Central Artery Hemodynamics in Angiotensin II-Induced Hypertension and Effects of Anesthesia.

Systemic hypertension is a strong risk factor for cardiovascular, neurovascular, and renovascular diseases. Central artery stiffness is both an initiator and indicator of hypertension, thus revealing a critical relationship between the wall mechanics and hemodynamics. Mice have emerged as a critical animal model for studying effects of hypertension and much has been learned. Regardless of the specific mouse model, data on changes in cardiac function and hemodynamics are necessarily measured under anesthesia. Here, we present a new experimental-computational workflow to estimate awake cardiovascular conditions from anesthetized data, which was then used to quantify effects of chronic angiotensin II-induced hypertension relative to normotension in wild-type mice. We found that isoflurane anesthesia had a greater impact on depressing hemodynamics in angiotensin II-infused mice than in controls, which led to unexpected results when comparing anesthetized results between the two groups of mice. Through comparison of the awake simulations, however, in vivo relevant effects of angiotensin II-infusion on global and regional vascular structure, properties, and hemodynamics were found to be qualitatively consistent with expectations. Specifically, we found an increased in vivo vascular stiffness in the descending thoracic aorta and suprarenal abdominal aorta, leading to increases in pulse pressure in the distal aorta. These insights allow characterization of the impact of regionally varying vascular remodeling on hemodynamics and mouse-to-mouse variations due to induced hypertension.

High content analysis of in vitro alveolar macrophage responses can provide mechanistic insight for inhaled product safety assessment.

Assessing the safety of inhaled substances in the alveolar region of the lung requires an understanding of how the respired material interacts with both physical and immunological barriers. Human alveolar-like macrophages in vitro provide a platform to assess the immunological response in the airways and may better inform the understanding of a response to an inhaled challenge being adaptive or adverse. The aim of this study was to determine if a morphometric phenotyping approach could discriminate between different inhaled nicotine products and indicate the potential mechanism of toxicity of a substance. Cigarette smoke (CS) and e-liquids extracted into cell culture medium were applied to human alveolar-like macrophages in mono-culture (ImmuONE™) and co-culture (ImmuLUNG™) to test the hypothesis. Phenotype profiling of cell responses was highly reproducible and clearly distinguished the different responses to CS and e-liquids. Whilst the phenotypes of untreated macrophages were similar regardless of culture condition, macrophages cultured in the presence of epithelial cells were more sensitive to CS-induced changes related to cell size and vacuolation processes. This technique demonstrated phenotypical observations typical for CS exposure and indicative of the established mechanisms of toxicity. The technique provides a rapid screening approach to determine detailed immunological responses in the airways which can be linked to potentially adverse pathways and support inhalation safety assessment.

Ageing and Mental Health in Canada: Perspectives from Law, Policy, and Longitudinal Research.

Canada is a relatively young, geographically-diverse country, with a larger proportion of the population aged over 65 than under 15. Increasing alongside the number of ageing Canadians is the number of older adults that live with mental health challenges. Across the life course, one in five Canadians will experience a mental health disorder with many more living with subclinical symptoms. For these individuals, their lived experience may be directly impacted by the contemporary laws and policies governing mental illness. Examining and reviewing the historical context of mental health and older adults, we provide insights into the evolving landscape of Canadian mental health law and policy, paternalistic roots in the infancy of the country, into modern foci on equity and diversity. Progressing in parallel to changes in mental health policy has been the advancement of mental health research, particularly through longitudinal studies of ageing. Although acting through different mechanisms, the evolution of Canadian mental health law, policy, and research has had, and continues to have, considerable impacts on the substantial proportion of Canadians living with mental health challenges.

Landscape and taxon age are associated with differing patterns of hybridization in two Eucalyptus (Myrtaceae) subgenera.

