Survival of transfused red blood cells from a donor with alpha-thalassemia trait in a recipient with sickle cell disease.

BACKGROUND: Post-transfusion survival of donor red blood cells (RBCs) is important for effective chronic transfusion therapy in conditions including sickle cell disease (SCD). Biotin labeling RBCs allows direct in vivo measurement of multiple donor RBC units simultaneously post-transfusion. STUDY DESIGN AND METHODS: In an observational trial of patients with SCD receiving monthly chronic transfusion therapy, aliquots of RBCs from one transfusion episode were biotin-labeled and infused along with the unlabeled RBC units. Serial blood samples were obtained to measure RBC survival. Donor units were tested for RBC indices, hemoglobin fractionation, and glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. For microcytic donor RBCs (MCV < 70 fL), HBA1 and HBA2 genetic testing was performed on whole blood. RESULTS: We present one recipient, a pediatric patient with SCD and splenectomy who received two RBC units with aliquots from each unit labeled at distinct biotin densities (2 and 18 µg/mL biotin). One donor unit was identified to have microcytosis (MCV 68.5 fL after biotinylation); whole blood sample obtained at a subsequent donation showed 2-gene deletion alpha-thalassemia trait (ɑ-3.7kb/ɑ-3.7kb) and normal serum ferritin. G6PD activity was >60% of normal mean for both. The RBCs with alpha-thalassemia RBC had accelerated clearance and increased surface phosphatidylserine post-transfusion, as compared with the normocytic RBC (half life 65 vs. 86 days, respectively). DISCUSSION: Post-transfusion RBC survival may be lower for units from donors with alpha-thalassemia trait, although the impact of thalassemia trait donors on transfusion efficacy requires further study.

Post-transfusion biotin-labeled red blood cell survival studies in pediatric sickle cell disease with antibodies of uncertain significance.

BACKGROUND: Red blood cell (RBC) antibodies are common in multiply transfused patients with sickle cell disease (SCD). Unlike RBC alloantibodies, the potential of autoantibodies to cause post-transfusion hemolysis may be uncertain. Biotin-labeling provides a direct measurement of red cell survival (RCS) over time, thus can be used to assess the clinical significance of RBC antibodies. Antibodies to biotinylated RBC (B-RBC) occasionally are detected after exposure, which may impact B-RBC survival in subsequent RCS studies. STUDY DESIGN AND METHODS: Pediatric patients with SCD receiving monthly chronic transfusions underwent RCS studies, receiving aliquots of allogeneic RBC labeled at distinct densities of biotin (2-18 µg/mL). B-RBC survival was followed for 4 months post-transfusion, and B-RBC antibody screening for 6 months. Patients with warm autoantibodies (WAA) or B-RBC antibodies are reported here. RESULTS: RBC antibodies were detected during RCS in four patients: one with WAA, one with WAA followed by B-RBC-specific antibodies, and two with transient B-RBC antibodies within the first 5 weeks of exposure. B-RBC half-lives (T50) ranged 37.6-61.7 days (mean 47.8 days). There was no evidence of increased hemolysis or accelerated B-RBC clearance in the presence of WAA or B-RBC antibodies. DISCUSSION: Biotinylation of allogenic RBC can be used to assess the possible effects of RBC antibodies on transfusion survival in individual cases, particularly when it is uncertain if the detected antibodies may result in hemolysis. In the cases presented here, neither WAA nor B-RBC antibodies were associated with significant shortening of B-RBC survival in individuals with SCD.

Identification of Protein Targets of S-Nitroso-Coenzyme A-Mediated S-Nitrosation Using Chemoproteomics.

