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Antibiotic resistance is a major global health threat. Agricultural use of antibiotics is considered to be a main contributor to the issue, influencing both animals and humans as defined by the One Health approach. The purpose of the present study was to determine the abundance of antibiotic-resistant bacterial populations and the overall bacterial diversity of cattle farm soils that have been treated with animal manure compost. Soil and manure samples were collected from different sites at Tullimba farm, NSW. Cultures were grown from these samples in the presence of 11 commonly used antibiotics and antibiotic-resistant bacteria (ARB) colonies were identified. Soil and manure bacterial diversity was also determined using 16S ribosomal RNA next-generation sequencing. Results showed that ARB abundance was greatest in fresh manure and significantly lower in composted manure. However, the application of composted manure on paddock soil led to a significant increase in soil ARB abundance. Of the antibiotics tested, the number of ARB in each sample was greatest for antibiotics that inhibited the bacterial cell wall and protein synthesis. Collectively, these results suggest that the transfer of antibiotic resistance from composted animal manure to soil may not be solely mediated through the application of live bacteria and highlight the need for further research into the mechanism of antibiotic resistance transfer., (© 2023 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Group A streptococcal (GAS) infection is associated with a spectrum of autoimmune diseases including acute rheumatic fever/rheumatic heart disease (ARF/RHD) and neurobehavioral abnormalities. Antibodies against GAS M proteins cross-react with host tissue proteins in the heart and brain leading to the symptomatology observed in ARF/RHD. As throat carriage of Streptococcus dysgalactiae subspecies equisimilis (SDSE) has been reported to be relatively high in some ARF/RHD endemic regions compared with GAS, and both SDSE and GAS express coiled-coil surface protein called M protein, we hypothesized that streptococci other than GAS can also associated with ARF/RHD and neurobehavioral abnormalities. Neurobehavioral assessments and electrocardiography were performed on Lewis rats before and after exposure to recombinant GAS and SDSE M proteins. Histological assessments were performed to confirm inflammatory changes in cardiac and neuronal tissues. ELISA and Western blot analysis were performed to determine the cross-reactivity of antibodies with host connective, cardiac and neuronal tissue proteins. Lewis rats injected with M proteins either from GAS or SDSE developed significant cardiac functional and neurobehavioral abnormalities in comparison to control rats injected with phosphate-buffered saline. Antibodies against GAS and SDSE M proteins cross-reacted with cardiac, connective and neuronal proteins. Serum from rats injected with streptococcal antigens showed higher immunoglobulin G binding to the striatum and cortex of the brain. Cardiac and neurobehavioral abnormalities observed in our experimental model were comparable to the cardinal symptoms observed in patients with ARF/RHD. Here for the first time, we demonstrate in an experimental model that M proteins from different streptococcal species could initiate and drive the autoimmune-mediated cardiac tissue damage and neurobehavioral abnormalities., (© 2022 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Hypertension is a major risk factor for kidney and cardiovascular disease. The treatment of hypertensive individuals by selected ACE inhibitors and certain di-and tripeptides halts the progression of renal deterioration and extends life-span. Renal reabsorption of these low molecular weight substrates are mediated by the PEPT1 and PEPT2 cotransporters. This study aims to investigate whether hypertension and ageing affects renal PEPT cotransporters at gene, protein expression and distribution as well as function in the superficial cortex and the outer medulla of the kidney. Membrane vesicles from the brush border (BBMV) and outer medulla (OMMV) were isolated from the kidneys of young Wistar Kyoto (Y-WKY), young spontaneously hypertensive (Y-SHR), and middle aged SHR (M-SHR) rats. Transport activity was measured using the substrate, β-Ala-Lys (AMCA). Gene expression levels of PEPT genes were assessed with qRT-PCR while renal localisation of PEPT cotransporters was examined by immunohistochemistry with Western Blot validation. The K m and V max of renal PEPT1 were decreased significantly in SHR compared to WKY BBMV, whilst the Vmax of PEPT2 showed differences between SHR and WKY. By contrast to the reported cortical distribution of PEPT1, PEPT1-staining was detected in the outer medulla, whilst PEPT2 was expressed primarily in the cortex of all SHR; PEPT1 was significantly upregulated in the cortex of Y-SHR. These outcomes are indicative of a redistribution of PEPT1 and PEPT2 in the kidney proximal tubule under hypertensive conditions that has potential repercussions for nutrient handling and the therapeutic use of ACE inhibitors in hypertensive individuals., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Non-conventional feed ingredients are receiving more interest in their ability to increase farming efficiency, sustainability and animal performance. The objective of this study was to determine the optimal rate of inclusion level of the full-fat black soldier fly larvae (BSFL) in broiler diets and to evaluate their impact on performance, nutrient digestibility, and the immune system (blood cells and intraepithelial lymphocytes). A total of 400 male day-old Ross 308 broilers were randomly assigned to 5 treatment groups with 8 replicates each. Five inclusion levels of full-fat BSFL were investigated across starter (0, 2.5%, 5%, 7.5% and 10%), grower and finisher diets (0, 5%, 10%, 15% and 20%). All diets were formulated based on digestible amino acid values according to the Aviagen (2016) recommendations. A polynomial regression at different degrees was performed to analyse broiler performance parameters (body weight, body weight gain, feed intake, and feed conversion ratio), nutrient digestibility, and blood cell count. Intraepithelial lymphocyte population data was analysed performing univariate linear regression. During the entire experimental period (from 2 to 42 d), BSFL inclusion levels decreased the feed conversion ratio by 10% in broilers that received 20% BSFL in their diets ( P  < 0.05). Lymphocytes and white blood cell count decreased linearly by 47.7% and 35.9%, respectively, with up to 20% BSFL inclusion ( P  < 0.001) . A 4-fold decrease in CD3+ T lymphocytes and a 9.7-fold decrease of CD3+CD8+ intestinal cytotoxic T lymphocytes occurred in broilers fed 20% BSFL compared to the control group. These findings suggest that the inclusion of BSFL can improve broiler performance and potentially reduce immune response energy expenditure in birds fed 20% BSFL for 42 d., Competing Interests: We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper., (© 2021 Chinese Association of Animal Science and Veterinary Medicine. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd.)

