Effect of bovine freeze-dried amniotic membrane (Amnisite-BA™) on uncomplicated canine corneal erosion

Objective   To evaluate the effectiveness of bovine freeze-dried amniotic membrane (FD-AM) (Amnisite-BA™) in the surgical treatment of corneal ulceration in dogs.
Animals studied   Eight normal Shih-tzu dogs.
Procedures   The corneas of 16 eyes were scored with an 8.0-mm trephine under general anesthetic and 100% ethanol was applied to remove a standardized button of corneal epithelium. The eyes were treated as described below and the corneas were evaluated 48 h later. The dogs were divided into four treatment groups: (i) control, (ii) amniotic membrane transplantation (AMT), (iii) nictitating membrane flap and (iv) contact lens. The proportion of the corneal wound that healed was calculated and all eyes were enucleated. Histological sections of cornea were assessed with the proliferating cell nuclear antigen (PCNA) assay.
Results   The proportion of corneas healed in the different treatment groups was (i) 38.02%, (ii) 89.15%, (iii) 52.31%, and (iv) 60.56%. Epithelial healing was significantly increased in the AMT group (ii) ( P  = 0.001) while groups (iii) and (iv) were not significantly different from the control group ( P  = 0.537 and P  = 0.198, respectively). The number of PCNA positive cells was (i) 275.00, (ii) 740.50, (iii) 285.75 and (iv) 420.59, these varying compared with the control group with statistical significance of (ii) P  = 0.002, (iii) P  = 0.999, and (iv) P  = 0.467. The greatest healing rate and epithelial cell proliferation was achieved with AMT compared to the other treatment regimes.
Conclusions  The results of this study show that FD-AM transplantation is an effective treatment for enhancing canine corneal wound healing and suggest that the approach will provide superior results compared to conventional treatments for the condition.     

The effects of moxifloxacin ophthalmic solution 0.5% or gatifloxacin ophthalmic solution 0.3% treatment on corneal wound healing in pigmented rabbits following anterior keratectomy

Purpose These studies examined corneal healing rates, Type‐IV collagen and zonula occludens membrane‐associated protein (ZO‐1) expression, as well as aqueous PGE₂ and IL‐1β concentrations in pigmented rabbits treated with either moxifloxacin 0.5%, gatifloxacin 0.3% or BSS^® following anterior keratectomy. Methods Anterior keratectomy surgery was followed by topical administration with commercial ophthalmic formulations of either moxifloxacin or gatifloxacin or BSS^® (TID for 96 h). Images of the fluorescein‐stained healing corneas were analyzed for wound area. At 48 or 96 h following surgery, aqueous humor samples were collected and analyzed for the inflammatory mediators PGE₂ and IL‐1β using an ELISA. The corneas were subsequently evaluated using both scanning and transmission electron microscopy. In a second parallel study, corneas were evaluated at both 48 and 96 h for Type‐IV collagen and ZO‐1 expression using immunohistochemistry. Results Fluorescein‐stained corneal images at 96 h postsurgery demonstrated that 90% ± 8% re‐epithelialization for moxifloxacin, 81% ± 14% for gatifloxacin, and 88 ± 6% for BSS^® (P > 0.05). PGE₂ levels in the aqueous humor of fluoroquinolone treated eyes were reduced at 48 h compared to BSS^® treated eyes. IL‐1β was undetectable in all samples. No differences in Type‐IV collagen or ZO‐1 expression were observed between any treatment groups. There were no differences between groups in histological appearance or in ultrastructural healing processes. Conclusions These studies demonstrated that the commercial ophthalmic formulations of moxifloxacin and gatifloxacin were similar to each other in their effects on the levels of aqueous humor PGE₂ and rates of corneal wound re‐epithelialization.     

Canine amniotic membrane transplantation for corneal reconstruction after the excision of dermoids in dogs

Transplantation of canine amniotic membrane (AM) in conjunction with a third eyelid flap was performed after the removal of large dermoids by keratectomy and conjunctivectomy on 7 eyes of 7 dogs. Corneal epithelialization was completed within 2 weeks after the transplantation. Five eyes attained normal transparency of the cornea within 5 weeks. Slight pigmentation of the bulbar conjunctiva at the limbus was observed in 1 dog that had pre-existing pigmentary keratitis. Neovascularization and scarring of the cornea and impaired vision were not found in any dogs at 8 weeks after the transplantation. In conclusion, transplantation of canine AM can promote corneal healing after the excision of large dermoids in dogs.     

