Alexander J. Indrikovs

miRNA : The new gene silencer

MicroRNAs (miRNAs) can be defined as small, noncoding sets of 19 to 24 nucleotides that have been associated with messenger RNA expression. miRNAs are members of a class of small regulatory RNAs that includes small interfering RNAs (siRNAs). miRNAs regulate the expression of downstream gene targets, including transcription factors, oncogenes, and tumor suppressor genes. Transcriptional profiling using genomic microarrays and beads has enabled the discovery of numerous miRNAs that are differentially expressed in normal tissues vs tumors and associated with cancer development, diagnosis, and prognosis. miRNA signatures can be used to detect and classify cancer and predict the severity of disease, with certain profiles of miRNA expression linked to aggressive cancers with advanced disease present at diagnosis. miRNAs have also become targets of novel anticancer gene therapy with antisense molecules that can inhibit miRNA activity currently being tested for their efficacy in a strategy of reducing miRNA activity on reporter genes bearing miRNA-binding sites. In the future, sophisticated genomic and proteomic techniques combined with complex bioinformatics data analyses will be required to translate these recent basic science discoveries to clinically useful diagnostic tests.

Human IgA-inducing protein from dendritic cells induces IgA production by naive IgD+ B cells.

Over the last several years, there has been a great deal of progress in characterizing the role of dendritic cells (DCs) in the activation and modulation of B cells. DC-secreted chemokines can induce B cell trafficking to the lymph nodes. DC-produced survival factors such as B cell-activating factor of the TNF family and a proliferation-inducing ligand have been shown to be essential for B cell maturation, but have also been implicated in class-switch recombination and B cell lymphoma survival. Recently added to this list of DC-derived factors effecting B cells is IgA-inducing protein (IGIP). In this study, we characterize production of IGIP by human DCs, and examine its capacity to induce IgA class switching and differentiation of naive B cells in vitro. Monocyte-derived DCs were cultured in vitro with TLR agonists (TLR3, 4, 5, and 9) and other factors, including CD40 ligand, GM-CSF, and IL-4 as well as the neuropeptide vasoactive intestinal peptide. Under in vitro stimulation with vasoactive intestinal peptide and CD40L, IGIP mRNA expression could be up-regulated as much as 35-fold above nonstimulated samples within 12–48 h. Naive B cells cultured with exogenous recombinant human IGIP produced IgA in greater quantities than nonstimulated controls. Finally, we demonstrate that IGIP stimulation drives the production of μ-α switch circles from IgM+IgD+ naive human B cells, indicating its role as an IgA switch factor.

A Prospective Audit Program to Determine Blood Component Transfusion Appropriateness at a Large University Hospital: A 5-Year Experience

Predetermined transfusion guidelines, pretransfusion approval, and transfusion audits are useful tools in the education of those ordering blood components, potentially resulting in the reduction of inappropriate use of blood components. Our institution requires mandatory prospective audits for a proportion (10%) of packed red blood cell unit orders and all orders for fresh frozen plasma, platelets, and cryoprecipitate. Cases where the blood bank physician recommends against a transfusion and the ordering physician concurs, or when blood components are released against blood banks recommendation, are referred to the transfusion committee. Transfusion committee members review the medical records to determine the circumstances surrounding the transfusion request as well as patient outcomes relating to their receiving or not receiving the transfusion. We analyzed 220 transfusion episodes brought before the transfusion committee from 2001 to 2005. The most requested blood component denied or changed was fresh frozen plasma. With only a few exceptions, the denial or change of blood components had no adverse effect on the patient. Nonetheless, these interventions were deemed appropriate by the transfusion committee. In most cases, blood components released based on the demand of the ordering physician, despite the advice of the blood bank physician, were deemed as inappropriate transfusions. This study therefore suggests that prospective audits of blood component orders can help reduce inappropriate transfusions and can be a valuable educational tool for the ordering physicians as well as for residents in training.

Transplantation of High Panel-Reactive Antibody Left Ventricular Assist Device Patients Without Crossmatch Using On-Bypass Pheresis and Alemtuzumab

BACKGROUND Highly sensitized (HS) left ventricular assist device (LVAD) patients with high panel-reactive antibody (PRA) levels present a challenge. Alemtuzumab, a potent depleting agent for T and B lymphocytes (months to years), and plasmapheresis, offer an opportunity for heart transplantation to these patients who might die of VAD complications on the transplant waiting list. This study compared rates of acute rejection and survival of a HS LVAD cohort with a contemporaneous control group after heart transplant. METHODS Clinical courses of 31 consecutive patients who underwent transplantation between January 2006 and January 2011 were reviewed. Eight patients with a T or B PRA of 70 or more (HS+) received non-crossmatched, ABO-compatible hearts using intraoperative plasmapheresis and alemtuzumab induction. Controls (HS-) received basiliximab induction. Acute rejection was defined as International Society for Heart and Lung Transplantation grades 2R or higher, or antibody-mediated rejection. RESULTS The difference in survival between HS+ and HS- groups at 1 year (100% vs 94%) or at a mean follow-up of 2.3 and 2.4 years (75% vs 70%) was not significant. Retrospective lymphocytotoxic crossmatches were positive in 7 of 8 HS+ patients (6 T+ and B+, 1 B+) vs none in the HS- group (p < 0.001). There was a trend toward increased risk of cellular rejection per 100 patient-days beyond 1 year in the HS+ group (p = 0.07). Risk of humoral rejection was significantly increased in the HS+ group (38% vs 4%; p = 0.04). CONCLUSIONS Heart transplantation with plasmapheresis and alemtuzumab in HS LVAD patients, most with a positive crossmatch, does not compromise midterm survival. The expected higher rates of rejection, especially beyond the first postoperative year, demand adjustments in surveillance strategies and immunosuppressive management.