S-Nitrosation is a cysteine post-translational modification fundamental to cellular signaling. This modification regulates protein function in numerous biological processes in the nervous, cardiovascular, and immune systems. Small molecule or protein nitrosothiols act as mediators of NO signaling by transferring the NO group (formally NO+) to a free thiol on a target protein through a transnitrosation reaction. The protein targets of specific transnitrosating agents and the extent and functional effects of S-nitrosation on these target proteins have been poorly characterized. S-nitroso-coenzyme A (CoA-SNO) was recently identified as a mediator of endogenous S-nitrosation. Here, we identified direct protein targets of CoA-SNO-mediated transnitrosation using a competitive chemical-proteomic approach that quantified the extent of modification on 789 cysteine residues in response to CoA-SNO. A subset of cysteines displayed high susceptibility to modification by CoA-SNO, including previously uncharacterized sites of S-nitrosation. We further validated and functionally characterized the functional effects of S-nitrosation on the protein targets phosphofructokinase (platelet type), ATP citrate synthase, and ornithine aminotransferase.

Engineering a Therapeutic Protein to Enhance the Study of Anti-Drug Immunity.

The development of anti-drug antibodies represents a significant barrier to the utilization of protein-based therapies for a wide variety of diseases. While the rate of antibody formation can vary depending on the therapeutic employed and the target patient population receiving the drug, the antigen-specific immune response underlying the development of anti-drug antibodies often remains difficult to define. This is especially true for patients with hemophilia A who, following exposure, develop antibodies against the coagulation factor, factor VIII (FVIII). Models capable of studying this response in an antigen-specific manner have been lacking. To overcome this challenge, we engineered FVIII to contain a peptide (323-339) from the model antigen ovalbumin (OVA), a very common tool used to study antigen-specific immunity. FVIII with an OVA peptide (FVIII-OVA) retained clotting activity and possessed the ability to activate CD4 T cells specific to OVA323-339 in vitro. When compared to FVIII alone, FVIII-OVA also exhibited a similar level of immunogenicity, suggesting that the presence of OVA323-339 does not substantially alter the anti-FVIII immune response. Intriguingly, while little CD4 T cell response could be observed following exposure to FVIII-OVA alone, inclusion of anti-FVIII antibodies, recently shown to favorably modulate anti-FVIII immune responses, significantly enhanced CD4 T cell activation following FVIII-OVA exposure. These results demonstrate that model antigens can be incorporated into a therapeutic protein to study antigen-specific responses and more specifically that the CD4 T cell response to FVIII-OVA can be augmented by pre-existing anti-FVIII antibodies.

Quantitative proteomic screen identifies annexin A2 as a host target for Salmonella pathogenicity island-2 effectors SopD2 and PipB2.

Intracellular pathogens need to establish an intracellular replicative niche to promote survival and replication within the hostile environment inside the host cell. Salmonella enterica serovar Typhimurium (S. Typhimurium) initiates formation of the unique Salmonella-containing vacuole and an extensive network of Salmonella-induced tubules in order to survive and thrive within host cells. At least six effectors secreted by the type III secretion system encoded within Salmonella pathogenicity island-2 (SPI-2), namely SifA, SopD2, PipB2, SteA, SseJ, and SseF, purportedly manipulate host cell intracellular trafficking and establish the intracellular replicative niche for S. Typhimurium. The phenotypes of these effectors are both subtle and complex, complicating elucidation of the mechanism underpinning host cell manipulation by S. Typhimurium. In this work we used stable isotope labeling of amino acids in cell culture (SILAC) and a S. Typhimurium mutant that secretes increased amounts of effectors to identify cognate effector binding partners during infection. Using this method, we identified the host protein annexin A2 (AnxA2) as a binding partner for both SopD2 and PipB2 and were able to confirm its binding to SopD2 and PipB2 by reciprocal pull down, although there was a low level of non-specific binding of SopD2-2HA and PipB2-2HA to the Ni-Sepharose beads present. We further showed that knockdown of AnxA2 altered the intracellular positioning of the Salmonella containing vacuole (SCV). This suggests that AnxA2 plays a role in the subcellular positioning of the SCV which could potentially be mediated through protein-protein interactions with either SopD2 or PipB2. This demonstrates the value of studying effector interactions using proteomic techniques and natural effector delivery during infection rather than transfection.

Quinazolin-derived myeloperoxidase inhibitor suppresses influenza A virus-induced reactive oxygen species, pro-inflammatory mediators and improves cell survival.