The pathogenesis of Acute Rheumatic Fever/Rheumatic Heart Disease (ARF/RHD) and associated neurobehavioral complications including Sydenham's chorea (SC) is complex. Disease complications triggered by Group A streptococcal (GAS) infection are confined to human and determining the early events leading to pathology requires a robust animal model that reflects the hallmark features of the disease. However, modeling these conditions in a laboratory animal, of a uniquely human disease is challenging. Animal models including cattle, sheep, pig, dog, cat, guinea pigs rats and mice have been used extensively to dissect molecular mechanisms of the autoimmune inflammatory responses in ARF/RHD. Despite the characteristic limitations of some animal models, several rodent models have significantly contributed to better understanding of the fundamental mechanisms underpinning features of ARF/RHD. In the Lewis rat autoimmune valvulitis model the development of myocarditis and valvulitis with the infiltration of mononuclear cells along with generation of antibodies that cross-react with cardiac tissue proteins following exposure to GAS antigens were found to be similar to ARF/RHD. We have recently shown that Lewis rats injected with recombinant GAS antigens simultaneously developed cardiac and neurobehavioral changes. Since ARF/RHD is multifactorial in origin, an animal model which exhibit the characteristics of several of the cardinal diagnostic criteria observed in ARF/RHD, would be advantageous to determine the early immune responses to facilitate biomarker discovery as well as provide a suitable model to evaluate treatment options, safety and efficacy of vaccine candidates. This review focuses on some of the common small animals and their advantages and limitations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rafeek, Sikder, Hamlin, Andronicos, McMillan, Sriprakash and Ketheesan.)

Background: The neuropsychiatric disorders due to post-streptococcal autoimmune complications such as Sydenham's chorea (SC) are associated with acute rheumatic fever and rheumatic heart disease (ARF/RHD). An animal model that exhibits characteristics of both cardiac and neurobehavioral defects in ARF/RHD would be an important adjunct for future studies. Since age, gender, strain differences, and genotypes impact on the development of autoimmunity, we investigated the behavior of male and female Wistar and Lewis rat strains in two age cohorts (<6 weeks and >12 weeks) under normal husbandry conditions and following exposure to group A streptococcus (GAS)., Methods: Standard behavioral assessments were performed to determine the impairments in fine motor control (food manipulation test), gait and balance (beam walking test), and obsessive-compulsive behavior (grooming and marble burying tests). Furthermore, electrocardiography, histology, and behavioral assessments were performed on male and female Lewis rats injected with GAS antigens., Results: For control Lewis rats there were no significant age and gender dependent differences in marble burying, food manipulation, beam walking and grooming behaviors. In contrast significant age-dependent differences were observed in Wistar rats in all the behavioral tests except for food manipulation. Therefore, Lewis rats were selected for further experiments to determine the effect of GAS. After exposure to GAS, Lewis rats demonstrated neurobehavioral abnormalities and cardiac pathology akin to SC and ARF/RHD, respectively., Conclusion: We have characterised a new model that provides longitudinal stability of age-dependent behavior, to simultaneously investigate both neurobehavioral and cardiac abnormalities associated with post-streptococcal complications., Competing Interests: No conflicts of interest relevant to this manuscript were reported., (© 2021 The Authors. Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.)

Background: Recent anthropomorphic disturbances are occurring at an increasing rate leading to organisms facing a variety of challenges. This change is testing the information processing capacity (IPC) of all animals. Brain function is widely accepted to be influenced by a variety of factors, including relative size, number of neurons and neuronal densities. Therefore, in order to understand what drives an animals IPC, a methodological approach to analyze these factors must be established., New Method: Here we created a protocol that allowed for high-throughput, non-biased quantification of neuronal density and size across six regions of the brain. We used the Isotropic Fractionator method in combination with flow cytometry to identify neuronal and non-neuronal cells in the brains of adult rats., Comparison With Existing Methods: The results obtained were comparable to those identified using stereological counting methods., Results: By employing this new method, the number of nuclei in a specific brain region can be compared between replicate animals within an experiment. By calibrating the forward scatter channel of the flow cytometer with size standard beads, neuronal and non-neuronal nuclear sizes can be estimated simultaneously with nuclei enumeration. These techniques for nuclear counting and size estimation are technically and biologically reproducible., Conclusion: Use of flow cytometry provides a methodological approach that allows for consistency in research, so that information on brain morphology, and subsequent function, will become comparable across taxa., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: This study examined whether circulating C-reactive protein (CRP) is elevated in obstructive sleep apnoea (OSA) independent of the confounding effects of comorbidities, smoking, body mass index (BMI), age and gender. Methods: A systematic review of the literature was performed using PubMed, Embase and Cochrane databases from 1 January 1997 to 1 November 2017 using the key words obstructive sleep apnoea and C-Reactive protein to identify full text English language studies that compared CRP in adult non-smoking OSA participants without comorbidities and adult healthy non-smoking control participants matched for BMI, age and gender. Data from eligible studies were subjected to meta-analysis using RevMan version 5.3. Results: Five studies (219 OSA participants, 116 controls) met the selection criteria. The total standard mean difference for circulating high sensitivity CRP was 0.61 mg/dL higher in OSA participants than in control participants (confidence interval: 0.38 to 0.84, p  < 0.00001), with low between-studies heterogeneity (df = 7, p  = 0.16, I 2  = 33%) and minimal evidence of publication bias. Conclusions : CRP levels in non-smoking OSA participants without comorbidities were increased relative to levels in healthy matched non-smoking control participants, suggesting that pharyngeal or systemic inflammatory effects attributable to OSA may elevate CRP.

Renal PEPT1 and PEPT2 cotransporters play an important role in the balance of circulating body oligopeptides and selected peptidomimetic drugs. We aim to comprehensively characterise age-related changes of the renal PEPT cotransporters at the gene, protein, and functional level. Brush border membrane vesicles (BBMV) and outer medulla membrane vesicles (OMMV) were isolated from the kidneys of young, middle-aged and old rats. The protein expression of PEPT1 was not only increased in BBMV from old rats, but PEPT1 also appeared in OMMV from middle-aged and old rats. SLC15A1 gene expression in the renal cortex increased in middle-aged group. PEPT2 protein expression was not only increased with ageing, but PEPT2 also was found in BBMV from middle-aged and old groups. SLC15A2 gene expression in the renal outer medulla increased in the old group. These changes in the expressions and localisations of PEPT1 and PEPT2 could explain the changes to transport activity in BBMV and OMMV. These findings provide novel insights that would be useful for maintaining protein nutrition and optimising the delivery of some peptidomimetic drugs in elderly individuals.