The use of xenologous amniotic membrane to repair canine corneal perforation created by penetrating keratectomy

This study was performed to evaluate the use of glycerol-preserved equine amniotic membrane as replacement for full-thickness corneal defects in dogs. Eighteen mixed-breed dogs were used. A perilimbal, full-thickness, 5 mm square corneal defect was created surgically, and a donor implant of equine amniotic membrane of the same size and shape sutured in place with 10–0 nylon simple interrupted sutures. Corneal edema was observed near the implant 24 h after surgery, but was absent after 1 week. Granulation tissue and corneal vascularization superficial to the implant were noticed on postoperative day 7, but were absent on day 30. Corneal vascularization persisted until the end of the experiment. There was no fluorescein retention by postoperative day 30. There was slight clearing of the corneal implant by postoperative 30, and slight pigmentation of the donor implant observed at postoperative day 180. An acute inflammatory process as well as fibroblasts were present at early postoperative stages. At postoperative day 60 there was no inflammatory cellular infiltrate, but fibroblasts and fibrosis were present. Corneal architecture was restored at the end of the experiment, with a layering of the epithelium–stroma–debris of amniotic membrane–stroma–endothelium present, and pigmentation and vascularization present in the deep layers of the cornea. Although vascularization indicated some degree of graft rejection, the clinical and histological evidence indicates that the xenologous amniotic membrane can be useful as a tectonic graft in the repair of full-thickness lesions of the cornea of dogs.     

Successful treatment of an unusually large corneal epithelial inclusion cyst using equine amniotic membrane in a dog

A 10‐year‐old intact male Yorkshire Terrier was referred for investigation of a large raised and nonpainful corneal lesion oculus dexter. Clinical examination revealed a pale, translucent corneal mass, which occupied half of the corneal surface and measured 11 mm × 11 mm × 13 mm. The mass was removed by superficial keratectomy and equine amniotic membrane (AM) was transplanted into the large corneal defect to cover the wound and provide tectonic support for the remaining cornea. The mass was histologically confirmed as a corneal epithelial inclusion cyst. There was no evidence of recurrence or complication at the surgical site 100 days postoperatively. Corneal epithelial inclusion cysts are uncommon in dogs. Although superficial keratectomy is the recommended treatment for corneal inclusion cyst, the combination of superficial keratectomy and AM transplantation had to be considered as an alternative for repair of large corneal defects. This is the first case report of the combined application of AM and superficial keratectomy to successfully treat a corneal inclusion cyst in a dog.     

Microstructure and glycosaminoglycan ratio of canine cornea after reconstructive transplantation with glycerin-preserved porcine amniotic membranes

Objective  Although amniotic membranes of canine, feline, and equine species have some advantages as corneal transplantation material in many canine ocular diseases, their softness, thinness, and low availability can pose problems. As an alternative, the more abundant porcine amniotic membranes may be used. This paper describes the use of glycerin-preserved porcine amniotic membranes in corneal transplantation in eight normal dogs.
Method  A 0.4-mm deep recipient bed in the axial cornea of the OS of all dogs was created using an 8-mm Barron radial vacuum trephine. The recipient bed was then filled with amnion, and the entire cornea was covered with another piece of the glycerin-preserved membrane. The ocular signs evaluated were corneal opacity and corneal vascularization. The dogs were euthanized on days 5, 10, 20, or 40 after surgery, and samples were collected to evaluate corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content and the glycosaminoglycan (GAG) ratio.
Results  Corneal opacity was observed immediately after surgery. Restoration of corneal transparency, regression of corneal vascularization, and visualization of the pupil and iris were noted on day 40.
Conclusions  The clinical observations were supported histologically by regained corneal thickness, parenchymal cell number, mean collagen fibril diameter, collagen fibril content, and GAG ratio, suggesting that this technique may be a novel method for the treatment of ocular surface disorders.     

Identification of putative orthologs of clinically relevant antimicrobial peptides in the equine ocular surface and amniotic membrane

Objectives

This study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies.

Procedures

A targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression.

Results

Equine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins.

Conclusions

This study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis.     

