Clinical outcomes and cost-effectiveness of superficial parotidectomy versus extracapsular dissection of the parotid gland: a single-centre retrospective study of 161 patients.

Improvements in preoperative diagnostics and intraoperative techniques have made the surgical excision of benign parotid gland tumours less invasive. Extracapsular dissection (ECD) has become more popular in comparison to superficial parotidectomy (SP), the gold standard. Although clinical outcomes have been reported, reports on cost-effectiveness are limited. The aim of this retrospective study was to analyse the surgical outcomes and cost-effectiveness of ECD versus SP in benign parotid tumour surgery. A retrospective cohort of 161 patients treated between 2012 and 2020 was collected. Data concerning demographics, clinical outcomes, and cost-efficiency were recorded. Analysis of the 161 unilateral parotidectomy cases (59 SP, 102 ECD) showed a significantly longer operation time, anaesthesia time, and length of stay for SP patients (all P < 0.001). Regarding postoperative complications, transient facial nerve weakness (P < 0.001) and haematoma formation (P = 0.016) were more prevalent in the SP patients. The frequency of positive margins was lower for SP (P = 0.037). No case of recurrence was identified with either technique. ECD showed excellent clinical outcomes as well as a reduction in complications when compared to SP. ECD is a viable alternative for superficial benign parotid gland tumours after thorough preoperative clinical, pathological, and radiological examination. The reduction in operation, anaesthesia, and hospitalization times with ECD is likely to result in a gain in cost-effectiveness.

Antibiotics in orthognathic surgery: a retrospective analysis and identification of risk factors for postoperative infection.

This study was undertaken to evaluate the infection rate following orthognathic surgery and to identify possible risk factors. A retrospective study was conducted. Patients undergoing orthognathic surgery from August 1, 2017 to July 31, 2018 were included. The outcome variable was surgical site infection (SSI). All data were analysed with respect to demographics and procedure specifications. A total of 137 patients (mean age 28.5±12.69 years) were included in this study, of whom 20 (14.6%) developed a SSI. The only risk factor identified was the type of surgery: those undergoing mandibular osteotomies (in bilateral sagittal split osteotomy (BSSO) or bimaxillary osteotomies) were far more likely to develop infections. Third molar teeth were removed during orthognathic surgery in 28.5% of the procedures, and a genioplasty was performed in 10.9%. Removal of osteosynthesis material because of infectious reasons was necessary in 10.2% of patients, with a strong association to previous SSI. In conclusion, this study showed an infection rate of 14.6% with no link to any demographic risk factor. Neither the simultaneous removal of third molar teeth nor genioplasty was found to be a risk factor for SSI. For literature comparison purposes, there is a clear need for the international guidelines defining SSI to be used.

A multicentre retrospective clinico-histopathological review of 250 patients after parotidectomy.

A clinicopathological review of parotid tumours treated surgically in two oral and maxillofacial surgery departments was conducted. The performance of fine needle aspiration cytology (FNAC) was also assessed. This retrospective study included 250 consecutive patients treated surgically for parotid gland-related tumours. Benign tumours (n=211, 84.4%) were more prevalent than malignancies (n=39, 15.6%). A predominance of pleomorphic adenoma (48.8%) was identified, and epithelial-myoepithelial carcinoma (3.6%) was the most common malignant tumour. Overall, the sensitivity and specificity of FNAC were 64% and 99%, respectively. Subgrouping resulted in sensitivity and specificity of 50% and 100% for clinically assisted FNAC versus, 72% and 99% for ultrasound guidance. Surgically, 31.6% underwent complete superficial parotidectomy and 28.4% underwent extracapsular dissection. Overall, facial nerve palsy was the most prevalent postoperative complication, affecting 29.2% (70/240); loss of function was transient in 21.2% (51/240) and permanent in 7.9% (19/240). Extracapsular dissection and superficial parotidectomy with facial nerve preservation were the treatments of choice when a benign tumour was suspected. Facial nerve palsy was quite frequent; treatment options however are scarce. Preoperative diagnostic workup using imaging and ultrasound-guided FNAC was essential in identifying malignancy so that surgical planning could be adapted.

Microdiversity of Echinococcus granulosus sensu stricto in Australia.