BACKGROUND AND AIMS: Hybridization is an important evolutionary process that can have a significant impact on natural plant populations. Eucalyptus species are well known for weak reproductive barriers and extensive hybridization within subgenera, but there is little knowledge of whether patterns of hybridization differ among subgenera. Here, we examine eucalypts of Western Australia's Stirling Range to investigate how patterns of hybridization are associated with landscape and taxon age between the two largest Eucalyptus subgenera: Eucalyptus and Symphyomyrtus. In doing so, we tested a hypothesis of OCBIL (old, climatically buffered, infertile landscape) theory that predicts reduced hybridization on older landscapes. METHODS: Single nucleotide polymorphism markers were applied to confirm the hybrid status, parentage and genetic structure of five suspected hybrid combinations for subg. Eucalyptus and three combinations for subg. Symphyomyrtus. KEY RESULTS: Evidence of hybridization was found in all combinations, and parental taxa were identified for most combinations. The older parental taxa assessed within subg. Eucalyptus, which are widespread on old landscapes, were identified as well-defined genetic entities and all hybrids were exclusively F1 hybrids. In addition, many combinations showed evidence of clonality, suggesting that the large number of hybrids recorded from some combinations is the result of long-term clonal spread following a few hybridization events rather than frequent hybridization. In contrast, the species in subg. Symphyomyrtus, which typically occur on younger landscapes and are more recently evolved, showed less distinction among parental taxa, and where hybridization was detected, there were high levels of introgression. CONCLUSIONS: Reduced hybridization in subg. Eucalyptus relative to extensive hybridization in subg. Symphyomyrtus affirmed the hypothesis of reduced hybridization on OCBILs and demonstrate that clade divergence times, landscape age and clonality are important drivers of differing patterns of speciation and hybridization in Eucalyptus.

Paternity analysis reveals wide pollen dispersal and high multiple paternity in a small isolated population of the bird-pollinated Eucalyptus caesia (Myrtaceae).

Optimal foraging behaviour by nectavores is expected to result in a leptokurtic pollen dispersal distribution and predominantly near-neighbour mating. However, complex social interactions among nectarivorous birds may result in different mating patterns to those typically observed in insect-pollinated plants. Mating system, realised pollen dispersal and spatial genetic structure were examined in the bird-pollinated Eucalyptus caesia, a species characterised by small, geographically disjunct populations. Nine microsatellite markers were used to genotype an entire adult stand and 181 seeds from 28 capsules collected from 6 trees. Mating system analysis using MLTR revealed moderate to high outcrossing (tm=0.479-0.806) and low estimates of correlated paternity (rp=0.136±s.e. 0.048). Paternity analysis revealed high outcrossing rates (mean=0.72) and high multiple paternity, with 64 different sires identified for 181 seeds. There was a significant negative relationship between the frequency of outcross mating and distance between mating pairs. Realised mating events were more frequent than expected with random mating for plants <40 m apart. The overall distribution of pollen dispersal distances was platykurtic. Despite extensive pollen dispersal within the stand, three genetic clusters were detected by STRUCTURE analysis. These genetic clusters were strongly differentiated yet geographically interspersed, hypothesised to be a consequence of rare recruitment events coupled with extreme longevity. We suggest that extensive polyandry and pollen dispersal is a consequence of pollination by highly mobile honeyeaters and may buffer E. caesia against the loss of genetic diversity predicted for small and genetically isolated populations.

Duration of disease influences survival to discharge of Thoroughbred mares with surgically treated large colon volvulus.

