The importance of controls in immunohistochemistry: how to validate an antibody, from research to diagnosis

The specificity of an immunohistochemical reaction is guaranteed by two sets of controls. Positive controls verify the specificity of the primary antibody and demonstrate that it binds only to the protein which was used as an immunogen. Negative controls ensure that the labelling technique is specific and that the primary antibody is responsible for generation of the immunostaining. In fact, the production of a labelling may also be related to cross reactivity or to non-specific physical or chemical interactions. This paper reviews the characteristics of various epitopes and antibodies, describes different strategies which prove the specificity of the immunohistochemical reaction in research or diagnostic pathology and point towards the essential information which should be reported in a paper.     

BNP/NT-proBNP: what is the best choice in an emergency laboratory?

BNP and NT-proBNP are both well established as diagnostic and prognostic markers for congestive heart failure (CHF). However it remains for the biologist to choose between these two biomarkers depending on his equipment availability. The aim of this study was to compare results obtained with the Biosite Triage BNP assay and the Dade Behring NT-proBNP assay with regards to the clinical status. One hundred twelve patients (average age 76 +/- 13 years) with acute dyspnea were including and stratified by diagnosis at presentation into 3 groups: patients without acute CHF (group I, n=50), patients with non-cardiac dyspnea and CHF history (group II, n=22) and patients with acute CHF (group III, n=40). Levels of both BNP and NT-proBNP were higher among patients with cardiac dyspnea (group III) than among patients with a non-cardiac dyspnea (BNP=740 pg/mL versus 84 pg/mL; p<0.001 / NT-proBNP=7.502 pg/mL versus 499 pg/mL; p<0.001). ROC analysis for BNP or NT-proBNP were not statistically different in patients with acute CHF (group III) compared with patients with a non-cardiac dyspnea (group I + II) (AUC=0.927 versus AUC=0.930, p=0.90). Neither there was a difference between ROC analysis for BNP or NT-proBNP in patients with cardiac dyspnea (group III) compared to patients with a non cardiac dyspnea (group I) (AUC=0.981 versus AUC=0.975, p=0.76).Measurement of BNP or NT-proBNP is of identical interest for the diagnosis of acute CHF in acute dyspnea. The BNP Biosite assay was faster because analysis is performed on whole blood. With regards to analytical performance, the NT-proBNP Dade Behring assay had a higher accuracy and is highly recommended for the follow-up of CHF treatment.     

CD14 and CD169 expression in human lymph nodes and spleen: specific expansion of CD14+CD169- monocyte-derived cells in diffuse large B-cell lymphomas

The mononuclear phagocyte system of human lymphoid tissue comprises macrophages and dendritic cells (DCs). The heterogeneity of the non-DC mononuclear phagocyte population in human lymphoid tissue has been little addressed. Here, we studied the expression of 2 monocyte-derived markers, CD14 and CD169 (sialoadhesin), in reactive human lymphoid tissue as well as in a series of 51 B-cell lymphomas by immunohistochemistry on paraffin-embedded tissue. We confirmed that lymph node sinusoidal monocyte-derived cells were the only population staining for CD169. Although most sinusoidal histiocytes also expressed CD14, monocyte-derived cells with phagocytosis such as erythrophagocytosis, anthracosis, or tingible bodies macrophage lacked CD14 and CD169. Among B-cell lymphomas, splenic marginal zone lymphoma was the only one associated with an expansion of the CD14(+)CD169(+) cells in the cords. With respect to nodal B-cell lymphomas, CD14(+) cells were rare among B-chronic lymphocytic leukemia, follicular lymphoma (FL), mantle cell lymphoma (MCL). However, strikingly, we found a strong expansion of CD14(+)CD169(-) cells in numerous diffuse large B-cell lymphomas (DLBCLs), except in cases associated with numerous mitoses, apoptotic bodies, and tingible bodies macrophages. When cultivated in granulocyte/macrophage colony stimulating factor/interleukin 4, DLBCL purified CD14(+) cells differentiate into plasmacytoid cells, expressing DC-specific intercellular adhesion molecule 3-grabbing nonintegrin, suggesting dendritic cell differentiation potential. Our observation fits well with the lymph node and host response cluster signatures described in the gene profiling signatures of DLBCL. However, the role of this CD14(+) population that may constitute a microenvironment-related marker of this subgroup of DLBCL remains to be determined.     