Echinococcus granulosus (sensu lato) is now recognized as an assemblage of cryptic species, which differ considerably in morphology, development, host specificity (including infectivity/pathogenicity for humans) and other aspects. One of these species, E. granulosus sensu stricto (s.s.), is now clearly identified as the principal agent causing cystic echinococcosis in humans. Previous studies of a small section of the cox1 and nadh1 genes identified two variants of E. granulosus s.s. to be present in Australia; however, no further work has been carried out to characterize the microdiversity of the parasite in its territory. We have analysed the sequence of the full length of the cox1 gene (1609 bp) from 37 isolates of E. granulosus from different hosts and geographic regions of Australia. The analysis shows that seven haplotypes of E. granulosus s.s. not previously described were found, together with five haplotypes known to be present in other parts of the world, including the haplotype EG01 which is widespread and present in all endemic regions. These data extend knowledge related to the geographical spread and host range of E. granulosus s.s. in a country such as Australia in which the parasite established around 200 years ago.

Assessment of reference genes for reliable analysis of gene transcription by RT-qPCR in ovine leukocytes.

With the availability of genetic sequencing data, quantitative reverse transcription PCR (RT-qPCR) is increasingly being used for the quantification of gene transcription across species. Too often there is little regard to the selection of reference genes and the impact that a poor choice has on data interpretation. Indeed, RT-qPCR provides a snapshot of relative gene transcription at a given time-point, and hence is highly dependent on the stability of the transcription of the reference gene(s). Using ovine efferent lymph cells and peripheral blood mono-nuclear cells (PBMCs), the two most frequently used leukocytes in immunological studies, we have compared the stability of transcription of the most commonly used ovine reference genes: YWHAZ, RPL-13A, PGK1, B2M, GAPDH, HPRT, SDHA and ACTB. Using established algorithms for reference gene normalization "geNorm" and "Norm Finder", PGK1, GAPDH and YWHAZ were deemed the most stably transcribed genes for efferent leukocytes and PGK1, YWHAZ and SDHA were optimal in PBMCs. These genes should therefore be considered for accurate and reproducible RT-qPCR data analysis of gene transcription in sheep.

Pulmonary delivery of ISCOMATRIX influenza vaccine induces both systemic and mucosal immunity with antigen dose sparing.

Using a large animal model, we evaluated whether delivery of influenza vaccine via its mucosal site of infection could improve vaccine effectiveness. Unexpectedly, pulmonary immunization with extremely low antigen doses (0.04 microg influenza) induced serum antibody levels equivalent to those resulting from a current human vaccine equivalent (15 microg unadjuvanted influenza, subcutaneously) and vastly superior lung mucosal antibodies. Induction of this potent response following lung vaccination was dependent on addition of ISCOMATRIX adjuvant and deep lung delivery. Functional antibody activity, marked by hemagglutination inhibition, was only present in the lungs of animals that received adjuvanted vaccine via the lungs, suggesting this approach could potentially translate to improved protection. The 375-fold reduction in antigen dose and improved mucosal antibody responses, compared to the current vaccine, suggests that mucosal delivery via the pulmonary route may be particularly relevant in the event of an influenza pandemic, when vaccine supplies are unlikely to meet demand.

Activation of NF-kappaB transcription factor in the preterm ovine brain and placenta after acute LPS exposure.

Intrauterine infection may be causally related to inflammation and injury of the fetal brain, however the mechanisms by which this occurs are unclear. We have investigated whether nuclear factor (NF)-kappaB, a transcription factor for proinflammatory cytokines, is activated in the fetal brain after acute LPS-exposure. At 95 days of gestation (term = approximately 147 days), 5 fetuses received a single intravenous bolus dose of LPS (1 microg/kg); 6 fetuses served as controls. Fetal blood samples were taken hourly for 6 hr post LPS-exposure to assess physiological status. Ewes and fetuses were then euthanased, placental and brain tissue examined histologically, and NF-kappaB activation assessed in several regions of the fetal brain using an electromobility shift assay (EMSA). Oxidative stress was measured using lipid peroxidation and 8-isoprostane biochemical assays and brain cytokine concentrations analysed by enzyme linked immunosorbent assay (ELISA). LPS-exposed fetuses (relative to controls) were hypoxemic and the haematocrit and lactate levels had increased. In the brains of LPS-exposed fetuses compared to controls, NF-kappaB binding activity was elevated in the hippocampus and the thalamus/basal ganglia; 8-isoprostane levels were elevated overall (P < 0.05) in the parietal/occipital/temporal lobes and thalamus/basal ganglia. TNF-alpha and IL-6 concentrations were not elevated, however, there was a tendency for an elevation of IFN-gamma concentrations in the thalamus/basal ganglia. IFN-gamma concentration was elevated (P < 0.05) in the plasma 4 hr after LPS-exposure. In the placenta, NF-kappaB binding activity was increased (P < 0.05). We conclude that acute systemic administration of LPS leads to increased binding activity of NF-kappaB subunits in specific regions of the fetal brain and in the placenta, but that there is no clear-cut relationship between this elevation and vulnerability to endotoxic damage.