REASONS FOR PERFORMING STUDY: Large colon volvulus results in strangulating obstruction requiring surgical treatment. Duration of this disease prior to surgical treatment is likely to influence survival directly. OBJECTIVES: The primary objective of this study was to evaluate the influence of duration of large colon volvulus on patient survival to discharge. Relationships between other factors and survival following large colon volvulus were also examined. STUDY DESIGN: Retrospective case analysis. METHODS: Medical records of Thoroughbred mares aged ≥2 years undergoing surgical treatment for large colon volvulus between 1 March 1986 and 28 February 2011 were reviewed. Multivariable logistic regression was used to identify influence of duration and other factors on survival to discharge. RESULTS: Within the study period, 1039 surgeries were performed to correct large colon volvulus in 896 Thoroughbred mares. Median duration of colic signs prior to admission was 2 h (interquartile range [IQR] 1-4 h). Median time from admission to anaesthetic induction was 25 min (IQR 15-45 min). Median surgical time was 70 min (IQR 55-85 min). Primary surgical treatment was simple correction of the large colon volvulus followed by replacement of the colon. The overall survival to discharge was 88%. The final model identified risk factors significantly associated with survival and included colic duration prior to admission, packed cell volume at admission, surgery length, duration of hypotension while under anaesthesia, heart rate 48 h post operatively, post operative manure consistency and days hospitalised. CONCLUSIONS: Duration of colic prior to admission was statistically associated with patient survival to discharge. Other factors significantly associated with survival in mares with large colon volvulus were related to disease severity and degree of colonic compromise. Mares with large colon volvulus have a good prognosis for recovery with early referral and prompt surgical treatment.

Factors influencing foaling rate following colic surgery in pregnant Thoroughbred mares in Central Kentucky.

REASONS FOR PERFORMING STUDY: Clinical experience has suggested that foaling rates following colic surgery in the pregnant mare are influenced by days of gestation. This premise has not been supported in previous studies. We also aimed to determine the effects of other potential influencing factors. OBJECTIVE: To determine the influence of age of gestation on foaling rates in pregnant mares following colic surgery and evaluate the relationships between other factors and foaling rates. METHODS: Medical records of Thoroughbred mares, which had colic surgery performed from 1993 to 2007 at the Rood and Riddle Equine Hospital, were reviewed. Mares identified in the record as pregnant or bred within the previous 15 days were included in this study. Age of the mare, date of surgery, gestational age, duration of colic at admission, packed cell volume at admission, surgical diagnosis, duration of general anaesthesia, intraoperative hypotension, intraoperative hypoxaemia and post operative signs of endotoxaemia were recorded. A mare was considered to have a live foal if that foal was registered with the North American Jockey Club. RESULTS: Of the 228 mares, where pregnancy had been confirmed, 152 (66.7%) had a live foal registered after surgery. Mares bred <40 days before surgery had a lower foaling rate compared with mares undergoing surgery ≥40 days after breeding: 48.7% vs. 69.8% (odds ratio [OR] = 0.41 [95% confidence interval (CI) 0.20-0.83], P = 0.012). Foaling rate was also influenced by mare's age (P = 0.008) and duration of colic signs before surgery (P = 0.03). CONCLUSIONS: The prognosis for a live foal after colic surgery in the pregnant Thoroughbred mare is significantly better if the mare is ≤15 years of age and ≥40 days of gestation. POTENTIAL RELEVANCE: The results of this study are useful for clinicians offering a prognosis for a live foal following colic surgery in pregnant mares.

Complications after two transphyseal bridging techniques for treatment of angular limb deformities of the distal radius in 568 Thoroughbred yearlings.

REASONS FOR PERFORMING STUDY: Surgical correction of carpal angular limb deformities by growth retardation is commonly undertaken with a screws and tension band wire loop technique (S&W) or a single transphyseal screw (STS). This study compares complications after S&W and STS bridging in the distal radius of Thoroughbred yearlings. OBJECTIVE: To compare the prevalence of complications serious enough to require follow-up radiographs following either S&W or STS surgery for growth manipulation in the distal radius of Thoroughbred yearlings. METHODS: Medical records and radiographs from Thoroughbred yearlings (age range 261-457 days) treated for carpal angular limb deformities at a single hospital over 2 years were reviewed. Each of the techniques was used exclusively during a single year. The complication threshold criterion for inclusion was the need for nonroutine radiographs of the operated site anytime after implant insertion or removal. RESULTS: Of 568 horses, 253 received S&W and 315 received STS. Horses were of similar age at the time of surgery for STS and S&W. Single transphyseal screws were left in place for a significantly shorter amount of time (16 days). Sex, the limb(s) treated and medial vs. lateral placement were not significantly different between techniques. Complications included physitis post implant removal, metaphyseal collapse post implant removal, infection, overcorrection and seroma formation severe enough to require radiography. Physitis and metaphyseal collapse occurred significantly more frequently with STS compared with S&W. Infection, overcorrection and seromas were not significantly different between techniques. CONCLUSION: The STS and S&W techniques are both viable treatment options for correction of carpal angular limb deformities. However, horses treated with the STS technique have a significantly increased risk of developing physitis or metaphyseal collapse. POTENTIAL RELEVANCE: Horses treated with STS bridging have a significantly increased risk of developing the post correction complications of moderate to severe physitis and metaphyseal collapse compared with horses treated with S&W bridging.

