Immunohistochemical Characterization of Canine Prostatic Intraepithelial Neoplasia

The development of prostate cancer is believed to be a multistep process, progressing sequentially from normal epithelium, to prostatic intraepithelial neoplasia (PIN) and, finally, to invasive neoplasia. Malignant stem cells within the basal cell layer of the prostatic epithelium are believed to play an important role in the failure of androgen-ablation therapy that occurs in the most advanced form of prostate cancer. The aim of the present study was to immunohistochemically characterize the lesions of canine PIN. Prostatic tissue from five dogs with PIN was compared with normal prostate tissue from nine further dogs. There was an increase in the number of basal epithelial cells in lesions consistent with PIN as defined by expression of the nuclear protein p63. These lesions had elevated expression of proliferating cell nuclear antigen (PCNA) and heterogeneous labelling for the nuclear androgen receptor (AR). These findings suggest that the basal cells present in PIN may play a role in canine prostate carcinogenesis and that the proliferation of these cells occurs despite the heterogeneous expression of the AR. (C) 2009 Elsevier Ltd. All rights reserved.

Associations of dietary dark-green and deep-yellow vegetables and fruits with cervical intraepithelial neoplasia: modification by smoking

Smoking has been positively and fruit and vegetable intake has been negatively associated with cervical cancer, the second most common cancer among women worldwide. However, a lower consumption of fruits and reduced serum carotenoids have been observed among smokers. It is not known whether the smoking effect on the risk of cervical neoplasia is modified by a low intake of fruits and vegetables. The present study examined the combined effects of tobacco smoking and diet using a validated FFQ and serum carotenoid and tocopherol levels on cervical intraepithelial neoplasia grade 3 (CIN3) risk in a hospital-based case-control study conducted in Sao Paulo, Brazil, between 2003 and 2005. The sample comprised 231 incident, histologically confirmed cases of CIN3 and 453 controls. A low intake (<= 39 g) of dark-green and deep-yellow vegetables and fruits without tobacco smoking had a lesser effect on CIN3 (OR 1.14; 95% CI 0.49, 2.65) than among smokers with higher intake (>= 40 g; OR 1.83; 95% CI 0.73, 4.62) after adjusting for confounders. The OR for the joint exposure of tobacco smoking and low intake of vegetables and fruits was greater (3.86; 95% CI 1.74, 8.57; P for trend < 0.001) compared with non-smokers with higher intake after adjusting for confounding variables and human papillomavirus status. Similar results were observed for total fruit, serum total carotene (including beta-, alpha-and gamma-carotene) and tocopherols. These findings suggest that the effect of nutritional factors on CIN3 is modified by smoking.

Diet and serum micronutrients in relation to cervical neoplasia and cancer among low-income Brazilian women

Cervical cancer is a leading cancer among women in developing countries. Infection with oncogenic human papillomavirus (HPV) types has been recognized as a necessary cause of this disease. Serum carotenoids and tocopherols have also been associated with risk for cervical neoplasia, but results from previous studies were not consistent. We evaluated the association of serum total carotene and tocopherols, and dietary intakes with the risk of newly diagnosed, histologically confirmed cervical intraepithelial neoplasia (CIN) grades 1, 2, 3 and invasive cancer in a hospital-based case-control study in Sao Paulo, Brazil. The investigation included 453 controls and 4 groups of cases (CIN1, n = 140; CIN2, n = 126; CIN3, n = 231; invasive cancer, n = 108) recruited from two major public clinics between 2003 and 2005. Increasing concentrations of serum lycopene were negatively associated with CIN1, CIN3 and cancer, with odds ratios (OR) (95% CI) for the highest compared to the lowest tertile of 0.53 (0.27-1.00, p for trend = 0.05), 0.48 (0.22-1.04, p for trend = 0.05) and 0.18 (0.06-0.52, p for trend = 0.002), respectively, after adjusting for confounding variables and HPV status. Increasing concentrations of serum alpha- and gamma-tocopherols, and higher dietary intakes of dark green and deep yellow vegetables/fruit were associated with nearly 50% decreased risk of CIN3. These results support the evidence that a healthy and balanced diet leading to provide high serum levels of antioxidants may reduce cervical neoplasia risk in low-income women.