Sequestration of anti-Cra in the placenta : serologic demonstration by placental elution

BACKGROUND: Few cases of antibodies to the Cromer (a) antigen have been described during pregnancy. Interestingly, the anti‐Cra titers decreased during pregnancy, and although the newborns were Cra+, the direct antiglobulin tests (DATs) were negative and hemolytic disease of the newborn (HDN) was not observed. Cromer antigens reside on the decay‐accelerating factor (DAF), which is expressed on the fetal derived syncytiotrophoblast layer of the placenta. It has been postulated that Cromer antibodies are not transported to the fetus, but are bound to placental DAF, thereby protecting the fetus from HDN and causing the disappearance of Cromer antibody in maternal plasma. This report is the first to demonstrate Cromer antibody sequestration by the placenta.

Case Scenario: Emergency Reversal of Oral Anticoagulation

REVERSING warfarin-induced anticoagulation quickly and effectively can be challenging in medically compromised patients presenting for emergency surgery. Anticoagulation reversal poses a delicate balance between increasing the risk of clotting and decreasing the risk of intraoperative blood loss. Furthermore, variable individual responses and warfarin’s adverse effect profile render particular dosing challenges. Consequently, meticulous monitoring of anticoagulated patients with prothrombin time and international normalized ratio (INR) must guide titration. Traditionally, fresh frozen plasma (FFP) has been the mainstay of warfarin reversal. Herein, we present a case in which we administered prothrombin complex concentrate (PCC) instead of FFP to hasten INR correction, reduce volume requirements, and diminish immune-related risks.

Closed-Loop– and Decision-Assist–Guided Fluid Therapy of Human Hemorrhage

Objectives: We sought to evaluate the efficacy, efficiency, and physiologic consequences of automated, endpoint-directed resuscitation systems and compare them to formula-based bolus resuscitation. Design: Experimental human hemorrhage and resuscitation. Setting: Clinical research laboratory. Subjects: Healthy volunteers. Interventions: Subjects (n = 7) were subjected to hemorrhage and underwent a randomized fluid resuscitation scheme on separate visits 1) formula-based bolus resuscitation; 2) semiautonomous (decision assist) fluid administration; and 3) fully autonomous (closed loop) resuscitation. Hemodynamic variables, volume shifts, fluid balance, and cardiac function were monitored during hemorrhage and resuscitation. Treatment modalities were compared based on resuscitation efficacy and efficiency. Measurements and Main Results: All approaches achieved target blood pressure by 60 minutes. Following hemorrhage, the total amount of infused fluid (bolus resuscitation: 30 mL/kg, decision assist: 5.6 ± 3 mL/kg, closed loop: 4.2 ± 2 mL/kg; p < 0.001), plasma volume, extravascular volume (bolus resuscitation: 17 ± 4 mL/kg, decision assist: 3 ± 1 mL/kg, closed loop: –0.3 ± 0.3 mL/kg; p < 0.001), body weight, and urinary output remained stable under decision assist and closed loop and were significantly increased under bolus resuscitation. Mean arterial pressure initially decreased further under bolus resuscitation (–10 mm Hg; p < 0.001) and was lower under bolus resuscitation than closed loop at 20 minutes (bolus resuscitation: 57 ± 2 mm Hg, closed loop: 69 ± 4 mm Hg; p = 0.036). Colloid osmotic pressure (bolus resuscitation: 19.3 ± 2 mm Hg, decision assist, closed loop: 24 ± 0.4 mm Hg; p < 0.05) and hemoglobin concentration were significantly decreased after bolus fluid administration. Conclusions: We define efficacy of decision-assist and closed-loop resuscitation in human hemorrhage. In comparison with formula-based bolus resuscitation, both semiautonomous and autonomous approaches were more efficient in goal-directed resuscitation of hemorrhage. They provide favorable conditions for the avoidance of over-resuscitation and its adverse clinical sequelae. Decision-assist and closed-loop resuscitation algorithms are promising technological solutions for constrained environments and areas of limited resources.

Effect of Freezing Plasma at −20°C for 2 Weeks on Prothrombin Time, Activated Partial Thromboplastin Time, Dilute Russell Viper Venom Time, Activated Protein C Resistance, and d-Dimer Levels

To assess the impact of preanalytical variables of time and temperature on prothrombin time (PT), activated partial thromboplastin time (aPTT), dilute Russell viper venom time (DRVVT), activated protein C resistance (APCR), and d-dimer, samples from 23 healthy individuals and 18 patients having coagulopathy with known abnormal PT and aPTT were collected. Plasma from each individual was separately pooled and aliquoted; the first 2 aliquots were stored at room temperature then analyzed at 2 hours (baseline) and 4 hours postcollection. The remaining aliquots were stored at −20°C and thawed for analysis at 48 hours, 1, and 2 weeks. In both healthy participants and participants with coagulopathy, PT, aPTT, APCR, DRVVT, and D-dimer had no significant changes at 4 and 48 hours, and 1 and 2 weeks postcollection compared to baseline, or the changes were less than 10%. The results indicate PT, aPTT, DRVVT, APCR, and d-dimer can be stored for 2 weeks at −20°C without compromising clinical interpretation in both healthy individuals and individuals with coagulopathy. Increasing storage time will facilitate sample processing from off-site clinics.