Superoxide radicals and other reactive oxygen species (ROS) are implicated in influenza A virus-induced inflammation. In this in vitro study, we evaluated the effects of TG6-44, a novel quinazolin-derived myeloperoxidase-specific ROS inhibitor, on influenza A virus (A/X31) infection using THP-1 lung monocytic cells and freshly isolated peripheral blood mononuclear cells (PBMC). TG6-44 significantly decreased A/X31-induced ROS and virus-induced inflammatory mediators in THP-1 cells (IL-6, IFN-γ, MCP-1, TNF-α, MIP-1ß) and in human PBMC (IL-6, IL-8, TNF-α, MCP-1). Interestingly, TG6-44-treated THP-1 cells showed a decrease in percent cells expressing viral nucleoprotein, as well as a delay in translocation of viral nucleoprotein into the nucleus. Furthermore, in influenza A virus-infected cells, TG6-44 treatment led to suppression of virus-induced cell death as evidenced by decreased caspase-3 activation, decreased proportion of Annexin V+PI+ cells, and increased Bcl-2 phosphorylation. Taken together, our results demonstrate the anti-inflammatory and anti-infective effects of TG6-44.

Marginal zone B cells mediate a CD4 T-cell-dependent extrafollicular antibody response following RBC transfusion in mice.

Red blood cell (RBC) transfusions can result in alloimmunization toward RBC alloantigens that can increase the probability of complications following subsequent transfusion. An improved understanding of the immune mechanisms that underlie RBC alloimmunization is critical if future strategies capable of preventing or even reducing this process are to be realized. Using the HOD (hen egg lysozyme [HEL] and ovalbumin [OVA] fused with the human RBC antigen Duffy) model system, we aimed to identify initiating immune factors that may govern early anti-HOD alloantibody formation. Our findings demonstrate that HOD RBCs continuously localize to the marginal sinus following transfusion, where they colocalize with marginal zone (MZ) B cells. Depletion of MZ B cells inhibited immunoglobulin M (IgM) and IgG anti-HOD antibody formation, whereas CD4 T-cell depletion only prevented IgG anti-HOD antibody development. HOD-specific CD4 T cells displayed similar proliferation and activation following transfusion of HOD RBCs into wild-type or MZ B-cell-deficient recipients, suggesting that IgG formation is not dependent on MZ B-cell-mediated CD4 T-cell activation. Moreover, depletion of follicular B cells failed to substantially impact the anti-HOD antibody response, and no increase in antigen-specific germinal center B cells was detected following HOD RBC transfusion, suggesting that antibody formation is not dependent on the splenic follicle. Despite this, anti-HOD antibodies persisted for several months following HOD RBC transfusion. Overall, these data suggest that MZ B cells can initiate and then contribute to RBC alloantibody formation, highlighting a unique immune pathway that can be engaged following RBC transfusion.

Detailed Case Reports of Surgical Treatment for Intraneural Ganglion Cysts.

Intraneural ganglion cysts are nonneoplastic mucinous cysts that form by the accumulation of thick mucinous fluid in the epineurium of peripheral nerves. Symptoms arise from mechanical compression of adjacent nerve fascicles from the intraneural ganglion cyst, and include local or radiating pain, paresthesias, weakness, and muscle atrophy. METHODS: Retrospective review of three cases of symptomatic intraneural ganglion cysts affecting the upper and lower extremity. RESULTS: In our cases, the intraneural ganglion cysts were completely decompressed with resection of the articular branches, leading to improvement in the patient's symptoms. CONCLUSIONS: Treatment of intraneural ganglion cysts requires an understanding of the underlying anatomy and pathophysiology; accurate early diagnosis is important and can lead to timely treatment and better outcomes.

Both maternal and embryonic exposure to mild hypoxia influence embryonic development of the intertidal gastropod Littorina littorea.

