Clinical significance of soluble interleukin-2 receptor measurement in immune-mediated diseases.

A soluble form of the interleukin-2 receptor (sIL-2R) is secreted upon T-cell activation. Increased blood levels of sIL-2R occur in a variety of immunological diseases. Although the biological function of sIL-2R is incompletely understood, both in health and disease, sIL-2R serum measurements are commonly conducted in clinical practice as it may help to facilitate diagnosis of specific immune-mediated diseases, such as haemophagocytic lymphohistiocytosis and sarcoidosis. In these, and in other immune-diseases, sIL-2R levels may be used as a biomarker to monitor/predict disease activity and treatment response. In this review, we will give a brief overview of the biology of the IL-2/IL-2R system and will subsequently discuss the clinical utility of sIL-2R measurement, especially in the context of haemophagocytic lymphohistiocytosis, sarcoidosis, rheumatoid arthritis, systemic lupus erythematosus, juvenile idiopathic arthritis, adult-onset Still's disease, ANCA-associated vasculitis, and IgG4-related disease.

A multicentre verification study of the QuantiFERON®-TB Gold Plus assay.

OBJECTIVES: The aim of this verification study was to compare the QuantiFERON®-TB Gold Plus (QFT-Plus) to the QuantiFERON®-TB Gold In Tube (QFT-GIT). The new QFT-Plus test contains an extra antigen tube which, according to the manufacturer additionally elicits a CD8+ T-cell response above the CD4+ T-cell response. We assessed the value of this tube in detecting recent latent tuberculosis infections. METHODS: Between May 2015 and December 2016, 1031 subjects underwent QFT-Plus and QFT-GIT test. Overall agreement between both tests and performance for different test indications and/or immune states was assessed. A difference of >0.6 IU/mL interferon-γ release between the two antigen tubes of the QFT-Plus assay was considered a true difference and used as estimation for CD8+ T-cell response. RESULTS: Analysis of the QuantiFERON tests resulted in an overall agreement between assays of 95%. Subjects considered to be recently exposed to tuberculosis had significantly more often a true difference in interferon-γ release compared to all other subjects (p = 0.029). CONCLUSION: Results of QFT-Plus are highly comparable to QFT-GIT. Although there is an indication that a true difference in interferon-γ release between the antigen tubes is associated with recent latent tuberculosis infection, the QFT-Plus could not be used to exclude recent exposure.

The clinical utility of JC virus antibody index measurements in the context of progressive multifocal leukoencephalopathy.

In natalizumab-treated patients without previous immunosuppressive treatment, the JCV antibody index is used to stratify PML risk. A high index value indicates that the risk to develop PML is significantly elevated, although probably about 99% of patients with this index value will not develop PML. This minireview aimed to provide an overview of the basic virology and immunology relevant to understanding JCV infections in MS patients, with a focus on what is presently known about antibodies to JCV and how they could be of use to predict and diagnose PML.

A study to investigate the factors that influence the prescribing habits of non-consultant hospital doctors in Ireland.

BACKGROUND: The Health Service Executive estimates it spent just under €2 billion on medicines in 2013 following a fivefold increase in the cost of medicines over the preceding decade. With this increasing cost, it is important to understand what factors affect doctors prescribing. AIMS: To investigate the influencing factors on prescribing of non-consultant hospital doctors (NCHDs) in Irish hospitals and to provide data regarding the sources of information NCHD's use for commonly prescribed drugs. METHODS: All medical manpower offices of adult public hospitals in the Republic of Ireland were emailed with our survey for distribution to NCHDs. It contained demographic information and questions regarding factors which most influence their prescribing of particular drug groups. Tests of significance were carried out using Chi-square. RESULTS: One hundred and seventy-nine surveys were returned out of a possible 8987 (0.02 %). Consultant preference was the biggest overall influencing factor on junior doctors prescribing (27 %). This was closely followed by local departmental policies (26 %). Evidence-based prescribing only influenced 14 % of the total prescribing of NCHDs with the pharmaceutical representative influence only a fraction behind (13 %). Knowledge obtained during medical school greater influenced postgraduate prescribing than undergraduate (34 vs 14 %, p = 0.046). Registrars were significantly more likely to prescribe using evidence-based medicine than intern and SHOs (p = 0.03). CONCLUSIONS: The prescription of medications in Ireland by NCHDs varies greatly depending not only on drug group, but it is also affected by the doctors' previous education and experience. This information is key in leading to sensible cost-effective prescribing.

