Putative ß-Barrel Outer Membrane Proteins of the Bovine Digital Dermatitis-Associated Treponemes: Identification, Functional Characterization, and Immunogenicity.

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative ß-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted ß-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as ß-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly ß-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal ß-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.

'The current mental health status of children and young people with JIA, and their wider family': a charity partner collaboration survey.

BACKGROUND: This paper presents insight into the scale of mental health concerns for families who have a child or young person with a diagnosis of Juvenile Idiopathic Arthritis (JIA) living in any of the four nations of the United Kingdom (UK). The study's objective is to share the current experiences of those that responded to a charity survey and consider future work to improve mental health support. METHODS: This work was initiated and led by five UK charity partner organisations working with families affected by JIA. Parents/carers of a child or young person with JIA, and young people with JIA, submitted self-completion online questionnaires. The questionnaire asked 19 core questions, with a focus on the mental health impact of having and living with a JIA diagnosis. Questionnaires were delivered via charity partner UK-wide mailing lists and social media. RESULTS: Questionnaire were completed by 291 participants over a 3-week period in February 2022. The majority of respondents were parents (229, 79%), 103 children had been diagnosed for over six years (35%), and 131 (45%) received shared care between paediatric rheumatology centres. In total, 168 (59%) children and young people with JIA had received, were currently receiving or were waiting for mental health support. Parents reported that their child's diagnosis impacted their own mental health (218, 82%). Children and young people reported never being offered mental health support during appointments for JIA (157, 54%), and 71 (50%) of these had never received support. CONCLUSION: Children and young people with JIA have significant mental health sequelae from their diagnosis. Our findings found that nearly 60% of our respondents have had or are requiring mental health support, with significant numbers of parents/carers reporting difficulties in accessing care for their child's mental health or their own mental health, due to their child's diagnosis. This unique collaborative charity-led study, illustrates the importance of timely and accessible mental health support. Further work is needed to understand why best practice guidance for mental health support is not being met consistently and to identify how to embed it into standard rheumatology care.

Convergent evolution of pain-inducing defensive venom components in spitting cobras.

Convergent evolution provides insights into the selective drivers underlying evolutionary change. Snake venoms, with a direct genetic basis and clearly defined functional phenotype, provide a model system for exploring the repeated evolution of adaptations. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of cobras have independently evolved the ability to spit venom at adversaries. Using gene, protein, and functional analyses, we show that the three spitting lineages possess venoms characterized by an up-regulation of phospholipase A2 (PLA2) toxins, which potentiate the action of preexisting venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by selection for defense.

Studies on the pathogenesis of acute inflammation. IX. The influence of hyperosomolality secondary to hyperglycemia upon the acute inflammatory response induced by thermal injury to ear chambers of rabbits.

The inflammatory reaction induced in ear chambers of rabbits by heat injury was studied in nondiabetic animals made hyperglycemic with continuous infusions of glucose. Hypertonic solutions of glucose administered intravenously to rabbits induced the following triad: hyperglycemia, hyperosmolality, and metabolic lactic acidosis. It was found that relatively short periods of this metabolic abnormality were associated with a significant reduction in the intensity of the inflammatory reaction. There was no evidence microscopically of circulatory impairment within ear chambers; and since neither arterial hypotension nor oxygen deficit was recorded during experiments, it appeared most likely that decreased leucocytic margination per se best explained the inhibited exudative response. Hyperglycemia seemed the dominant factor responsible for this anti-inflammatory effect. It was found subsequently that hyperglycemia and hyperosmolality without metabolic acidosis impaired cellular exudation just as well.

Percutaneous central venous catheters versus peripheral cannulae for delivery of parenteral nutrition in neonates.

