Multiple protein extract microarray for profiling human food-specific immunoglobulins A, M, G and E.

Existing food immunoglobulin (Ig) tests require large volumes of serum, are limited to one immunoglobulin class, are not amenable to high throughput analysis and only give a limited picture of the immunological response to food antigens. Conversely a new generation of Component Resolved Diagnostic systems using pure proteins is highly specific and totally dependent on the availability of the protein in its recombinant or natural origin form. Here we demonstrate a proof-of-concept of a microarray test based on protein extracts of food components. Our approach relies on innovations on three different fronts: the novelty of using arrayed food samples sequentially extracted with detergent and chaotropic agents, the ability to measure four different Ig classes simultaneously and the ability to analyse the generated data via a suitable bioinformatics/statistical analysis interface. This approach combines high numerical power of microarrays with automation, high throughput analysis and enables detailed investigation of the Ig profiles to food antigens. The prototype shown contains extracts of approximately 350 food ingredients that cover most of the food products found in the UK. Here we showed that the use of a sequential extraction technique to solubilise and then denature food samples has its benefits in the assessment of variations in antigenicity when tested with human sera. A patient dependent degree of class specificity was observed with human sera (IgG specificity correlates well with IgA>IgM>>>>>IgE). Besides generating a simultaneous profile for IgA, IgM, IgG and IgE the array system has shown good discrimination between challenge responders in atopic and non-atopic individuals. Poly- and mono-specific IgE responders were easily identified. The mathematical modelling of specific IgE content showed good correlations when compared with established IgE antibody testing assay (UniCAP). Although in its proof-of-principle stages, the immune profiling technique described here has the potential to provide unique insights into exposure/sensitization and establish relationships between specific immunoglobulin classes and subclasses against food protein antigens. In further developments, the immune profiling technique could also be extended to other related areas such as parasite and bacterial gut infection. Full analyses of large longitudinal and retrospective clinical trials are on going to determine the positive and negative predictive values of the technique.

Post-splenectomy splenic artery aneurysm rupture in an atypical presentation of pre-eclampsia.

Splenic artery aneurysm rupture in pregnancy is an uncommon catastrophic event. We report a patient who presented at 15 3/7 weeks with atypical pre-eclampsia. After termination was recommended, the patient chose to continue the pregnancy. Reversal of clinical and laboratory abnormalities occurred and the patient was discharged. The patient presented again at 24 weeks with severe pre-eclampsia and residual splenic artery aneurysm rupture, at the site of a splenectomy that had been performed 24 years previously.

Initial fetal platelet counts predict the response to intravenous gammaglobulin therapy in fetuses that are affected by PLA1 incompatibility.

OBJECTIVE: Fetal alloimmune thrombocytopenia is the result of maternal fetal platelet antigen incompatibility; intracranial hemorrhage is its most serious complication. Our previous studies have demonstrated an inability to accurately predict fetal platelet counts in this disorder. The goal of the present investigation was to identify factors that would predict the response of the fetal platelet count to therapy so that use of fetal blood sampling could be minimized. STUDY DESIGN: Patients who were eligible for the study were all those who (1) had alloimmune thrombocytopenia secondary to Pl(A1) (HPA-1a, Zw(A)) platelet antigen incompatibility, (2) were treated with maternally administered intravenous immunoglobulin at 1 g/kg of body weight per week, with or without low dose steroids, and (3) had percutaneous fetal blood sampling before the initiation of therapy (first fetal blood sampling) and again 3 to 7 weeks afterwards (second fetal blood sampling). RESULTS: In this retrospective review, 74 patients who were affected by alloimmune thrombocytopenia had a median platelet count of 21,000 per microliter at the first fetal blood sampling and 47,000 per microliter at the second fetal blood sampling, with a median increase in platelet count of 24,000 per microliter. Response to treatment was defined as either (1) an improvement in platelet count (the second fetal blood sampling greater than the first fetal blood sampling, and second fetal blood sampling > 20,000 per microliter) or (2) a minimal decline in platelet count (the first fetal blood sampling > or = 40,000 per microliter and the difference between the first and second fetal blood sampling < or = 10,000 per microliter). The first fetal blood sampling had prognostic value for the second fetal blood sampling (P = .0001), although the previous sibling birth platelet count and history of sibling intracranial hemorrhage did not predict the platelet count at the first or second fetal blood sampling or the change in platelet count between the samplings. When the patients were segregated to first fetal blood sampling of > 20,000 per microliter versus < or = 20,000 per microliter, the response rates for the 2 groups were 89% (33/37 patients) versus 51% (19/37 patients; P = .001). CONCLUSION: In fetal alloimmune thrombocytopenia secondary to Pl(A1) platelet antigen incompatibility, fetuses with platelet counts > 20,000 per microliter at the initiation of therapy were predicted to maintain their platelet count at the second fetal blood sampling at > 20,000 per microliter. The characteristics of the previous sibling, as previously reported, did not predict the initial fetal blood sampling, the second fetal blood sampling, or the response to treatment.