Characterisation of the rapid release of activin A following acute lipopolysaccharide challenge in the ewe.

A series of experiments were conducted in adult ewes to delineate the release profile of activin A and its relationship to other cytokines following an i.v. injection of the bacterial cell wall component, lipopolysaccharide (LPS). Following this challenge, plasma activin A increased rapidly and appeared to be released in a biphasic manner, slightly preceding the release of tumour necrosis factor-alpha (TNFalpha) and before elevation of interleukin (IL)-6 and follistatin levels. The concentration of activin A was correlated with body temperature during the response to LPS. A second experiment compared cytokine concentrations in matched blood and cerebrospinal fluid (CSF) samples. This revealed that activin A was not released centrally in the CSF following a peripheral LPS injection, nor was TNFalpha or the activin binding protein, follistatin, but IL-6 showed a robust elevation. In a third experiment, the stimulus for activin A release was examined by blocking prostaglandin synthesis. Flurbiprofen, a prostaglandin synthesis inhibitor, effectively attenuated the fever response to LPS and partly inhibited cortisol release, but the cytokine profiles were unaffected. Finally, the bioactivity of TNFalpha and/or IL-1 was blocked using soluble receptor antagonists. These treatments did not affect the initial release of activin A, but blockade of TNFalpha depressed the second activin peak. These studies define more rigorously the release of activin A into the circulation following acute inflammatory challenge. The response is rapid and probably biphasic, independent of prostaglandin- mediated pathways and does not depend upon stimulation by TNFalpha or IL-1. The data suggest that activin A release is an early event in the inflammatory cascade following the interaction of LPS with its cellular receptor.

In vivo electroporation improves immune responses to DNA vaccination in sheep.

In vivo electroporation was utilised to enhance plasmid DNA expression in sheep muscle to improve the immune response to DNA vaccination. DNA encoding enhanced green fluorescence protein expressed at higher levels in sheep muscle following in vivo electroporation which caused minimal muscle damage. Groups of seven sheep were then given three intramuscular injections of plasmids encoding two Haemonchus contortus Ag, with and without electroporation at 0, 3 and 7 weeks. Humoral responses were enhanced in electroporated sheep. Four weeks after vaccination, all groups were injected subcutaneously with recombinant Ag formulated in Quil A. Induction of vaccine-specific immune memory was demonstrated in DNA-vaccinated sheep.

Local immune responses to influenza antigen are synergistically enhanced by the adjuvant ISCOMATRIX.

The peripheral (draining) lymph node, as the primary site of immune induction, determines the course of systemic responses to an injected antigen. Lymphatic duct cannulation procedures in sheep were used to investigate local immunoreactivity to human influenza virus antigen (Flu ag) admixed with the adjuvant ISCOMATRIX (IMX). Compared to Flu ag or IMX alone, the co-administration of Flu ag and IMX (Flu ag+IMX) synergistically enhanced a number of immunological responses (lymphocyte and blast migration from the node, antigen-specific antibody levels and IL6 output in efferent lymph, and antigen-induced proliferation in cultured efferent lymph cells). Together, these results demonstrate that IMX is an immune modulator, and that lymphatic duct cannulation procedures may be used to evaluate antigen/adjuvant combinations for vaccine development.

Evidence for activin A and follistatin involvement in the systemic inflammatory response.

The inflammatory cascade is a multifactorial process regulated by interwoven cytokine and growth factor networks. This review summarizes the emerging evidence that implicate activin A and follistatin in inflammatory processes. Our recent studies have determined that activin A is released early in the cascade of circulatory cytokines during systemic inflammatory episodes, roughly coincident with tumour necrosis factor (TNF)-alpha and before interleukin (IL)-6 and follistatin. The source(s) of this activin A are not yet established, but prime candidates are monocytes/macrophages, other immune cell types or vascular endothelial cells. Clinical data are limited, but activin beta(A) subunit mRNA or activin A protein is elevated in inflammatory bowel diseases and inflammatory arthropathies, and circulating concentrations of follistatin are elevated in patients with sepsis. In more mechanistic approaches, in vitro studies show that activin A can have both pro- and anti-inflammatory actions on key inflammatory mediators such as TNFalpha, IL-1beta and IL-6. Furthermore, there is emerging understanding of how the intracellular signaling pathway for activin A, incorporating Smads, may interact with and be modulated by other key regulatory cytokines and growth factors.

