Blimp-1 and c-Maf regulate immune gene networks to protect against distinct pathways of pathobiont-induced colitis.

Intestinal immune responses to microbes are controlled by the cytokine IL-10 to avoid immune pathology. Here, we use single-cell RNA sequencing of colon lamina propria leukocytes (LPLs) along with RNA-seq and ATAC-seq of purified CD4+ T cells to show that the transcription factors Blimp-1 (encoded by Prdm1) and c-Maf co-dominantly regulate Il10 while negatively regulating proinflammatory cytokines in effector T cells. Double-deficient Prdm1fl/flMaffl/flCd4Cre mice infected with Helicobacter hepaticus developed severe colitis with an increase in TH1/NK/ILC1 effector genes in LPLs, while Prdm1fl/flCd4Cre and Maffl/flCd4Cre mice exhibited moderate pathology and a less-marked type 1 effector response. LPLs from infected Maffl/flCd4Cre mice had increased type 17 responses with increased Il17a and Il22 expression and an increase in granulocytes and myeloid cell numbers, resulting in increased T cell-myeloid-neutrophil interactions. Genes over-expressed in human inflammatory bowel disease showed differential expression in LPLs from infected mice in the absence of Prdm1 or Maf, revealing potential mechanisms of human disease.

Mouse transcriptome reveals potential signatures of protection and pathogenesis in human tuberculosis.

Although mouse infection models have been extensively used to study the host response to Mycobacterium tuberculosis, their validity in revealing determinants of human tuberculosis (TB) resistance and disease progression has been heavily debated. Here, we show that the modular transcriptional signature in the blood of susceptible mice infected with a clinical isolate of M. tuberculosis resembles that of active human TB disease, with dominance of a type I interferon response and neutrophil activation and recruitment, together with a loss in B lymphocyte, natural killer and T cell effector responses. In addition, resistant but not susceptible strains of mice show increased lung B cell, natural killer and T cell effector responses in the lung upon infection. Notably, the blood signature of active disease shared by mice and humans is also evident in latent TB progressors before diagnosis, suggesting that these responses both predict and contribute to the pathogenesis of progressive M. tuberculosis infection.

Lung adenocarcinoma promotion by air pollutants.

A complete understanding of how exposure to environmental substances promotes cancer formation is lacking. More than 70 years ago, tumorigenesis was proposed to occur in a two-step process: an initiating step that induces mutations in healthy cells, followed by a promoter step that triggers cancer development1. Here we propose that environmental particulate matter measuring ≤2.5 µm (PM2.5), known to be associated with lung cancer risk, promotes lung cancer by acting on cells that harbour pre-existing oncogenic mutations in healthy lung tissue. Focusing on EGFR-driven lung cancer, which is more common in never-smokers or light smokers, we found a significant association between PM2.5 levels and the incidence of lung cancer for 32,957 EGFR-driven lung cancer cases in four within-country cohorts. Functional mouse models revealed that air pollutants cause an influx of macrophages into the lung and release of interleukin-1ß. This process results in a progenitor-like cell state within EGFR mutant lung alveolar type II epithelial cells that fuels tumorigenesis. Ultradeep mutational profiling of histologically normal lung tissue from 295 individuals across 3 clinical cohorts revealed oncogenic EGFR and KRAS driver mutations in 18% and 53% of healthy tissue samples, respectively. These findings collectively support a tumour-promoting role for  PM2.5 air pollutants  and provide impetus for public health policy initiatives to address air pollution to reduce disease burden.

PKN2 deficiency leads both to prenatal 'congenital' cardiomyopathy and defective angiotensin II stress responses.

