Memory B cells at successive stages of differentiation. Affinity maturation and the role of IgD receptors

The following evidence, mainly presented here, suggests that IgD receptors play a crucial role in determining the potential for affinity maturation in memory B cell populations. IgD receptors are present on the first memory B cells to appear after priming. These memory cells give rise to more-mature memory cells that have lost their IgD receptors. The proportions of early (IgD(+)) and mature (IgD(-)) memory cells found in individual donors vary with time, priming conditions, and the availability of T cell help, and both populations frequently coexist for long periods of time. IgD(+) and IgD(-) memory cells carry IgG receptors and give rise to IgG responses with identical isotype representation in adoptive recipients. IgD(+) memory cells, however, always give rise to predominantly low-affinity antibody responses, whereas IgD(-) memory cells consistently generate responses of substantially higher average affinity. This affinity differential is maintained between early and mature memory populations in the same donor and does not appear to be a result of selective differentiation of higher-affinity IgD(+) memory cells into the IgD(-) memory pool. Thus, the selective forces responsible for affinity maturation appear to operate mainly in mature memory cell populations that have already lost IgD receptors; or, stated conversely, little or no selection towards high-affinity memory appears to occur among memory cells that retain IgD receptors. In discussing these findings, we suggest that the IgD receptors themselves are responsible for maintaining early memory populations at a lower average affinity than IgD(-) populations in the same animal. The IgD receptors, we argue, serve to increase the antigen-binding capacity of lower-affinity memory cells so that these cells can survive, expand, and differentiate (to IgD(-)) at antigen concentrations that select against expansion of low- affinity memory cells no longer carrying IgD receptors. Thus, when antigen is limiting, IgD(-) memory populations will be selectively expanded to higher average affinities, whereas coexisting IgD(+) populations will retain their initial affinity profile. This hypothesis suggests that mechanisms that regulate expression and loss of IgD receptors are central to the adaptability of the immune system in its response to invading pathogens. Two related roles can be envisioned for the IgD receptors in this regard. First, they extend the lower boundary of the affinity range of early memory cell populations induced by a given antigenic stimulus and therefore broaden the diversity of responses obtainable from these populations. Secondly, they support the persistence of low-affinity memory populations under conditions where antigen becomes limiting and eventually disappears. These persisting populations then serve as a diversely reactive reservoir from which mature memory populations can be drawn with higher affinities either for the original antigen or, more importantly, for related antigens that the animal may subsequently encounter. Thus the existence of IgD receptors on early memory cells maintains the full range of response diversity despite ongoing selective expansion of (mature) memory populations to produce antibodies with high combining affinities for individual antigens. The flexibility inherent in such an organizational system, we believe, could be expected to account for the evolutionary development of IgD receptors and the regulatory capabilities that support operation of the system.     

Augmentation of spontaneous macrophage-mediated cytolysis by eosinophil peroxidase

Eosinophil peroxidase (EPO), a cationic protein purified from horse blood, adhered to four different types of tumor cells, markedly potentiating their lysis by preformed or enzymatically generated H(2)0(2) (up to 76-fold, as assayed in serum-containing tissue culture medium without supplemental halide). Similarly, compared with uncoated tumor cells, EPO-coated tumor cells were up to 32 times more sensitive to lysis when incubated with macrophages or granulocytes whose respiratory burst was triggered by PMA. However, EPO-coated tumor cells were also readily lysed by bacillus Calmette- Guerin-activated macrophages in the absence of exogenous triggering agents. This spontaneous cytolysis was rapid (50 percent at 2 h) and potent (50 percent lysis at macrophage/tumor cell ratios of 1.5 to 4.6), and was observed with both a peroxide-sensitive tumor (TLX9) and a peroxide-resistant tumor (NK lymphoma). Under the conditions used, neither EPO alone nor macrophages alone were spontaneously cytolytic. Neither EPO nor EPO-coated tumor cells triggered a detectable increment in H(2)0(2) release from macrophages. Nonetheless, spontaneous macrophage-mediated cytolysis of EPO- coated tumor cells was completely inhibitable by catalase (50 percent inhibition, 23 U/ml), although not by heated catalase, indicating a requirement for H(2)0(2). Cytolysis was also completely inhibitable by azide (50 percent inhibition, 2.6 X 10 -5 M), indicating a requirement for enzymatic activity of EPO. Thus, a cytophilic peroxidase from eosinophils and H(2)0(2) spontaneously released from activated macrophages interacted synergistically in a physiologic medium to destroy tumor cells.     

