Bone regeneration in critical-sized mandibular symphysis defects using bioceramics with or without bone marrow mesenchymal stem cells in healthy, diabetic, osteoporotic, and diabetic-osteoporotic rats.

OBJECTIVES: To compare new bone formation in mandibular critical-sized bone defects (CSBDs) in healthy, diabetic, osteoporotic, and diabetic-osteoporotic rats filled with bioceramics (BCs) with or without bone marrow mesenchymal stem cells (BMSCs). METHODS: A total of 64 adult female Sprague-Dawley rats were randomized into four groups (n = 16 per group): Group 1 healthy, Group 2 diabetic, Group 3 osteoporotic, and Group 4 diabetic-osteoporotic rats. Streptozotocin was used to induce type 1 diabetes in Group 2 and 4, while bilateral ovariectomy was used to induce osteoporosis in Group 3 and 4. The central portion of the rat mandibular symphysis was used as a physiological CSBD. In each group, eight defects were filled with BC (hydroxypatatite 60% and ß-tricalcium phosphate 40%) alone and eight with BMSCs cultured on BC. The animals were sacrificed at 4 and 8 weeks, and the mandibles were processed for micro-computed tomography to analyze radiological union and bone mineral density (BMD); histological analysis of the bone union; and immunohistochemical analysis, which included immunoreactivity of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2). RESULTS: In all groups (healthy, diabetics, osteoporotics, and diabetics-osteoporotics), the CSBDs filled with BC + BMSCs showed greater radiological bone union, BMD, histological bone union, and more VEGF and BMP-2 positivity, in comparison with CSBDs treated with BC alone (at 4 and 8 weeks). CONCLUSIONS: Application of BMSCs cultured on BCs improves bone regeneration in CSBDs compared with application of BCs alone in healthy, diabetic, osteoporotic, and diabetic-osteoporotic rats.

Use of buccal fat pad-derived stem cells cultured on bioceramics for repair of critical-sized mandibular defects in healthy and osteoporotic rats.

OBJECTIVE: To compare new bone formation in mandibular symphysis critical-sized bone defects (CSBDs) in healthy and osteoporotic rats filled with bioceramics (BCs) with or without buccal fat pad mesenchymal stem cells (BFPSCs). MATERIALS AND METHODS: Thirty-two adult female Sprague-Dawley rats were randomized to two groups (n = 16 per group): group 1 healthy and group 2 osteoporotic (with bilateral ovariectomy). The central portion of the rat mandibular symphysis was used as a physiological CSBD. In each group, eight defects were filled with BC (hydroxyapatite 60% and ß-tricalcium phosphate 40%) alone and eight with BFPSCs cultured on BC. The animals were sacrificed at 4 and 8 weeks, and the mandibles were processed for micro-computed tomography to analyze radiological union and bone mineral density (BMD); histological analysis of the bone union; and immunohistochemical analysis, which included immunoreactivity of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2). RESULTS: In both groups, CSBDs filled with BC + BFPSCs showed greater radiological bone union, BMD and histological bone union, and more VEGF and BMP-2 positivity, compared with CSBDs treated with BC alone at 4 and 8 weeks. CONCLUSIONS: The application of BFPSCs cultured on BCs improves bone regeneration in CSBDs compared with BCs alone in healthy and osteoporotic rats. CLINICAL RELEVANCE: Our results may aid bone regeneration of maxillofacial CSBDs of both healthy and osteoporotic patients, but further studies are necessary.

First report of a hawfinch (Coccothraustes coccothraustes) parasitized by Harpirhynchus nidulans in the Iberian Peninsula.

Some avian Harpirhynchidae mites live under the skin and develop cutaneous cysts. Despite the obvious lesions that these parasites can produce, little is currently known about the behavioural disturbances that cyst-forming mites may cause in infected wild birds. We report an infection by Harpirhynchidae mites in a hawfinch (Coccothraustes coccothraustes) in southeast Spain. The bird was easily captured due to its inability to fly. During clinical examination it was found to have multiple severe traumatic injuries, possibly due to a blow or a fall, as result of which the bird was euthanized. At necropsy, the hawfinch was found to be in good body condition. Two yellowish and friable mite-filled cysts were detected in the subalar region of both wings. Mites were morphologically identified as Harpirhynchus nidulans, and histological analysis of the cystic lesions was also performed. This is the first time that the occurrence of a hawfinch infected by H. nidulans in the Iberian Peninsula has been reported.