The protein kinase PKN2 is required for embryonic development and PKN2 knockout mice die as a result of failure in the expansion of mesoderm, cardiac development and neural tube closure. In the adult, cardiomyocyte PKN2 and PKN1 (in combination) are required for cardiac adaptation to pressure-overload. The specific role of PKN2 in contractile cardiomyocytes during development and its role in the adult heart remain to be fully established. We used mice with cardiomyocyte-directed knockout of PKN2 or global PKN2 haploinsufficiency to assess cardiac development and function using high resolution episcopic microscopy, MRI, micro-CT and echocardiography. Biochemical and histological changes were also assessed. Cardiomyocyte-directed PKN2 knockout embryos displayed striking abnormalities in the compact myocardium, with frequent myocardial clefts and diverticula, ventricular septal defects and abnormal heart shape. The sub-Mendelian homozygous knockout survivors developed cardiac failure. RNASeq data showed up-regulation of PKN2 in patients with dilated cardiomyopathy, suggesting an involvement in adult heart disease. Given the rarity of homozygous survivors with cardiomyocyte-specific deletion of PKN2, the requirement for PKN2 in adult mice was explored using the constitutive heterozygous PKN2 knockout. Cardiac hypertrophy resulting from hypertension induced by angiotensin II was reduced in these haploinsufficient PKN2 mice relative to wild-type littermates, with suppression of cardiomyocyte hypertrophy and cardiac fibrosis. It is concluded that cardiomyocyte PKN2 is essential for heart development and the formation of compact myocardium and is also required for cardiac hypertrophy in hypertension. Thus, PKN signalling may offer therapeutic options for managing congenital and adult heart diseases.

Treponema spp. spirochetes and keratinopathogenic fungi isolated from keratomas in donkeys.

Keratoma is an aberrant keratin mass thought to originate from epidermal horn-producing cells interposed between the stratum medium of the hoof wall and the underlying third phalanx. The cause is unknown, although the presence of keratomas is frequently associated with chronic irritation, focal infection, or trauma. A total of 167 donkeys with keratomas were presented in this study. The diagnosis of a keratoma was based on clinical signs, radiography, and histopathologic examination. Surgical excision was attempted on all donkeys with lameness unless euthanasia was advised. Histopathologic examination, including Giemsa, periodic acid Schiff, and Young's silver special histochemical stains, was performed and showed the presence of fungal hyphae and spirochete bacteria within the degenerate keratin. Polymerase chain reaction (PCR) for treponeme bacteria was performed on 10 keratoma lesions and 9 healthy pieces of hoof (controls). All healthy donkey tissues were negative for the 3 recognized digital dermatitis (DD) treponeme phylogroups, whereas 3 of 10 (30%) donkey keratoma samples were positive for one of the DD treponeme phylogroups. Routine fungal culture and PCR for fungi were performed on 8 keratoma lesions and 8 healthy pieces of hoof (controls). Keratinopathogenic fungi were detected in 1 of 8 (12.5%) keratomas, while only non-keratinopathogenic, environmental fungi were detected in 8 control healthy hoof samples. This is the first time the DD treponemes phylogroup and keratinopathogenic fungi have been detected in keratomas. Further studies are required to assess the significance of this finding.

Histopathological description of iridophoromas resembling skin nodule syndrome in Siamese fighting fish Betta splendens.

Intensive breeding of the Siamese fighting fish Betta splendens, a species that inhabits marshlands in Thailand and other countries in south-east Asia, was traditionally focused on game fighting because the males are very territorial, but has evolved to become mainly devoted to ornamental purposes. Recently, a preliminarily named 'skin nodule syndrome' (SNS) has been described affecting this fish species in Thailand, with multiple bacterial infections suggested, including 4 species of Mycobacterium spp., although the etiology remains elusive. Here we describe the histopathological and immunohistochemical characteristics of 2 iridophoromas in 2 male Siamese fighting fish that strongly resemble the lesions described for SNS. Immunohistochemical analysis yielded negative results for Melan-A, PNL-2, and S-100, likely due to species-specific reasons. The results for molecular detection of mycobacterial DNA were also negative in both cases. The published histological lesions of SNS are very similar to the 4 chromatophoromas that have been reported affecting this species since 2015. Taken together with the present cases, we hypothesize that the preliminarily named SNS in fighting fish may be characterized as invasive chromatophoromas, although the true etiology remains to be elucidated and could include infectious and non-infectious causes. Further studies are necessary to examine whether commercial breeding of Siamese fighting fish has resulted in a possible genetic origin.

Comparison of the prevalence of Domestic Cat Hepadnavirus in a population of cats with uveitis and in a healthy blood donor cat population in the United Kingdom.

