Clearance of HIV-1 or SIV reservoirs by promotion of apoptosis and inhibition of autophagy: Targeting intracellular molecules in cure-directed strategies.

The reservoirs of the HIV display cellular properties resembling long-lived immune memory cells that could be exploited for viral clearance. Our interest in developing a cure for HIV stems from the studies of immunologic memory against infections. We and others have found that long-lived immune memory cells employ prosurvival autophagy and antiapoptotic mechanisms to protect their longevity. Here, we describe the rationale for the development of an approach to clear HIV-1 by selective elimination of host cells harboring replication-competent HIV (SECH). While reactivation of HIV-1 in the host cells with latency reversing agents (LRAs) induces viral gene expression leading to cell death, LRAs also simultaneously up-regulate prosurvival antiapoptotic molecules and autophagy. Mechanistically, transcription factors that promote HIV-1 LTR-directed gene expression, such as NF-κB, AP-1, and Hif-1α, can also enhance the expression of cellular genes essential for cell survival and metabolic regulation, including Bcl-xL, Mcl-1, and autophagy genes. In the SECH approach, we inhibit the prosurvival antiapoptotic molecules and autophagy induced by LRAs, thereby allowing maximum killing of host cells by the induced HIV-1 proteins. SECH treatments cleared HIV-1 infections in humanized mice in vivo and in HIV-1 patient PBMCs ex vivo. SECH also cleared infections by the SIV in rhesus macaque PBMCs ex vivo. Research efforts are underway to improve the efficacy and safety of SECH and to facilitate the development of SECH as a therapeutic approach for treating people with HIV.

The Innate Immune Response to Mycobacterium tuberculosis Infection.

Infection with Mycobacterium tuberculosis causes >1.5 million deaths worldwide annually. Innate immune cells are the first to encounter M. tuberculosis, and their response dictates the course of infection. Dendritic cells (DCs) activate the adaptive response and determine its characteristics. Macrophages are responsible both for exerting cell-intrinsic antimicrobial control and for initiating and maintaining inflammation. The inflammatory response to M. tuberculosis infection is a double-edged sword. While cytokines such as TNF-α and IL-1 are important for protection, either excessive or insufficient cytokine production results in progressive disease. Furthermore, neutrophils-cells normally associated with control of bacterial infection-are emerging as key drivers of a hyperinflammatory response that results in host mortality. The roles of other innate cells, including natural killer cells and innate-like T cells, remain enigmatic. Understanding the nuances of both cell-intrinsic control of infection and regulation of inflammation will be crucial for the successful development of host-targeted therapeutics and vaccines.

The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages.

The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1ß production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.

Rapid infusion of rituximab is well tolerated in children with hematologic, oncologic, and rheumatologic disorders.

Traditional administration of rituximab requires careful titration and may involve many hours to minimize the risk of reactions. The objective of this study was to evaluate the safety of rapid infusions of rituximab in a pilot group of children with hematologic, oncologic, and rheumatologic disorders, and to determine the incidence of rate-related infusion reactions. Twenty patients enrolled in the study. All patients tolerated the rapid infusion of rituximab and no patient had an infusion-related reaction. We conclude that rapid infusions of rituximab are well tolerated and safe in our pilot group of patients.

Diagnosis and management of 365 ureteric injuries following obstetric and gynecologic surgery in resource-limited settings.

INTRODUCTION: Ureteric injuries are among the most serious complications of pelvic surgery. The incidence in low-resource settings is not well documented. METHODS: This retrospective review analyzes a cohort of 365 ureteric injuries with ureterovaginal fistulas in 353 women following obstetric and gynecologic operations in 11 countries in Africa and Asia, all low-resource settings. The patients with ureteric injury were stratified into three groups according to the initial surgery: (a) obstetric operations, (b) gynecologic operations, and (c) vesicovaginal fistula (VVF) repairs. RESULTS: The 365 ureteric injuries in this series comprise 246 (67.4%) after obstetric procedures, 65 (17.8%) after gynecologic procedures, and 54 (14.8%) after repair of obstetric fistulas. Demographic characteristics show clear differences between women with iatrogenic injuries and women with obstetric fistulas. The study describes abdominal ureter reimplantation and other treatment procedures. Overall surgical results were good: 92.9% of women were cured (326/351), 5.4% were healed with some residual incontinence (19/351), and six failed (1.7%). CONCLUSIONS: Ureteric injuries after obstetric and gynecologic operations are not uncommon. Unlike in high-resource contexts, in low-resource settings obstetric procedures are most often associated with urogenital fistula. Despite resource limitations, diagnosis and treatment of ureteric injuries is possible, with good success rates. Training must emphasize optimal surgical techniques and different approaches to assisted vaginal delivery.

Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells.

Mitochondrial reactive oxygen species (mtROS) generated continuously under physiological conditions have recently emerged as critical players in the regulation of immune signaling pathways. In this study we have investigated the regulation of antiviral signaling by increased mtROS production in plasmacytoid dendritic cells (pDCs), which, as major producers of type I interferons (IFN), are the key coordinators of antiviral immunity. The early phase of type I IFN production in pDCs is mediated by endosomal Toll-like receptors (TLRs), whereas the late phase of IFN response can also be triggered by cytosolic retinoic acid-inducible gene-I (RIG-I), expression of which is induced upon TLR stimulation. Therefore, pDCs provide an ideal model to study the impact of elevated mtROS on the antiviral signaling pathways initiated by receptors with distinct subcellular localization. We found that elevated level of mtROS alone did not change the phenotype and the baseline cytokine profile of resting pDCs. Nevertheless increased mtROS levels in pDCs lowered the TLR9-induced secretion of pro-inflammatory mediators slightly, whereas reduced type I IFN production markedly via blocking phosphorylation of interferon regulatory factor 7 (IRF7), the key transcription factor of the TLR9 signaling pathway. The TLR9-induced expression of RIG-I in pDCs was also negatively regulated by enhanced mtROS production. On the contrary, elevated mtROS significantly augmented the RIG-I-stimulated expression of type I IFNs, as well as the expression of mitochondrial antiviral-signaling (MAVS) protein and the phosphorylation of Akt and IRF3 that are essential components of RIG-I signaling. Collectively, our data suggest that increased mtROS exert diverse immunoregulatory functions in pDCs both in the early and late phase of type I IFN responses depending on which type of viral sensing pathway is stimulated.

Iatrogenic genitourinary fistula: an 18-year retrospective review of 805 injuries.

INTRODUCTION AND HYPOTHESIS: Genitourinary fistula poses a public health challenge in areas where women have inadequate access to quality emergency obstetric care. Fistulas typically develop during prolonged, obstructed labor, but providers can also inadvertently cause a fistula when performing obstetric or gynecological surgery. METHODS: This retrospective study analyzes 805 iatrogenic fistulas from a series of 5,959 women undergoing genitourinary fistula repair in 11 countries between 1994 and 2012. Injuries fall into three categories: ureteric, vault, and vesico-[utero]/-cervico-vaginal. This analysis considers the frequency and characteristics of each type of fistula and the risk factors associated with iatrogenic fistula development. RESULTS: In this large series, 13.2 % of genitourinary fistula repairs were for injuries caused by provider error. A range of cadres conducted procedures resulting in iatrogenic fistula. Four out of five iatrogenic fistulas developed following surgery for obstetric complications: cesarean section, ruptured uterus repair, or hysterectomy for ruptured uterus. Others developed during gynecological procedures, most commonly hysterectomy. Vesico-[utero]/-cervico-vaginal fistulas were the most common (43.6 %), followed by ureteric injuries (33.9 %) and vault fistulas (22.5 %). One quarter of women with iatrogenic fistulas had previously undergone a laparotomy, nearly always a cesarean section. Among these women, one quarter had undergone more than one previous cesarean section. CONCLUSIONS: Women with previous cesarean sections are at an increased risk of iatrogenic injury. Work environments must be adequate to reduce surgical error. Training must emphasize the importance of optimal surgical techniques, obstetric decision-making, and alternative ways to deliver dead babies. Iatrogenic fistulas should be recognized as a distinct genitourinary fistula category.

Outcome of intracranial electroencephalography monitoring and surgery in magnetic resonance imaging-negative temporal lobe epilepsy.