Bone union formation in the rat mandibular symphysis using hydroxyapatite with or without simvastatin: effects on healthy, diabetic, and osteoporotic rats.

OBJECTIVE: The objective is to compare new bone formation in critical defects in healthy, diabetic, and osteoporotic rats filled with hydroxyapatite (HA) alone and HA combined with simvastatin (SV). MATERIALS AND METHODS: A total of 48 adult female Sprague-Dawley rats were randomized into three groups (n = 16 per group): Group, 1 healthy; Group 2, diabetics; and Group 3, osteoporotics. Streptozotocin was used to induce type 1 diabetes in Group 2, while bilateral ovariectomy was used to induce osteoporosis in Group 3. The central portion of the rat mandibular symphysis was used as a physiological critical bone defect. In each group, eight defects were filled with HA alone and eight with HA combined with SV. The animals were sacrificed at 4 and 8 weeks, and the mandibles were processed for micro-computed tomography to analyze radiological union and bone mineral density (BMD); histological analysis of the bone union; and immunohistochemical analysis, which included immunoreactivity of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2). RESULTS: In all groups (healthy, diabetics, and osteoporotics), the defects filled with HA + SV presented greater radiological bone union, BMD, histological bone union, and more VEGF and BMP-2 positivity, in comparison with bone defects treated with HA alone. CONCLUSIONS: Combined application of HA and SV improves bone regeneration in mandibular critical bone defects compared with application of HA alone in healthy, diabetic, and osteoporotic rats. CLINICAL RELEVANCE: This study might help to patients with osteoporosis or uncontrolled diabetes type 1, but future studies should be done.

Effect of female sex hormones on the developmental cycle of Chlamydia abortus compared to a penicillin-induced model of persistent infection.

BACKGROUND: Chlamydia abortus, an obligate intracellular pathogen with an affinity for placenta, causes reproductive failure. In non-pregnant animals, an initial latent infection is established until the next gestation, when the microorganism is reactivated, causing abortion. The precise mechanisms that trigger the awakening of C. abortus are still unknown. Sexual hormones such as estradiol and progesterone have been shown to affect the outcome of infection in other species of the family Chlamydiaceae, while estrogens increase chlamydial infection, progesterone has the opposite effect. To try to establish whether there is a relationship between these events and the latency/ reactivation of C. abortus in the reproductive tract of small ruminants, ovine endometrial (LE) and trophoblastic (AH-1) cells were treated with estradiol or progesterone prior to their infection with C. abortus. The results are compared with those obtained for treatment with penicillin prior to infection, which is a well-established model for studying persistent infection in other chlamydial species. Cells were examined by transmission electron microscopy, and an mRNA expression analysis of 16 genes related to the chlamydial developmental cycle was made. RESULTS: The changes observed in this study by the action of sex hormones seem to depend on the type of cell where the infection develops. In addition, while the changes are morphologically similar to those induced by treatment with penicillin, the patterns of gene expression are different. Gene expression patterns therefore, seem to depend on the persistence induced models of C. abortus used. Hormone treatments induced aberrant forms in infected endometrial cells but did not affect the chlamydial morphology in trophoblast cells. At the genetic level, hormones did not induce significant changes in the expression of the studied genes. CONCLUSIONS: The results suggest that penicillin induces a state of persistence in in vitro cultured C. abortus with characteristic morphological features and gene transcriptional patterns. However, the influence of hormones on the C. abortus developmental cycle is mediated by changes in the host cell environment. Furthermore, a persistent state in C. abortus cannot be characterised by a single profile of gene expression pattern, but may change depending on the model used to induce persistence.

Effect of progesterone on the vaccination and immune response against Chlamydia abortus in sheep.