Cultivation of corneal epithelial cell sheets on canine amniotic membrane

Objective To develop and assess canine corneal epithelial cell sheets cultivated from limbal stem cells on amniotic membrane. Procedures Canine corneal limbal segments were obtained from six beagle dogs. Cryopreserved denuded amniotic membranes (obtained from Miniature Dachshund and Cavalier King Charles Spaniel breeds) from which the epithelial cells were removed were used as scaffolds. The limbal segments were cultured on these amniotic membranes with 3T3 feeder cells for 2 weeks. The harvested corneal epithelial cell sheets were stained with H&E for histologic analysis. The harvested sheets were analyzed immunohistochemically using a corneal epithelium‐specific marker keratin 3(K3) and putative stem cell markers ABCG2, p63, and vimentin. Results Cultivated cells from the corneal limbal tissues reached confluency in 7–8 days. The cultivated cells adhered to the denuded amniotic membrane and formed a sheet. The cultivated cell sheet was transparent and consisted of five to eight layers. K3 was observed in all layers and ABCG2, p63, and vimentin were notably present in the basal layer of the cultivated canine epithelium by immunofluorescence. Conclusions Canine corneal epithelial cells were successfully cultivated on the canine amniotic membrane. The cultivated epithelial sheets contained putative stem cells in the basal layer and had a stratified epithelium.     

Amniotic membrane transplantation for the reconstruction of the ocular surface in three cases

This paper describes the use of amniotic membrane in the reconstructive surgical repair of generalized keratomalacia, ankyloblepharon, and after fibrous histiocytoma removal in two dogs and a cat. Case 1 was an 11-year-old female Yorkshire terrier with severe bullous keratomalacia in the left eye (OS). A frozen canine amniotic membrane graft and a third eyelid flap were applied. At day 80 postoperatively, only a mild scar and corneal vascularization were present. Case 2 was a 4-year-old female Siamese cat with symblepharon of both eyes after rhinotracheitis. Resection of the conjunctiva and frozen canine amniotic membrane transplant were performed. One month later, there was a little corneal scarring and corneal vascularization. Case 3 was a 6-year-old female terrier with a scleral and corneal mass at the 11-12 o'clock position (OS). Resection of the mass and amniotic membrane transplantation were performed. The mass was a fibrous histiocytoma localized to the sclera and cornea. This eye healed with mild conjunctivalization and no pigmentation. Amniotic membrane transplantation can be used as a method of reconstruction of the ocular surface with good repair of the cornea and minimal scarring in small animals.     

Elevated Plasma Levels of Interleukin 1β, Tumour Necrosis Factor α and Monocyte Chemotactic Protein 1 Are Associated with Pregnancy Toxaemia in Ewes

Pregnancy toxaemia is a metabolic disorder that results from an inadequate energy supply to the growing maternal–fetal unit. The mechanism underlying the pathogenesis of the syndrome has not been fully clarified; however, a key role for cytokines and chemokines including interleukin 1 β (IL-1β), tumour necrosis factor α (TNF-α) and monocyte chemotactic protein 1 (MCP-1) has been indicated in women and experimental animals. However, information on the maternal plasma levels of IL-1β, TNF-α and MCP-1 in ewes with pregnancy toxaemia is limited. Thus, the present study was designed to determine plasma IL-1β, TNF-α and MCP-1 concentrations in ewes with severe (n = 6) and mild (n = 4) naturally occurring pregnancy toxaemia and in uncomplicated pregnant ewes (n = 10) using enzyme-linked immunosorbent assay (ELISA). All ewes with pregnancy toxaemia had significantly lower body temperature and respiratory rate than uncomplicated pregnant ewes (p < 0.05). With the highest concentrations in severe cases, heart rate, proteinuria and serum uric acid levels as well as plasma IL-1β, TNF-α and MCP-1 were significantly different among all three groups (p < 0.05). The plasma concentrations of IL-1β in control ewes and ewes with mild and severe toxaemia were 15.81 ± 3.90 pg/ml, 23.83 ± 2.42 pg/ml and 34.55 ± 8.03 pg/ml, respectively. The plasma concentrations of TNF-α in control ewes and ewes with mild and severe toxaemia were 7.71 ± 1.61 pg/ml, 16.13 ± 3.63 pg/ml, and 22.85 ± 3.64 pg/ml, respectively. The plasma concentrations of MCP-1 in control ewes and ewes with mild and severe toxaemia were 101.70 ± 9.86 pg/ml, 134.75 ± 6.24 pg/ml, and 157.67 ± 9.69 pg/ml, respectively. Moreover, plasma IL-1β, TNF-α and MCP-1 levels were positively correlated with clinical and well-establish biochemical parameters of pregnancy toxaemia, serum uric acid and proteinuria (p < 0.01). Concomitant increase of plasma IL-1β, TNF-α and MCP-1 concentrations along with serum uric acid, proteinuria, and worsening of the clinical signs indicates that such cytokines are involved in the aetiopathogenesis and in perpetuation of the local and systemic inflammatory reactions in pregnancy toxaemia in ewes. Hence, plasma IL-1β, TNF-α and MCP-1 may potentially serve as markers to monitor prognosis of pregnancy toxaemia in ewes.     