We evaluated the outcomes of intracranial electroencephalography (iEEG) recording and subsequent resective surgery in patients with magnetic resonance imaging (MRI)-negative temporal lobe epilepsy (TLE). Thirty-two patients were identified from the Mayo Clinic Epilepsy Surgery Database (Arizona, Florida, and Minnesota). Eight (25.0%) had chronic iEEG monitoring that recorded neocortical temporal seizure onsets; 12 (37.5%) had mesial temporal seizure onsets; 5 (15.6%) had independent neocortical and mesial temporal seizure onsets; and 7 (21.9%) had simultaneous neocortical and mesial seizure onsets. Neocortical temporal lobe seizure semiology was the only factor significantly associated with neocortical temporal seizure onsets on iEEG. Only 33.3% of patients who underwent lateral temporal neocorticectomy had an Engel class 1 outcome, whereas 76.5% of patients with iEEG-guided anterior temporal lobectomy that included the amygdala and the hippocampus had an Engel class 1 outcome. Limitations in cohort size precluded statistical analysis of neuropsychological test data.

Aloe vera downregulates LPS-induced inflammatory cytokine production and expression of NLRP3 inflammasome in human macrophages.

Aloe vera has been used in traditional herbal medicine as an immunomodulatory agent inducing anti-inflammatory effects. However, its role on the IL-1ß inflammatory cytokine production has not been studied. IL-1ß production is strictly regulated both at transcriptional and posttranslational levels through the activity of Nlrp3 inflammasome. In this study we aimed to determine the effect of Aloe vera on the molecular mechanisms of Nlrp3 inflammasome-mediated IL-1ß production in LPS-activated human THP-1 cells and monocyte-derived macrophages. Our results show that Aloe vera significantly reduced IL-8, TNFα, IL-6 and IL-1ß cytokine production in a dose dependent manner. The inhibitory effect was substantially more pronounced in the primary cells. We found that Aloe vera inhibited the expression of pro-IL-1ß, Nlrp3, caspase-1 as well as that of the P2X7 receptor in the LPS-induced primary macrophages. Furthermore, LPS-induced activation of signaling pathways like NF-κB, p38, JNK and ERK were inhibited by Aloe vera in these cells. Altogether, we show for the first time that Aloe vera-mediated strong reduction of IL-1ß appears to be the consequence of the reduced expression of both pro-IL-1ß as well as Nlrp3 inflammasome components via suppressing specific signal transduction pathways. Furthermore, we show that the expression of the ATP sensor P2X7 receptor is also downregulated by Aloe vera that could also contribute to the attenuated IL-1ß cytokine secretion. These results may provide a new therapeutic approach to regulate inflammasome-mediated responses.

Solvent detergent vs. fresh frozen plasma in cirrhotic patients undergoing liver transplant surgery: a prospective randomized control study.

BACKGROUND: Although orthotopic liver transplantation (OLT) is nowadays considered standard practice at experienced centres, it can still be affected by a significant risk of massive bleeding and its related complications. Solvent/detergent plasma (S/D Plasma) has been proposed as an alternative to fresh frozen plasma (FFP) to curtail such complications. This study aimed at evaluating the efficacy of S/D Plasma in OLT patients by comparing it to FFP. MATERIALS AND METHODS: Sixty-three OLT patients were randomized into two groups depending on whether they were transfused with FFP or S/D plasma. A thromboelastography-based protocol aimed at achieving and maintaining predetermined coagulation goals was used to guide plasma transfusions. At the beginning and the end of surgery, standard laboratory coagulation tests were performed together with the assessment of the VII, VIII, V, XII factors and S protein blood levels. RESULTS: The two study groups equally achieved the thromboelastography goals but with a reduced amount of transfusions in the S/D plasma group (P < 0.0001). At the end of surgery, factors V and XII and S protein blood levels were lower in the S/D plasma patients who also showed lower INR, aPTT and antithrombin III levels. CONCLUSION: In cirrhotic patients undergoing OLT, the use of S\D plasma associated with thromboelastography allows the same clinical results but with a significant reduction in the amount of plasma transfusions.

Ragweed pollen extract intensifies lipopolysaccharide-induced priming of NLRP3 inflammasome in human macrophages.

Ragweed pollen extract (RWE) possesses intrinsic NADPH oxidase activity that induces oxidative stress by initiating the production of intracellular reactive oxygen species (ROS). The ROS are important contributors to the manifestation of allergic inflammation; furthermore, concomitant exposure to an allergen and an endotoxin trigger a stronger inflammatory response. One of the main pro-inflammatory cytokines produced in inflammatory responses is interleukin-1ß (IL-1ß), and its production is associated with caspase-1-containing inflammasome complexes. Intracellular ROS have been implicated in NLRP3 inflammasome-mediated IL-1ß production, therefore, we aimed to study whether RWE influences the function of NLRP3 inflammasome. Here we describe that, in the presence of NADPH, RWE significantly elevates lipopolysaccharide-induced IL-1ß production of THP-1 cells as well as human primary macrophages and dendritic cells. We also demonstrate that increased IL-1ß production is mediated through NLRP3 inflammasome in THP-1 macrophages. We provide evidence that RWE elevates cytosolic ROS level in these cells, and ROS inhibitors abolish IL-1ß production. Furthermore, we show that RWE enhances lipopolysaccharide-induced gene transcription/expression of pro-IL-1ß and key components of the inflammasome via a ROS-dependent mechanism.

