Fatal Acute Hemolytic Transfusion Reaction due to Anti-Wra.

BACKGROUND: The Wra blood group antigen is a low-frequency antigen. Antibody screening sets used in pretransfusion laboratory investigations usually do not contain a Wr(a+) cell. If subsequent cross-matching is performed without indirect antiglobulin test (IAT), Wra antibodies reacting with donor red blood cells (RBCs) will be missed. For reasonable economic and time-saving arguments the risk of missing the detection of a potential clinically relevant antibody is worldwide accepted. CASE REPORT: A 66-year-old women with a negative antibody screen rapidly deteriorated after she received two units of RBCs for symptomatic anemia after hip surgery. Diagnosis of a transfusion reaction was obscured by pre-existing and nonspecific symptoms. Laboratory investigation indicated acute hemolysis. Cross-matching in IAT was positive for the first unit, and an extended antibody identification panel showed reactivity with Wr(a+) cells. The patient did not respond to supportive therapy and died within 48 h after the start of transfusion. CONCLUSION: This dramatic case provides further evidence on the clinical relevance of Wra blood group antibodies. In addition, it underlines the clinical importance of risk awareness in the blood transfusion chain and the possible complexity in relation to patient monitoring in daily transfusion practice.

Cetrorelix suppression test to assess the source of androgen overproduction in postmenopausal hirsutism.

UNLABELLED: A 75-year-old woman presenting with recent onset hirsutism and severely elevated serum androgen levels was evaluated to assess the source of excessive androgen production. Commonly recommended hormonal stimulation and suppression tests, and the usually employed imaging techniques were non-diagnostic. In this report, we describe a new suppression test based on the use of the GnRH receptor antagonist, cetrorelix, to determine whether androgen production was LH-dependent. Cetrorelix, administered in a daily dose of 250 microg subcutaneously, suppressed serum LH within 24 h and reduced serum androgen levels to normal within 48-72 h, indicating that androgen overproduction was of ovarian origin. This diagnosis was confirmed by laparoscopic ovariectomy. CONCLUSION: The cetrorelix suppression test is a simple procedure that provides valuable information regarding the source of androgen excess in postmenopausal hirsutism.

The small GTPase Rap1 is activated by turbulence and is involved in integrin [alpha]IIb[beta]3-mediated cell adhesion in human megakaryocytes.

The small GTPase Rap1, which is activated by a large variety of stimuli, functions in the control of integrin-mediated cell adhesion. Here we show that in human megakaryocytes and several other commonly used hematopoietic cell lines such as K562, Jurkat, and THP-1, stress induced by gentle tumbling of the samples resulted in rapid and strong activation of Rap1. This turbulence-induced activation could not be blocked by inhibitors previously shown to affect Rap1 activation in human platelets, such as the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and various protein kinase C inhibitors. Also inhibition of actin cytoskeleton dynamics did not influence this activation of Rap1, suggesting that this activation is mediated by cell surface receptors. Human platelets, however, were refractory to turbulence-induced activation of Rap1. To determine the consequences of Rap1 activation we measured adhesion of megakaryocytes to fibrinogen, which is mediated by the integrin alphaIIbbeta3, in the presence of inhibitors of Rap1 signaling. Introduction of both Rap1GAP and RalGDS-RBD in the megakaryoblastic cell line DAMI strongly reduced basal adhesion to immobilized fibrinogen. This inhibition was partially rescued by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate but not by alpha-thrombin. From these results we conclude that in megakaryocytes turbulence induces Rap1 activation that controls alphaIIbbeta3-mediated cell adhesion.

Binding of protein kinase B to the plakin family member periplakin.

The serine/threonine kinase protein kinase B (PKB/c-Akt) acts downstream of the lipid kinase phosphoinositide 3-kinase (PI3K) and functions as an essential mediator in many growth-factor-induced cellular responses such as cell cycle regulation, cell survival and transcriptional regulation. PI3K activation generates 3'-phosphorylated phosphatidylinositol lipids (PtdIns3P) and PKB activation requires PtdIns3P-dependent membrane translocation and phosphorylation by upstream kinases. However PKB activation and function is also regulated by interaction with other proteins. Here we show binding of PKB to periplakin, a member of the plakin family of cytolinker proteins. Interaction between PKB and periplakin was mapped to part of the pleckstrin homology (PH) domain of PKB, which is probably not involved in lipid binding, and indeed binding to periplakin did not affect PKB activation. We therefore investigated the possibility that periplakin may act as a scaffold or localization signal for PKB. In cells endogenous periplakin localizes to different cellular compartments, including plasma membrane, intermediate filament structures, the nucleus and mitochondria. Overexpression of the C-terminal part of periplakin, encompassing the PKB binding region, results in predominant intermediate filament localization and little nuclear staining. This also resulted in inhibition of nuclear PKB signalling as indicated by inhibition of PKB-dependent Forkhead transcription factor regulation. These results suggest a possible role for periplakin as a localization signal in PKB-mediated signalling.

The small GTPase Rap1 is required for Mn(2+)- and antibody-induced LFA-1- and VLA-4-mediated cell adhesion.

In T-lymphocytes the Ras-like small GTPase Rap1 plays an essential role in stimulus-induced inside-out activation of integrin LFA-1 (alpha(L)beta(2)) and VLA-4 (alpha(4)beta(1)). Here we show that Rap1 is also involved in the direct activation of these integrins by divalent cations or activating antibodies. Inhibition of Rap1 either by Rap GTPase-activating protein (RapGAP) or the Rap1 binding domain of RalGDS abolished both Mn(2+)- and KIM185 (anti-LFA-1)-induced LFA-1-mediated cell adhesion to intercellular adhesion molecule 1. Mn(2+)- and TS2/16 (anti-VLA-4)-induced VLA-4-mediated adhesion were inhibited as well. Interestingly, both Mn(2+), KIM185 and TS2/16 failed to induce elevated levels of Rap1GTP. These findings indicate that available levels of GTP-bound Rap1 are required for the direct activation of LFA-1 and VLA-4. Pharmacological inhibition studies demonstrated that both Mn(2+)- and KIM185-induced adhesion as well as Rap1-induced adhesion require intracellular calcium but not signaling activity of the MEK-ERK pathway. Moreover, functional calmodulin signaling was shown to be a prerequisite for Rap1-induced adhesion. From these results we conclude that in addition to stimulus-induced inside-out activation of integrins, active Rap1 is required for cell adhesion induced by direct activation of integrins LFA-1 and VLA-4. We suggest that Rap1 determines the functional availability of integrins for productive binding to integrin ligands.