Histo-morphological effects on equine synovium after arthroscopic synovectomy using two different motorized synovial resectors and two different intensities.

The purpose of the study was to determine the histo-morphological effects on villous synovium after synovectomy using two different motorized synovial resectors and two different intensities ex-vivo. Thirty-three (n = 33) equine metacarpophalangeal/metatarsophalangeal joints were used. Synovectomy was performed along the dorsomedial/dorsolateral synovium (n = 66) using two motorized synovial resectors (aggressive full radius resector, AFRR, used at two intensities: single treatment, n = 24 vs. triple treatment, n = 21 vs. aggressive meniscus side cutter, AMSC, n = 21). Arthroscopic images were evaluated blindly for resector type and intensity. Histological images were evaluated descriptive for synovial morphology and the extent of tissue loss using a microscopic scale. Scanning electron microscopy described the synovial morphology. The synovectomized areas were specific for each resector used and distinguishable from arthroscopic images. The AFRR demonstrated a clear demarcation between treated and non-treated areas and removed the stratum synoviale completely including parts of the underlying stratum fibrosum. In contrast, the AMSC showed less clear demarcation, villous scaffolds and no involvement of the stratum fibrosum. Triple intense treated AFFR samples resulted in significantly deeper lesions compared to single treatments (p = 0.037) but could not be distinguished on arthroscopic images. The morphological effects on villous synovium differ according to the resector type used. The extent of synovial tissue loss cannot be estimated from arthroscopic images but histologically. The type and use of motorized synovial resector determines the morphological alterations of the treated synovium. Arthroscopic control is considered unsuitable to control synovectomy depth.

Quantitative analysis of infiltrating immune cells and bovine papillomavirus type 1 E2-positive cells in equine sarcoids.

Sarcoids are the most frequently observed skin tumours in equids and consist of cutaneous accumulations of transformed fibroblasts. Their aetiopathogenesis is closely linked to a presumably abortive infection by bovine papillomavirus (BPV) types 1 and 2. In cattle, dermal fibropapillomas induced by BPV1/2 usually regress spontaneously due to a local, cell-mediated, immune response; however, equids appear to lack an effective immune response to BPV1/2 and mechanisms of immune evasion have been postulated. As a consequence, equine sarcoids tend to persist and are prone to recur. In this study, cryosections were analysed by immunofluorescent staining and a high content analysis system to determine the presence and distribution of CD4(+), CD8(+), FoxP3(+), RORγt(-), CD206(+) and CD14(+) cells, along with expression of the BPV1 early regulatory protein E2. A higher density of cells was positive for BPV1 E2(+) within the transformed tissue than in perilesional tissue or normal skin of horses with sarcoids and control horses. The proportion of CD8(+) cytotoxic T cells was significantly increased in perilesional and lesional tissues, whereas CD4(+) T helper cells were present in higher density only in lesional tissue compared to normal skin from horses with and without sarcoids. The proportion of pro-inflammatory CD4(+)FoxP3(+)RORγt(+) regulatory T cells was decreased in sarcoid tissue compared to perilesional, distant and control tissue. There were no significant differences in densities of CD4(+)FoxP3(+) RORγt(-) regulatory T cells between sarcoids and control tissues. Equine sarcoids are characterised by infiltrations of CD8(+) and CD4(+) T cells, with decreased representation by pro-inflammatory CD4(+)FoxP3(+)RORγt(+) regulatory T cells.

High-content analysis of factors affecting gold nanoparticle uptake by neuronal and microglial cells in culture.

Owing to their ubiquitous distribution, expected beneficial effects and suspected adverse effects, nanoparticles are viewed as a double-edged sword, necessitating a better understanding of their interactions with tissues and organisms. Thus, the goals of the present study were to develop and present a method to generate quantitative data on nanoparticle entry into cells in culture and to exemplarily demonstrate the usefulness of this approach by analyzing the impact of size, charge and various proteinaceous coatings on particle internalization. N9 microglial cells and both undifferentiated and differentiated SH-SY5Y neuroblastoma cells were exposed to customized gold nanoparticles. After silver enhancement, the particles were visualized by epipolarization microscopy and analysed by high-content analysis. The value of this approach was substantiated by assessing the impact of various parameters on nanoparticle uptake. Uptake was higher in microglial cells than in neuronal cells. Only microglial cells showed a distinct size preference, preferring particles with a diameter of 80 nm. Positive surface charge had the greatest impact on particle uptake. Coating with bovine serum albumin, fetuin or protein G significantly increased particle internalization in microglial cells but not in neuronal cells. Coating with wheat germ agglutinin increased particle uptake in both N9 and differentiated SH-SY5Y cells but not in undifferentiated SH-SY5Y cells. Furthermore, internalization was shown to be an active process and indicators of caspase-dependent apoptosis revealed that gold nanoparticles did not have any cytotoxic effects. The present study thus demonstrates the suitability of gold nanoparticles and high-content analysis for assessing numerous variables in a stringently quantitative and statistically significant manner. Furthermore, the results presented herein showcase the feasibility of specifically targeting nanoparticles to distinct cell types.