In vitro inhibitory effect of trichostatin A on canine grade 3 mast cell tumor

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect skin and soft tissue in dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. Trichostatin A (TSA), an antifungal antibiotic, has shown inhibitory effects on the proliferation and induction of apoptosis in various types of cancer cells. In order to evaluate the potential of trichostatin A as a therapeutic drug, cells of grade 3 MCT were cultured and treated with concentrations of 1 nM to 400 nM of TSA. MTT assay and trypan blue exclusion assays were performed to estimate cell growth and cell viability, and cell cycle analysis was evaluated. TSA treatment showed a reduction in numbers of viable cells and an increase of cell death by apoptosis. The cell cycle analysis showed an increase of hypodiploid cells and a reduction of G0/G1 and G2/M -phases. According to these results, trichostatin A may be an interesting potential chemotherapeutic agent for the treatment of canine MCT.

Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis

The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1). B. canis non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of canine brucellosis. (c) 2008 Elsevier Ltd. All rights reserved.

Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis

In the present study, the validation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of canine brucellosis is described. Two different antigenic extracts, obtained by heat or ultrasonic homogenization of microbial antigens from a wild isolate of Brucella canis bacteria, were compared by ELISA and Western blot (WB). A total of 145 canine sera were used to define sensitivity, specificity and accuracy of the ELISA as follows: (1) sera from 34 animals with natural B. canis infection, confirmed by blood culture and PCR, as well as 51 sera samples from healthy dogs with negative results by the agar-gel immunodiffusion (ACID) test for canine brucellosis, were used as the control panel for B. cants infection; and (2) to scrutinize the possibility of cross reactions with other common dog infections in the same geographical area in Brazil, 60 sera samples from dogs harboring known infections by Leptospira sp., Ehrlichia canis, canine distemper virus (CDV), Neospora caninum, Babesia canis and Leishmania chagasi (10 in each group) were included in the study. The ELISA using heat soluble bacterial extract (HE-antigen) as antigen showed the best values of sensitivity (91.18%), specificity (100%) and accuracy (96.47%). In the WB analyses, the HE-antigen showed no cross-reactivity with sera from dogs with different infections, while the B. canis sonicate had various protein bands identified by those sera. The performance of the ELISA standardized with the heat soluble B. canis antigen indicates that this assay can be used as a reliable and practical method to confirm infection by this microorganism, as well as a tool for seroepidemiological studies. (C) 2010 Elsevier Ltd. All rights reserved.

Expression of Connexins in Normal and Neoplastic Canine Bone Tissue

Previous studies showed that intercellular communication by gap junctions has a role in bone formation. The main connexin involved in the development, differentiation, and regulation of bone tissue is connexin (Cx) 43. In addition, Cx46 is also expressed, mostly localized within the trans-Golgi region. Alterations in the expression pattern and aberrant location of these connexins are associated with oncogenesis, demonstrating a deficient gap junctional intercellular communication (GJIC) capacity in neoplastic tissues. In this study, we evaluated normal and neoplastic bone tissues regarding the expression of Cx43 and Cx46 by immunofluorescence, gene expression of these connexins by real-time PCR, and their correlation with cell proliferation index and deposition of collagen. Fourteen neoplastic bone lesions, including 13 osteosarcomas and I multilobular tumor of bone, were studied. The mRNA levels of Cx43 were similar between normal and neoplastic bone tissue. In normal bone tissue, the Cx43 protein was found mainly in the intercellular membranes. However, in all bone tumors studied here, the Cx43 was present in both cell membranes and also aberrantly in the cytoplasm. Regarding only tumor samples, we determined a possible inverse correlation between Cx43 expression and cellular proliferation, although a positive correlation between Cx43 expression and collagen deposition was also noted. In contrast, Cx46 had lower levels of expression in neoplastic bone tissues when compared with normal bone and was found retained in the perinuclear region. Even though there are differences between these two connexins regarding expression in neoplastic versus normal tissues, we concluded that there are differences regarding the subcellular location of these connexins in normal and neoplastic dog bone tissues and suggest a possible correlation between these findings and some aspects of cellular proliferation and possibly differentiation.