OBJECTIVE: Domestic Cat Hepadnavirus (DCH) is a novel virus recently identified in the domestic cat. Currently, little is known regarding its clinical significance. The hepadnaviridae family includes the Hepatitis B Virus (HBV). Co-infection of HBV and Hepatitis C in humans increases the risk of uveitis. We aimed to determine whether DCH is present in the United Kingdom (UK) and whether DCH warrants investigation as a potential cause of uveitis in cats. PROCEDURES: Clinical records from the Royal Veterinary College (RVC) and the Animal Health Trust (AHT) were reviewed for feline cases diagnosed with endogenous uveitis. A healthy control group was identified from cats presented to the RVC as blood donors. DNA was extracted from stored blood samples using commercially available kits. Polymerase chain reaction assays were performed to confirm the presence of feline DNA and to detect the presence of DCH DNA using previously described protocols. RESULTS: Blood samples were available from 65 cats with endogenous uveitis and 43 healthy control cats. Two blood samples from cats with endogenous uveitis tested positive for the presence of DCH DNA. DCH DNA was not detected in the control group. There was no statistically significant difference between the prevalence of DCH between the groups. CONCLUSIONS: Domestic Cat Hepadnavirus is present in the UK. This study failed to demonstrate a conclusive link between DCH and uveitis in cats, although further studies to investigate an association with other feline diseases are warranted.

Dispersion of adeleid oocysts by vertebrates in Gran Canaria, Spain: report and literature review.

Within the family Adeleidae, Adelina spp. belong to a group of arthropod pathogens. These parasites have been reported to have a wide geographic distribution, however, there are no reports of these protists in the Canary Islands, Spain. One of the peculiarities of the life cycle of Adelina spp. is the participation of a predator, because fecundation and sporulation occur inside the body cavity, and so necessitate destruction of the definitive host. The involvement therefore of a 'dispersion host', which eats the definitive host and spreads the oocysts through its faeces, is critical for the maintenance of certain Adelina spp. On the island of Gran Canaria, adeleid oocysts have been found in stool samples from four animals, three California kingsnakes (Lampropeltis californiae), and one feral cat. These animals were part of a larger coprological study of vertebrate parasites (117 snakes, 298 cats), where pseudoparasitic elements were also recorded. L. californiae and feral cats are invasive species which are widespread across the island and this novel finding of Adelina spp. oocysts in their faeces suggests that they could also serve as potential sentinel species for arthropod parasites.

Parasitological findings in the invasive California kingsnake (Lampropeltis californiae) in Gran Canaria, Spain.

The California kingsnake (Lampropeltis californiae), native to North America, is a significant threat to the conservation of endemic species in the Spanish Macaronesian island of Gran Canaria. However, its role disseminating potential invasive parasites, such as zoonotic pentastomids, has not been proven. Among its parasitic fauna, only protistans have been documented, in contrast to other Lampropeltis spp., which are known to carry pentastomids. Thus, a parasitological study is urgently required. Between 2016 and 2018, a total of 108 snakes were necropsied and stool samples examined. A single snake was infested with Ophionyssus natricis, and another individual with Serpentirhabdias sp. Only this latter snake presented gross lesions, characterized by granulomatous pneumonia. No Pentastomida were found. By contrast, almost the entire population (98.5%) was infested with larval helminths (three different nematode and two cestode species), characterized by granulomatous gastrointestinal serositis. This suggests the snake poses a 'dead end' host for local parasites. Based on these findings, snakes in Gran Canaria carry potential zoonotic mites, which along with Serpentirhabdias sp. could represent a threat to endemic lizards. The presence of metazoan parasites and their lesions are reported for the first time in the California kingsnake.

Suspensory Ligament Desmitis Caused by Onchocerca sp. in Three Donkeys.

Three donkeys were presented with progressive lameness and distal suspensory ligament breakdown in multiple limbs. Treatment with nonsteroidal anti-inflammatory drugs was only partially effective and eventually the donkeys were euthanized due to further progression of the lameness and concerns for their welfare. At necropsy, the distal part of the suspensory ligaments in multiple limbs, including the suspensory ligament branches, was markedly thickened, enlarged, and mottled white and brown on cut section. In one case, adult Onchocerca sp. nematodes were grossly identified embedded within the suspensory ligaments. Histopathologic examination revealed chronic, multifocal to coalescing, moderate to severe, lymphoplasmacytic, eosinophilic, and fibrosing desmitis and tendinitis with intralesional, coiled adult nematodes of Onchocerca sp., accompanied by osseous and cartilaginous metaplasia. To the authors' knowledge, this is the first histopathologic description of suspensory ligament desmitis and tendinitis associated with Onchocerca sp. in donkeys.