Tear production in dogs with epiphora and corneal epitheliopathy

Measurement of tear volume by phenol red thread tear test (PRT), Schirmer's tear test (STT-1) and Schirmer's tear test with topical anesthesia (STT-2), and vital staining with a combination of fluorescein and rose Bengal of the cornea were performed in dogs with normal eyes and those with epiphora. The breeds included the Shih-Tzu ( n  = 26), and five other breeds ( n  = 50). The PRT, STT-1 and STT-2 results from the five breeds of normal dogs were 26.9 ± 3.0 mm, 17.4 ± 4.3 mm, and 8.8 ± 3.2 mm, respectively. The PRT, STT-1, and STT-2 results for the Shih-Tzu eyes were (mean ± standard deviation): 28.2 ± 4.3 mm, 19.5 ± 4.1 mm, and 9.2 ± 4.5 mm, respectively. In the five breeds, corneal epitheliopathy, as evidenced by the retention of topical fluorescein and rose Bengal, occurred in 97% of dogs with epiphora and in 55% of the dogs without epiphora. Also in the dogs with corneal epitheliopathy the STT-2-value was low (4.0 ± 2.8 mm) compared with those eyes without corneal epitheliopathy (8.8 ± 3.1 mm). In the Shih-Tzu breed the STT-2 results were not significantly different between those dogs with corneal epitheliopathy and those with normal corneas. In the non-Shih-Tzu breeds the decrease in basic tear secretion, as measured by the STT-2, is associated with the corneal epitheliopathy.     

Basic fibroblast growth factor stimulates epithelial cell growth and epithelial wound healing in canine corneas

Objective  To evaluate the effect of basic fibroblast growth factor (bFGF) on the proliferation of canine corneal epithelial cells and epithelial wound healing.
Animal studied  Canine corneal epithelial cells from the corneas of euthanized dogs and corneal epithelial wounds on one eye from each of 24 dogs.
Procedures  The proliferation of corneal epithelial cells in vitro was measured using the methylthiazolyl-tetrazolium (MTT) assay. A corneal wound on one eye of each dog was made with a corneal trephine (6 mm diameter). Four concentrations of bFGF, 0, 100, 500, and 1000 ng/mL, were applied to the affected eyes of dogs, t.i.d. Fluorescein staining was used to assess closure of the corneal epithelial wound.
Results  The addition of bFGF resulted in a significant increase in epithelial proliferation at 24 h after culture, except 1 ng/mL bFGF. Cells with all bFGF treatments proliferated significantly at 48 and 96 h compared to those in the non-bFGF group. bFGF at a concentration of 10 ng/mL promoted cell proliferation maximally. The wound healing rate in the bFGF-treated groups was greater than that in the control. All corneal wounds in bFGF-treated corneas closed by day 7, whereas two of six corneal wounds in the control showed poor healing. None of the eyes developed corneal clouding or neovascularization during the experiment.
Conclusions  Basic fibroblast growth factor accelerated the proliferation of canine epithelial cells and effectively promoted corneal epithelial wound healing.     