BACKGROUND: Parenteral nutrition for neonates may be delivered via a short peripheral cannula or a central venous catheter. The latter may either be inserted via the umbilicus or percutaneously. Because of the complications associated with umbilical venous catheter use, many neonatal units prefer to use percutaneously inserted catheters following the initial stabilisation period. The method of parenteral nutrition delivery may affect nutrient input and consequently growth and development. Although potentially more difficult to place, percutaneous central venous catheters may be more stable than peripheral cannulae, and need less frequent replacement. These delivery methods may also be associated with different risks of adverse events, including acquired systemic infection and extravasation injury. OBJECTIVES: To determine the effect of infusion via a percutaneous central venous catheter versus a peripheral cannula on nutrient input, growth and development, and complications including systemic infection, or extravasation injuries in newborn infants who require parenteral nutrition. SEARCH STRATEGY: The standard search strategy of the Cochrane Neonatal Review Group was used. This included searches of the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, Issue 1, 2007), MEDLINE (1966 - February 2007), EMBASE (1980 - February 2007), conference proceedings, and previous reviews. SELECTION CRITERIA: Randomised controlled trials that compared the effect of delivering parenteral nutrition via a percutaneous central venous catheter versus a peripheral cannulae in neonates. DATA COLLECTION AND ANALYSIS: Data were extracted the data using the standard methods of the Cochrane Neonatal Review Group, with separate evaluation of trial quality and data extraction by each author, and synthesis of data using relative risk, risk difference and mean difference. MAIN RESULTS: Four trials eligible for inclusion were found. These trials recruited a total of 368 infants and reported a number of different outcomes. One study showed that the use of a percutaneous central venous catheter was associated with a decreased risk of cumulative nutritional deficit during the trial period: Mean difference in the percentage of the prescribed nutritional intake actually received: -7.1% (95% confidence interval -11.02, -3.2). In another trial, infants in the percutaneous central venous catheter group needed significantly fewer catheters/cannulae per infant during the trial period: Mean difference in the number of catheters/cannulae per infant: -3.2 (95% confidence interval -5.13, -1.27). Meta-analysis of data from all four trials did not find any evidence of an effect on the incidence of systemic infection: Typical relative risk: 0.94 (95% confidence interval 0.70, 1.25); typical risk difference: -0.02 (95% confidence interval -0.12, 0.08). AUTHORS' CONCLUSIONS: Data from one small study suggest that the use of a percutaneous central venous catheter to deliver parenteral nutrition in newborn infants improves nutrient input. The significance of this in relation to long-term growth and developmental outcomes is unclear. Another study suggested that the use of a percutaneous central venous catheter rather than a peripheral cannula decreases the number of catheters/cannulae needed to deliver the nutrition. No evidence was found to suggest that percutaneous central venous catheter use increased the risk of adverse events, particularly systemic infection.

A pilot interventional study to evaluate the impact of cholecalciferol treatment on HbA1c in type 1 diabetes (T1D).

BACKGROUND: Higher 25(OH)D3 levels are associated with lower HbA1c, but there are limited UK interventional trials assessing the effect of cholecalciferol on HbA1c. AIMS: (1) To assess the baseline 25(OH)D3 status in a Manchester cohort of children with type 1 diabetes (T1D). (2) To determine the effect of cholecalciferol administration on HbA1c. METHODS: Children with T1D attending routine clinic appointments over three months in late winter/early spring had blood samples taken with consent. Participants with a 25(OH)D3 level <50 nmol/L were treated with a one-off cholecalciferol dose of 100,000 (2-10 years) or 160,000 (>10 years) units. HbA1c levels before and after treatment were recorded. RESULTS: Vitamin D levels were obtained from 51 children. 35 were Caucasian, 11 South Asian and 5 from other ethnic groups. 42 were vitamin D deficient, but 2 were excluded from the analysis. All South Asian children were vitamin D deficient, with mean 25(OH)D3 of 28 nmol/L. In Caucasians, there was a negative relationship between baseline 25(OH)D3 level and HbA1c (r = -0.484, P < 0.01). In treated participants, there was no significant difference in mean HbA1c at 3 months (t = 1.010, P = 0.328) or at 1 year (t = -1.173, P = 0.248) before and after treatment. One-way ANCOVA, controlling for age, gender, ethnicity, BMI and diabetes duration showed no difference in Δ HbA1c level. CONCLUSION: We report important findings at baseline, but in children treated with a stat dose of cholecalciferol, there was no effect on HbA1c. Further studies with larger sample sizes and using maintenance therapy are required.

Thrombocytopenia among extremely low birth weight neonates: data from a multihospital healthcare system.