Size and geometry of apical sesamoid fracture fragments as a determinant of prognosis in Thoroughbred racehorses.

REASON FOR PERFORMING STUDY: Analysis was performed to examine a method for refining the preoperative prognosis for horses that had surgery to remove apical fractures of the proximal sesamoid bones (PSBs). OBJECTIVES: To determine if: 1) there was a difference in size or configuration of apical fractures between the different anatomical locations of the PSBs, which have been shown to affect the prognosis; and 2) the size or configuration could predict the prognosis for racehorses with these fractures. METHODS: The study included 110 weanlings and yearlings and 56 training racehorses that underwent surgery to remove apical PSB fractures. Radiographs of the fractures were used for measurement of the abaxial and axial proportion and the abaxial to axial ratio, and race records were used to determine average earnings per start (AEPS) and total post operative starts. Analysis of variance and regression statistics were used to compare the fragment sizes between the specific PSBs on each of the limbs and compare size and configuration of the fractures to prognosis. RESULTS: There was a significantly larger abaxial to axial ratio (more transverse fracture) for the forelimb medial sesamoids than for all other sesamoids in untrained racehorses (P = 0.03). There were no other significant differences in size. There was no relationship between fracture size or configuration and AEPS nor total post operative starts. CONCLUSIONS: Apical fractures in weanlings and yearlings tend to be more transverse in the forelimb medial PSBs than the other PSBs. Apical fracture size and geometry does not determine prognosis for apical sesamoid fractures. POTENTIAL RELEVANCE: Horses that undergo surgery to remove larger apical fractures of the PSBs do not have a worse outcome than those horses with smaller fractures.

Adolescents in mental health crisis: the role of routine follow-up calls after emergency department visits.

To improve care of adolescents in mental health crisis, the role of routine follow-up calls in discharged patients with referral plans after emergency department (ED) presentation to a children's hospital was explored. Main outcome measure was patient attendance at referral sites. In 113 mental health patients with follow-up appointments, either patient/carers or corresponding referral services could be contacted. Median age was 14 years, 77% were girls, and most presentations were after self-harm/depression (61%). Eighty-three per cent (95% CI 75% to 90%) were compliant with the discharge plan without prompting from the ED staff. Fourteen per cent (95% CI 8% to 22%) did not comply after being called by ED staff, and only 3% (95% CI 1% to 7%) were persuaded to attend their outpatient care after being prompted by ED staff. Routine follow-up calls for adolescent mental health patients after ED care are not warranted in all settings.

Management and assessment of prognosis after gastrocnemius disruption in Thoroughbred foals: 28 cases (1993-2007).