Evaluation of transformation growth factor beta1, interleukin-10, and interferon-gamma in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi

The aims of this study were to evaluate the immunomodulatory role of TGF-β₁, IL-10, and INF-γ in spleen and liver extracts and supernatant cultures of white spleen cells from male symptomatic and asymptomatic dogs, naturally infected by Leishmania (Leishmania) chagasi. Thirty dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniosis area, were selected by positive ELISA serological reaction for Leishmania sp. and divided into two groups: asymptomatic (n = 15) and symptomatic (n = 15) consisting of animals with at least three characteristic signs (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). After euthanasia, spleen and liver fragments were collected for ex vivo quantification of TGF-β₁, IL-10, and INF-γ. Naturally active in vitro produced TGF-β₁ was also evaluated in spleen cell culture supernatant. Spleen and liver extract of asymptomatic dogs had higher mean TGF-β₁ levels than symptomatic dogs. High concentrations of IL-10 were found in spleen, and mainly in liver extract of both groups. Higher INF-γ concentrations were found in spleen extracts of symptomatic dogs, and in liver extracts of asymptomatic dogs. Extract of this cytokine was lower in spleen extract. Although INF-γ is being produced in canine infection, mean levels of TGF-β₁ and IL-10 from spleen and liver extracts were quantitatively much higher; suggesting that immune response in both asymptomatic and symptomatic dogs was predominantly type Th2.     

Serum Acute Phase Protein Concentrations in Dogs with Autoimmune Hemolytic Anemia

Background: Serial monitoring of acute phase protein (APP) concentrations in canine autoimmune hemolytic anemia (AIHA) has not been reported.
Hypotheses: Acute canine AIHA is accompanied by an acute phase response (APR) characterized by increased C-reactive protein (CRP) and α1-acid glycoprotein (AAG) concentrations and decreased albumin concentrations.
Animals: Twenty-seven dogs with AIHA and 11 control dogs.
Methods: Prospective, cohort study. CRP, AAG, and albumin concentrations, white blood cell (WBC) count, and packed cell volume (PCV) were determined at admission (day 1), every 48 hours until death or discharge, and on days 30, 90, 180, and 365.
Results: Compared with controls, CRP and AAG concentrations were increased and albumin concentration was decreased in dogs with AIHA (days 1–7; P < .002) and normalized with disease stabilization (days 9–365; P > .05). APP concentrations on day 1 were not predictive of survival, duration of hospitalization, or number of blood transfusions ( P = .153–.940). PCV correlated with APP concentrations over time (CRP r =−.600, AAG r =−.665, albumin r = .533; P < .0001) as did WBC count (CRP r = .253, AAG r = .486, albumin r =−.246; P < .006). Day 1 CRP concentration was lower for dogs that received corticosteroids before referral (115.3 μg/mL) compared with dogs that did not (191.2 μg/mL; P = .02).
Conclusions: An APR occurs in canine AIHA. Initial APP concentrations are not predictive of acute survival, correlate with hematologic markers of remission, and normalize rapidly with disease stabilization.     

Evaluation of topical nalbuphine or oral tramadol as analgesics for corneal pain in dogs: a pilot study

Objective To evaluate the effectiveness of topical nalbuphine or oral tramadol in the treatment of corneal pain in dogs. Animals studied Fourteen male Beagle dogs. Procedures Dogs were divided into three treatment groups and sedated with dexmedetomidine (5 μ/kg IV). A 4 mm corneal epithelial wound was created in the right eye (OD) of all dogs. Sedation was reversed with atipamazole IM. All dogs received pre/post ophthalmic examinations. Post operatively, Group NB (n = 5) received topical 1% preservative‐free nalbuphine OD q8 h and an oral placebo PO q8 h. Group TR (n = 5) received tramadol (4 mg/kg) PO q8 h and topical sterile saline OD q8 h. Group CNTRL (n = 4) received topical sterile saline OD q8 h and an oral placebo q8 h. All dogs received topical 0.3% gentamicin OD TID until healed. Dogs were pain scored using a pain scoring system modified from the University of Melbourne pain scale at 0, 1, 2, 4, and 6 h, then every 6 h by observers masked to treatment, until corneal wounds were healed. Treatment failure was recorded if cumulative pain scores were above a minimum threshold of acceptable pain and rescue analgesia of morphine (1.0 mg/kg IM) was administered subsequently. Result Four dogs in Group NB, one dog in Group TR, and two dogs in Group CNTRL required rescue analgesia. There was no significant difference in the incidence of treatment failure between groups (P = 0.184). Mean time to rescue was 9.16 h. All corneal wounds were healed by 84 h. Conclusions The results of this study suggest tramadol rather than nalbuphine should be further investigated for the treatment of corneal pain.     