There is growing evidence that maternal exposure to environmental stressors can alter offspring phenotype and increase fitness. Here, we investigate the relative and combined effects of maternal and developmental exposure to mild hypoxia (65 and 74% air saturation, respectively) on the growth and development of embryos of the marine gastropod Littorina littorea Differences in embryo morphological traits were driven by the developmental environment, whereas the maternal environment and interactive effects of maternal and developmental environment were the main driver of differences in the timing of developmental events. While developmental exposure to mild hypoxia significantly increased the area of an important respiratory organ, the velum, it significantly delayed hatching of veliger larvae and reduced their size at hatching and overall survival. Maternal exposure had a significant effect on these traits, and interacted with developmental exposure to influence the time of appearance of morphological characters, suggesting that both are important in affecting developmental trajectories. A comparison between embryos that successfully hatched and those that died in mild hypoxia revealed that survivors exhibited hypertrophy in the velum and associated pre-oral cilia, suggesting that these traits are linked with survival in low-oxygen environments. We conclude that both maternal and developmental environments shape offspring phenotype in a species with a complex developmental life history, and that plasticity in embryo morphology arising from exposure to even small reductions in oxygen tensions affects the hatching success of these embryos.

Revisions to Limits for Methanol in the Air of Spacecraft.

INTRODUCTION: The previous Spacecraft Maximal Allowable Concentrations (SMACs) for methanol were established by characterizing minor effects upon cognitive functions as a no-observable adverse effects level (NOAEL). However, an increasing awareness of the risk posed by Space-Associated Neuro-ocular Syndrome (SANS) has caused NASA Toxicology to reexamine SMACs for methanol because exposure to it can also adversely affect ocular health. An updated review of the literature indicates that no adjustments to the SMACs due to SANS complications were required, while confirming that effects upon the central nervous system remain the appropriate basis for the SMACs for methanol. Our review, however, identified several issues that provide justification for modest SMAC reductions. It has recently been recognized that inhaled methanol may reach the brain via the olfactory system and be absorbed there into the highly toxic metabolite formaldehyde. A benchmark dose (BMD) for an extra risk of 10%, derived from an analysis of the incidences of neurological lesions in monkeys chronically exposed to methanol, is an order of magnitude less than the Environmental Protection Agency's (EPA's) reference concentration for chronic inhalation of methanol. Reports calling attention to the relative insensitivity of traditional methods of assessing cognitive function erode confidence that adverse effects at the concentration reported as a NOAEL would have been recognizable. Therefore, an additional modest safety factor of three is applied to SMACs for methanol.Scully RR, Garcia H, McCoy JT, Ryder VE. Revisions to limits for methanol in the air of spacecraft. Aerosp Med Hum Perform. 2019; 90(9):807-812.

Guidelines for the Detection of NADPH Oxidases by Immunoblot and RT-qPCR.

The identification of NADPH oxidase (NOX) isoforms in tissues is essential for interpreting experiments and for next step decisions regarding cell lines, animal models, and targeted drug design. Two basic methods, immunoblotting and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), are important to monitor NOX protein and messenger RNA (mRNA) levels, respectively, for a range of investigations from understanding cell signaling events to judging NOX inhibitor efficacies. For many other genes that are expressed in high abundance, these methods may seem rather simple. However, detecting the low expression levels of endogenous NOX/DUOX is difficult and can be frustrating, so some guidelines would be helpful to those who are facing difficulties. One reason why detection is so difficult is the limited availability of vetted NOX/DUOX antibodies. Many of the commercial antibodies do not perform well in our hands, and dependable antibodies, often generated by academic laboratories, are in limited supply. Another problem is the growing trend in the NOX literature to omit end-user validation of antibodies by not providing appropriate positive and negative controls. With regard to NOX mRNA levels, knockdown of NOX/DUOX has been reported in cell lines with very low endogenous expression (C q values ≥30) or in cell lines devoid of the targeted NOX isoform (e.g., NOX4 expression in NCI-60 cancer cell panel cell line 786-0). These publications propagate misinformation and hinder progress in understanding NOX/DUOX function. This chapter provides overdue guidelines on how to validate a NOX antibody and provides general methodologies to prepare samples for optimal detection. It also includes validated methodology to perform RT-qPCR for the measurement of NOX mRNA levels, and we suggest that RT-qPCR should be performed prior to embarking on NOX protein detection.