Chlamydia abortus produces ovine enzootic abortion (OEA). Symptoms are not observed until the organism colonises the placenta, eventually causing abortion. Infected animals become carriers and will shed the organism in the following oestruses. This process suggests that sex hormones might play an important role in the physiopathology of OEA, affecting the success of chlamydial clearance and also jeopardising the effectiveness of vaccination. However, the mechanisms through which sex hormones are involved in chlamydial pathogenicity remain unclear. The aim of this study, therefore, was to determine the effect of progesterone on the immune response against C. abortus and on the protection conferred by an experimental inactivated vaccine in sheep. Eighteen sheep were ovariectomised and divided into four groups: vaccinated and progesterone-treated (V-PG), vaccinated and non-treated (V-NT), non-vaccinated and non-treated (NV-NT) and non-vaccinated and progesterone-treated sheep (NV-PG). Animals from both PG groups were treated with commercial medroxyprogesterone acetate impregnated intravaginal sponges before and during the vaccination (V-PG) or just before challenge (NV-PG). The animals from both V groups were subcutaneously immunised with an experimental inactivated vaccine, which was seen to confer high protection in previous studies. All sheep were challenged intratracheally with C. abortus strain AB7 and were sacrificed on day 8 post-infection. Morbidity was measured as the variation in rectal temperature and samples of sera were collected for antibody and cytokine (IFN-γ and IL-10) analysis by commercial ELISA. In addition, lung and lymph node samples were collected for chlamydial detection by qPCR and for histopathological and immunohistochemical analyses. Sheep from the V-PG group showed less severe or no lesions and lower morbidity than the other groups. They also had the highest abundance of regulatory T-cells. The sheep from V-NT also manifested high antibody levels against C. abortus and less severe lesions than those observed in non-vaccinated sheep, which showed high morbidity, low antibody levels and severe lesions, especially in NV-NT. These results confirm the effectiveness of the experimental vaccine employed and suggest that progesterone could enhance the effect.

N-cadherin expression in feline mammary tumors is associated with a reduced E-cadherin expression and the presence of regional metastasis.

The cadherin family of adhesion molecules regulates cell-cell interactions. N-cadherin is expressed by neural and fibroblast cells but not by normal epithelial cells. In human medicine, the role of N-cadherin in breast cancer remains controversial, but some studies have described the switch from E-cadherin to N-cadherin as a critical step in the malignant progression of neoplastic cells. The present study was carried out on 160 feline mammary tumors (21 adenomas and 139 carcinomas). The relationship between the immunohistochemical expression of N-cadherin in neoplastic epithelial cells and 2 established prognostic factors such as regional metastasis and tumor grade was examined. The results of the study showed a statistically significant relation between the expression of N-cadherin and the 2 prognostic factors, and also a reduced expression of E-cadherin in tumors that expressed N-cadherin.

Reduced expression of E-cadherin and ß-catenin and high expression of basal cytokeratins in feline mammary carcinomas with regional metastasis.

Feline mammary carcinomas are highly aggressive neoplasms. Several mechanisms are thought to be involved in their progression, including the loss of epithelial adhesion molecules. The present study was carried out on 21 adenomas and 139 mammary carcinomas. Of the carcinomas, 66 were not reported to have metastasized, while the remaining 73 had evidence of regional lymph node metastasis at the moment of diagnosis. The relationship was examined between the expression of the E-cadherin-ß-catenin complex and basal (CK5/6, CK14) and luminal (CK8/18) cytokeratin expression. In the medical literature, carcinomas expressing basal cytokeratins are reported as having a poor prognosis in human breast cancer. Results revealed that preservation of the expression of E-cadherin and ß-catenin is a significant feature of carcinomas without metastasis, whereas carcinomas with metastasis reveal the loss of one or both adhesion molecules. Additionally, basal cytokeratin expression was statistically associated with the presence of regional metastasis. Furthermore, the expression of E-cadherin-ß-catenin was significantly correlated with the high expression of CK18 and low expression of CK5/6.