OBJECTIVES: Thrombocytopenia is common in neonatal intensive care units (NICU), with 18 to 35% of patients developing this problem before hospital discharge. It might be even more common among extremely low birth weight neonates (ELBW, < or = 1000 g birth weight). However, little is known about thrombocytopenia in the ELBW population. We sought to determine the incidence, timing, causes, platelet transfusions given, and outcomes of thrombocytopenia among ELBW neonates. STUDY DESIGN: We performed a cohort analysis of all 284 ELBW neonates born during 2003 and 2004 cared for in any of the Intermountain Healthcare level III NICUs. RESULTS: Multiple platelet counts were obtained in all 284 (range, 4 to 441 platelet counts/patient). Of the 284, 208 (73%) had one or more platelet counts < or =150 000/microl. Most were detected during the first days of life; 80% were detected during the first week and only 20% were detected thereafter. Thrombcytopenia was more common among the smallest patients; 85% incidence among those < or =800 g, 60% among those 801 to 900 g, and 53% among those 901 to 1000 g. Platelet transfusions were given to 129 of the 208 thrombocytopenic neonates. More than 90% were given prophylactically (the patient was not bleeding). The mortality rate among those that received platelet transfusions was twice that of those that received no platelet transfusions (P < 0.01). In 48% of cases, the cause of the thrombocytopenia went undiagnosed. The most common explanations were being small for gestational age or delivered to a hypertensive mother, DIC, bacterial infection, fungal infection, and necrotizing enterocolitis, respectively. CONCLUSIONS: We observed thrombocytopenia among ELBW neonates at a rate more than twice that reported among the general NICU population. Much remains to be discovered about the etiology and best treatments of thrombocytopenia among ELBW neonates.

The affinity of rabbit muscle fructose-bisphosphatase for fructose bisphosphate.

This paper reports that microM concentrations of fructose bisphosphate are titrated by rabbit muscle fructose-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) when the enzyme concentration is varied in the range which secures measurable initial velocities of reaction: a result that can only be explained by supposing that the enzyme has a greater affinity for fructose bisphosphate than suggested by Fernando, J., Enser, M., Pontremoli, S. and Horecker, B.L. (1968) Arch. Biochem. Biophys. 126, 599-606. The results also suggest that the keto form of the substrate may be the preferred configuration and that the enzyme is inhibited by magnesium-bound fructose bisphosphate.

The analysis of kinetic data for regulatory enzymes as a function of pH.

A new method is described for the analysis of kinetic data for regulatory enzymes as a function of pH. The method makes use of the limiting hyperbolae predicted by the exponential model for a regulatory enzyme. The pH dependence of the parameters that describe these hyperbolae can be analysed with the Michaelis pH functions to give the pK values of the enzyme-substrate complex and those of the free enzyme in its limiting conformational states. The procedure is illustrated by application to data for the effect of pH on the kinetic properties of aspartate transcarbamylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2).

Protein A immunoadsorption in the treatment of malignant disease.

Circulating immune complexes (CIC) are known to be present in cancer patients and are responsible for much of the cancer-associated immunosuppression. Removal or modulation of these "blocking factors" can reverse the immunosuppression. Protein A from Staphylococcus aureus has the unusual property of binding to CIC with high avidity. Use of protein A as an immunoadsorbent in extracorporeal immunotherapy affinity columns has resulted in antitumor and antiviral responses in animals. Our group developed a multicenter trial to assess toxicity and antitumor response with this biologic response modifier alone. Overall, 24% (21 of 87 patients) had objective tumor regressions including both partial responses (PR) and less than PR. No complete responses (CR) were observed. Responses were observed in acquired immune deficiency syndrome (AIDS)-related Kaposi's sarcoma (six of 17 PR; two of 17 less than PR; overall, 47%), breast adenocarcinoma (five of 22 PR; three of 22 less than PR; overall response, 36%), colon adenocarcinoma, (one PR, one less than PR; overall response, 11%), and non-oat cell lung carcinoma (two of seven less than PR). The procedure was well tolerated and could be performed on an outpatient basis. No adverse reaction was observed in 735 of 1,113 treatments (66%). The most common adverse effect was an "influenza-like" syndrome consisting of fever and chills. Pain was present in 12% of the patients. There were no study-related deaths. Serum IgG and CIC levels did not statistically change due to therapy in responding or nonresponding patients. Complement levels remained within the normal range. Liver and renal tests remained stable throughout the study. In summary, protein A immunoadsorption of plasma is well tolerated in the outpatient clinic, has demonstrated antitumor activity in resistant solid tumors, and functions as a biologic response modifier.

Treatment of patients with HIV thrombocytopenia and hemolytic uremic syndrome with protein A (Prosorba column) immunoadsorption.