Bronchoalveolar lavage cell pattern from healthy human lung.

Bronchoalveolar lavage (BAL) is widely accepted as a key diagnostic procedure in interstitial lung diseases (ILD). We performed a study to obtain reference intervals of differential cell patterns in BAL fluid with special attention to the origin of lavage fluid, e.g. bronchial/alveolar, to atopy and smoking status and to age of the healthy people. We performed bronchoalveolar lavage in 55 healthy subjects with known atopy status (age: 18-64 years, non-smokers/smokers: 34/21) and determined differential cell counts and lymphocyte subsets in BAL fluid and blood. Moreover, in a subgroup of non-smoking healthy individuals we measured the expression of the regulatory T cell marker forkhead box protein 3 (FoxP3) on blood and BAL fluid lymphocytes in addition to a comprehensive set of activation markers. Differential cell counts from the alveolar lavage fraction differed significantly from calculated pooled fractions (n = 11). In contrast, marginal differences were found between atopic and non-atopic subjects. Interestingly, the BAL fluid CD4(+) /CD8(+) ratio correlated strongly with age (r(2) = 0·50, P < 0·0001). We consider the bronchial and alveolar fraction to be lavage fluid from fundamentally different compartments and recommend analysis of the alveolar fraction in diagnostic work-up of ILD. In addition, our data suggest that age corrected BAL fluid CD4(+) /CD8(+) ratios should be used in the clinical evaluation of patients with interstitial lung diseases.

Genetic variability in the IL1RN gene and the balance between interleukin (IL)-1 receptor agonist and IL-1ß in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a rapidly progressive interstitial lung disease of unknown aetiology. Interleukin (IL)-1ß plays an important role in inflammation and has been associated with fibrotic remodelling. We investigated the balance between IL-1ß and IL-1 receptor antagonist (IL-1Ra) in bronchoalveolar lavage fluid (BALF) and serum as well as the influence of genetic variability in the IL1B and IL1RN gene on disease susceptibility and cytokine levels. In 77 IPF patients and 349 healthy controls, single nucleotide polymorphisms (SNPs) in the IL1RN and IL1B genes were determined. Serum and BALF IL-1Ra and IL-1ß levels were measured using a multiplex suspension bead array system and were correlated with genotypes. Both in serum and BALF a significantly decreased IL-1Ra/IL-1ß ratio was found in IPF patients compared to healthy controls. In the IL1RN gene, one SNP was associated with both the susceptibility to IPF and reduced IL-1Ra/IL-1ß ratios in BALF. Our results show that genetic variability in the IL1RN gene may play a role in the pathogenesis of IPF and that this role may be more important than thought until recently. The imbalance between IL-1Ra and IL-1ß might contribute to a proinflammatory and pro-fibrotic environment in their lungs.

Objective assessment of the cryoprotective effects of dimethylformamide for freezing goat semen.

The aim of this work was to assess the cryoprotective effects of dimethylformamide (DMF) for freezing goat semen, using an objective analysis by computer-assisted sperm analysis (CASA). Twenty-one ejaculates (seven per animal) were collected from three stud bucks with the aid of an artificial vagina and immediately evaluated for gross and microscopic characteristics. The semen was diluted in two steps with a Tris-egg yolk extender containing 6% glycerol or 6% DMF, frozen in 0.50-mL straws, and stored in liquid nitrogen. Samples were accessed for sperm morphology, sperm membrane structural and functional integrity, and by CASA, immediately after thawing. There were differences (P<0.05) between glycerol and DMF with regard to subjective progressive motility (23.9±2.2% vs. 16.6±2.0%), objective progressive motility (3.5±0.4% vs. 1.8±0.3%), linearity (53.9±1.6% vs. 48.1±1.4%) and amplitude of lateral head (2.3±0.1 vs. 2.9±0.1 mm), which confirmed the efficiency of glycerol. In conclusion, dimethylformamide could be used as an alternative cryoprotectant for goat semen freezing. However it was showed that no benefits were derived by using dimethylformamide to replace glycerol at an equal 6% concentration.

