Improved quantification of protein in vaccines containing aluminum hydroxide by simple modification of the Lowry method

Aluminum (Al) components in vaccines are known to act as adsorbents that interfere with accurate protein quantification by the Lowry method. Therefore, certain modifications based on the characteristics and compositions of the vaccine are required for determination of protein contents.We investigated the effects of an additional centrifugal separation and found that protein contents were overestimated by up to 238% without centrifugation through a collaborative study performed with hepatitis B vaccines containing Al. However, addition of a centrifugation step yielded protein concentrations that were similar to the actual values, with small coefficients of variation (CVs). Proficiency testing performed in 11 laboratories showed that four laboratories did not have satisfactory results for vaccines containing aluminum hydroxide, although all laboratories were proficient in protein analysis when samples did not contain aluminum hydroxide. Incomplete resuspension of aluminum hydroxide solution with alkaline copper solution was the major cause of insufficient proficiency in these laboratories.     

The development and validation of methods for evaluating the immune system in preweaning piglets

The preweaning piglet has been found to be a valuable research model for testing ingredients used in infant formula. As part of the safety assessment, the neonates' immune system is an important component that has to be evaluated. In this study three concurrent strategies were developed to assess immune system status. The methods included (1) immunophenotying to assess circulating innate immune cell populations, (2) monitoring of circulating cytokines, particularly in response to a positive control agent, and (3) monitoring of localized gastrointestinal tissue cytokines using immunohistochemistry (IHC), particularly in response to a positive control agent. All assays were validated using white papers and regulatory guidance within a GLP environment. To validate the assays precision, accuracy and sample stability were evaluated as needed using a fit for purpose approach. In addition animals were treated with proinflammtory substances to detect a positive versus negative signal. In conclusion, these three methods were confirmed to be robust assays to evaluate the immune system and GIT-specific immune responses of preweaning piglets.     

Monitoring minimal residual disease in peripheral blood in B-lineage acute lymphoblastic leukaemia

The use of peripheral blood rather than marrow has potential advantages for monitoring minimal residual disease during the treatment of leukaemia. To determine the feasibility of using blood, we used a sensitive polymerase chain reaction method to quantify leukaemia in the blood and marrow in 35 paired samples from 15 children during induction treatment. Leukaemic cells in the blood ranged from 1.1 × 10⁻² to < 9.4 × 10⁻⁷ leukaemic cells/total cells, corresponding to 1.3 × 10⁷ to < 2 × 10³ leukaemic cells/l. In 15 paired samples, leukaemia could be quantified in both tissues and in 20 paired samples, leukaemia was not detected in one or both tissues so that only upper level limits could be set. In the former 15 pairs, the level of leukaemia in peripheral blood was directly proportional to that in marrow but was a mean of 11.7-fold lower. Leukaemia in blood was detected in 10/12 pairs in which the level in marrow was > 10⁻⁴, but in only two of 13 pairs in which the level in marrow was < 10⁻⁵. Patients studied at multiple time-points showed parallel declines in the number of leukaemic cells in both tissues. The results showed that leukaemia could be monitored in peripheral blood during induction therapy, and quantitative considerations based on the results suggest that monitoring of blood during post-induction therapy may be of value in detecting molecular relapse.     

Jejunal secretion of secretory immunoglobulins and gliadin antibodies in celiac disease