Isolation, Characterization, and Differentiation Potential of Canine Adipose-Derived Stem Cells

Adipose tissue may represent a potential source of adult stem cells for tissue engineering applications in veterinary medicine. It can be obtained in large quantities, under local anesthesia, and with minimal discomfort. In this study, canine adipose tissue was obtained by biopsy from subcutaneous adipose tissue or by suction-assisted lipectomy (i.e., liposuction). Adipose tissue was processed to obtain a fibroblast-like population of cells similar to human adipose-derived stem cells (hASCs). These canine adipose-derived stem cells (cASCs) can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of cASCs are of mesodermal or mesenchymal origin. cASCs are able to differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirate, canine adipose tissue may also contain multipotent cells and represent an important stem cell source both for veterinary cell therapy as well as preclinical studies.

Objective Detection and Delineation of Oral Neoplasia Using Autofluorescence Imaging

Although the oral cavity is easily accessible to inspection, patients with oral cancer most often present at a late stage, leading to high morbidity and mortality. Autofluorescence imaging has emerged as a promising technology to aid clinicians in screening for oral neoplasia and as an aid to resection, but current approaches rely on subjective interpretation. We present a new method to objectively delineate neoplastic oral mucosa using autofluorescence imaging. Autofluorescence images were obtained from 56 patients with oral lesions and 11 normal volunteers. From these images, 276 measurements from 159 unique regions of interest (ROI) sites corresponding to normal and confirmed neoplastic areas were identified. Data from ROIs in the first 46 subjects were used to develop a simple classification algorithm based on the ratio of red-to-green fluorescence; performance of this algorithm was then validated using data from the ROIs in the last 21 subjects. This algorithm was applied to patient images to create visual disease probability maps across the field of view. Histologic sections of resected tissue were used to validate the disease probability maps. The best discrimination between neoplastic and nonneoplastic areas was obtained at 405 nm excitation; normal tissue could be discriminated from dysplasia and invasive cancer with a 95.9% sensitivity and 96.2% specificity in the training set, and with a 100% sensitivity and 91.4% specificity in the validation set. Disease probability maps qualitatively agreed with both clinical impression and histology. Autofluorescence imaging coupled with objective image analysis provided a sensitive and noninvasive tool for the detection of oral neoplasia.

In Vitro Evaluation of Acellular Dermal Matrix as a Three-Dimensional Scaffold for Gingival Fibroblasts Seeding

Background: Tissue engineering principles could improve the incorporation of acellular dermal matrix (ADM). The aim of this study is to verify if ADM is a suitable three-dimensional matrix for gingival fibroblasts and cancerous cells ingrowth, and also if cultured medium conditioned in ADM affect cellular behavior. Methods: Canine gingival fibroblasts (CGF), human gingival fibroblasts (HGF), and murine melanoma cell line (B16F10) were seeded on ADM for up to 14 days. The following parameters were assessed: morphology and distribution of CGF, HGF, and B16F10; CGF and HGF viability; and the effect of ADM conditioned medium (CM) on CGF viability. Results: Epifluorescence revealed that CGF were unevenly distributed on the ADM surface, showing no increase in cell number over the periods of study; HGF formed a monolayer on the ADM surface in a higher number at 14 days (P<0.05); B16F10 exhibited an increase in cell number within 7 days (P<0.05), and were mainly arranged in cell aggregates on the ADM, forming a continuous layer at 14 days. A higher percentage of cells on the ADM surface (P<0.05) compared to inside was observed for all cell types. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MU) values indicated higher cell viability in samples cultured with HGF compared to CGF (P=0.024). A significantly lower cell viability for CGF grown in CM compared to cells grown in non-CM was observed at 48 and 72 hours (P<0.05). Conclusions: ADM is not suitable as a three-dimensional matrix for gingival fibroblasts ingrowth. Gingival fibroblasts and highly proliferative cells as B16F10 can only be superficially located on ADM, and CGF are negatively affected by culture medium conditioned in ADM, reducing its viability. J Periodontol 2011;82:293-301.

Tissue damage after sodium hypochlorite extrusion during root canal treatment

Sodium hypochlorite solution is toxic to vital tissues, causing severe effects if extruded during endodontic treatment. This paper presents a report on the tissue damage related to inadvertent extrusion of concentrated sodium hypochlorite solution during root canal treatment. A 65-year-old woman was referred with moderate pain, ecchymosis, and severe swelling of the right side of the face. These symptoms appeared immediately after a root canal treatment of the maxillary right canine, which had been started 21 hours earlier. It was diagnosed as air emphysema related to sodium hypochlorite solution extravasation during the endodontic treatment. To avoid this, an initial radiograph should be taken to determine the correct canal working length and confirm root canal integrity. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: e46-e49)