Genetic variation in GREM1 is a risk factor for fibrosis in pulmonary sarcoidosis.

Sarcoidosis is a granulomatous systemic disorder most often affecting the lung. Pulmonary fibrosis develops in approximately 10%-15% of patients with sarcoidosis. The human gene GREM1 encodes gremlin, a member of the bone morphogenetic protein antagonist family. Bone morphogenetic proteins are essential for the maintenance of tissue homeostasis and regeneration after injury. We examined associations between genetic variation in GREM1 and pulmonary disease outcome in patients with pulmonary sarcoidosis. Four common tag single nucleotide polymorphisms spanning GREM1 were genotyped in 483 controls and in 237 sarcoidosis patients with radiographic data at pulmonary disease outcome, defined by chest X-ray after a minimum of 4 years follow-up. Highly significant differences were found between GREM1 genotype frequencies in sarcoidosis patients without chest X-ray abnormalities (stage 0) (n = 116) versus patients who had fibrosis on chest X-ray (stage IV) (n = 59) at pulmonary disease outcome. The most significant association was with GREM1 rs1919364. The recessive model resulted in an increased risk of fibrosis development for homozygous carriers of the C allele at GREM1 rs1919364 versus carriers of the G allele [P = 9.3 × 10⁻7, χ² = 24.1, odds ratio (OR) = 6.37 (2.89-14.1)]. This study is the first to suggest that genetic variation of GREM1 predisposes to pulmonary fibrosis in sarcoidosis patients. Carriers of the GREM1 CC genotype at position rs1919364 were at 6.4 times greater risk for developing fibrosis.

T-cell activation profiles in different granulomatous interstitial lung diseases--a role for CD8+CD28(null) cells?

Lymphocytes play a crucial role in lung inflammation. Different interstitial lung diseases may show distinct lymphocyte activation profiles. The aim of this study was to examine the expression of a variety of activation markers on T lymphocyte subsets from blood and bronchoalveolar lavage fluid (BALF) of patients with different granulomatous interstitial lung diseases and healthy controls. Bronchoalveolar lavage cells and blood cells from 23 sarcoidosis patients, seven patients with hypersensitivity pneumonitis and 24 healthy controls were analysed. Lymphocyte activation status was determined by flow cytometry. Lymphocytes were stained with antibodies against CD3, CD4, CD8, CD25, CD28, CD69, very late antigen-1 (VLA)-1, VLA-4 and human leucocyte antigen D-related (HLA-DR). In general, CD28, CD69 and VLA-1 expression on BALF CD4+ lymphocytes and HLA-DR expression on BALF CD8+ lymphocytes was different in patients with hypersensitivity pneumonitis and sarcoidosis patients with parenchymal involvement. This BALF lymphocyte phenotype correlated with carbon monoxide diffusing lung capacity (Dlco) values across interstitial lung diseases (ILD) (r2 = 0.48, P = 0.0002). In sarcoidosis patients, CD8+CD28(null) blood lymphocytes correlated with lower Dlco values (r = -0.66, P = 0.004), chronic BALF lymphocyte activation phenotype (r2 = 0.65, P < 0.0001), radiographic staging (stage I versus stage II and higher, P = 0.006) and with the need for corticosteroid treatment (P = 0.001). Higher expression of CD69, VLA-1 and HLA-DR and lower expression of CD28 on BALF lymphocytes suggests prolonged stimulation and chronic lymphocyte activation in patients with ILD. In sarcoidosis, blood CD8+CD28(null) cells might be a new biomarker for disease severity but needs further investigation.

Variation in IL7R predisposes to sarcoid inflammation.