Evaluating the efficacy of cinnamaldehyde and Echinacea purpurea plant extract in broilers against Eimeria acervulina.

Coccidiostats could be phased out as feed additives before 1 January 2013 for public health and food safety reasons, and, as a replacement, bioactive compounds found in plants are currently being investigated since they are more likely to be found acceptable by consumers. The aim of our study was to evaluate the effect of cinnamaldehyde (CIN) and Echinacea purpurea plant extract (EP) as additives by analyzing the performance traits, oocyst excretion and intestinal lesions following experimental infection with Eimeria acervulina. A total of 72 Ross male broilers were raised from 1 to 35 d and randomly assigned to four dietary treatments: control, without additives (C); 150 mg kg(-1) cinnamaldehyde (CIN); 1000 mg kg(-1)E. purpurea plant extract (EP); 150 mg kg(-1) cinnamaldehyde plus 1000 mg kg(-1)E. purpurea plant extract (CIN+EP). At 25 d, 12 chickens per treatment were orally infected with E. acervulina. Coccidia infestation led to lower performance but with no significant differences between the infected groups. Oocyst output reached its peak from 6 to 9 d post-infection in all treatments. At duodenal level, gross lesion scores were lower for cinnamaldehyde diets (P<0.05). A similar trend was observed in the microscopic lesion scores, with a non-significant reduction as a result of cinnamaldehyde addition (P>0.05). Scoring methods for macro- and microscopic lesions showed a positive linear relationship (G=+0.70). Further studies are necessary to assess the possible anticoccidian action of the cinnamaldehyde and its value as an alternative or adjunct in therapeutic or prophylactic strategies.

Microscopical and immunological features of tuberculoid granulomata and cavitary pulmonary tuberculosis in naturally infected goats.

Caprine tuberculosis is caused by bacteria of the Mycobacterium tuberculosis complex (Mycobacterium bovis and Mycobacterium caprae). Although typical tuberculoid granulomata are usually observed in the lungs and lymph nodes of infected goats, the presence of cavitary lesions with exuberant mycobacterial growth is also a common feature in this species. The aim of this study was to characterize the immunological mechanisms that lead to liquefaction and cavity formation by comparing granulomata and cavitary lesions. Samples from animals positive by skin testing were collected for microscopical and immunohistochemical examination. Samples were also collected for analysis of cytokine gene expression in the lesions by real time polymerase chain reaction. There were marked differences between granulomata and cavitary lesions. In cavitary lesions there was a substantial population of neutrophils and a significant decrease in the number of CD4(+) T cells, with concomitant increases in other T-cell populations (CD8(+) and cells expressing the γδ form of the T-cell receptor). The enzyme iNOS was strongly expressed by macrophages in the cavitary lesions. There was no difference in the balance of pro- and anti-inflammatory cytokine gene expression in the lesions. These findings suggest that cavitary lesions are reactivation sites, where conditions are optimal for Mycobacterium proliferation and that immunological mechanisms may underlie the severe destruction of lung tissue that characterizes the cavitary pathology.

Cyclooxygenase-2 expression is related with localization, proliferation, and overall survival in canine melanocytic neoplasms.

A direct relationship has been firmly established between cyclooxygenase-2 (COX-2) expression and malignant behavior in human melanoma. This report examines the relationship between COX-2 expression and tumor location, mitotic and proliferative indices, degree of T CD3(+) lymphocyte infiltration, overall survival, and frequency of recurrence and metastasis of 57 melanocytic tumors (25 oral and 32 cutaneous). COX-2 was highly or moderately expressed in 88% of oral neoplasms (22 of 25), whereas for their cutaneous counterparts, COX-2 expression was low or insignificant in 75% of cases (24 of 32). High and moderate COX-2 expression levels were observed in 73% of melanocytic tumors with a mitotic index ≥ 3 per 10 high-power fields (26 of 36), whereas in 81% of tumors with a mitotic index < 3 (17 of 21), expression was mild or absent. There were 41 cases with known clinical outcomes; of those showing high, moderate, and mild COX-2 expression, 83.3% (10 of 12), 37.5% (3 of 8), and 25% (2 of 8) died, respectively, whereas 100% of animals showing no COX-2 expression (13 of 13) were still alive at the last follow-up. COX-2 expression was statistically correlated with tumor location, mitotic and percentage Ki-67 proliferative indices, and overall survival, frequency of neoplastic recurrence and metastasis. Regression analysis also showed disease-specific predictive value for COX-2 expression for subjects with melanocytic neoplasms. Additionally, only high COX-2 expression showed significant differences in overall survival, in comparison with moderate, mild, or absent expression. These results suggest that high COX-2 expression may be considered a prognostic biomarker and potentially as a target for therapeutic and preventive strategies in canine melanocytic neoplasms.