The aim of this study was to determine the secretion of secretory immunoglobulins and gliadin antibodies in the small bowel in celiac disease. Twenty-four patients were investigated by perfusion of a defined jejunal segment. Four of the patients studied had a serum IgA deficiency and had no measurable amounts of secretory IgA in the perfusion fluid. The other patients demonstrated a significant increase in the jejunal concentration of secretory IgA (median 28.5 mg/liter) compared with healthy controls (median 16 mg/liter,N=16) and of IgM, celiac (median 12.3 mg/liter) compared to healthy controls (median 6.8 mg/liter,N=16). Jejunal IgA gliadin antibodies were detected in all patients except those with an IgA deficiency. All patients had jejunal IgM gliadin antibodies, but none of the patients had measurable jejunal IgG gliadin antibodies. A positive correlation was detected between serum and jejunal IgA gliadin antibody levels in the celiac patients, (P<0.01). Calculation of the ratio between gliadin antibodies and total levels of IgA and IgM in serum and jejunal perfusate demonstrated that the jejunal synthesis of gliadin antibodies of IgA and IgM type is both more pronounced and persistent than the systemic humoral immune response to gliadin.This work was supported by grants from the Swedish Medical Research Council, the Swedish Life Insurance Companies' Trust for Medical Research, and Th.C. Berghs Foundation and Pharmacia AB, Sweden.     

Imaging Risk in Multisystem Inflammatory Diseases

Rheumatic diseases are immune-mediated inflammatory multisystem diseases with frequent cardiovascular manifestations including perimyocarditis, valvular disease, coronary artery disease, heart failure with or without preserved ejection fraction, pulmonary hypertension, aneurysms, and thrombosis. Echocardiography, carotid ultrasonography, cardiac computed tomography, cardiac magnetic resonance imaging, and positron emission tomography are valid diagnostic tools for the detection of the cardiovascular complications of the multisystem diseases that frequently determine prognosis. Furthermore, the findings of these methods may offer additive risk stratification in asymptomatic patients over the conventional risk scores used to assess cardiovascular risk in the primary prevention setting. Finally, the imaging methods offer a unique opportunity to monitor the effects of treatment on atherosclerotic lesions, coronary microcirculatory dysfunction, myocardial inflammation and fibrosis. However, studies are needed to investigate whether improvement of imaging markers by treatment or selection of treatment according to its effects on surrogate imaging markers is linked to improved prognosis.     

Prognostic Impact of Ischemic Mitral Regurgitation Severity and Myocardial Infarct Quantification by Cardiovascular Magnetic Resonance

ObjectivesThis study sought to evaluate the role of cardiac magnetic resonance (CMR) for the quantification of ischemic mitral regurgitation (IMR) and myocardial infarct size (MIS) in patients with ischemic cardiomyopathy (ICM). This study also sought to explore the interaction between IMR severity and MIS and its association with outcomes in patients with ICM.BackgroundIMR occurs secondary to a disease of the left ventricle and is associated with poor outcomes. The role of CMR for the evaluation and risk stratification of patients with ICM and IMR remains uncertain.MethodsConsecutive patients with ICM who underwent baseline CMR were included. MIS was quantified on late gadolinium enhancement imaging as the proportion of left ventricular mass. IMR was quantified with CMR by calculating the mitral regurgitant fraction (MRFraction). Cox proportional hazards models were built to assess the association of IMR and MIS quantification with the combined endpoint of all-cause death or heart transplant.ResultsWe evaluated 578 patients (mean age: 62 ± 11 years, 76% males). The mean left ventricular ejection fraction was 25 ± 11%, with an MIS of 24 ± 16% and MRFraction of 18 ± 17%. Over a median follow-up time of 4.9 years, 198 (34%) patients experienced death or cardiac transplant. On multivariable analysis, after comprehensive medical risk score, subsequent revascularization, implantable cardioverter-defibrillator insertion, and surgical mitral valve intervention were controlled for, the interaction of IMR severity and MIS emerged as a powerful predictor of adverse outcomes (p = 0.008). For patients with significant IMR (MRFraction: ≥35%), the hazard ratio comparing moderate MIS (15% to 29%) versus small MIS (<15%) was 1.51 (0.57 to 3.98), and the hazard ratio comparing large MIS (≥30%) versus small MIS was 5.41 (2.34 to 12.7).ConclusionsRisk associated with IMR is more comprehensively described as an interaction between IMR severity and MIS. Further studies in patients IMR using comprehensive CMR evaluation are needed to verify whether this approach can improve patient selection and procedural outcomes to address IMR.