Pododermatitis in Captive and Free-Ranging Greater Flamingos (Phoenicopterus roseus).

Pododermatitis is frequent in captive flamingos worldwide, but little is known about the associated histopathologic lesions. Involvement of a papillomavirus or herpesvirus has been suspected. Histopathologic evaluation and viral assessment of biopsies from 19 live and 10 dead captive greater flamingos were performed. Selected samples were further examined by transmission electron microscopy and immunohistochemistry. Feet from 10 dead free-ranging greater flamingos were also evaluated. The histologic appearance of lesions of flamingos of increasing age was interpreted as the progression of pododermatitis. Mild histologic lesions were seen in a 3-week-old flamingo chick with no macroscopic lesions, and these were characterized by Micrococcus-like bacteria in the stratum corneum associated with exocytosis of heterophils. The inflammation associated with these bacteria may lead to further histologic changes: irregular columnar proliferations, papillary squirting, and dyskeratosis. In more chronic lesions, hydropic degeneration of keratinocytes, epidermal hyperplasia, and dyskeratosis were seen at the epidermis, as well as proliferation of new blood vessels and increased intercellular matrix in the dermis. Papillomavirus DNA was not identified in any of the samples, while herpesvirus DNA was seen only in a few cases; therefore, these viruses were not thought to be the cause of the lesions. Poor skin health through suboptimal husbandry may weaken the epidermal barrier and predispose the skin to invasion of Micrococcus-like bacteria. Histologic lesions were identified in very young flamingos with no macroscopic lesions; this is likely to be an early stage lesion that may progress to macroscopic lesions.

In vitro rescue of FGA deletion by lentiviral transduction of an afibrinogenemic patient's hepatocytes.

BACKGROUND: Congenital afibrinogenemia is a rare inherited autosomal recessive disorder in which a mutation in one of three genes coding for the fibrinogen polypeptide chains Aα, Bß and γ results in the absence of a functional coagulation protein. A patient with congenital afibrinogenemia, resulting from an FGA homozygous gene deletion, underwent an orthotopic liver transplant that resulted in complete restoration of normal hemostasis. The patient's explanted liver provided a unique opportunity to further investigate a potential novel treatment modality. OBJECTIVE: To explore a targeted gene therapy approach for patients with congenital afibrinogenemia. METHODS AND RESULTS: At the time of transplant, the patient's FGA-deficient hepatocytes were isolated and transduced with lentiviral vectors encoding the human fibrinogen Aα-chain. FGA-transduced hepatocytes produced fully functional fibrinogen in vitro. CONCLUSIONS: Orthotopic liver transplantation is a possible rescue treatment for failure of on-demand fibrinogen replacement therapy. In addition, we provide evidence that hepatocytes homozygous for a large FGA deletion can be genetically modified to restore Aα-chain protein expression and secrete a functional fibrinogen hexamer.

Intestinal atresia and ectopia in a bovine fetus.

A 2-year-old Red Holstein cow was presented with uterine torsion at 235 days of pregnancy. The fetus extracted by cesarean section had weak vital signs and marked abdominal distention. An edematous pouch that contained tubular structures with peristaltic activity was associated with the umbilical cord. Because of poor prognosis, both dam and fetus were euthanized. At necropsy, the fetus had severe distention of the forestomachs, abomasum, and proximal small intestine; absence of distal small intestine, cecum, and proximal colon; atresia of the 2 blind ends of the intestine; and atrophy of distal colon and rectum. The tubular structures associated with the umbilical cord were identified as the segments of intestine that were absent in the fetus. Intestinal atresia combined with ectopia may be caused by local ischemia during temporary herniation and rotation of the fetal gut into the extraembryonic coelom. The close connection between ectopic intestine and amniotic sheath of the umbilical cord in this case may have facilitated vascularization and allowed development and viability of the ectopic intestine.