Cognitive-behavioral therapy for women with lifelong vaginismus: a randomized waiting-list controlled trial of efficacy

Women with lifelong vaginismus (N=117) were randomly assigned to cognitive-behavioral group therapy, cognitive-behavioral bibliotherapy, or a waiting list. Manualized treatment comprised sexual education, relaxation exercises, gradual exposure, cognitive therapy, and sensate focus therapy. Group therapy consisted of ten 2-hr sessions with 6 to 9 participants per group. Assistance with minimal-contact bibliotherapy consisted of 6 biweekly, 15-min telephone contacts. Twenty-one percent of the participants left the study before posttreatment assessment. Intent-to-treat analysis revealed that successful intercourse at posttreatment was reported by 14% of the treated participants compared with none of the participants in the control condition. At the 12-month follow-up 21% of the group therapy participants and 15% of the bibliotherapy participants, respectively, reported successful intercourse. Cognitive-behavioral treatment of lifelong vaginismus was thus found to be efficacious, but the small effect size of the treatment warrants future efforts to improve the treatment.     

Kinetics of gene expression in murine cutaneous graft-versus-host disease

The kinetics of gene expression associated with the development of cutaneous graft-versus-host disease (GVHD) were examined in a mouse model of MHC-matched allogeneic hematopoietic stem cell transplantation. Ear skin was obtained from recipient mice with or without GVHD between 7 and 40 days after transplantation for histopathological analysis and gene expression profiling. Gene expression patterns were consistent with early infiltration and activation of CD8(+) T and mast cells, followed by CD4(+) T, natural killer, and myeloid cells. The sequential infiltration and activation of effector cells correlated with the histopathological development of cutaneous GVHD and was accompanied by up-regulated expression of many chemokines and their receptors (CXCL-1, -2, -9, and -10; CCL-2, -5, -6, -7, -8, -9, -11, and -19; CCR-1 and CCR-5), adhesion molecules (ICAM-1, CD18, Ly69, PSGL-1, VCAM-1), molecules involved in antigen processing and presentation (TAP1 and TAP2, MHC class I and II, CD80), regulators of apoptosis (granzyme B, caspase 7, Bak1, Bax, and BclII), interferon-inducible genes (STAT1, IRF-1, IIGP, GTPI, IGTP, Ifi202A), stimulators of fibroblast proliferation and matrix synthesis (interleukin-1beta, transforming growth factor-beta1), and markers of keratinocyte proliferation (keratins 5 and 6), and differentiation (small proline-rich proteins 2E and 1B). Many acute-phase proteins were up-regulated early in murine cutaneous GVHD including serum amyloid A2 (SAA2), SAA3, serpins a3g and a3n, secretory leukocyte protease inhibitor, and metallothioneins 1 and 2. The kinetics of gene expression were consistent with the evolution of cutaneous pathology as well as with current models of disease progression during cutaneous GVHD.     

The experience of mental death: the core feature of complex posttraumatic stress disorder

Exposure to extreme interpersonal stress, exemplified by the experience of torture, represents a threat to the psychological integrity of the victim. The experience is likely to result in mental death, in the loss of the victim's pretrauma identity. Mental death is characterized by loss of core beliefs and values, distrust, and alienation from others, shame and guilt, and a sense of being permanently damaged. Mental death is a primary feature of a distinct posttrauma syndrome, complex posttraumatic stress disorder (PTSD), which is refractory to standard exposure therapies. We identify cognitive mechanisms that mediate the symptoms of complex PTSD, and suggest how current treatments need to be modified to obtain enhanced treatment outcomes.     