Shortened gestational age following multifetal pregnancy reduction: can chronic placental inflammation be the explanation?

OBJECTIVE: This study tests the hypothesis that chronic inflammatory foci in the placentas of siblings that undergo multifetal pregnancy reduction are associated with shortened gestational length. METHODS: Among 446 patients who underwent multifetal pregnancy reduction (MPR), 56 delivered at Mount Sinai Hospital, 37 (66%) had their placentas referred to surgical pathology and 29 (78%) of the 37 patients had tissue sampled from the placenta of the reduced sibling. Slides were reviewed (by C.M.S.) blinded to clinical data. Lesions were diagnosed using previously published criteria. Specifically, inflammatory lesions were correlated with the various perinatal parameters. Non-parametric testing considered p < 0.05 to be significant. RESULTS: Ten (35%) of 29 patients had chronic inflammation in the reduced placenta. Their gestational age at delivery was 33.1 +/- 3.2 weeks, compared to 35.8 +/- 2.3 weeks in those without chronic inflammation (Z = -2.53, p = 0.01). There was no difference between the cases with and those without chronic inflammation in the reduced placenta, in regard to past reproductive history or clinical assessment of the MPR procedure (e.g. the number of attempts, duration of the procedure, or post-procedural complications). CONCLUSION: The majority of patients who underwent MPR did not develop a chronic inflammatory response to the process of 'resorbing' the placental tissues of the reduced sibling. However, a significant number (35%) of women who delivered viable offspring after MPR had chronic inflammation in the placenta, and had a shortened gestational length.

Prenatal diagnosis and management of abnormalities in the urologic system.

We have reviewed the prenatal diagnosis and management of abnormalities in the urologic system. Urologic anomalies may be caused by embryologic aberrations, genetic disease, or a nonrandom association with other structural abnormalities. There is a wide range of prognoses, depending on the cause and the impact of the anomaly on the production of amniotic fluid. Management focuses on obtaining an accurate prenatal diagnosis, providing appropriate counseling, and ensuring the proper surveillance or treatment before and after birth.

Successful pregnancy in severe methylmalonic acidaemia.

Methylmalonic acidaemia is an inborn error of metabolism characterized by recurrent episodes of life-threatening ketoacidosis. With improved and intensive treatment, these patients are living into adulthood, but many experience late-onset disease complications such as chronic renal failure, chronic pancreatitis and osteopenia. We report the successful delivery of a healthy baby to a 20-year-old woman with vitamin B12-unresponsive methylmalonic acidaemia who has these late-onset manifestations of the disease and had plasma methylmalonic acid concentrations of 1900 mumol/L during the first trimester of pregnancy.

Human T cells express specific binding sites for C1q. Role in T cell activation and proliferation.

Although the receptor that binds to the collagen-like domain of human C1q (C1qR) is expressed on a wide variety of cell types, the presence or absence of this receptor on human T lymphocytes has been debatable. The current studies were undertaken to re-examine whether human T cells possess specific binding sites for C1q by using a combination of techniques, including radioligand binding studies, flow cytometric analysis, and epifluorescence imaging techniques. Radioligand binding studies indicate that both peripheral T cells and the cultured T cell line, MOLT4, bind 125I-labeled C1q in a specific and apparently saturable manner, reaching equilibrium within 30 min at 37 degrees C under conditions of subphysiologic (90 mM NaCl) ionic strength. Western blot analysis with anti-C1qR of membrane proteins derived from Raji and MOLT4 cells showed an apparent single band of approximately 60 kDa under nonreducing conditions. Furthermore, when peripheral blood T cells were stimulated with 12,-o-tetradecanoyl phorbol-13-ester acetate for 5 days at 37 degrees C and assessed by FACS for their ability to bind anti-C1qR, the mitogen-induced cells were found to bind 40 to 50% more than their unstimulated counterparts. In addition, both CD4+ and CD8+ T cells were found to bind anti-C1qR. When the cells were mitogen induced with either 12,-o-tetradecanoyl phorbol-13-ester acetate, Con A, or PWM for 48 h in the presence or absence of 50 micrograms/ml C1q then pulsed with 1 microCi [3H]thymidine for 16 h at 37 degrees C, proliferation was significantly inhibited (40 to 80%, n = 7) as assessed by reduced [3H]thymidine incorporation. Taken together, the data suggest that: 1) Human T cells express C1qR in which immunoblots reveal a 60-kDa single chain protein. 2) C1qR expression is up-regulated by mitogens that induce T cell proliferation. 3) The primary ligand, C1q, induces an antiproliferative signal, which suggests that the C1qR plays a role in T cell activation and proliferation. In addition, the data contribute to the characterization of C1qRs on cells in peripheral blood and indicate that all cells, with the exception of erythrocytes, bear functional C1q receptors.