Sarcoidosis is a chronic granulomatous disorder characterized by a massive influx of Th1 lymphocytes. Both naive and memory T cells express high levels of interleukin 7 receptor-alpha (IL7R alpha), encoded by the IL7R gene. The purpose of this study was to investigate the role of the IL7R gene region in susceptibility to sarcoidosis. Six common single-nucleotide polymorphisms (SNPs) spanning IL7R were genotyped and analyzed in 475 sarcoidosis patients and 465 healthy controls. Replication of one significant associated SNP was carried out in 206 independent sarcoidosis patients, 127 controls and 126 patients with Löfgren's disease. The rs10213865 SNP was associated with sarcoidosis (P=0.008), and in silico analysis showed a complete linkage (r(2)=1, D'=1) with a functional nonsynonymous coding SNP in exon 6 (rs6897932, T244I). Combined analysis of 663 individuals with sarcoidosis and 586 controls (homozygous carriers of risk allele, P=5 x 10(-4), odds ratio=1.49 (1.19-1.86)) provided strong statistical support for a genuine association of IL7R with the risk of sarcoidosis. In addition, we report the same trend between variation in the IL7R gene and patients with Löfgren's disease, suggesting that variation in IL7R may confer general risk for developing granulomatous lung disease.

Effect of variation in ITGAE on risk of sarcoidosis, CD103 expression, and chest radiography.

The integrin alpha(E)beta(7) is believed to play a key role in retention of lymphocytes in mucosal tissues of gut, urogenital tract and lung. Five common single nucleotide polymorphisms spanning ITGAE, the gene encoding the alpha(E) (CD103) unit, were genotyped in 556 sarcoidosis patients and 465 controls. The -1088 A/G polymorphism was associated with sarcoidosis (P=0.004). An increased risk of disease was found for homozygous carriers of the A allele vs. carriers of the G allele (P=0.001, odds ratio=1.63 [1.22-2.17]). Analysis of lymphocytes from bronchoalveolar lavage and in vitro functional tests showed higher percentages of CD103+CD4+ T cells for the sarcoidosis risk genotype. Radiographic staging at disease outcome revealed prevalence of -1088 AA genotype in patients with fibrosis (P=0.01). A higher proportion of CD103+CD4+ T cells and ITGAE -1088 AA genotype might be associated with fibrosis formation in pulmonary sarcoidosis.

The importance of hand anatomy in the accident and emergency department: assessment of hand anatomy knowledge in doctors in training.

Good anatomical knowledge is essential for the early recognition of severe or significant hand injuries in the Accident and Emergency (A&E) department, in particular nerve, vascular or tendon injuries. In 1992, Murphy and Olney assessed hand anatomy knowledge in junior doctors. We have repeated this study 16 years on. The 2008 cohort performed worse in response to every question asked and in some areas significantly so. We discuss the results in relation to the recognition of serious injuries and also with regards to anatomy teaching in medical schools and at postgraduate level.

Trypanosoma vivax nos tecidos testicular e epididimário de ovinos experimentalmente infectados / Trypanosoma vivax in testicular and epidydimal tissues of experimentally infected sheep

Quatro ovinos machos, com cerca de 12 meses de idade (Ovinos 1-4), foram infectados por via intravenosa com aproximadamente 1,25x10(5) tripomastigotas de Trypanosoma vivax, outros quatro ovinos (Ovinos 5-8) destinaram-se ao grupo controle. Após a infecção, exames clínicos visando avaliar temperatura retal, freqüências cardíaca e respiratória e parasitemia foram realizados diariamente por 30 dias, tempo estabelecido para o término do experimento. A avaliação do hematócrito foi realizada a cada cinco dias. Ao final do período experimental, os animais foram castrados e os testículos e epidídimos submetidos ao exame anatomopatológico. Amostras destes órgãos dos Ovinos 1, 4 e 5 foram tomadas para a realização da reação em cadeia da polimerase (PCR). Os parâmetros clínicos (hipertermia, aumento das freqüências cardíaca e respiratória, aumento de volume dos linfonodos e palidez das mucosas) mantiveram-se para o grupo infectado acima dos valores mostrados pelo grupo controle durante todo o período experimental. A parasitemia foi observada a partir do 3º dia pós-infecção (dpi) com picos nos 6-10os dpi e nos 15-18os dpi. Os Ovinos 1 e 4 apresentaram, a partir do 25º dpi, anemia acentuada. Macroscopicamente, todos os testículos dos animais do grupo infectado apresentaram-se flácidos e com coloração pálida. Microscopicamente, observaram-se degeneração testicular moderada a acentuada, epididimite multifocal e hiperplasia do epitélio epididimário. A análise por PCR de T. vivax nos tecidos testicular e epididimário resultou em 100% de positividade para ovinos infectados experimentalmente. As lesões epididimárias e testiculares associadas à presença do parasita nesses órgãos, detectada por PCR, sugerem a participação do parasita no mecanismo etiopatogênico de danos reprodutivos.(AU)