Background and Aim: The diagnosis of celiac disease autoimmune pathology relies on the subjective histological assignment of biopsies into Marsh score categories. It is hypothesized that Marsh score categories have unique gene expression signatures. The aims were as follows: first, to develop a celiac disease quantitative reverse transcription-polymerase chain reaction (RT-PCR) array; second, define gene expression signatures associated with Marsh score categories; and third, develop equations that classify biopsies into Marsh score categories and to monitor the efficacy of patient treatment., Methods: Gene targets for inclusion in the celiac RT-PCR (qRT-PCR) array were identified using systematic analysis of published celiac transcriptomic data. The array was used to assess the gene expression associated with histological changes in duodenal biopsies obtained from adult patients. Finally, Marsh score classification equations were defined using discriminant analysis., Results: The array contained 87 genes. The expression of 26 genes were significantly (p < 0.06) associated with the discrete Marsh score categories. As the Marsh score pathology of biopsies increased, there was a progression of innate immune gene expression through adaptive Th1-specific gene expression with a concurrent decrease in intestinal structural gene expression in high Marsh score samples. These 26 genes were used to define classification equations that accounted for 99% of the observed experimental variation and which could classify biopsies into Marsh score categories and monitor patient treatment progression., Conclusions: This proof-of-concept study successfully developed a celiac RT-PCR array and has provided evidence that discriminant equations defined using gene expression data can objectively and accurately classify duodenal biopsies into Marsh score categories., (© 2018 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Background/aim: The gold standard diagnostic for coeliac disease (CD) is subjective histological assignment of biopsies into the Marsh score categories. It is hypothesized that discrete Marsh score categories can be quantitatively resolved into a continuum using discriminant equations defined using histological and gene expression data. Therefore, the aim of this study was to use a combination of histological and gene expression data to develop equations that classify CD patient biopsies into a quantitative Marsh score continuum which could be used by clinicians to monitor CD treatment efficacy., Methods: Both empirical and simulated gene expression and histological data were used to define predictive Marsh score equations. The distances of treated sample biopsies from the Marsh score standards were determined using the Mahalanobis distance calculation., Results: Three function, high resolution discriminant equations derived from simulated data were used to accurately classify 99.6% of simulated and empirically derived biopsy data. The first function resolved active (Marsh type 3) CD from mild (Marsh type 1) CD. The second function resolved normal (no specific pathology) biopsies from mild CD. The third function resolved active Marsh score 3 into a and b subcategories. Finally, measuring the Mahalanobis distance enabled the conversion of discrete Marsh score categories into a continuum., Conclusions: This proof-of-concept study successfully demonstrated that the discrete Marsh score scale can be converted into a quantitative continuum capable of high resolution monitoring of patient treatment efficacy using equations defined by gene expression and histology data., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: Some prostate cancer (PCa) patients become clinically anxious or depressed after diagnosis and treatment. Some also show the physiological signs of chronic stress. However, there are currently no data describing how these particular patients might be identified at intake. This study tested the individual and combined predictive power of a psychological factor and a genetic factor as potential predictors of anxiety, depression, and chronic stress in a sample of PCa patients., Methods: Ninety-five PCa patients completed psychological inventories for anxiety, depression, and psychological resilience (PR) and also gave a saliva sample for cortisol and a mouthwash sample for genetic testing for the presence of the BDNF Val66Met polymorphism., Results: High PR patients had significantly lower anxiety and depression than low PR patients, but showed no significant differences in their salivary cortisol. Carriers of the Met allele of the BDNF Val66Met polymorphism had significantly higher salivary cortisol concentrations than patients who did not carry this allele., Conclusions: Each of these two factors may provide valuable information regarding the vulnerability of PCa patients to anxiety, depression, or chronic stress. Suggestions are made for their inclusion in clinical settings.

Although germ cells from donor rams transplanted into irradiated recipient testes have produced donor derived offspring, efficiency is low. Further optimization of recipient irradiation protocols will add precision to the depletion of recipient spermatogonia prior to germ cell transplant. Three irradiation doses (9,12,15 Gy) were administered to ram lambs aged 14 weeks (Group 1) and 20 weeks (Group 2), then testicular biopsies were collected 1, 2 and 3 months after irradiation. At 1 month after irradiation of Group 1, only the largest dose (15 Gy) reduced spermatogonia numbers below 10% of non-irradiated controls, whereas in Group 2 lambs, each irradiation dose reduced spermatogonia below 10% of controls. In both Groups, fewer differentiated germ cells were present in seminiferous tubules compared to controls. At 2 months after irradiation, spermatogonia numbers in both Groups increased more than sixfold to be similar to controls, whereas fewer differentiated germ cells were present in the tubules of both Groups. At 3 months in Group 1, each irradiation dose reduced spermatogonia numbers to <30% of controls and fewer tubules contained differentiated germ cells. Lesser expression of spermatogonial genes, VASA and UCHL-1, was observed in the 15 Gy group. In Group 2, only 12 Gy treated tubules contained fewer spermatogonia. Knowledge of these subtle differences between age groups in the effect of irradiation doses on spermatogonia or differentiated germ cell numbers and the duration of recovery of spermatogonia numbers after irradiation will aid the timing of germ cell transplants into prepubertal recipient lambs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study was conducted to determine the effect of Ascaridia galli infection on free-range laying hens. Lohmann Brown laying hens (n = 200) at 17 wk of age were allocated to 4 treatment groups (n = 50 per group), each with 5 replicate pens of 10 hens. Hens in 3 treatment groups were orally inoculated with different doses of embryonated A. galli eggs: low (250 eggs), medium (1,000 eggs), and high (2,500 eggs) levels, whereas hens of the control group were not infected. Infection levels were monitored using excreta egg counts and mature A. galli worm counts in the intestine. Anti A. galli antibody titers (IgY) in the serum were measured prior to infection, and at 6, 11, 15, and 20 wk post infection (PI) and in egg yolk at 11 and 20 wk PI. Parameters evaluated included feed intake, egg production, egg weight, egg mass, FCR, liver weight, liver fat, and intra epithelial immune cell infiltration. The results showed no difference in feed intake, body weight, or FCR among any treatment groups (P > 0.05). Egg production was lower in the low infection group compared to other groups at 20 wk of age (P < 0.01). Serum IgY was higher in the infected groups' hens at 20 wk PI compared to control group hens (P < 0.01). Yolk IgY increased significantly over time and was higher in infected hens compared to hens of the control group at 11 and 20 wk PI (P < 0.001). No differences were observed in liver lipid content or intraepithelial lymphocytes infiltration among treatment groups. Ascaridia galli eggs in the coprodeum content and adult A. galli worm count were higher in infected hens compared to hens of the control group (P < 0.01). In conclusion, the effects of artificial infection with A. galli on the parameters investigated were minor, and egg yolk antibody may be a more reliable indicator of A. galli infection than serum antibody or excreta egg count., (© 2017 Poultry Science Association Inc.)