The immune response to a DNA vaccine can be modulated by co-delivery of cytokine genes using a DNA prime-protein boost strategy.

A large-scale DNA vaccination trial was performed in sheep to investigate whether co-delivery of the cytokine genes IL-4, IL-5, IL-15, GM-CSF or IFN-gamma could modulate the immune response generated to an antigen, in a DNA prime-recombinant protein boost regime. Vaccination with the recombinant EG95 protein has been shown to induce protection in sheep from Echinococcus granulosus infection, the causative agent of hydatid disease. Here we demonstrate that vaccination with DNA encoding EG95 effectively primed the humoral response, as judged by high IgG anti-EG95 titres detected one-week after a boost with the recombinant protein. However, by two weeks after protein-boost the titres in the control group had reached levels similar to the groups primed with EG95 DNA. Priming with two doses of DNA vaccine followed by boosting with recombinant protein induced a predominantly IgG1 response. In contrast, priming and boosting with the protein vaccine generated a strong IgG2 response. Co-delivery of the EG95 DNA vaccine with DNA encoding GM-CSF enhanced the antibody titre to EG95 while co-delivery of IFN-gamma or IL-4 encoding DNA appeared to reduce the ability of the DNA vaccine to prime an IgG antibody response. This study has demonstrated the efficacy of the co-delivery of cytokines to modulate immune responses generated in a DNA prime-protein boost strategy.

Genetic adjuvants for DNA vaccines.

The relatively low efficacy of DNA vaccines in inducing immune responses, especially in large animal species and humans, has impaired their practical use. Despite considerable effort expended on improving DNA vaccine delivery, only minute amounts of Ag are available for immune induction following DNA vaccination. Two complementary strategies have been used to improve and modulate the immune response induced by DNA vaccines: (i) supplementing DNA vaccines with plasmids encoding cytokines and (ii) targeting the Ag encoded by DNA vaccine through genetically fusing the Ag to molecules binding cell surface receptors. This paper reviews recent progress in these two areas and possible mechanisms responsible for the observed effects.

Immune responses to ISCOM formulations in animal and primate models.

ISCOMs are typically 40 nm cage-like structures comprising antigen, saponin, cholesterol and phospholipid. ISCOMs have been shown to induce antibody responses and activate T helper cells and cytolytic T lymphocytes in a number of animal species, including non-human primates. Recent clinical studies have demonstrated that ISCOMs are also able to induce antibody and cellular immune responses in humans. This review describes the current understanding of the ability of ISCOMs to induce immune responses and the mechanisms underlying this property. Recent progress in the characterisation and manufacture of ISCOMs will also be discussed.

Trichostrongylus colubriformis extract upregulates TNF-alpha receptor expression and enhances TNF-alpha sensitivity of L929 cells.

Many pathogens have developed strategies to avoid the host's immune system and hence improve their long-term survival. These strategies include antigenic variation, mimicry of host regulatory proteins and production of immunoregulatory molecules. The ruminant gastrointestinal nematode Trichostrongylus colubriformis produces several factors with homology to human immunoregulatory proteins. However, direct immunomodulation by T. colubriformis proteins has not yet been unequivocally demonstrated. Results in the present paper demonstrate that soluble T. colubriformis factors promote proliferation of the TNF-susceptible mouse fibrosarcoma cell line L929, while inhibiting proliferation of all other cell types tested. In addition, T. colubriformis homogenate enhanced the susceptibility of L929 cells to the cytotoxic action of ovine TNF-alpha. Within 1 h of exposure, T. colubriformis factors bind L929 cells in a stable fashion, yet it takes up to 24 h for the cells to become sensitised to TNF-alpha. Interestingly, the increase of both TNF-alpha sensitivity and proliferation of treated L929 cells correlated with an upregulation in expression of TNF-alpha p55 and p75 receptors.

Induction of lymphocyte recruitment in the absence of a detectable immune response.