Canine Respiratory Coronavirus: A Naturally Occurring Model of COVID-19?

Discovered in 2003 at the Royal Veterinary College, London, canine respiratory coronavirus (CRCoV) is a betacoronavirus of dogs and major cause of canine infectious respiratory disease complex. Generally causing mild clinical signs of persistent cough and nasal discharge, the virus is highly infectious and is most prevalent in rehoming shelters worldwide where dogs are often closely housed and infections endemic. As the world grapples with the current COVID-19 pandemic, the scientific community is searching for a greater understanding of a novel virus infecting humans. Similar to other betacoronaviruses, SARS-CoV-2 appears to have crossed the species barrier, most likely from bats, clearly reinforcing the One Health concept. Veterinary pathologists are familiar with coronavirus infections in animals, and now more than ever this knowledge and understanding, based on many years of veterinary research, could provide valuable answers for our medical colleagues. Here I review the early research on CRCoV where seroprevalence, early immune response, and pathogenesis are some of the same key questions being asked by scientists globally during the current SARS-CoV-2 pandemic.

Nonocular Melanocytic Neoplasia in Cats: Characterization and Proposal of a Histologic Classification Scheme to More Accurately Predict Clinical Outcome.

Nonocular melanocytic neoplasia is considered uncommon in cats yet is routinely encountered in diagnostic pathology and recognized to exhibit a wide variation in biological behavior. Accurate prediction of clinical outcomes is challenging with no widely recognized prognostic criteria. Signalment and tumor location were retrospectively evaluated in 324 cats diagnosed with nonocular melanocytic neoplasia. Histologic features were described in 141 neoplasms and outcome data were available in 79 cases. Immunohistochemistry using Melan-A, PNL-2, cyclooxygenase 2 (COX-2), and E-cadherin was performed in a subset (n = 24). Multivariate analysis identified tumor site, mitotic count, and the presence of intratumoral necrosis to be independent predictors of tumor-related death. On the basis of these findings, we propose a novel histologic grading scheme in which nonocular melanocytic neoplasms involving the lips, oral or nasal mucosa, or nasal planum are considered high grade if they fulfill 1 or both of the following criteria: at least 4 mitoses in 10 high-power fields (HPF) or presence of intratumoral necrosis; those arising elsewhere are considered high grade if they fulfill both of the above criteria. Of 79 tumors with outcome data, 43 (54%) were low grade and 36 (46%) were high grade. The grading system had an 80% sensitivity and 92% specificity for predicting tumor-related death in this population of cats. Median survival for cats with low-grade tumors was not reached, and the median survival was 90 days for those with a high-grade tumor. PNL-2 and Melan-A were sensitive markers for feline nonocular melanocytic neoplasia, and although not significantly associated with prognosis, a large proportion expressed COX-2, suggesting a potential therapeutic role for COX-2 inhibitors.

Canine Ependymoma: Diagnostic Criteria and Common Pitfalls.

Reports of canine ependymoma are generally restricted to single case reports with tumor incidence estimated at 2% to 3% of primary central nervous system (CNS) tumors. While most commonly reported in the lateral ventricle, tumors can occur anywhere in the ventricular system and in extraventricular locations. Rosettes and pseudorosettes are a common histologic feature; however, these features can be mimicked by other CNS neoplasms. Thirty-seven potential ependymoma cases were identified in a retrospective database search of 8 institutions, and a histologic review of all cases was conducted. Of 37 cases, 22 candidate cases were further subjected to a consensus histologic and immunohistochemical review, and only 5 of 37 (13.5%) were conclusively identified as ependymoma. The neuroanatomic locations were the lateral ventricle (3/5), third ventricle (1/5), and mesencephalic aqueduct (1/5). Subtypes were papillary (4/5) and tanycytic (1/5). Histologic features included rosettes (5/5), pseudorosettes (5/5), ependymal canals (2/5), tanycytic differentiation (1/5), blepharoplasts (1/5), ciliated cells (1/5), and high nuclear to cytoplasmic ratio (5/5). Immunolabeling for GFAP (4/4) and CKAE1/3 (3/4) was found in pseudorosettes, rosettes, and scattered individual neoplastic cells. Diffuse but variably intense cytoplasmic S100 immunolabeling was detected in 3 of 4 cases. Olig2 intranuclear immunolabeling was observed in less than 1% of the neoplastic cells (3/3). Tumors that had pseudorosettes and mimicked ependymoma included oligodendroglioma, choroid plexus tumor, pituitary corticotroph adenoma, papillary meningioma, and suprasellar germ cell tumor. These findings indicate that canine ependymoma is an extremely rare neoplasm with histomorphologic features that overlap with other primary CNS neoplasms.