Doppler sonography of the medial arterial blood supply to the coxofemoral joints of 36 medium to large breed dogs and its relationship with radiographic signs of joint disease.

The medial arterial supply to 68 of the 72 coxofemoral joints of 36 medium to large breed dogs was examined ultrasonographically. The medial circumflex femoral artery and three branches were identified; the artery and its transverse branch were identified in all 68 joints, and the deep branch was identified in 61 joints, and the ascending branch was identified in 63. However, the acetabular and obturator branches were not identified. The pulsatility index, the mean velocity and the peak systolic velocity of the medial circumflex femoral artery were determined and associated with a radiographic score of degenerative coxofemoral joint disease and a lath distraction index (LDI). In joints with a LDI greater than 0.35, the pulsatility index was significantly lower (P=0.023) and its mean velocity was higher (P=0.005). However, no significant associations were observed in individual dogs when the measurements in both joints were taken into account.

Exposure of rainbow trout (Oncorhynchus mykiss) to nonylphenol is associated with an increased chloride cell fractional surface area.

Nonylphenol is a biodegradation product of a widely used group of non-ionic detergents. Because of its ubiquitous distribution and persistence, nonylphenol is present in surface waters as a pollutant. Little is known about its biological effects at environmentally relevant concentrations other than its action as a xenoestrogen. The goal of the present paper was to study the trout gill surface epithelium as the major interface between fish and water in view of possible morphological alterations due to exposure to nonylphenol. Rainbow trout were intermittently exposed to 10 micrograms/l nonylphenol and gill samples from experimental and control animals were investigated by scanning electron microscopy. Gill surface epithelium was scrutinised for changes in chloride cell density and their status regarding cell surface modifications. In addition, chloride cell fractional surface area (CCFA) was determined by morphometrical methods. Statistical analysis revealed a highly significant increase of CCFA in animals exposed to nonylphenol as compared to control animals (P = 0.0001). Semi-quantitative assessment of the other parameters suggested a higher chloride cell density and a larger proportion of chloride cells bearing microvilli. Taken together, these results provide evidence that exposure of trout to nonylphenol is associated with a substantial increase in the active interface of chloride cells with water. We interpret these findings as being a means to further the fish's capacity for calcium exchange.

Ultrastructural validation of an improved culture system for boar efferent duct epithelium.

A tissue culture system in which cells retain defined ultrastructural and functional characteristics was established to provide a basis for functional investigations of the efferent ductules in boars. A currently used culture protocol for rat epididymal epithelium was used as a starting point and was subsequently modified because of unsatisfactory results. Epithelial plaques were isolated by mechanical uncoiling of the ductules and two sequential enzymatic digestion steps. Plaques were seeded onto extracellular matrix-coated permeable membranes and maintained in a two-chamber system. Samples taken before seeding and after 7 days in culture were processed for transmission and scanning electron microscopy. Perfusion-fixed material from earlier studies served as a reference to assess ultrastructural preservation. In addition, endocytotic activity was investigated by adding cationized ferritin to the culture medium on day 8 before fixation. At the end of the disaggregation procedure, cells were cuboidal, while cilia, microvilli and cell organelles were well preserved. After 7 days in culture, three types of cell formation were observed: cysts, pseudotubules and epithelial sheets. Cell sheets were made up of closely juxtaposed cells bearing motile kinocilia and exhibiting well-developed polar differentiation, as judged from the localization of cell junctions and organelles. Although it did not return to the values of native material, cell height was greater than that of cells grown according to the pre-existing protocol. Furthermore, preferential uptake of ferritin by principal cells after 7 days in culture was demonstrated. Preservation of these fundamental characteristics of the in vivo state corroborates that our in vitro system will furnish reliable information.

Transport of biologically active material in laser cutting.

The transport of biologically active material during laser cutting with CO2 and Er lasers is demonstrated. This transport mechanism removes particles from the surface of gelatin, agar, and liver samples into the depth of the laser-formed craters. The transport phenomenon is explained by a contraction and condensation of enclosed hot water vapor. We show by cultivating transported bacteria in agar that biological particles can survive the shock of the transport. Determination of the numbers of active cells evidences a more pronounced activity of the cultivated bacteria after impact with an Er laser than with a CO2 laser.