Can the reading for serologic reactivity following 37 degrees C incubation be omitted?

The need to detect antibodies that agglutinate and/or hemolyze red cells (RBCs) directly at 37 degrees C, but do not react in subsequently performed indirect antiglobulin tests (IATs), is of concern relative to the streamlining and automation of antibody detection methods. To determine incidence and significance of such reactions, data from 87,480 tests, which used low-ionic-strength saline, 10-minute incubation at 37 degrees C, and anti-IgG, were analyzed for unexpected antibodies. There were 3590 positive tests, of which 475 showed reactions at 37 degrees C but not in subsequently performed IATs (37 + IAT-). Of these, 196 reactions were due to autoantibodies or other factors usually considered insignificant with respect to the survival of transfused incompatible RBCs, 176 were due to alloantibodies of questionable clinical significance (M, Lea, P1, etc.), and 103 were associated with alloantibodies of potential clinical significance (63 E, 27 K, 5 Jka, 4 D, 3 cE, and 1 C). This latter reaction was seen in 72 patients, with two 37 + IAT-antibodies occurring in each of 3 patients. Of the 75 potentially significant 37 + IAT-antibodies, 57 were seen in patients recently exposed to homologous RBCs, 13 in patients with a history of transfusion and/or pregnancy, and 5 in patients with no known exposure to homologous RBCs. IAT reactivity was observed in subsequent samples with 27 of these antibodies. The predictive value of a 37 + IAT-test was 21.7 percent for a potentially significant antibody. The incidence was 0.12 percent of all tests for unexpected antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment of a system to standardize acceptability criteria for alanine aminotransferase activity in donated blood

A multilaboratory study was conducted to develop a system for standardizing alanine aminotransferase (ALT) acceptability criteria ("cutoffs") for donated blood. Without standardized cutoffs, each laboratory must develop its own cutoff, and this may not make optimal use of ALT testing to reduce transmission of non-A, non-B hepatitis (NANB). Defining an ALT acceptability criterion in absolute terms is necessary because relative cutoffs based on local donor populations may be affected by the prevalence of NANB in each community. This study involved 16 laboratories using 23 different analytic systems. The ALT results of the analysis of a plasma reference sample could be used to translate mathematically a single, absolute cutoff to units applicable to each analytic system. The distribution of ALT results in 1.4 million donations from across the country was established; basing the cutoff on this sample avoids the problems inherent in using a local donor base to establish a cutoff. We propose the implementation of a system to standardize ALT acceptability criteria to an activity level defined by analysis of a nationwide donor sample.     

Procedure‐related pain among adult patients with hematologic malignancies

Background: Cancer patients undergo numerous invasive diagnostic procedures. However, there are only sparse data on the characteristics and determinants for procedure‐related pain among adult cancer patients. Methods: In this prospective study, we evaluated the characteristics and determinants of procedure‐related pain in 235 consecutive hematologic patients (M/F:126/109; median age 62 years, range 20–89 years) undergoing a bone marrow aspiration/biopsy (BMA) under local anesthesia. Questionnaires were used to assess patients before‐, 10 min and 1–7 days post BMA. Using logistic regression models, we calculated odds ratios (ORs) and 95% confidence intervals (CIs). Results: 165/235 (70%) patients reported pain during BMA; 92 (56%), 53 (32%) and 5 (3%) of these indicated moderate [visual analogue scale (VAS)≥30 mm], severe (VAS>54 mm) and worst possible pain (VAS=100 mm), respectively. On multivariate analyses, pre‐existing pain (OR=2.60 95% CI 1.26–5.36), anxiety about the diagnostic outcome of BMA (OR=3.17 95% CI 1.54–6.52), anxiety about needle‐insertion (OR=2.49 95% CI 1.22–5.10) and low employment status (sick‐leave/unemployed) (OR=3.14 95% CI 1.31–7.55) were independently associated with an increased risk of pain during BMA. At follow‐up 10 min after BMA, 40/235 (17%) patients reported pain. At 1, 3, 6 and 7 days post BMA, pain was present in 137 (64%), 90 (42%), 43 (20%) and 25 (12%) patients, respectively. Conclusions: We found that 3/4 of hematologic patients who underwent BMA reported procedural pain; one third of these patients indicated severe pain. Pre‐existing pain, anxiety about the diagnostic outcome of BMA or needle‐insertion, and low employment status were independent risk factors.     