Characterization of the ATP4 ion pump in Toxoplasma gondii.

The Plasmodium falciparum ATPase PfATP4 is the target of a diverse range of antimalarial compounds, including the clinical drug candidate cipargamin. PfATP4 was originally annotated as a Ca2+ transporter, but recent evidence suggests that it is a Na+ efflux pump, extruding Na+ in exchange for H+ Here we demonstrate that ATP4 proteins belong to a clade of P-type ATPases that are restricted to apicomplexans and their closest relatives. We employed a variety of genetic and physiological approaches to investigate the ATP4 protein of the apicomplexan Toxoplasma gondii, TgATP4. We show that TgATP4 is a plasma membrane protein. Knockdown of TgATP4 had no effect on resting pH or Ca2+ but rendered parasites unable to regulate their cytosolic Na+ concentration ([Na+]cyt). PfATP4 inhibitors caused an increase in [Na+]cyt and a cytosolic alkalinization in WT but not TgATP4 knockdown parasites. Parasites in which TgATP4 was knocked down or disrupted exhibited a growth defect, attributable to reduced viability of extracellular parasites. Parasites in which TgATP4 had been disrupted showed reduced virulence in mice. These results provide evidence for ATP4 proteins playing a key conserved role in Na+ regulation in apicomplexan parasites.

Nasal delivery of H5N1 avian influenza vaccine formulated with GenJet™ or in vivo-jetPEI® induces enhanced serological, cellular and protective immune responses.

Avian influenza virus infection is a serious public health threat and preventive vaccination is the most cost-effective public health intervention strategy. Unfortunately, currently available unadjuvanted avian influenza vaccines are poorly immunogenic and alternative vaccine formulations and delivery strategies are in urgent need to reduce the high risk of avian influenza pandemics. Cationic polymers have been widely used as vectors for gene delivery in vitro and in vivo. In this study, we formulated H5N1 influenza vaccines with GenJet™ or in vivo-jetPEI®, and showed that these formulations significantly enhanced the immunogenicity of H5N1 vaccines and conferred protective immunity in a mouse model. Detailed analyses of adaptive immune responses revealed that both formulations induced mixed TH1/TH2 antigen-specific CD4 T-cell responses, antigen-specific cytotoxic CD8 T-cell and memory B-cell responses. Our findings suggest that cationic polymers merit future development as potential adjuvants for mucosal delivery of poorly immunogenic vaccines.

Margins for Benign Salivary Gland Neoplasms of the Head and Neck.

The proper ablation of any neoplasm of the head and neck requires the inclusion of linear and anatomic barrier margins surrounding the neoplasm. Extirpative surgery of the major and minor salivary glands is certainly no exception to this surgical principle. To this end, the selection and execution of the most appropriate ablative surgical procedure for a major or minor benign salivary gland neoplasm is an essential exercise in oral and maxillofacial surgery. Of equal importance is the intraoperative identification and preservation of the pseudocapsule surrounding the benign neoplasm. This article reviews these important elements specifically related to ablative surgery of benign neoplasms of the parotid, submandibular and minor salivary glands with strict attention to observed nomenclature.

Assembly, structure, function and regulation of type III secretion systems.

Type III secretion systems (T3SSs) are protein transport nanomachines that are found in Gram-negative bacterial pathogens and symbionts. Resembling molecular syringes, T3SSs form channels that cross the bacterial envelope and the host cell membrane, which enable bacteria to inject numerous effector proteins into the host cell cytoplasm and establish trans-kingdom interactions with diverse hosts. Recent advances in cryo-electron microscopy and integrative imaging have provided unprecedented views of the architecture and structure of T3SSs. Furthermore, genetic and molecular analyses have elucidated the functions of many effectors and key regulators of T3SS assembly and secretion hierarchy, which is the sequential order by which the protein substrates are secreted. As essential virulence factors, T3SSs are attractive targets for vaccines and therapeutics. This Review summarizes our current knowledge of the structure and function of this important protein secretion machinery. A greater understanding of T3SSs should aid mechanism-based drug design and facilitate their manipulation for biotechnological applications.