Four adult sheep (number 1, 2, 3 and 4), all males, were inoculated intravenously with 1ml of blood containing 1.25x10(5) trypomastigotes of Trypanosoma vivax, and Sheep 5, 6, 7 and 8 were used as control. After infection, clinical exams considering rectal temperature, respiratory and cardiac frequencies, and parasitaemia were recorded daily for a 30-day experiment period. Blood samples were obtained for 5-day intervals to hematocrit analysis. At the end of the experimental period, the sheep were orquiectomized. Testes and epididymides from these animals were studied anatomopathologically. Samples from these tissues of Sheep 1, 4 and 5 were taken to polymerase chain reaction (PCR). Clinical parameters remained for the infected group above the values observed in the control group during the experimental period. Parasitaemia was observed on day 3 post-infection, and the highest values occurred between day 6 and 10, and day 15 and 18 post-infection. Sheep 1 and 4 showed severe anemia on day 25 post-infection. All sheep of the infected group showed flabby and palid testes. Histologically, moderate to severe testicular degeneration, multifocal epididymitis and hyperplasia of epididymal epithelium were observed. The result of T. vivax PCR analysis in the testes and epididymal tissues was positive in 100% of the samples of the experimentally infected sheep. Epididymal and testicular lesions associated with the presence of the parasite in these tissues, shown by PCR, suggest the participation of T. vivax in the pathophysiological mechanism of reproductive damage.(AU)

Angiotensin-converting enzyme genotype may predict survival following major trauma.

BACKGROUND: As a key component of the endocrine renin-angiotensin system (RAS), angiotensin-converting enzyme (ACE) regulates circulatory homeostasis. Meanwhile, the local RAS influences tissue growth, inflammatory and metabolic responses. The absence (deletion, D) rather than the presence (insertion, I) of a 287 base pair fragment in the ACE gene is associated with higher circulating and tissue ACE activity, with excess mortality in critical illness (including adult acute respiratory distress syndrome and paediatric meningococcal infection) and with worse functional outcome from traumatic brain injury. OBJECTIVE: To determine if the ACE genotype is associated with mortality following major trauma. METHODS: 41 subjects with major trauma admitted to the Royal London Hospital over a 2-year period via the Helicopter Emergency Medical Service were enrolled. ACE genotype was available in 36. Injury Severity Score (ISS), Revised Trauma Score (RTS), age, sex and outcome data were recorded for each. ACE genotype was determined from leucocyte DNA using well described techniques. RESULTS: The presence of one or more D alleles was associated with a mortality of 36.4% compared with 7.1% for II alleles (p = 0.048). Age (p = 0.044) also predicted mortality whereas RTS (p = 0.08) and ISS (p = 0.46) did not. ACE genotype was significantly associated with RTS but not age or ISS. CONCLUSION: The ACE D allele may be associated with mortality from major trauma. Replication of these findings in larger studies may aid definition of high-risk subgroups that would benefit from early intensive management. New therapeutic targets might also be suggested.

Linkage between Toll-like receptor (TLR) 2 promotor and intron polymorphisms: functional effects and relevance to sarcoidosis.