Review of the Lynch syndrome: history, molecular genetics, screening, differential diagnosis, and medicolegal ramifications

Platelets have a central role in the development of arterial thrombosis and subsequent cardiovascular events. An appreciation of this complex process has made antiplatelet therapy the cornerstone of cardiovascular disease management. However, numerous patients will experience a recurrent atherothrombotic vascular event despite adequate antiplatelet therapy. Individual differences in the rate of platelet activation and reactivity markedly influence normal hemostasis and the pathological outcome of thrombosis. Such an individual variability is largely determined by environmental and genetic factors. These are known to either hamper platelets' response to agonists, and thereby mimic the pharmacological modulation of platelet function or mask therapy effect and sensitize platelets. In this article, we reviewed the antiplatelet mechanisms of aspirin and clopidogrel and the possible role of different polymorphisms, which may affect the efficacy of antiplatelet therapy. Heterogeneity in the way patients respond to aspirin and clopidogrel may in part reflect variation in cyclooxygenase (COX)-1, COX-2, glycoprotein (GP) Ib alpha, GP Ia/IIa, GP IIb/IIIa, UGT1A6*2, P2Y₁, P2Y₁₂, CYP2C9, CYP3A4 and CYP3A5 genotypes.     

Pharmacogenetic characterization of indacaterol,a novel β2-adrenoceptor agonist

Background and purpose:

Indacaterol is a novel β₂-adrenoceptor agonist in development for the treatment of chronic obstructive pulmonary disease. The aim of this study was to investigate the comparative pharmacology of indacaterol in recombinant cells expressing the common polymorphic variants of the human β₂-adrenoceptor and in human primary airway smooth muscle (ASM) cells.

Experimental approach:

Chinese hamster ovarian-K1 cell lines expressing high and low levels of the common human β₂-adrenoceptor variants were generated [Gly16-Glu27-Val34-Thr164(GEVT), RQVT, GQVT] and also the rare GQVI variant. Human primary ASM cells were isolated from explants of trachealis muscle. Adenosine-3′,5′-cyclic-monophosphate production was used as an outcome measure.

Key results:

In both the low- and high-expression recombinant GEVT ‘wild type’ cell lines indacaterol is a high-efficacy agonist. Salmeterol and formoterol were identified as low- and high-efficacy agonists, respectively, and showed similar potencies to indacaterol irrespective of the β₂-adrenoceptor genotype. The I164 variant cell line was associated with a reduced capacity to generate adenosine-3′,5′-cyclic-monophosphate in response to β₂-adrenoceptor agonist. In the human primary ASM cells indacaterol gave a maximal response intermediate between that of salmeterol and formoterol.

Conclusions and implications:

These data demonstrate that indacaterol is a high-efficacy agonist in recombinant cell systems but acts with lower efficacy in human primary ASM cells. No marked genotype-dependent effects were observed for common variants; however, changes in I164 receptor activity were identified, which were dependent on the level of expression of β₂-adrenoceptors.     

Functional entrapment of the popliteal artery

A case of functional entrapment missed at the initial angiogram is presented. The imaging of popliteal artery entrapment syndrome and functional entrapment is discussed. The importance of appropriate imaging is emphasized. The classification of popliteal artery entrapment syndrome is discussed and it is proposed that functional entrapment is added to the existing classification in the interest of consistent reporting.