Objective: To investigate the effect of chronic stress as measured in cortisol concentrations upon the association between psychological resilience (PR) and depression in prostate cancer (PCa) patients., Methods: A total of 104 men with PCa completed inventories on PR, depression, and background factors, plus gave a sample of their saliva for cortisol assay., Results: The inverse correlation between PR and depression was present only for PCa patients with low or moderate concentrations of salivary cortisol (when classified as more than 1.0 SD below the mean vs within 1.0 SD of the group mean) but not for those men whose cortisol was >1.0 SD from the group mean. Specific PR factors and behaviours that made the greatest contribution to depression were identified for the low and moderate cortisol groups., Conclusions: These results suggest that there are particular aspects of PR that are most strongly related to depression, but that PR's inverse association with depression may be absent in participants with extreme chronic physiological stress., (Copyright © 2017 John Wiley & Sons, Ltd.)

Broilers commonly suffer from necrotic enteritis (NE). Other gastrointestinal infectious diseases affect poultry, including nematode infections which are considered a re-emerging disease in barn and free-range systems. The aim of this study was to characterize the immune response of broilers after artificial infection with NE and contrast these with responses to the nematode Ascaridia galli and determine whether immune parameters measured during the course of infection can be used to distinguish infected from uninfected birds. A total of 96 one-day-old male Ross 308 broiler chickens were used in this study. At 10 days of age, broilers were randomly assigned to one of the following treatment groups: control birds (n = 32), A. galli infected birds (n = 32), or NE infected birds (n = 32) and inoculated with the appropriate infective agents. The immune response of birds was monitored through evaluation of haematology parameters, acute phase protein production, and intraepithelial intestinal lymphocyte population changes at 11, 16, 20, and 32 days of age. T-helper cells (CD4 + CD8 - ) increased significantly over time, and were significantly higher in A. galli and NE compared to day 10 controls. In conclusion, α-1 glycoprotein levels can distinguish birds with NE from other birds, including those infected with A. galli; also T-helper cell numbers can distinguish both NE and A. galli from uninfected birds and thirdly, 10 days post infection is the best time point to evaluate the bird's immune response for A. galli infections.

To measure the prevalence and severity of Generalised Anxiety Disorder (GAD), hypo- and hypercortisolaemia, and their association in a sample of prostate cancer (PCa) patients, 97 Australian PCa patients completed a background questionnaire and the GAD-7, and provided a sample of saliva collected 30-45 min after waking. The mean GAD7 score was 9.67 (SD = 3.09), and prevalence rates for current anxiety were higher than those reported for non-PCa males of a similar age. Mean salivary cortisol concentrations (30.78 nmol/L, SD = 13.97 nmol/L) were also higher than for age-comparative non-PCa men. There was a significant inverse correlation between GAD and cortisol (r = -. 209, p < .05), and four subgroups of GAD-cortisol patients were able to be identified, with evidence of both hyper- and hypocortisolaemia. These findings provide initial neurobiological evidence of the chronic and profound nature of stress experienced by PCa patients, and also suggest a possible measure that might be used to identify most at-risk PCa patients., (© 2017 John Wiley & Sons Ltd.)

The comparative strength of the 5-HTTLPR polymorphism as a 'predictor' of depression after major stress, versus the 'protective' effect of psychological resilience (PR) against depression after major stress, was tested in a homogeneous sample of older men who had all received a diagnosis and treatment for prostate cancer. Results supported the association between PR and lower depression after stress, but did not support the association between the 5-HTTLPR and elevated depression after stress. Examination of PR at scale, factor, and item level identified the specific PR-related behaviour that was the most powerful predictor of low depression. These data suggest that the carriage of the short form of the 5-HTTLPR may negate the protective effect of PR against depression in these men, or that PR may nullify the depression vulnerability of this form of the 5-HTTLPR. These findings may explain some of the 'null' findings regarding the link between the 5-HTTLPR and depression in the wider literature by arguing for an interaction between these two factors in the association between major stress and depression., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Aggression is a major problem in children with Autism Spectrum Disorder (ASD) but little is known about the possible contributors to this behaviour., Aims: To determine the relative strength of the relationships between developmental, cognitive, symptomatic, hormonal and mood factors and 'Aggression towards Others' in boys with ASD., Method: Predictors of Aggression towards Others were investigated in a sample of 136 boys with Autism Spectrum Disorder (M age=11.3yr, SD=3.2yr, range=6yr to 17yr). Data were collected from the boys themselves and their parents (14 fathers, 122 mothers)., Results: Results indicated that age and Low Registration on the Sensory Profile were the only significant correlates of this form of aggression. Importantly, testosterone levels did not account for level of social aggression., Conclusions: These data suggest that these boys may have learnt more effective methods of dealing with their frustration as they grew older or benefitted from cognitive maturation, and that having a high neurological threshold may be a source of frustration for these boys. The relationship between Aggression towards Others and Low Registration is discussed and clinical implications of the findings explicated., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: The aim of this pilot study was to attempt to define a set of equations from histological observations of tissue affected with coeliac disease (CD) to predict Marsh score., Material/methods: Tissue from 15 patients with untreated CD, 6 patients with treated CD and 9 healthy control patients were stained using the standard H&E, Giemsa's staining for tissue sections and Alcian Blue protocols. A number of histological measures were then taken from each section and the data was used to ultimately design a set of linear predictive algorithms to calculate Marsh score., Results: Using MANOVA and discriminant analysis, two linear functions were defined which could accurately predict the Marsh score of patients 62.5% (full Marsh score) to 79.2% (simplified Marsh score) of the time., Conclusions: This pilot study has shown that a set of objective histological measures can be used to define algorithms to predict Marsh score in CD patients and also monitor treatment compliance and progression., (Copyright © 2016 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.)