REASONS FOR PERFORMING STUDY: Few studies have evaluated the athletic prognosis of foals affected by gastrocnemius disruption. OBJECTIVE: To examine the diagnosis and management of gastrocnemius disruption in Thoroughbred (TB) foals, determine short-term survival rate and assess future racing performance. The hypothesis was that Thoroughbred foals with gastrocnemius disruption are able to perform as racehorses comparably to their age matched maternal siblings. METHODS: The medical records of foals diagnosed with gastrocnemius muscle disruption were reviewed. Information on training and racing was acquired from published works and race records. Paired t tests were utilised to compare performance variables of affected racehorses to their maternal siblings in starts, earnings and earnings/start for their 2- and 3-year-old racing seasons. Fisher's exact tests were employed to determine the association between sex, limb affected, age on admission, degree of caudal reciprocal apparatus dysfunction, concurrent disease, antibiotic therapy, complications, abscess formation and likelihood of entering training or starting a race. RESULTS: Sample size was too small to detect significant differences in performance variables between affected horses and controls. Of 28 foals, 17 (61%) presented with concomitant illness; foals without concurrent disease were more likely to achieve race training or start a race (P = 0.04); 23 (82%) were short-term survivors defined as survival to discharge. Of these 23 survivors, 7 were aged <2 years at the time of the study. Eighty-one percent (13/16) of the survivors that were of racing age were in training or had started a race. CONCLUSIONS: In this population, 82% of TB foals affected with gastrocnemius disruption were able to achieve training or start a race. Foals presenting for gastrocnemius disruption have a high prevalence of concurrent disease processes. POTENTIAL RELEVANCE: The assessment of athletic prognosis and treatment complications provides useful information to clinicians treating gastrocnemius muscle disruption in foals and making recommendations to clientele.

Racing performance after arthroscopic removal of apical sesamoid fracture fragments in Thoroughbred horses age < 2 years: 151 cases (1989--2002).

REASONS FOR PERFORMING STUDY: Studies on arthroscopic removal of apical proximal sesamoid fracture fragments in Thoroughbred (TB) horses age > or = 2 years have reported a high success rate. However, there are no reports documenting the racing prognosis of TB horses that undergo such surgery as weanlings or yearlings. OBJECTIVES: To describe the incidence of apical proximal sesamoid fractures in immature TB horses, age < 2 years and determine probability and quality of racing performance after arthroscopic removal of such fractures in 151 TB weanlings and yearlings. METHODS: The medical records of TB horses age < 2 years that underwent arthroscopic surgery for removal of apical proximal sesamoid fracture fragments were reviewed. Follow-up information was obtained from race records. Student's t tests were used to compare performance variables of operated racehorses to that of their maternal siblings. RESULTS: Ninety-two percent (139/151) of fractures occurred in the hindlimbs and 8% (11/151) in the forelimbs (fracture of both fore- and hindlimb, n = 1). Horses with forelimb fractures had a greatly reduced probability of racing (55%) compared to those with hindlimb fractures (86%). Overall, 84% of the horses raced post operatively and had performance records similar to that of their maternal siblings, 78% (787/1006) of which raced. CONCLUSIONS: Arthroscopic removal of apical proximal sesamoid fracture fragments in TB weanlings and yearlings carries an excellent prognosis for racing in horses with hindlimb fractures and a reduced prognosis in those with forelimb fractures. Medial fractures of the forelimb carry the worst prognosis. POTENTIAL RELEVANCE: The determination of prognosis increases knowledge on apical sesamoid bone fractures and potential for arthroscopic restoration of the ability to race; and enables the value of yearlings for subsequent sale to be established.

Racing performance after arthroscopic removal of apical sesamoid fracture fragments in Thoroughbred horses age > or = 2 years: 84 cases (1989-2002).

REASONS FOR PERFORMING STUDY: Studies have shown that surgical removal of apical fracture fragments in Standardbred racehorses carries the best prognosis for return to racing performance, but there are no reports involving mature Thoroughbred (TB) racehorses. OBJECTIVES: To describe the incidence of apical proximal sesamoid fractures in TB racehorses and determine probability and quality of racing performance after arthroscopic removal of such fractures in TB racehorses age > or = 2 years. METHODS: Medical records and pre- and post operative race records of TB racehorses age > or = 2 years that underwent arthroscopic surgery for removal of apical proximal sesamoid fracture fragments were reviewed. RESULTS: Sixty-four percent of fractures occurred in the hindlimbs and 36% in the forelimbs. Horses with forelimb fractures had a reduced probability of return to racing (67%) compared to those with hindlimb fractures (83%), but the majority (77%) of treated horses recovered to return to race post operatively. Horses with medial forelimb fractures raced at only a 47% rate; those with suspensory desmitis at 63%. Unlike Standardbreds, there was no difference in probability of racing post operatively between horses that had, and had not, raced preoperatively. CONCLUSIONS: Data show that arthroscopic removal of apical proximal sesamoid fracture fragments is successful at restoring ability to race in skeletally mature TB horses without evidence of severe suspensory ligament damage. Prognosis for return to racing is excellent (83%) in horses with hindlimb fractures and good (67%) in those with forelimb fractures. Medial fractures of the forelimb have the worst prognosis. POTENTIAL RELEVANCE: The determination of prognosis for differing sites in TB racehorses should increase knowledge of apical proximal sesamoid bone fractures and improve communication from veterinarian to owner, and trainer, on the potential for arthroscopic restoration of the ability to race.