The intracellular pathogens Propionibacterium acnes and Mycobacterium tuberculosis have been leading suspects as the cause of sarcoidosis, a systemic disorder characterized by the formation of non-caseating granulomas. Toll-like receptor (TLR) 2 is important in the innate immune response against both pathogens, and is therefore of interest in sarcoidosis research. In the present study, three single nucleotide polymorphisms and one dinucleotide repeat polymorphism in the TLR-2 gene were genotyped in 419 sarcoidosis patients, divided into a study cohort and a validation cohort, and 196 healthy controls. In the study cohort we found a significant increase in prevalence of the AA-genotype at promotor location -16934 in patients with chronic disease compared to patients with acute/self-remitting sarcoidosis (34.5% versus 15.9%, respectively, P = 0.006, P(c) = 0.019). These results could not be confirmed in our validation cohort, implicating a possible role for TLR-2 genetics in only a small percentage of sarcoidosis patients. Furthermore, linkage was found between the promotor polymorphism -16934 A/T and the number of GT repeats in intron 1 (P < 0.0001). After in vitro stimulation of peripheral blood mononuclear cells (PMBCs) with different TLR-2 agonists, a correlation between induction of TNF-alpha (P = 0.008), interleukin (IL)-12 (P = 0.008) as well as IL-6 (P = 0.02), and the number of GT repeats was observed. In conclusion, the data show that polymorphisms in TLR-2 might be important in a small group of sarcoidosis patients and that their functional consequences explain partly some of the variance in cytokine pattern observed in different clinical phenotypes of this disease.

Impairment of coagulation by commonly used resuscitation fluids in human volunteers.

BACKGROUND: This study compared the effects of two commonly used resuscitation fluids on whole blood coagulation. METHODS: 1000 ml of two resuscitation fluids each (saline and Gelofusine) were given to eight volunteers in a crossover design with a 2-week washout period. The effect on whole blood coagulation was assessed using the Sonoclot analyzer, a conventional coagulation screen and coagulation markers. RESULTS: No significant effect was found on whole blood coagulation by giving saline (time to peak clot increased by a mean of 106 s; (95% confidence interval (CI) -140 to 354), whereas Gelofusine delayed the time to peak by a mean of 845 s (95% CI 435 to 1255). By contrast, there was no change in the conventional coagulation screen with either fluid. CONCLUSION: It was concluded that some resuscitation fluids have an effect on clot formation that is not shown by the conventional coagulation screen, but is disclosed only if the whole coagulation process is studied.

The effects of commonly used resuscitation fluids on whole blood coagulation.

OBJECTIVES: Evidence on the effect of crystalloid and colloid resuscitation fluids on coagulation is confusing, with contradictory results from previous studies. This study was performed to test the effect on whole blood coagulation of a range of resuscitation fluids in vitro using a single method at a single dilution. METHODS: Seven resuscitation fluids were tested in vitro at a dilution of 40%. Whole blood coagulation was measured using a Sonoclot analyser. RESULTS: A crystalloid/colloid split of effect on coagulation in vitro was not seen. The time to clot formation with Gelofusine, dextran and hydroxyethyl starch was a greatly increased, whereas saline and Haemaccel had little effect, or were slightly procoagulant. CONCLUSIONS: Some resuscitation fluids have a profound effect on coagulation. The confusion in the literature may result from the effect on coagulation being both fluid and dilution dependent, with no simple crystalloid/colloid split.

Does calcium cause the different effects of Gelofusine and Haemaccel on coagulation?

BACKGROUND: Gelofusine (which does not contain calcium) has a greater effect on coagulation than Haemaccel (which contains 6.25 mmol/l of calcium). This in vitro study was performed to assess whether calcium might be the cause of the different effects on coagulation. METHODS: Three solutions were compared; (a) Gelofusine, (b) Gelofusine with calcium added to 6.25 mmol/l, and (c) Haemaccel. Thromboelastography (Sonoclot) was used to examine whole blood coagulation, with time to peak clot weight as the primary outcome measure. RESULTS: There was no significant difference between the Gelofusine containing solutions. Both Gelofusine solutions gave a greater impairment of coagulation than the Haemaccel solution. CONCLUSIONS: The different effect of Gelofusine on coagulation compared with Haemaccel does not seem to be related to the different calcium contents of the solutions.