Should we treat congenital heart block with fluorinated corticosteroids?

Cardiac involvement is the most severe manifestation of neonatal lupus. It is usually diagnosed as an advanced (second or third degree) atrio-ventricular (AV) congenital heart block (CHB) starting around 20 weeks of gestation. At this gestational time, a window of opportunity might exist, and fluorinated corticosteroids (FS) have been attempted as a therapeutic option. Unfortunately the quality of the studies is methodologically low, due to the retrospective design and to the rarity of autoimmune CHB. Some case reports and small case series basically suggested a possible utility of FS, mainly in the context of incomplete CHB, while larger observational retrospective studies did not find any efficacy of FS either on mortality, on the rate of pacemaker implantation, to reduce the degree of incomplete CHB or to reduce the development of cardiomyopathy. Of note the distinction between complete and incomplete AV block may be very difficult and time consuming in utero. Overall FS should not be recommended in CHB, but might be considered for a short time for a recent incomplete CHB.     

Identification of a new cluster of T-cell receptor delta recombining elements

Within the human T-cell receptor delta (TCRD) gene we have identified a new cluster of seven delta recombining elements (deltaRec2.1-2.7), located 2.6-5.2 kilobases downstream of the Vdelta2 gene segment. The deltaRec2 elements are isolated recombining signal sequences (RSS), which were shown to rearrange with the Ddelta3 and Jdelta1 segments of the TCRD gene as well as with the psiJalpha of the TCRA gene. Rearrangements involving the deltaRec2 elements were found in all peripheral blood (PB) samples from 10 healthy individuals, although their frequency was about 100-fold lower than that of classical deltaRec rearrangements. The total frequency of deltaRec2 rearrangements was lower in PB T lymphocytes, as compared with thymocytes, suggesting that they are deleted during T-cell development. The decrease of the frequency of the deltaRec2-Ddelta3 rearrangements was most prominent: 11 times lower in PB T lymphocytes than in thymocytes. Since the deltaRec2-Jdelta1 rearrangements contained the Ddelta3 segment in the junctional region, we assume that they are derived from the deltaRec2-Ddelta3 rearrangements. In contrast, the majority of deltaRec2-psiJalpha rearrangements did not contain the Ddelta3 segment, indicating that they are single step rearrangements. The deltaRec2-Jdelta1 and deltaRec2-psiJalpha rearrangements seem to be T-lineage specific, but the deltaRec2-Ddelta3 rearrangements were also found at very low frequencies in B lymphocytes and natural killer cells. Our results suggest that deltaRec2 rearrangements are transient steps in the recombinatorial process of the TCRAD locus and are probably deleted by subsequent Valpha-Jalpha rearrangements. We hypothesize, that in a similar manner to the classical deltaRec rearrangements, the deltaRec2 rearrangements might also contribute to T-cell differentiation towards the TCR-alphabeta lineage.     

Heme oxygenase: evolution,structure, and mechanism

Heme oxygenase has evolved to carry out the oxidative cleavage of heme, a reaction essential in physiological processes as diverse as iron reutilization and cellular signaling in mammals, synthesis of essential light-harvesting pigments in cyanobacteria and higher plants, and the acquisition of iron by bacterial pathogens. In all of these processes, heme oxygenase has evolved a similar structural and mechanistic scaffold to function within seemingly diverse physiological pathways. The heme oxygenase reaction is catalytically distinct from that of other hemoproteins such as the cytochromes P450, peroxidases, and catalases, but shares a hemoprotein scaffold that has evolved to generate a distinct activated oxygen species. In the following review we discuss the evolution of the structural and functional properties of heme oxygenase in light of the recent crystal structures of the mammalian and bacterial enzymes.