Lymphocyte recruitment from blood into the lymph node is thought to be initiated by the presence of antigen. In this study, we have used lymphatic cannulation in sheep to demonstrate that the adjuvant ISCOMATRIX can induce dramatic lymph node activation in the absence of antigen. Consistent patterns of node shutdown (decreased output) and cell recruitment (increased output) with minimal blast cell responses were observed indicating that an antigen-specific immune response is not required. Production of IL-6, IL-8 and IFN-gamma, and the transient presence of red blood cells and neutrophils in the efferent lymph were associated with changes in efferent lymph cell trafficking. These early events may facilitate the screening of low frequency antigen-specific cells for binding to antigen and the subsequent amplification of the immune response.

Ovine interleukin-12: analysis of biologic function and species comparison.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced mainly by phagocytic and antigen-presenting cells (APC). The cDNA encoding the ovine IL-12 (OvIL-12) subunits, p40 and p35, were generated from concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC). The ovine genes encoded proteins that had the highest amino acid identity to caprine p40 (99% amino acid identity) and p35 (97% amino acid identity) and also displayed a high degree of identity with human p40 (84%) and p35 (79%) homologs. To ensure the equal expression of both subunits, we used the self-cleaving properties of the 2A oligopeptide from foot-and-mouth disease virus (FMDV) to express IL-12 as a single, long open reading frame (ORF) encoding p402Ap35. Using an in vitro transcription/translation system, we demonstrated that this 2A oligopeptide mediated cleavage of the p402Ap35 into p402A and p35, in a manner similar to the processing of the FMDV polypeptide. Moreover, when expressed in COSm6 cells, this self-processing polypeptide encoded a functional heterodimer, which elicited biologic activities associated with IL-12 in other species.

The expression and biologic effects of ovine interleukin-4 on T and B cell proliferation.

Using the reverse-transcriptase polymerase chain reaction (RT-PCR), cDNA encoding ovine (Ov) interleukin-4 (OvIL-4) was generated from mitogen-stimulated peripheral blood mononuclear cells (PBMC). Two identical clones generated from separate RT-PCR reactions differed from a published OvIL-4 sequence, although they had a high degree of identity with the bovine and human homologs. We show by sequence analysis that the OvIL-4 cDNA retained the four alpha-helix structure and disulfide bonds identified in human IL-4 (HuIL-4). Moreover, the cDNA encoding OvIL-4 was expressed in insect cells using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) as a vector. Supernatants from insect cells infected with the recombinant virus secreted an additional protein with a relative molecular mass of 17,000. This protein was recognized by an anticervine IL-4 monoclonal antibody (mAb) in a Western blot and did not react with any proteins in supernatants from uninfected insect cells or cells infected with the wild-type AcMNPV. Supernatants from insect cells infected with the recombinant virus induced the proliferation of activated B cells in a dose-dependent manner and typically demonstrated 5 x 105 dilution U/ml of activity. However, OvIL-4 had no effect on the proliferation of resting T cells isolated from efferent lymph and actually inhibited the ability of a mitogen to stimulate these resting lymphocytes. In contrast, OvIL-4 induced the proliferation of mitogen-activated lymphoblast, demonstrating the complex role(s) OvIL-4 plays in the regulation of B and T cells.

Characterisation of monoclonal antibodies to ovine interleukin-6 and the development of a sensitive capture ELISA.

A purified recombinant ovine (rOv) interleukin-6 (IL-6) was used to generate specific murine monoclonal antibodies (mAbs) and a polyclonal rabbit antisera to this cytokine. From the 31 initial hybridoma cell lines generated, three stable clones were established which secreted mAbs to rOvIL-6, as judged by a direct enzyme-linked immunosorbent assay (ELISA) and Western blotting. Their specificity was further confirmed by demonstrating that none of the mAbs recognised any of the six other irrelevant recombinant ovine cytokines tested by direct ELISA. All three mAbs displayed cross-reactivity with human and African green monkey IL-6 as demonstrated by direct ELISA and Western blotting. In contrast, the polyclonal antibodies only cross-reacted with bovine IL-6 and not with either of the human or monkey homologues. By combining a mAb with the polyclonal antisera a sensitive, IL-6-specific, capture ELISA was developed that had a sensitivity of 150 pg/ml. This detection system was unequivocally validated by demonstrating that native OvIL-6 could be detected in efferent lymph draining from a stimulated popliteal lymph node. In addition, one of the mAbs was shown to allow the detection of OvIL-6 by intracellular cytokine staining and flow cytometry.