in myasthenia gravis (MG). While achieving the same surgical goal, this approach offers advantages of improved cosmesis, shorter recovery time and minimal chest wall disruption over the gold standard of sternotomy. Better patient acceptance of this minimally invasive technique may result in wider application of the benefits of thymectomy in MG.

Ultrastructural study of the early development and localization of Loma salmonae in the gills of experimentally infected rainbow trout.

The early ultrastructural stages of Loma salmonae were studied in the gills of experimentally infected rainbow trout. No parasitic stages were identified during the first 2 wk of the infection. By week 3 postexposure (PE), uninucleate and binucleate meronts were recognized within host cells (no xenomas) associated with the capillary channels of secondary lamellae and lamellar arteries. An inflammatory reaction was absent. In secondary lamellae, infected cells were isolated from the capillary lumen, and some were recognized as pillar cells. In lamellar arteries, infected cells were localized beneath the endothelium and not in the lumen. Inflammatory reaction and destruction of parasites inside blood cells in the lumen of secondary lamellae were observed by week 4 PE. Three hypotheses, i.e., isolation, internalization, and evasion, are proposed to explain the localization of the infected cells in the gills. It is concluded that meronts are the earliest parasitic stage observed by week 3 PE, pillar cells are secondarily infected by phagocytosis of infected cells in the blood, endothelial cells of gills are not infected, and inflammatory response to the parasite starts by week 4 PE.

Localization of the initial developmental stages of Loma salmonae in rainbow trout (Oncorhynchus mykiss).

The intracellular microsporidian parasite Loma salmonae affects salmonids of the genus Oncorhynchus and is a significant cause of economic losses in pen-reared Chinook salmon (O. tshawytscha) in British Columbia. Loma salmonae infection is easily recognized by the xenomas that form in the gills, but early stages of infection are difficult to detect in histologic sections. In situ hybridization (ISH), using an L. salmonae-specific digoxigenin-labeled single-stranded DNA probe, was used to detect the parasite during the early stages of infection. Loma salmonae was detected in the gut mucosal epithelium as early as 24 hours postexposure (PE), and it localized in the lamina propria of the intestine within 24 hours of infection. After the parasite was detected in the lamina propria, dividing merogonic stages in infected cells in the heart were detected by ISH as early as 2 days PE, providing the first evidence of parasitaemia and hematogenous distribution of this parasite in infected blood cells. The parasites inside the infected cells appeared to be undergoing merogony as they passed through the heart, indicating that proliferation may start at the site of infection, before the parasite arrives to the gills for their final developmental phase. This is the first time that L. salmonae passage through the intestinal wall and migration to the heart has been visualized; however, the identity of the cells harboring the parasite has yet to be determined.