A forward genetic screen identifies a negative regulator of rapid Ca2+-dependent cell egress (MS1) in the intracellular parasite Toxoplasma gondii.

Toxoplasma gondii, like all apicomplexan parasites, uses Ca2+ signaling pathways to activate gliding motility to power tissue dissemination and host cell invasion and egress. A group of "plant-like" Ca2+-dependent protein kinases (CDPKs) transduces cytosolic Ca2+ flux into enzymatic activity, but how they function is poorly understood. To investigate how Ca2+ signaling activates egress through CDPKs, we performed a forward genetic screen to isolate gain-of-function mutants from an egress-deficient cdpk3 knockout strain. We recovered mutants that regained the ability to egress from host cells that harbored mutations in the gene Suppressor of Ca2+-dependent Egress 1 (SCE1). Global phosphoproteomic analysis showed that SCE1 deletion restored many Δcdpk3-dependent phosphorylation events to near wild-type levels. We also show that CDPK3-dependent SCE1 phosphorylation is required to relieve its suppressive activity to potentiate egress. In summary, our work has uncovered a novel component and suppressor of Ca2+-dependent cell egress during Toxoplasma lytic growth.

NADPH Oxidase 1 Is Associated with Altered Host Survival and T Cell Phenotypes after Influenza A Virus Infection in Mice.

The role of the reactive oxygen species-producing NADPH oxidase family of enzymes in the pathology of influenza A virus infection remains enigmatic. Previous reports implicated NADPH oxidase 2 in influenza A virus-induced inflammation. In contrast, NADPH oxidase 1 (Nox1) was reported to decrease inflammation in mice within 7 days post-influenza A virus infection. However, the effect of NADPH oxidase 1 on lethality and adaptive immunity after influenza A virus challenge has not been explored. Here we report improved survival and decreased morbidity in mice with catalytically inactive NADPH oxidase 1 (Nox1*/Y) compared with controls after challenge with A/PR/8/34 influenza A virus. While changes in lung inflammation were not obvious between Nox1*/Y and control mice, we observed alterations in the T cell response to influenza A virus by day 15 post-infection, including increased interleukin-7 receptor-expressing virus-specific CD8+ T cells in lungs and draining lymph nodes of Nox1*/Y, and increased cytokine-producing T cells in lungs and spleen. Furthermore, a greater percentage of conventional and interstitial dendritic cells from Nox1*/Y draining lymph nodes expressed the co-stimulatory ligand CD40 within 6 days post-infection. Results indicate that NADPH oxidase 1 modulates the innate and adaptive cellular immune response to influenza virus infection, while also playing a role in host survival. Results suggest that NADPH oxidase 1 inhibitors may be beneficial as adjunct therapeutics during acute influenza infection.

Regulation of Starch Stores by a Ca(2+)-Dependent Protein Kinase Is Essential for Viable Cyst Development in Toxoplasma gondii.

Transmissible stages of Toxoplasma gondii store energy in the form of the carbohydrate amylopectin. Here, we show that the Ca(2+)-dependent protein kinase CDPK2 is a critical regulator of amylopectin metabolism. Increased synthesis and loss of degradation of amylopectin in CDPK2 deficient parasites results in the hyperaccumulation of this sugar polymer. A carbohydrate-binding module 20 (CBM20) targets CDPK2 to amylopectin stores, while the EF-hands regulate CDPK2 kinase activity in response to Ca(2+) to modulate amylopectin levels. We identify enzymes involved in amylopectin turnover whose phosphorylation is dependent on CDPK2 activity. Strikingly, accumulation of massive amylopectin granules in CDPK2-deficient bradyzoite stages leads to gross morphological defects and complete ablation of cyst formation in a mouse model. Together these data show that Ca(2+) signaling regulates carbohydrate metabolism in Toxoplasma and that the post-translational control of this pathway is required for normal cyst development.