Cytokine expression in an ex vivo culture system of duodenal samples from dogs with chronic enteropathies: modulation by probiotic bacteria

There is evidence that probiotics have immune-modulating effects on intestinal inflammation during chronic enteropathies (CE). In an ex vivo culture system we investigated the influence of probiotics on mRNA and protein expression levels of cytokines in intestinal samples from dogs suffering from CE. Duodenal samples of client-owned dogs with CE (group CE; n = 12) were collected during diagnostic endoscopy. Additional duodenal samples of gastrointestinally healthy dogs (group C; n = 4) from an unrelated study were available. Based on histopathological analyses, no pathological changes or only mild to moderate eosinophilic and/or lymphoplasmacytic duodenitis were diagnosed. Tissue samples were cultured: (1) with cell culture medium alone (negative control), (2) with a probiotic cocktail (PC), constituted of three Lactobacilli spp. from healthy canine fecal isolates, (3) with the individual strains of PC, and (4) with a placebo powder. Viability of intestinal tissue and probiotic bacteria before and after culture was evaluated. The mRNA abundance of interleukin (IL)-10, IL-12p40, interferon (IFN)-γ, tumor necrosis factor (TNF)-, and transforming growth factor (TGF)-β1 was analyzed by real-time polymerase chain reaction (RT-PCR). Protein concentrations of IFN-γ and IL-10 were measured in culture supernatant by ELISA. Results of RT-PCR were expressed as 2^{(−2ΔCrossing Point)} × 100 after normalization with β-actin. There was a loss of about 1 log CFU/mL of probiotic bacteria during the incubation period. Viability of tissue was maintained as confirmed by non-significant release of lactate dehydrogenase. In C, addition of PC increased IL-10 mRNA levels (P < 0.1). In CE, PC increased mRNA and protein levels of IL-10 (P < 0.05). On the mRNA level, the ratio of TNF-/IL-10, IFN-γ/IL-10, and IL-12p40/IL-10 decreased after addition of PC (P < 0.05). The results demonstrate favorable effects of PC on regulatory cytokines relative to inflammatory cytokines that might contribute to reduction of intestinal inflammation.     

FC-35 Real-time evaluation of cytokine and protease expression in flea-allergic and nonallergic dogs

Atopic dermatitis (AD) is very common in dogs, but its pathogenesis is not yet fully understood. It has been suggested that a Th2-dominant status may be associated with the occurrence of canine AD. IL-12 is thought to be important for the differentiation of Th1 cells. The IL-12 receptor β2 (IL-12Rβ2) gene is considered to play a critical role in signal transduction and is attracting attention as one of the causative genes of AD in humans. The purpose of this study was to investigate the relationship between IL-12Rβ2 gene expression and canine AD. The canine IL-12Rβ2 gene was cloned by RT-PCR and its nucleotide sequences were determined. Canine IL-12Rβ2 showed 76.8% homology at the amino acid level with human IL-12Rβ2, and its structural motifs were well conserved. cDNA with a 91 bp deletion including the transmembrane region was also cloned, which consequently produced a frame shift and an early stop codon. The deletion region corresponded to exon 14 of the human IL-12Rβ2 gene on chromosome 1. The expression of deleted canine IL-12Rβ2 mRNA in phytohemagglutinin-stimulated peripheral blood mononuclear cells was examined in seven healthy dogs and 11 AD dogs. Both deleted and intact mRNAs were expressed at constant ratios in healthy and AD dogs. The results indicate that the deletion of the transmembrane region is not associated with the occurrence of AD, and that the expression of the deleted mRNA may be constitutive and produced by alternative splicing.
Funding: Self-funded.     