Bone Formation Following Implantation of Titanium Sponge Rods into Humeral Osteotomies in Dogs: A Histological and Histometrical Study

Background: Titanium (Ti) is widely proven to enhance bone contact and growth on its surface. It is expected that bone defects could benefit from Ti to promote healing and to increase strength of the implanted area. Purpose: The present study aimed at comparing the potential of porous Ti sponge rods with synthetic hydroxyapatite (HA) for the healing of bone defects in a canine model. Material and Methods: Six mongrel dogs were submitted to three trephined osteotomies of 6.0 x 4.0 mm in one humerus and after 2 months another three osteotomies were performed in the contralateral humerus. A total of 36 defects were randomly filled either with Ti foam, particulate HA, or coagulum (control). The six animals were killed 4 months after the first surgery for histological and histometrical analysis. Results: The Ti-foam surface was frequently found in intimate contact with new bone especially at the defect walls. Control sites showed higher amounts of newly formed bone at 2 months - Ti (p = 0.000) and HA (p = 0.009) - and 4 months when compared with Ti (p = 0.001). Differently from HA, the Ti foam was densely distributed across the defect area which rendered less space for bone growth in the latter`s sites. The use of Ti foams or HA resulted in similar amounts of bone formation in both time intervals. Nevertheless, the presence of a Ti-foam rod preserved defect`s marginal bone height as compared with control groups. Also, the Ti-foam group showed a more mature bone pattern at 4 months than HA sites. Conclusion: The Ti foam exhibited good biocompatibility, and its application resulted in improved maintenance of bone height compared with control sites. The Ti foam in a rod design exhibited bone ingrowth properties suitable for further exploration in other experimental situations.

Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs

The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination.

Early Development and Putative Primordial Germ Cells Characterization in Dogs

Contents Previously, three distinct populations of putative primordial germ cells (PGCs), namely gonocytes, intermediate cells and pre-spermatogonia, have been described in the human foetal testis. According to our knowledge, these PGCs have not been studied in any other species. The aim of our study was to identify similar PGC populations in canine embryos. First, we develop a protocol for canine embryo isolation. Following our protocol, 15 canine embryos at 21-25 days of pregnancy were isolated by ovaryhysterectomy surgery. Our data indicate that dramatic changes occur in canine embryo development and PGCs specification between 21 to 25 days of gestation. At that moment, only two PGC populations with distinct morphology can be identified by histological analyses. Cell population 1 presented round nuclei with prominent nucleolus and a high nuclear to cytoplasm ratio, showing gonocyte morphology. Cell population 2 was often localized at the periphery of the testicular cords and presented typical features of PGC. Both germ cell populations were positively immunostained with anti-human OCT-4 antibody. However, at day 25, all cells of population 1 reacted positively with OCT-4, whereas in population 2, fewer cells were positive for this marker. These two PGCs populations present morphological features similar to gonocytes and intermediate cells from human foetal testis. It is expected that a population of pre-spermatogonia would be observed at later stages of canine foetus development. We also showed that anti-human OCT-4 antibody can be useful to identify canine PGC in vivo.

Use of extensible internal device in the femur of young dogs

An extensible internal device (EID) was developed to preserve growth plate during the treatment of fracture complications or segmental bone loss from tumour resection in children. Since this type of extensible, trans-physeal, internal fixation device has only been used in a few paediatric cases; the aim of this study was to evaluate an in vivo canine study, a surgical application of this device, and its interference with longitudinal growth of the non-fractured distal femur. Ton clinically healthy two- to three-month-old poodles weighing 1.5-2.3 kg were used. Following a medial approach to the right distal femur, one extremity of the EID, similar to a T-plate, was fixed in the femoral condyle with two cortical screws placed below the growth plate. The other extremity, consisting of an adaptable brim with two screw holes and a plate guide, was fixed in the third distal of the femoral diaphysis with two cortical screws. The EID was removed 180 days after application. All of the dogs demonstrated full weight-bearing after surgery. The values of thigh and stifle circumferences, and stifle joint motion range did not show any difference between operated and control hindlimbs. The plate slid in the device according to longitudinal bone growth, in all but one dog. In this dog, a 10.5% shortening of the femoral shaft was observed due to a lack of EID sliding. The other dogs had the some longitudinal lengths in both femurs. The EID permits longitudinal bone growth without blocking the distal femur growth plate if appropriately placed.