Histopathological lesions in 15 cats with leishmaniosis.

Recent research into the prevalence of Leishmania infantum infection in the Mediterranean basin points to the involvement of cats as a reservoir host, but only sporadic cases of feline leishmaniosis have been reported. Feline leishmaniosis presents primarily as cutaneous disease and diagnosis is based on the demonstration of the parasite by skin biopsy. The present report describes the microscopical changes in tissue biopsies from 15 cats with leishmaniosis. The biopsies were derived from the skin, ocular tissue and mucocutaneous junctions. The most common histopathological feature was diffuse granulomatous inflammation with macrophages containing numerous amastigotes. Other patterns included granulomatous perifolliculitis and lichenoid interface dermatitis, where there were fewer parasitized macrophages. The presence of amastigotes was confirmed by immunohistochemistry in each case. The results of the study confirm the value of histopathological and immunohistochemical techniques for the diagnosis of feline leishmaniosis.

Immunohistochemical characterization of a pulmonary large-cell carcinoma in a dog.

Primary pulmonary tumors are less common in dogs than secondary (metastatic) tumors. Most primary tumors are malignant and of epithelial origin. Pulmonary large-cell carcinoma is considered extremely rare in domestic animals, and some of the few reported cases actually may have been cases of malignant pulmonary histiocytosis. An 8-year-old female Wire Fox Terrier with diffuse alveolar pattern radiographically was euthanatized when it failed to respond to antibiotic therapy. Histologically, pulmonary alveolar spaces contained clusters of large round anaplastic cells with ample eosinophilic cytoplasm and large irregularly shaped nuclei with prominent nucleoli. Immunohistochemistry was used to distinguish large-cell carcinoma from malignant pulmonary histiocytosis. Tumor cells had strong immunoreactivity for cytokeratin, consistent with epithelial origin. However, a substantial percentage of the neoplastic cells co-expressed vimentin and MHC-II. The type II alveolar epithelial cell was considered the cell of origin of the neoplasm based on the presence of lamellar bodies in some neoplastic cells and immunoreactivity for surfactant protein A and thyroid transcription factor-1.

Immunohistochemical characterization and evaluation of prognostic factors in canine oral melanomas with osteocartilaginous differentiation.

Melanomas are the most common malignant oral neoplasm in dogs. Osteocartilaginous differentiation in oral melanomas is a rare feature described both in veterinary and human medicine. Here, 10 cases of this type of neoplasm were used to study their immunohistochemical, biological, and clinical characteristics. Reactivity for S100 and melan A antigen was evaluated, and 4 prognosis factors (mitotic index, invasiveness of epithelium, nuclear atypia, and proliferation index) were analyzed and correlated with the clinical course of the neoplasms after diagnosis. Immunohistochemical analysis of the studied neoplasms, including the osteocartilaginous areas, showed positive immunoreaction for S100 and melan A, except in one dog, which was negative for melan A. Analysis of the results showed that oral melamonas with osteocartilaginous differentiation have a clinical course similar to that of other melanomas in the oral cavity. Analysis of the mitotic index and the expression of proliferation marker Ki-67 could be useful tools for predicting the biological behavior of these neoplasms.

Canine carcinosarcomas in the head.