Dystocia in a referral hospital setting: approach and results.

REASONS FOR PERFORMING STUDY: Dystocia in the mare is an emergency in which duration has a profound effect on survival of the foal. Specific examination of the effects of dystocia duration on foal survival provides information to enable horse care personnel and veterinarians to manage these cases more effectively and maximise the chances of obtaining a live foal. HYPOTHESIS: Dystocia duration would have a negative impact on foal survival while method of dystocia resolution would not have an effect on foal survival. Additionally, we were interested in determining the effects of dystocia on subsequent fertility. METHODS AND RESULTS: In the years 1986-1999, 247 dystocias were admitted. Of these, 91 % resulted in survival and discharge of the mare, 42% in delivery of a live foal, and 29% of foals survived to discharge. Period from hospital arrival to delivery for foals alive at discharge (23.0 +/- 14.1 mins) was not significantly different than for foals not surviving (24.8 +/- 10.6 mins) (P > 0.05); and from chorioallantoic rupture to delivery for foals alive at discharge (71.7 +/- 343 mins) was significantly less than for foals not surviving (853 +/- 37.4 mins) (P < 0.05). Average predystocia live foaling rates for all mares with available records was 84%. Overall post dystocia live foaling rates over the entire period of this study were 67%. Of mares bred in the year of the dystocia, 59% had a live foal in the year following. CONCLUSIONS: Based on these results, dystocia duration has a significant effect on foal survival and resolution methods should be chosen to minimise this time, as the difference between mean dystocia duration for foals that lived and those that did not in this study was 13.6 mins. Post dystocia foaling rates reported here are higher than previously reported for both same-season and overall breedings, indicating same-season breeding may be rewarding for select dystocia cases. POTENTIAL RELEVANCE: Dystocia resolution methods that minimise delivery time may maximise foal survival. Post dystoicia breeding may be rewarding in select cases.

A phylogenetic analysis of Diurideae (Orchidaceae) based on plastid DNA sequence data.

DNA sequence data from plastid matK and trnL-F regions were used in phylogenetic analyses of Diurideae, which indicate that Diurideae are not monophyletic as currently delimited. However, if Chloraeinae and Pterostylidinae are excluded from Diurideae, the remaining subtribes form a well-supported, monophyletic group that is sister to a "spiranthid" clade. Chloraea, Gavilea, and Megastylis pro parte (Chloraeinae) are all placed among the spiranthid orchids and form a grade with Pterostylis leading to a monophyletic Cranichideae. Codonorchis, previously included among Chloraeinae, is sister to Orchideae. Within the more narrowly delimited Diurideae two major lineages are apparent. One includes Diuridinae, Cryptostylidinae, Thelymitrinae, and an expanded Drakaeinae; the other includes Caladeniinae s.s., Prasophyllinae, and Acianthinae. The achlorophyllous subtribe Rhizanthellinae is a member of Diurideae, but its placement is otherwise uncertain. The sequence-based trees indicate that some morphological characters used in previous classifications, such as subterranean storage organs, anther position, growth habit, fungal symbionts, and pollination syndromes have more complex evolutionary histories than previously hypothesized. Treatments based upon these characters have produced conflicting classifications, and molecular data offer a tool for reevaluating these phylogenetic hypotheses.