Both antibodies and circulating immune complexes (CIC), which bind to platelets and induce the destruction and clearance of platelets by the reticuloendothelial system, are found in patients with human immunodeficiency virus (HIV) and immune thrombocytopenic purpura (ITP). IgG and CIC were removed from patients' plasma by extracorporeal immunoadsorption using protein A-silica columns (PROSORBA columns). Of the 36 HIV-positive ITP patients treated, 29 received more than one treatment and were evaluated for response. Sixteen patients showed more than a 50% increase in their platelet counts. Platelet-associated IgG (PAIgG) and/or platelet-directed IgG and CIC were elevated in all patients. After four to eight treatments, 16 of 29 patients showed a 170% to 430% increase in platelet counts. A decrease in CIC and PAIgG was noted in responding patients. The median duration of response to date was 8 to 12 months. This treatment was associated with immune modulation and the development of an anti-F (ab')2 antibody response. The antibody functions by complexing with both platelet-binding IgG and CIC, neutralizing their binding capacity for platelets and enhancing their clearance from the circulation. Nine patients with mitomycin-C-induced hemolytic uremic syndrome (HUS) were also treated with PROSORBA columns. Pretreatment platelet counts were markedly reduced while a definite increase in platelet counts was observed upon completion of therapy. There was a decrease of hemolysis and stabilization of renal function in three patients. PROSORBA column treatment has demonstrated marked activity against both HIV-ITP and HUS, and has successfully freed patients from the bleeding diathesis associated with these syndromes.

Protein A immunotherapy in the treatment of cancer: an update.

Protein A, a naturally occurring Staphylococcus aureus cell surface protein, has the unusual property of binding circulating immune complexes and immunoglobulin G with high avidity. CIC have played a major role in cancer-associated immunosuppression. Thus, removal of the immunosuppressive agents, ie, the CIC, may lead to a modulation of the immunosuppression and a liberation of the immune system to perform an antitumor effect. In animal studies, protein A has been used in extracorporeal immunoadsorption columns and treatments have resulted in tumor shrinkage and antiviral responses. Our group developed a multicenter clinical trial to assess toxicity and antitumor responses with this biologic response modifier alone. This is an update of our original trial. We have now treated 142 patients for a total of 1,306 treatments. The patients consisted of 74 males and 68 females. Their age ranged from 7 to 83 years, with a mean of 50 years. The Karnofsky performance index values ranged from 40 to 95, with a mean of 80. Patients who received seven or more treatments were considered eligible for tumor response assessment, and all patients with one or more treatments were eligible for toxicity assessment. Thus, there were 101 patients eligible for tumor response and 142 eligible for toxicity response. The total response rate was 22 patients or 21.8% (partial remission [PR], 12 patients, 12%; less than PR, 10 patients, 10%). Response rates were similar in the 13 treatment centers. Toxicity was assessed in 142 patients. One thousand three hundred six treatments were assessed for treatment toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies in healthy subjects of different age groups.

Autoantibody determinations in 1284 healthy Caucasian subjects of various age groups were made by indirect immunofluorescence for anti-nuclear, anti-gastric, and anti-thyroglobulin antibodies. A sex-dependent relationship between age and prevalence of anti-gastric and anti-thyroglobulin antibodies was found. No association was found between age and anti-nuclear antibodies.

Association between fluorescent antinuclear antibodies, capillary patterns, and clinical features in scleroderma spectrum disorders.

Antinuclear antibody and in vivo capillary patterns were studied in 33 patients with Raynaud's phenomenon only and in 68 patients with scleroderma spectrum disorders; the results were correlated with clinical and laboratory findings. In addition, antinuclear antibody results in the groups with Raynaud's phenomenon only and scleroderma spectrum disorders were compared with those found in 70 patients with systemic lupus erythematosus (SLE). Distinct antinuclear antibody profiles were observed in the three diagnostic groups. Comparison of patients with anticentromere antibodies with others in the group with scleroderma spectrum disorders demonstrated that anticentromere antibody-positive patients tended to have a milder disease: less skin and visceral involvement, less frequent presence of hypertension, anemia, and elevated sedimentation rate. These differences did not, however, reach statistical significance. Comparison of patients with scleroderma spectrum disorders according to in vivo capillary patterns revealed that those with an "active" pattern had significantly more extensive skin involvement than those with a "slow" pattern. Visceral involvement tended to be greater in all organ systems in the group with an "active" pattern and reached statistical significance for muscle and kidney. Hypertension was also significantly more frequent in the group with an "active" pattern than in the group with a "slow" one. The latter was positively correlated with the presence of anticentromere antibody.

An ultrastructural method for the use of polyvinylpyrrolidone and dextrans as electron opaque tracers.