Background: Previous studies have reported correlations between various aspects of the behaviour and symptomatology of children with Autism Spectrum Disorder (ASD) and their parents' self-reports of stress via standardised scales., Aims: To extend that literature, a physiological index of parental chronic stress was used instead of their self-reports-dysregulation of the Diurnal Rhythm (DR) of the Hypothalamic-Pituitary-Adrenal (HPA) axis., Methods: A sample of 149 parents of a child with ASD provided salivary cortisol at the predicted time of daily maximum cortisol concentration and at a time of daily lower concentration. Adherence to the predicted DR was assessed via a dichotomous (present/not-present) as well as a continuous measure, and MANOVA and linear regression were used to detect significant associations between ASD-related variables in their children and parents' DR., Results: Identified only a single significant correlate of DR dysregulation in both statistical procedures-Self-Injurious Behaviour (SIB) exhibited by their child and observed by the parents., Conclusions and Implications: These findings extend previous data using self-report indices of parental stress and should be included in parent-support settings to alert parents to the long-term health effects of the stress they experience in regard to their child's SIB., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Salivary cortisol may be used as a biomarker of stress and anxiety in children with an autism spectrum disorder (ASD). Some suggestions have been made that the measurement of cortisol needs to be undertaken by repeated days' observations to ensure reliability of the data obtained. These requirements are discussed in regard to 14 studies of the test-retest agreement and stability in cortisol data across repeated daily measurements. Results of those studies almost universally fail to support the argument for repeated daily measurements of cortisol. Implications for the research protocols of studies using cortisol as an index of stress in children with ASD are discussed.

To further describe Hypothalamus-Pituitary-Adrenal (HPA) axis activity in children with Autism Spectrum Disorder (ASD), the Diurnal Fluctuation (DF) and Cortisol Awakening Response (CAR) were investigated in a sample of 39 high functioning girls with ASD. Although group mean data conformed to the DF and CAR models, over half of the participants showed inverse CAR and over 14% had inverted DF cortisol concentrations. Examination of three potential sets of predictor factors (physiological, ASD-related, and mood) revealed that only self-reported Major Depressive Disorder was significantly associated with CAR status, and that the girls' concern about dying or suicide was the most powerful contributor to the variance in CAR status. These findings add to the literature regarding the HPA axis dysfunction in children with ASD., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Salivary cortisol may be used as a biomarker of stress and anxiety in children with an Autism Spectrum Disorder (ASD) and is particularly valuable in studies of the association between stress-related cortisol concentrations and other factors such as comorbid disorders or aspects of the ASD phenotype. Although protocols for the collection of cortisol shortly after waking are often based on the assumption of the presence of a diurnal rhythm in cortisol, that rhythm may not be as reliable in children with an ASD as in non-ASD children. Alternatively, collecting cortisol during the afternoon may represent a more reliable procedure with less inter-participant variability., (Copyright © 2015. Published by Elsevier Inc.)

Background: Comorbidity of anxiety and depression predicts impaired treatment outcomes, poor quality of life and increased suicide risk. No study has reported on a combined measure of anxiety-depression in boys with an Autism Spectrum Disorder., Aims: To explore the prevalence, underlying factor structure and relationships between anxiety-depression, physiological stress and symptoms of Autism Spectrum Disorder (ASD)., Methods: 150 boys (aged 6-18 years; IQ M=94.9, range=73-132) with an ASD plus their parents (135 mothers, 15 fathers) completed scales about the boys' anxiety and depression, and the boys provided samples of their saliva in the morning and afternoon. Parents also completed the ASD Behaviour Checklist about the boys' ASD symptoms., Results: The two sources of ratings were not significantly different for prevalence of anxiety-depression but the factor structures varied between the parents' and boys' responses, with a four-factor solution for the boys' ratings and a three-factor solution for the parents' ratings. There were also differences in the correlations between cortisol and anxiety-depression and between ASD symptoms and anxiety depression across the boys' and parents' data., Conclusions: Assessment of anxiety and depression comorbidity from parents and from children with an ASD themselves could provide a valuable adjunct datum when diagnosing ASD., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

To identify if age influenced the relationship between one of the central symptoms of Autism Spectrum Disorder (ASD) and physiological stress, the association between stereotypic behaviour (SB) and stress-related cortisol concentrations was examined in a sample of 150 young males with an ASD. Parent-rated SB was significantly correlated with cortisol concentrations for boys aged 6 years to 12 years but not for adolescents aged 13 years to 18 years. This age-related difference in this association was not a function of cortisol concentrations but was related to differences in SB across these two age groups. IQ did not have a significant effect on this relationship, suggesting that age-related learning may have been a possible pathway for reduced SB during adolescence. The aspect of SB that was most powerfully related to cortisol was general repetitive behaviour rather than movements of specific body parts. Explanations of these findings are raised for further investigation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The agreement over time in morning salivary cortisol concentrations and also self- and parent-rated anxiety was investigated in a sample of 16 boys with an ASD. Cortisol and anxiety data were collected eight months apart. Results indicated that there were significant correlations between each pair of measures from the two occasions, suggesting that cortisol concentrations and anxiety did not vary much at all over that time, challenging the assumption that cortisol needs to be measured over multiple days to obtain reliable data from children with an ASD. Implications for research into the ways these children respond to chronic stressors are discussed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Gastrointestinal nematodes pose a major risk to the farming of small ruminants worldwide. Infections are typically controlled by anthelmintics, however as resistance to anthelmintics increases, it is necessary that the mechanism of host responses are understood in order to develop alternative control options. It is hypothesised that basophils are involved in the initiation of an anti-parasite immune response, independent of IgE. In this study, the in vitro activation states of CD203c(+) basophil-like KU812 cells were determined in the presence of Trichostrongylus colubriformis parasitised HT29 epithelial cells with or without mucin. Cell surface expression of CD164, CD107a and CD13 antigens on gated CD203(+) cells were determined and qRT-PCR was used to examine gene expression changes of IL33 (a Th2 cytokine) and the high affinity IgE receptor (FcɛRIα) within the co-culture. When KU812 basophils encountered T. colubriformis and/or mucin in a parasitised epithelium, the basophils increased cell surface expression of CD13 and CD164 antigens, independent of IgE. T. colubriformis also increased the number of CD203c(+) KU812 cells that expressed CD13 and CD164 antigens. These data support the in vivo observations of T. colubriformis primary infections in guinea pigs and sheep., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