In vitro comparison of transfixation and standard full-limb casts for prevention of displacement of a mid-diaphyseal third metacarpal osteotomy site in horses.

OBJECTIVE: To compare transfixation and standard full-limb casts for prevention of in vitro displacement of a mid-diaphyseal third metacarpal osteotomy site in horses. SAMPLE POPULATION: 6 forelimbs from 6 horses euthanatized for reasons not related to the musculoskeletal system. PROCEDURE: A 30 degrees osteotomy was performed in the mid-diaphysis of the third metacarpal bone. Two 4.5-mm cortical bone screws were placed across the osteotomy site to maintain alignment during casting. Two 6.35-mm Steinmann pins were placed from a lateral-to-medial direction in the distal aspect of the radius. A full-limb cast that incorporated the pins was applied. An extensometer was positioned in the osteotomy site through a window placed in the dorsal aspect of the cast, and after removal of the screws, displacement was recorded while the limb was axially loaded to 5,340 N (1,200 lb). Pins were removed, and the standard full-limb cast was tested in a similar fashion. RESULTS: The transfixation cast significantly reduced displacement across the osteotomy site at 445 N (100 lb), 1,112 N (250 lb), 2,224 N (500 lb), and 4,448 N (1,000 lb), compared with the standard cast. CONCLUSION AND CLINICAL RELEVANCE: A full-limb transfixation cast provides significantly greater resistance than a standard full-limb cast against axial collapse of a mid-diaphyseal third metacarpal osteotomy site when the bone is placed under axial compression. Placement of full-limb transfixation casts should be considered for the management of unstable fractures of the third metacarpal bone in horses.

Isolation of Neisseria gonorrhoeae mutants that show enhanced trafficking across polarized T84 epithelial monolayers.

Initiation of a gonococcal infection involves attachment of Neisseria gonorrhoeae to the plasma membrane of an epithelial cell in the mucosal epithelium and its internalization, transepithelial trafficking, and exocytosis from the basal membrane. Piliation and expression of certain Opa proteins and the immunoglobulin A1 protease influence the transcytosis process. We are interested in identifying other genetic determinants of N. gonorrhoeae that play a role in transcellular trafficking. Using polarized T84 monolayers as a model epithelial barrier, we have assayed an N. gonorrhoeae FA1090 minitransposon (mTn) mutant bank for isolates that traverse the monolayer more quickly than the isogenic wild-type (WT) strain. From an initial screen, we isolated four mutants, defining three genetic loci, that traverse monolayers significantly more quickly than their WT parent strain. These mutants adhere to and invade cells normally and do not affect the integrity of the monolayer barrier. Backcrosses of the mutations into the WT FA1090 strain yielded mutants with a similar fast-trafficking phenotype. In two mutants, the mTns had inserted 370 bp apart into the same locus, which we have named fit, for fast intracellular trafficker. Backcrosses of one of these mutants into the MS11A genetic background also yielded a fast-trafficking mutant. The fit locus contains two overlapping open reading frames, fitA and fitB, whose deduced amino acid sequences have predicted molecular weights of 8.6 and 15.3, respectively. Neither protein contains a signal sequence. FitA has a potential helix-turn-helix motif, while the deduced sequence of FitB offers no clues to its function. fitA or fitB homologues are present in the genomes of Pseudomonas syringae and Rhizobium meliloti, but not Neisseria meningitidis. Replication of the MS11A fitA mutant in A431 and T84 cells is significantly accelerated compared to that of the isogenic WT strain. In contrast, growth of this mutant in liquid media is normal. Taken together, these results strongly suggest that traversal of N. gonorrhoeae across an epithelial barrier is linked to intracellular bacterial growth.

Effects of the immunoglobulin A1 protease on Neisseria gonorrhoeae trafficking across polarized T84 epithelial monolayers.