A simple method has been developed whereby polymers of polyvinylpyrrolidone and dextran can be visualized at the ultrastructural level by postfixing tissue with osmium tetroxide partially reduced by potassium ferrocyanide. Further enhancement of polymer electron opacity is obtained by counterstaining thin sections with bismuth subnitrate chelated by alkaline tartrate. These tracers remain adequately dispersed in serum and, depending on molecular size and concentration, are easily detected at the ultrastructural level within vascular lumena, basement lamina, apical vacuoles of proximal and distal tubules and endothelial vesicles. This technique offers the potential of studying nephron and cell permeability with families of tracers, the members of which are similar in chemical affinities, charge density and molecular shape.

Gm allotypes in blacks with systemic lupus erythematosus.

Serum samples were collected from 328 healthy American Blacks and from 61 American Blacks with systemic lupus erythematosus (SLE). Sera were typed for the Gm1,2,3,5,6,13,17, and 21 allotypes as well as for the Km(1) allotype. The frequency of Gm phenotype 1,17;5,6,13 was significantly increased in the SLE patients (p = 0.0001, RR = 3.19, EF = 0.29). Our data suggest the existence of at least two immunoglobulin allotype associated genes that somehow interact to increase susceptibility to SLE in Blacks. To our knowledge, this is the first report of an association of Gm and SLE in Blacks.

Pulmonary function analysis in the rabbit following bronchochallenge to causative agents and mediators of the acute byssinotic response.

New Zealand White rabbits were acutely bronchochallenged for 5 min to ascertain airway responsiveness with six potential byssinogenic agents and mediators: 0.1 g/mL cotton dust extract (CDE), 0.1 g/mL cotton bract extract (CBE), 1 mg/mL endotoxin, 1 mg/mL n-formyl methionyl peptide (n-fMet), 10 mg/mL 5-hydroxytryptamine (5-HT), and 1 mg/mL prostaglandin F2 alpha (PGF2 alpha). Methacholine (MC), 10 mg/mL, was used as a control bronchoconstrictor. Clinically objective criteria were established using increases in resistance values compared to those obtained with saline controls. Animals were classified as: mild responders (Mi) = 125-149%; moderate responders (Mo) = 150-199%; or severe responders (S) = greater than 200%. Three of five (2Mo, 1S) rabbits showed increased pulmonary resistance to CDE bronchochallenge, 3/5 (1Mi, 1Mo, 1S) to CBE, 1/5 (Mo) to purified endotoxin, 4/5 (1Mo, 3S) to n-fMet, 3/5 (1Mi, 1Mo, 1S) to 5-HT, and 2/5 (1Mo, 1S) to PGF2 alpha. All five rabbits (1Mo, 4S) responded to MC bronchochallenge. Rabbits responded minimally to saline, the common solvent of all test agents; however, when challenged with methacholine, a known bronchoconstrictor, rabbits showed significant overt symptoms of acute respiratory distress with immediate and substantial increases in resistance over saline controls. CDE, CBE, and n-fMet inhalation challenge resulted in a majority or all animals showing increased resistance. 5-HT contained in CDE and CBE, exhibited similar resistance increases; however, endotoxin, also found in cotton dust, showed little airway reactivity. The rabbit is useful for characterizing changes in pulmonary function parameters seen in the acute byssinotic reaction. This study has demonstrated that bronchochallenge in the rabbit with potential byssinogenic agents (CDE, CBE, endotoxin, and n-fMet) and mediators (5-HT and PGF2 alpha) result in measurable changes in airway function, particularly increased resistance. Since bronchoconstriction is the major clinical manifestation of the acute byssinotic reaction in man and animals, it is likely that bronchoconstriction observed in cotton mill workers may be in part or totally the result of inherent dust constrictor substances or secondarily released mediators.

Diabetic glomerulonephropathy: histopathologic, immunofluorescent, and ultrastructural studies of 16 cases.

Sixteen cases of diabetic glomerulopathy are reported. Direct immunofluorescent and ultrastructural studies of renal biopsy tissues demonstrated that two patients had linear deposits of IgM and C'3 in the absence of IgG, four diabetic patients had sclerosis-induced entrapment of immunoglobulins and complement, and one patient had granular immune complexes in the subepithelial and intramembranous portion of the glomerular basement membrane. In one patient, who had nodular glomerular lesions, diffuse fibrillar deposits of electron-dense material were observed in the mesangium. In this mesangial infiltrate, light microscopy revealed the absence of amyloid and direct immunofluorescence revealed the absence of all immunoglobulins, complement components, and fibrinogen. Our study suggests that the morphologic alterations observed in diabetic glomerulopathy might be mediated by either immune mechanism or by abnormal biochemical or functional factors, such as impairment of the mesangial IgA clearance mechanism.