There is considerable evidence of a confound between symptoms of generalised anxiety disorder (GAD) and autism spectrum disorder (ASD) in children who have an ASD. Although there have been several attempts to describe how these two disorders overlap and interact to influence the assessment and diagnosis of children with an ASD, principally by reference to cortisol assayed from these children's saliva, the overall evidence is inconsistent. Because previous models of these relationships have focused upon cortisol and GAD to the exclusion of age, diurnal fluctuation in the HPA axis and the source of GAD data, these variables were examined in a sample of 150 young males with an ASD. Results indicated that there was a significant interaction between these variables, with the association between GAD and cortisol demonstrated for children but not for adolescents, with an interaction between the source of GAD information (self- vs parent-ratings) and whether the child's cortisol concentrations followed the expected diurnal reduction during the day. These data suggest that the validity of cortisol as a biomarker of GAD in children and adolescents with an ASD may be established for only selected subgroups of this population., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Gastrointestinal nematodes remain a major limitation to the productivity of livestock systems. Selective breeding to produce populations that have an enhanced ability to resist infection is a viable and ongoing option to reduce this impact. The development of new phenotypes that facilitate this process is therefore of great interest. For this reason we explored relationships between haematological parameters and the ability of sheep to resist nematode infection. A multivariate analytical approach was used to define algorithms based on the blood parameters that can be used to rank the ability of sheep to resist nematode infection in a single blood sample and can be applied independent of infection status. The algorithms were shown to classify susceptible sheep with a 100% accuracy and resistant sheep with 80% accuracy. Further development of this platform approach may be an important advance for small ruminant production systems worldwide and might also be applied to other diseases of livestock or even environmental stressors such as heat., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

When plasminogen binds to cells its activation to plasmin is markedly enhanced compared to the reaction in solution. Thus, cells become armed with the broad spectrum proteolytic activity of plasmin. Cell-surface plasmin plays a key role in macrophage recruitment during the inflammatory response. Proteins exposing basic residues on the cell surface promote plasminogen activation on eukaryotic cells. We have used a proteomics approach combining targeted proteolysis with carboxypeptidase B and multidimensional protein identification technology, MudPIT, and a monocyte progenitor cell line to identify a novel transmembrane protein, the plasminogen receptor, Plg-R(KT). Plg-R(KT) exposes a C-terminal lysine on the cell surface in an orientation to bind plasminogen and promote plasminogen activation. Here we review the characteristics of this new protein, with regard to membrane topology, conservation of sequence across species, the role of its C-terminus in plasminogen binding, its function in plasminogen activation, cell migration, and its role in macrophage recruitment in the inflammatory response.

Gastrointestinal nematodes represent a major production problem for ruminant livestock. Enhancing immunity to gastrointestinal nematodes through vaccination is desirable but mechanistic understanding of initial host responses that facilitate gastrointestinal nematode protective immunity is limited. We hypothesise that gastrointestinal nematode invasion induces mucosal epithelium damage and alarmin (e.g. IL33) release, thereby contributing to initiation of protective gastrointestinal nematode immunity. To test this, an in vitro air-liquid interface human HT-29 epithelial cell-Trichostrongylus colubriformis co-culture system was developed. Exsheathed L3 T. colubriformis exhibited both sinusoidal and burrowing motions in the co-culture system. Burrowing parasites, but not ivermectin-paralysed larvae, induced necrotic death of epithelial cells (annexin V(+)/propidium iodide(+)/caspase 3/7(-)). Microscopy confirmed that larvae consumed labelled necrotic epithelial cell contents. Trichostrongylus colubriformis larvae and their post-exsheathment antigens (excretory/secretory products) significantly induced IL33 mRNA expression in the epithelial cells. Immunoblot confirmed that IL33 was released from epithelial cells due to the damage caused by motile larvae. Exposure of HT-29 cells to alum or Sigma proprietary adjuvants induced significant epithelial cell IL33 mRNA expression without inducing cellular necrosis. Hence, the intracellular contents were not released externally where they might exert alarmin activity and this may limit their ability to trigger a protective anti-gastrointestinal nematode response. We conclude that T. colubriformis motion at the infection site induces intestinal epithelial cell necrosis which facilitates the release of intracellular contents, including IL33, and may be fundamental to the initiation of an appropriate host response to gastrointestinal nematodes. Our co-culture model is useful for studying initial epithelial cell-parasite interactions without conducting expensive animal trials., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