Correlating Gastrointestinal Histopathologic Changes to Clinical Disease Activity in Dogs With Idiopathic Inflammatory Bowel Disease.

Prior studies have failed to detect a convincing association between histologic lesions of inflammation and clinical activity in dogs with inflammatory bowel disease (IBD). We hypothesized that use of a simplified histopathologic scoring system would improve the consistency of interpretation among pathologists when describing histologic lesions of gastrointestinal inflammation. Our aim was to evaluate the correlation of histopathologic changes to clinical activity in dogs with IBD using this new system. Forty-two dogs with IBD and 19 healthy control dogs were enrolled in this retrospective study. Endoscopic biopsies from the stomach, duodenum, ileum, and colon were independently scored by 8 pathologists. Clinical disease activity was scored using the Canine Inflammatory Bowel Disease Activity Index (CIBDAI) or the Canine Chronic Enteropathy Clinical Activity Index (CCECAI), depending on the individual study center. Summative histopathological scores and clinical activity were calculated for each tissue (stomach, duodenum, ileum, and colon) and each tissue histologic score (inflammatory/morphologic feature). The correlation between CCECAI/CIBDAI and summative histopathologic score was significant ( P < .05) for duodenum ( r = 0.42) and colon ( r = 0.33). In evaluating the relationship between histopathologic scores and clinical activity, significant ( P < .05) correlations were observed for crypt dilation ( r = 0.42), lamina propria (LP) lymphocytes ( r = 0.40), LP neutrophils ( r = 0.45), mucosal fibrosis ( r = 0.47), lacteal dilation ( r = 0.39), and villus stunting ( r = 0.43). Compared to earlier grading schemes, the simplified scoring system shows improved utility in correlating histopathologic features (both summative histology scores and select histologic scores) to IBD clinical activity.

Immunohistochemical characterization of feline lymphoplasmacytic anterior uveitis.

OBJECTIVE: To characterize the immune cells present in different forms of feline anterior uveitis. SAMPLES: Eyes were obtained from 49 cats diagnosed with chronic idiopathic lymphoplasmacytic anterior uveitis, 7 cats with feline infectious peritonitis (FIP), and 9 cats euthanized for nonocular disease. METHODS: H&E sections were scored on the level of infiltrate in the anterior uvea. Immunohistochemistry was performed for FoxP3, CD3, and IL-17A, and positive cells were quantified in multiple images of each sample. A generalized estimating equation tested for an association between the level of inflammation and the prevalence of these cell types. RESULTS: Cells stained positive for IL-17A in idiopathic uveitis but not in FIP samples. We found significantly fewer FoxP3+ and CD3+ cells in low-grade compared with high-grade inflammation in idiopathic uveitis or FIP samples (P values all <.005), but no difference between FIP and high-grade samples. CONCLUSIONS: Idiopathic, but not FIP-associated, uveitis appears to have Th17 cell involvement. The numbers of FoxP3+ and CD3+ T-cells present appear directly correlated; thus, the severity of disease does not appear directly determined by the numbers of regulatory cells.

14-3-3σ Protein Expression in Canine Renal Cell Carcinomas.

14-3-3σ is a protein expressed in many epithelial tissues associated with essential cell functions, including cell-cycle control, apoptosis, and cytoskeletal integrity. There is a paucity of knowledge of the tumorigenesis of canine renal cell carcinomas (RCCs), and the histological origin of this tumor has not been established. This study analyzed the expression of 14-3-3σ, Ki-67, cytokeratins, and vimentin in 40 canine RCCs. Aberrant expression of 14-3-3σ was demonstrated in 15 (38%) cases and was associated with a significantly shorter survival time ( P < .002). In contrast to canine RCC, normal kidney did not express 14-3-3σ. The Ki-67 proliferation index did not show utility as a prognostic factor. The distal convoluted tubular epithelium in normal kidneys coexpressed cytokeratins and vimentin, and thus maintenance of this coexpression pattern in canine RCC suggests that most tumors arise from the distal segment of the nephron. These results suggest that 14-3-3σ is a potential negative prognostic factor and a possible therapeutic target.