Use of recombinant factor IX and thromboelastography in a patient with hemophilia B undergoing liver transplantation: a case report.

Hemophilia B is a congenital recessive disorder caused by deficiency of coagulation factor IX (FIX). Surgical procedures can be performed in patients with hemophilia using high-purity and/or recombinant FIX, which has been shown to be safe and effective in surgical hemostasis. Liver transplantation is the only potentially curative treatment available for these patients, providing a long-term phenotypic cure for hemophilia. End-stage liver disease together with hemophilia exposes patients to greater risks of bleeding complications during the perioperative period with consequent difficulties in managing coagulopathy. The limited experiences reported by different investigators and the various strategies for clotting factor replacement make it difficult to define a single approach with respect to the optimal dose and method of administering FIX to achieve perioperative hemostasis. The limits of plasma-based coagulation tests--prothrombin time, activated partial thromboplastin time--have made thromboelastography a valid alternative in this kind of surgery. It has been demonstrated to be a useful tool for real-time analysis of clot formation using a whole-blood assay format. Further, it accurately illustrates the clinical effects of procoagulant or anticoagulant interventions. In this article, we have described the usefulness of thromboelastography to monitor the ability of high-purity FIX supplementation to restore a normal coagulation state and to guide the perioperative administration of blood products in a successful orthotopic liver transplantation in a hemophilic patient with deficiencies of factors IX and X, presenting with hepatitis C virus-related cirrhosis and hepatocellular carcinoma.

A web-based registry of inherited bleeding disorders in the region of Emilia-Romagna: results at three and a half years.

A Registry of inherited bleeding disorders was set up in the Region of Emilia-Romagna (RER) to collect information about these diseases and to improve the quality of care. From January 2003, the eight Haemophilia Centres (HC) in the RER began to use computerized clinical records; every 6 months, they send data to Parma Hospital to be processed and published in a website (http://www.registroemofiliarer.it). Great efforts are made to ensure high quality of data. Results of general interest are included in a free 'public area' and more sensitive data in a 'reserved area' (open only to HC and to health authorities). A total of 610 individuals are included: 249 haemophilia A (HA), 63 haemophilia B (HB), 173 von Willebrand's disease, 69 rare bleeding disorders, seven platelet disorders and 49 haemophilia carriers; 131 were genotyped, 188 were tested for inhibitors (16 affected). The most frequent bleeding was haemarthrosis. The joint score (evaluated in 104 haemophiliacs) was higher in severe HA. There were 22 HIV-positive and 182 hepatitis C virus-positive patients (21% have chronic hepatitis, two hepatocellular carcinoma). In 2005, two patients received primary prophylaxis, 47 secondary prophylaxis, four children were on immune-tolerance induction. From 2003 to 2005 the use of recombinant products was greatly increased and the majority of patients received them. The mean clotting factor consumption for prophylaxis was higher than on-demand treatment. The main features of registry are to collect high quality and comprehensive data of all patients followed by HC, to improve quality of care and it's availability on the web.

Pathophysiology of bleeding in surgery.

Bleeding is a major surgical complication. Although mortality rates of 0.1% are observed for surgical procedures, it may be 5% to 8% for elective vascular surgery, and increase to 20% in the presence of severe bleeding. In major surgery for liver diseases, as well as in cardiac surgery, excessive blood loss is associated with increased mortality, morbidity, and intensive care stay. Approximately 75% to 90% of intraoperative and early postoperative bleeding is due to technical factors. However, in some cases either acquired or congenital coagulopathies may favor, if not directly cause, surgical hemorrhage. Uncontrolled bleeding leads to a combination of hemodilution, hypothermia, consumption of clotting factors, and acidosis, which in turn worsen the clotting process, further exacerbating the problem in a vicious bloody circle. At present, the standard treatment for surgical bleeding is the rapid control of the source of bleeding by either surgical or radiological techniques. Blood-derived products as well as hemostatic agents, such as aprotinin, tranexamic acid, and DDAVP, are widely used to improve hemostatic balance in bleeding patients. Recombinant activated factor VII (rFVIIa) has been reported to be effective for the treatment of surgical or traumatic massive bleeding unresponsive to conventional therapy. Although most reports are anecdotal, and therefore exposed to a "positive" selection bias, the number of cases is impressive, strongly suggesting that in such patients rFVIIa may afford a hemostatic advantage beyond that of conventional replacement therapy.