Four cases of neoplasms in the heads of old dogs were studied. All the dogs showed both carcinomatous and sarcomatous malignant components in an admixed growing pattern. Histologic analysis of the tumors showed that the carcinomatous cells resembled squamous cell carcinoma in all dogs except one, where an adenoid arrangement of the neoplastic cells was also observed. The sarcomatous component showed osteoid matrix produced by pleomorphic poorly differentiated cells, which is regarded as a typical feature of osteosarcomas. Immunohistochemical analysis demonstrated that the components were positive only for cytokeratin (carcinomatous component) or vimentin (sarcomatous component). This observation led us to classify the neoplasms as true carcinosarcomas, thus providing evidence of a new preferential location for this unusual tumor in dog.

Limited role of polymorphonuclear neutrophils in a pregnant mouse model of secondary infection by Chlamydophila abortus (Chlamydia psittaci serotype 1).

The aim of this work was to study the role of polymorphonuclear neutrophils (PMNs) in the clearance of infection, and in the development of specific immunity against Chlamydophila abortus (Chlamydia psittaci serotype 1) secondary infection. A pregnant mouse model depleted of neutrophils by the RB6-8C5 monoclonal antibody was used. No clinical signs were observed in depleted or non-depleted mice after secondary infection and no significant differences were observed in the litter size between the infected and control groups. In PMN-depleted mice C. abortus was not detected in the materno-fetal unit but merely produced low, persistent levels of infection in spleen and liver. In the non-depleted mice the level of infection was significantly lower, being resolved during the first few days post-reinfection. In both infected mice groups the immune response in the liver was quickly established and was seen to be composed mainly of CD4(+)T lymphocytes and macrophages. A Th1 response characterized by the presence of IFN-gamma and TNF-alpha in serum was observed during early infection, with significantly higher levels in the non-depleted animals. Our results suggest that PMNs have little influence on the control of C. abortus secondary infection, although they are a first line of defense and may influence the early production of TNF-alpha and IFN-gamma.

Differences in the immune response against ruminant chlamydial strains in a murine model.

CBA/J mice were used in the present study to establish differences between the immune response to three chlamydial strains: AB7 (Chlamydia psittaci wild-type strain), 1B (C. psittaci vaccinal strain) and iB1 (C. pecorum). The evolution of chlamydial infection was evaluated in each strain by studying the clinical signs, the number of bacteria isolated from the spleen and the pathology of the liver. Three aspects of the immune response were then studied: the characterization of the infiltrate of leukocytes in the liver, the percentages of T- and B-cells, macrophages and neutrophils in the spleen, and the presence of cytokines in the serum. Infection followed a different course in the C. psittaci-infected mice; 1B-infected mice showed milder levels in all the parameters analysed than their AB7-infected counterparts. The resolution of infection was earlier in 1B-infected mice and, although the immune response to both strains was Th1-like, a more intense CD8+ T-cell response and an earlier presence of TNF-alpha in serum were observed in this group. C. pecorum infection was controlled mainly by a non-specific immune response, since these mice showed no signs of a systemic specific immune response. Neutrophil depletion experiments showed that these cells play a very limited role in the non-specific response against C. pecorum.

Postnatal evolution of lymphocyte subpopulations in peripheral blood and lymphoid organs in the goat.

Monoclonal antibodies (mAbs) against bovine leukocyte antigens specific for T cells (CD2, CD4, CD8 and gammadelta receptor) and B cells (surface IgM) were used in samples from one week and one-, three- and seven-month-old goats to study the evolution of lymphocyte subsets by flow cytometry in peripheral blood and the lymphoid organs: thymus, jejunal (JPP) and ileal (IPP) Peyer's patches, spleen and mesenteric lymph nodes. An increase in the values of alpha/beta receptor T cells with age was recorded whereas the gammadelta receptor T cells fell in number. In peripheral blood and in all tissues, except IPP the values for B cells (sIgM+) were low. The CD4+ and CD8+ cells predominated in JPP while B cells were the most important subpopulation in IPP. In the spleen, as in JPP, the CD4/CD8 ratio was less than one and the gammadelta T cells values were high. In mesenteric lymph nodes, CD8+ and B(sIgM) cells predominated in the youngest animals.