We previously demonstrated that the Neisseria IgA1 protease cleaves LAMP1 (lysosome-associated membrane protein 1), a major integral membrane glycoprotein of lysosomes, thereby accelerating its degradation rate in infected A431 human epidermoid carcinoma cells and resulting in the alteration of lysosomes in these cells. In this study, we determined whether the IgA1 protease also affects the trafficking of Neisseria gonorrhoeae across polarized T84 epithelial monolayers. We report that N. gonorrhoeae infection of T84 monolayers, grown on a solid substrate or polarized on semiporous membranes, also results in IgA1 protease-mediated reduction of LAMP1. We demonstrate that iga mutants in two genetic backgrounds exited polarized T84 monolayers in fewer numbers than the corresponding wild-type strains. Finally, we present evidence that these mutants have a statistically significant and reproducible defect in their ability to traverse T84 monolayers. These results add to our previous data by showing that the IgA1 protease alters lysosomal content in polarized as well as unpolarized cells and by demonstrating a role for the protease in the traversal of epithelial barriers by N. gonorrhoeae.

Infection of epithelial cells by pathogenic neisseriae reduces the levels of multiple lysosomal constituents.

Members of our group reported recently that neisseria infection of human epithelial cells results in accelerated degradation of the major lysosomal integral membrane protein LAMP1 and that this is due to hydrolysis of this glycoprotein at its immunoglobulin A1 (IgA1)-like hinge by the neisseria type 2 IgA1 protease (L. Lin et al., Mol. Microbiol. 24:1083-1094, 1997). We also reported that the IgA1 protease plays a major role in the ability of the pathogenic neisseriae to survive within epithelial cells and hypothesized that this is due to alteration of lysosomes as a result of protease-mediated LAMP1 degradation. In this study, we tested the hypothesis that neisseria infection leads to multiple changes in lysosomes. Here, we report that neisseria infection also reduces the levels of three other lysosomal markers: LAMP2, lysosomal acid phosphatase (LAP), and CD63. In contrast, neither the epidermal growth factor receptor level nor the beta-tubulin level is affected. A detailed examination of LAMP2 indicated that the reduced LAMP2 levels are not the result of an altered biosynthetic rate or of cleavage by the IgA1 protease. Nevertheless, the protease plays a role in reducing LAMP2 and LAP activity levels, as these are partially restored in cells infected with an iga mutant. We conclude that neisseria infection results in multiple changes to the lysosomes of infected epithelial cells and that these changes are likely an indirect result of IgA1 protease-mediated cleavage of LAMP1.

Effect of three types of half-limb casts on in vitro bone strain recorded from the third metacarpal bone and proximal phalanx in equine cadaver limbs.

OBJECTIVE: To determine effect of 3 half-limb casts on bone strains recorded from the proximal phalanx (P-1) and third metacarpal bone (MCIII) of equine cadaver limbs, using a mechanical testing machine. ANIMALS: 12 equine cadaver limbs and 4 live horses. PROCEDURE: Bone strains were recorded at middorsal P-1 and the dorsal cortical aspect of the distal third of MCIII while limbs were variably loaded with 100 to 1,000 lb of force. To determine ability of the cast to protect the distal portion of the limb from weight-bearing loads, strains were recorded with the limb in 1 of the 3 casts and with it unsupported. To determine cast-induced discomfort, weight-supporting and transfixation pin casts were evaluated on 2 live horses RESULTS: All 3 casts significantly reduced bone strain at P-1. Significant differences were observed: mean 61% reduction for the standard half-limb cast, 84% for the transfixation pin cast, and 97% for the weight-supporting cast at weight-bearing force of 500 lb. Only the weight-supporting cast significantly reduced strains recorded from MCIII. The weight-supporting cast was not well tolerated by 2 live horses. CONCLUSIONS: The 3 casts significantly reduced transfer of weight-bearing forces to the distal portion of the limb. The weight-supporting cast effectively reduced strain on the P-1 to near 0, but was well tolerated by live horses. The transfixation pin cast reduced strain on the P-1 by > 80% at weight-bearing loads of 500 lb, and live horses were comfortable. Standard half-limb casts significantly reduced strains on the P-1, but to a lesser degree than did other casts.