猪圆环病毒Ⅱ型体外诱导3D4/2细胞炎症模型的建立

【目的】通过对猪圆环病毒Ⅱ型（PCV2）体外感染3D4/2细胞浓度、时间与细胞炎症水平进行探讨,建立PCV2体外感染3D4/2细胞炎症反应模型,以期为后期药物调控PCV2诱发3D4/2细胞炎症反应的研究奠定基础。【方法】将3D4/2细胞分为对照组及10⁰、10^-1、10^-2和10^-3 PCV2感染组,每组3个重复。对照组用DMEM培养,各PCV2感染组用不同稀释倍数PCV2液培养,2 h后均更换为含5%胎牛血清（FBS）的DMEM维持液进行培养,培养4、8、12和24 h后分别收集细胞及细胞上清液。采用Griess法检测一氧化氮（NO）水平,DCFH-DA荧光探针法检测活性氧（ROS）水平,酶标法检测还原型谷胱甘肽（GSH）水平,分光光度法检测黄嘌呤氧化酶（XOD）和髓过氧化物酶（MPO）活性,ELISA法测定白细胞介素-1β（IL-1β）、IL-6、肿瘤坏死因子-α（TNF-α）、IL-10、γ干扰素（IFN-γ）、IL-8、单核细胞趋化蛋白1（MCP-1）以及环氧合酶1（COX-1）和COX-2的分泌水平。【结果】10⁰至10^-3 PCV2作用4、8、12和24 h均能够成功感染3D4/2细胞。与对照组相比,10⁰ PCV2在感染3D4/2细胞4、8、12、24 h后ROS水平均极显著升高（P<0.01）,10^-1至10^-3 PCV2感染3D4/2细胞8、12、24 h后ROS水平显著或极显著升高（P<0.05;P<0.01）;10⁰至10^-3 PCV2感染3D4/2细胞8、12、24 h后,细胞内NO浓度及MPO活性显著提高（P<0.05）,细胞上清液中的IL-1β、IL-6、TNF-α、IL-10、IFN-γ、IL-8和MCP-1水平及COX-1活性均显著或极显著升高（P<0.05;P<0.01）,其中10⁰ PCV2感染3D4/2细胞后,各炎症因子水平上升最显著,且随着时间的延长,NO浓度逐渐升高,XOD活性逐渐降低。【结论】PCV2可诱导3D4/2细胞炎症反应,且10⁰ PCV2体外感染3D4/2细胞4~12 h是建立炎症模型的最佳条件。     

Serum nitrate and nitrite in dogs with spontaneous cardiac disease

The purpose of this study was to determine whether nitric oxide (NO) concentrations are high in dogs with chronic valvular disease (CVD) and dilated cardiomyopathy (DCM) compared to healthy controls and to determine whether NO concentrations are correlated with type of cardiac disease, disease severity, medical therapy, or serum tumor necrosis factor (TNF) and interleukin-1 (IL-1). Blood was collected from 32 dogs with DCM, from 10 dogs with CVD, and from 10 healthy controls. Indirect determination of NO concentrations was performed by a commercial photoabsorbance assay that uses a Greiss reagent to measure the concentration of nitrite and nitrate (NN), end products of NO metabolism. TNF and IL-1 activities were measured by bioassay. Mean NN concentrations were significantly higher in dogs with heart disease (median, 4.57 microM; range, 0.00-31.05 microM) than in controls (median, 0.00 microM; range, 0.00-6.16 microM; P = .04). NN concentrations in dogs with cardiac disease were not correlated with type or severity of cardiac disease, medication type, or TNF and IL-1 concentrations. NN concentrations were inversely correlated with fractional shortening. The results of this study suggest that metabolites of NO are increased in some dogs with cardiac disease, but these increases appear to be independent of disease severity, TNF and IL-1 concentrations, and type of pharmacologic intervention.     

Immunomodulatory effects of human amniotic membrane-derived mesenchymal stem cells

Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are capable of differentiating into several lineages and possess immunomodulatory properties. In this study, we investigated the soluble factor-mediated immunomodulatory effects of hAM-MSCs. Mitogen-induced peripheral blood mononuclear cell (PBMC) proliferation was suppressed by hAM-MSCs in a dose-dependent manner as well as hAM-MSC culture supernatant. Moreover, interferon-gamma and interleukin (IL)-17 production significantly decreased from PBMC, whereas IL-10 from PBMCs and transforming growth factor beta (TGF-β) production from hAM-MSCs significantly increased in co-cultures of hAM-MSCs and PBMCs. Production of several MSC factors, including hepatocyte growth factor (HGF), TGF-β, prostaglandin E2 (PGE2), and indoleamine 2, 3 dioxygenase (IDO), increased significantly in hAM-MSCs co-cultured with PBMCs. These results indicate that the immunomodulatory effects of hAM-MSCs may be associated with soluble factors (TGF-β, HGF, PGE2, and IDO), suggesting that hAM-MSCs may have potential clinical use in regenerative medicine.