This study reports on the molecular systems biology of gastrointestinal nematode (GIN) infection and potential biomarkers for GIN resistance in sheep. Microarray gene expression data were obtained for 3 different tissues at 4 time points from sheep artificially challenged with two types of nematodes, Haemonchus contortus (HC) and Trichostrongylus colubriformis (TC). We employed an integrated systems biology approach, integrating 3 main methods: standard differential gene expression analyses, weighted gene co-expression network analyses (WGCNA) and quantitative genetic analyses of gene expression traits of key biomarkers. Using standard differential gene expression analyses we identified differentially expressed genes (DE) which responded differently in sheep challenged with HC compared to those challenged with TC. These interaction genes (e.g. MRPL51, SMEK2, CAT, MAPK1IP1 and SLC25A20A) were enriched in Wnt receptor signalling pathway (p = 0.0132) and positive regulation of NFκβ transcription factor activity (p = 0.00208). We report FCER1A, a gene encoding a high-affinity receptor for the Fc region of immunoglobulin E, which is linked to innate immunity to GIN in sheep. Using weighted gene co-expression network analysis (WGCNA) methods, we identified gene modules that were correlated with the length of infection (disease modules). Hub genes (with high intramodular connectivity) were filtered further to identify biomarkers that are related to the length of infection (e.g. CAT, FBX033, COL15A1, IGFBP7, FBLN1 and IgCgamma). The biomarkers we found in HC networks were significantly associated with functions such as T-cell and B-cell regulations, TNF-alpha, interleukin and cytokine production. In TC networks, biomarkers were significantly associated with functions such as protein catabolic process, heat shock protein binding, protein targeting and localization, cytokine receptor binding, TNF receptor binding, apoptosis and IGF binding. These results provide specific gene targets for therapeutic interventions and provide insights into GIN infections in sheep which may be used to infer the same in related host species. This is also the first study to apply the concept of estimating breeding values of animals to expression traits and reveals 11 heritable candidate biomarkers (0.05 to 0.92) that could be used in selection of animals for GIN resistance.

Activation of plasminogen, the zymogen of the primary thrombolytic enzyme, plasmin, is markedly promoted when plasminogen is bound to cell surfaces, arming cells with the broad spectrum proteolytic activity of plasmin. In addition to its role in thrombolysis, cell surface plasmin facilitates a wide array of physiologic and pathologic processes. Carboxypeptidase B-sensitive plasminogen binding sites promote plasminogen activation on eukaryotic cells. However, no integral membrane plasminogen receptors exposing carboxyl terminal basic residues on cell surfaces have been identified. Here we use the exquisite sensitivity of multidimensional protein identification technology and an inducible progenitor cell line to identify a novel differentiation-induced integral membrane plasminogen receptor that exposes a C-terminal lysine on the cell surface, Plg-R(KT) (C9orf46 homolog). Plg-R(KT) was highly colocalized on the cell surface with the urokinase receptor, uPAR. Our data suggest that Plg-R(KT) also interacts directly with tissue plasminogen activator. Furthermore, Plg-R(KT) markedly promoted cell surface plasminogen activation. Database searching revealed that Plg-R(KT) mRNA is broadly expressed by migratory cell types, including leukocytes, and breast cancer, leukemic, and neuronal cells. This structurally unique plasminogen receptor represents a novel control point for regulating cell surface proteolysis.

An emerging area of research has documented a novel role for the plasminogen activation system in the regulation of neurotransmitter release. Prohormones, secreted by cells within the sympathoadrenal system, are processed by plasmin to bioactive peptides that feed back to inhibit secretagogue-stimulated release. Catecholaminergic cells of the sympathoadrenal system are prototypic prohormone-secreting cells. Processing of prohormones by plasmin is enhanced in the presence of catecholaminergic cells, and the enhancement requires binding of plasmin(ogen) to cellular receptors. Consequently, modulation of the local cellular fibrinolytic system of catecholaminergic cells results in substantial changes in catecholamine release. However, mechanisms for enhancing prohormone processing and cell-surface molecules mediating the enhancement on catecholaminergic cells have not been investigated. Here we show that plasminogen activation was enhanced >6.5-fold on catecholaminergic cells. Carboxypeptidase B treatment decreased cell-dependent plasminogen activation by approximately 90%, suggesting that the binding of plasminogen to proteins exposing C-terminal lysines on the cell surface is required to promote plasminogen activation. We identified catecholaminergic plasminogen receptors required for enhancing plasminogen activation, using a novel strategy combining targeted specific proteolysis using carboxypeptidase B with a proteomics approach using two-dimensional gel electrophoresis, radioligand blotting, and tandem mass spectrometry. Two major plasminogen-binding proteins that exposed C-terminal lysines on the cell surface contained amino acid sequences corresponding to beta/gamma-actin. An anti-actin monoclonal antibody inhibited cell-dependent plasminogen activation and also enhanced nicotine-dependent catecholamine release. Our results suggest that cell-surface-expressed forms of actin bind plasminogen, thereby promoting plasminogen activation and increased prohormone processing leading to inhibition of neurotransmitter release.

Localization of plasminogen and plasminogen activators on cell surfaces promotes plasminogen activation and serves to arm cells with the broad spectrum proteolytic activity of plasmin. Cell surface proteolysis by plasmin is an essential feature of physiological and pathological processes requiring extracellular matrix degradation for cell migration including macrophage recruitment during the inflammatory response, tissue remodeling, wound healing, tumor cell invasion and metastasis and skeletal myogenesis. Cell associated plasmin on platelets and endothelial cells is optimally localized for promotion of clot lysis. In more recently recognized functions that are likely to be independent of matrix degradation, cell surface-bound plasmin participates in prohormone processing as well as stimulation of intracellular signaling. This issue of Frontiers in Bioscience on Plasminogen Receptors encompasses chapters focusing on the kinetics of cell surface plasminogen activation and the regulation of plasminogen receptor activity as well as the contribution of plasminogen receptors to the physiological and pathophysiological processes of myogenesis, muscle regeneration and cancer. The molecular identity of plasminogen receptors is cell-type specific, with distinct molecular entities providing plasminogen receptor function on different cells. This issue includes chapters on the well studied plasminogen receptor functions.

Mycoplasma species bovine group 7 bound plasminogen at the cell surface in a lysine-dependent manner. Cell-bound plasminogen was rapidly activated to plasmin by exogenous urokinase, and this activity was associated with plasminogen binding capacity. Binding assays using plasminogen modified with a trifunctional cross-linking agent revealed several binding proteins.