Observational Study Design in Veterinary Pathology, Part 2: Methodology.

Observational studies are a basis for much of our knowledge of veterinary pathology, yet considerations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offered advice on planning and carrying out an observational study. Part 2 of the series focuses on methodology. Our general recommendations are to consider using already-validated methods, published guidelines, data from primary sources, and quantitative analyses. We discuss 3 common methods in pathology research-histopathologic scoring, immunohistochemistry, and polymerase chain reaction-to illustrate principles of method validation. Some aspects of quality control include use of clear objective grading criteria, validation of key reagents, assessing sample quality, determining specificity and sensitivity, use of technical and biologic negative and positive controls, blinding of investigators, approaches to minimizing operator-dependent variation, measuring technical variation, and consistency in analysis of the different study groups. We close by discussing approaches to increasing the rigor of observational studies by corroborating results with complementary methods, using sufficiently large numbers of study subjects, consideration of the data in light of similar published studies, replicating the results in a second study population, and critical analysis of the study findings.

Observational Study Design in Veterinary Pathology, Part 1: Study Design.

Observational studies are the basis for much of our knowledge of veterinary pathology and are highly relevant to the daily practice of pathology. However, recommendations for conducting pathology-based observational studies are not readily available. In part 1 of this series, we offer advice on planning and conducting an observational study with examples from the veterinary pathology literature. Investigators should recognize the importance of creativity, insight, and innovation in devising studies that solve problems and fill important gaps in knowledge. Studies should focus on specific and testable hypotheses, questions, or objectives. The methodology is developed to support these goals. We consider the merits and limitations of different types of analytic and descriptive studies, as well as of prospective vs retrospective enrollment. Investigators should define clear inclusion and exclusion criteria and select adequate numbers of study subjects, including careful selection of the most appropriate controls. Studies of causality must consider the temporal relationships between variables and the advantages of measuring incident cases rather than prevalent cases. Investigators must consider unique aspects of studies based on archived laboratory case material and take particular care to consider and mitigate the potential for selection bias and information bias. We close by discussing approaches to adding value and impact to observational studies. Part 2 of the series focuses on methodology and validation of methods.

Rabbits with naturally occurring cataracts referred for phacoemulsification and intraocular lens implantation: a preliminary study of 12 cases.

OBJECTIVE: To describe the presentation of 15 rabbits with naturally occurring cataracts referred for phacoemulsification surgery, the procedure in 13 cases and the follow-up in 12. ANIMALS INCLUDED: Fifteen rabbits (30 eyes), nine of which stopped following visual cues in association with cataract progression. MATERIALS AND METHODS: Rabbits underwent preoperative ophthalmic and ocular ultrasound examination. RESULTS: Thirteen rabbits (22 eyes) had mature cataracts. Ten were bilateral and three unilateral. Two rabbits had an anterior chamber abscess. The cataract in one of these was incipient. One rabbit had bilateral immature cataracts. One rabbit had a subluxated lens, and one had a retinal detachment. Thirteen rabbits (22 eyes) underwent phacoemulsification. Eighteen, 13.5-mm capsular tension rings (CTRs) and seventeen, 13-mm IOLs (Acrivet® , Berlin, Germany) were fitted including one 41D 60V-model, and three 49D and thirteen 58D 20S-models. Intraoperative complications included one unilateral posterior-capsular tear, one lens subluxation, and one expulsive choroidal hemorrhage. One rabbit died during anesthetic recovery. Nine cases were PCR-tested for Encephalitozoon cuniculi, and only three were positive. The median follow-up time was 12 months (4-24 months). Rabbits that were not following visual cues preoperatively did so postoperatively, and surgery resulted in a clear visual axis for the follow-up period in every case except in two, due to reasons other than the surgery. CONCLUSIONS: Phacoemulsification with CTR and IOL implantation offers good long-term results and can improve the quality of life of pet rabbits. Retinal detachment, lens luxation, expulsive choroidal hemorrhage, and anesthetic death are potential complications.