A simple and safe nomogram for the management of oral anticoagulation prior to minor surgery.

In 80 consecutive, anticoagulated patients scheduled for minor surgery, we reduced warfarin daily dosage by 50% on days 4, 3 and 2 before the surgery, restoring the original dose the day immediately before surgery. The evening after surgery, patients took a double warfarin dose, and then the usual maintenance dose was reintroduced. The mean International Normalized Ratio (INR) value assessed 1 week before surgery was 2.63 (range 1.88-3.87); it decreased at the moment of performing surgery to 1.68 (range 1.42-2.20; P < 0.05 with respect to the preoperative value), and returned to 2.43 7 days after (range 1.96-3.51, P = ns with respect to the preoperative value). No significant difference was found comparing prothrombin fragment 1.2 (F1.2) levels 1 week before surgery and on the morning of surgery (0.49 ng/ml vs 0.67 ng/ml, P = ns), suggesting that no activation of blood coagulation had taken place following the reduction of anticoagulant therapy. Patients developed neither major nor minor bleeding, nor thromboembolism during the procedures or up to 1 month after surgery. In our experience, this method for the management of anticoagulation before minor surgery has been shown to be safe and useful, avoiding the cumbersome shift to either intravenous or subcutaneous heparin.

Platelet responsiveness to L-arginine in hypertensive disorders of pregnancy.

OBJECTIVE: In chronically hypertensive (CH), preeclamptic (PE), and normotensive pregnant women (N), we investigated ex vivo platelet aggregation in response to L-arginine (L-Arg) and sodium nitroprusside (SN), which are respectively the substrate and donor of nitric oxide (NO). METHODS: Platelet aggregation was determined with a dual-channel aggregometer by measuring transmittance of light through the sample in comparison to platelet poor plasma, as a reference. Aggregation induced by adenosine diphosphate was continuously recorded for 3 min and measured before and after preincubation with L-Arg and SN. RESULTS: Preincubation with L-Arg significantly reduced platelet aggregation in N and CH patients (p < 0.05) but not in PE women. Preincubation with SN affected aggregation in PE women also (p < 0.001). No correlation was found between platelet response to L-Arg or SN stimuli and the severity of hypertensive disorders expressed as week of gestation at delivery or birth weight. CONCLUSIONS: The present study demonstrates that a decreased platelet sensitivity to L-Arg characterizes PE women, whereas SN maintains its antithrombotic power. This impairment seems to be specific for PE, because platelets of CH patients utilize L-Arg normally. This finding supports the involvement of the L-Arg-NO pathway in the pathogenesis of the procoagulative features of PE and probably in the onset of the disease. The maintained response to SN in PE patients suggests a possible therapeutical use of NO donors in the disease.

In vitro binding and partitioning of irinotecan (CPT-11) and its metabolite, SN-38, in human blood.

The binding of CPT-11 and SN-38 to human plasma proteins was studied by ultrafiltration at 37 degrees C and pH 7.4. In plasma, CPT-11 was 66-60% bound in the range 100-4000 ng/ml and SN-38 was 94-96% bound in the range 50-200 ng/ml. At these concentrations the plasma binding of CPT-11 was slightly saturable, but the plasma binding of SN-38 was concentration-independent. Albumin was the main carrier of CPT-11 and SN-38 in plasma. In blood, the binding of CPT-11 was moderate (80%), mainly to plasma proteins (47%) and erythrocytes (33%). The binding of SN-38 was high (99%) and most of SN-38 in blood was located in blood cells (approximately 66%) The simulation of a grade 3 hematotoxicity (according to National Cancer Institute's Common Toxicity Criteria grading) on the SN-38 blood distribution yielded an increase in fu (free fraction of drug in plasma) from 1.05 to 2.08 and a decrease in C(Bl)/C(P) from 1.66 to 1.14 (both resulting from a decreased cell binding).