The urokinase plasminogen activation system is a key modulator of the tissue remodeling processes required for tumor cell invasion and metastasis. Malignant progression is characterised by inappropriately high cell surface levels of receptor- bound active urokinase. This enhances the rate of plasminogen activation resulting in markedly increased levels of cell surface plasmin. The repercussions of this are significant and include the activation of growth factors and signaling pathways, and the degradation of extracellular matrices, either directly or indirectly, via the activation of matrix metalloproteinases. Native, circulating plasminogen binds in a lysine- and/or carbohydrate-dependent manner to tumor and endothelial cells with low affinity but high capacity and a heterogeneous group of plasminogen receptors have been identified. This heterogeneity underscores the complexity of the mechanisms responsible for the regulation of cell-surface plasminogen binding. This review summarizes the literature on known plasminogen receptor candidates and shows that they can be subdivided into three classes based on their mode of interaction with plasminogen. We also aim to emphasize the notion that in the tumor environment the known intrinsic functional relationship between plasminogen conformation and activation is essentially connected to cellular binding. This allows plasminogen to be co-localised in an activation-susceptible form with the enhanced uPA levels seen in malignancy and together furnishes tumor cells with elevated tissue remodeling capacity. In addition, some of the pitfalls and strategies encountered when conducting plasminogen receptor experiments are also addressed.

Lymphocytic hypophysitis is part of the spectrum of organ-specific autoimmune diseases, and although its histopathology is well documented, its pathogenesis is unclear. Serum autoantibodies directed against a 49-kDa cytosolic protein are detected by immunoblotting in 70% of patients with biopsy-proven lymphocytic hypophysitis. Here we report the purification and identification of this first target autoantigen in lymphocytic hypophysitis. The autoantigen has a molecular mass of 49 kDa, a cytosolic localization, and a ubiquitous tissue distribution. The 49-kDa protein was purified from monkey brain and human placental cytosol. Limited amino acid sequencing after proteolytic digestion of the human placental protein showed identity with alpha-enolase. The identification was confirmed using sera from patients with pituitary autoimmunity, which strongly reacted with recombinant human alpha-enolase and yeast enolase, but not with rabbit muscle beta- enolase. This indicates that the immunoreactive epitopes are largely conserved from yeast to human, but are not present in beta-enolase. alpha-Enolase autoantibodies are not specific to pituitary autoimmune disease and have been reported in other autoimmune diseases. However, this study is the first to indicate a role for alpha-enolase as an autoantigen in lymphocytic hypophysitis.

Metastasis is the principal cause of death in breast cancer patients. New and improved treatments for eradicating micrometastases are needed. To this end, a novel alpha-emitting protein construct, 213Bi-labelled plasminogen activator inhibitor type-2 (PAI-2) (alpha-PAI-2), was evaluated in vitro. This construct exploits: (a) the overexpression of the cell-surface receptor bound urokinase plasminogen activator (uPA) in the metastatic spread of breast cancer cells; (b) the binding and inhibition of receptor-bound uPA by PAI-2; and (c) the high cytotoxicity of alpha radiation. High labeling efficiencies and stability of 213Bi bound to human recombinant PAI-2 conjugated with cyclic diethylenetriaminepentaacetic acid anhydride were achieved (greater than 90%). The uPA inhibitory activity of the chelated PAI-2 was maintained as determined by complex formation with uPA and by inhibition of uPA activity. Furthermore, the reactivity of alpha-PAI-2 was confirmed in a cell assay as this construct was highly cytotoxic to breast cancer cell lines that express active, receptor bound uPA. The specificity of alpha-PAI-2 targeting was shown using several controls. Firstly, an active uPA blocking agent that limits PAI-2 binding significantly improved cell survival by a factor greater than three. Secondly, a non-specific alpha-BSA construct had minimal cytotoxic effect. Moreover, alpha-PAI-2 was not cytotoxic to freshly isolated normal human leukocytes, confirming that cells which do not contain active, receptor bound uPA cannot be targeted by alpha-PAI-2. In conclusion, we have validated, in vitro, the potential of alpha-PAI-2 as a novel therapeutic agent for breast cancer.

The urokinase-dependent activation of plasminogen by breast cancer cells plays an important role in metastasis. We have previously shown that the metastatic breast cancer cell line MDA-MB-231 over-expresses urokinase and binds and efficiently activates plasminogen at the cell surface compared to non-metastatic cells. The aim of this study was to further characterise plasminogen binding and determine the topology of cell surface-bound plasminogen in terms of its potential for activation. The lysine-dependent binding of plasminogen at 4 degrees C to MDA-MB-231 cells was stable and resulted in an activation-susceptible conformation of plasminogen. Topologically, a fraction of bound plasminogen was co-localised with urokinase on the surfaces of MDA-MB-231 cells where it could be activated to plasmin. At 37 degrees C plasmin was rapidly lost from the cell surface. Apart from actin, other candidate plasminogen receptors were either not expressed or did not co-localise with plasminogen at the cell surface. Thus, based on co-localisation with urokinase, plasminogen binding is partitioned into two functional pools on the surface of MDA-MB-231 cells. In conclusion, these results shed further light on the functional organisation of the plasminogen activation cascade on the surface of a metastatic cancer cell., (Copyright 2001 Cancer Research Campaign http://www.bjcancer.com.)

Plasminogen binds with low affinity in a lysine-dependent manner to many cell types. Previously, a 54 kDa plasminogen receptor found on the surface of U-937 cells was identified as an alpha-enolase-like molecule. The aims of this study were to determine whether recombinant alpha-enolase (r-alpha-enolase), encoded by ENO1, was a plasminogen binding protein and to generate polyclonal antibodies against this antigen. Plasminogen specifically bound r-alpha-enolase with a Kd 1.9 microM and approached saturation at 10 microM. Lysine-dependent plasminogen binding to r-alpha-enolase was demonstrated by a greater than 80% inhibition of binding by the lysine analogues epsilon-amino caproic acid and tranexamic acid, whilst only 14% inhibition occurred with the arginine analogue benzamidine. Removal of the C-terminal lysine residue of r-alpha-enolase with carboxy-peptidase B significantly reduced its plasminogen binding capacity, suggesting that binding required C-terminal lysine residue of r-alpha-enolase. Binding to r-alpha-enolase enhanced the activation rate of plasminogen by urokinase but prevented alpha 2-antiplasmin from binding plasminogen. Taken together, these data suggest that the gene product of human ENO1 encodes an authentic plasminogen binding protein.