Multiple stiffening effects of nanoscale knobs on human red blood cells infected with Plasmodium falciparum malaria parasite

During its asexual development within the red blood cell (RBC), Plasmodium falciparum (Pf), the most virulent human malaria parasite, exports proteins that modify the host RBC membrane. The attendant increase in cell stiffness and cytoadherence leads to sequestration of infected RBCs in microvasculature, which enables the parasite to evade the spleen, and leads to organ dysfunction in severe cases of malaria. Despite progress in understanding malaria pathogenesis, the molecular mechanisms responsible for the dramatic loss of deformability of Pf-infected RBCs have remained elusive. By recourse to a coarse-grained (CG) model that captures the molecular structures of Pf-infected RBC membrane, here we show that nanoscale surface protrusions, known as “knobs,” introduce multiple stiffening mechanisms through composite strengthening, strain hardening, and knob density-dependent vertical coupling. On one hand, the knobs act as structural strengtheners for the spectrin network; on the other, the presence of knobs results in strain inhomogeneity in the spectrin network with elevated shear strain in the knob-free regions, which, given its strain-hardening property, effectively stiffens the network. From the trophozoite to the schizont stage that ensues within 24–48 h of parasite invasion into the RBC, the rise in the knob density results in the increased number of vertical constraints between the spectrin network and the lipid bilayer, which further stiffens the membrane. The shear moduli of Pf-infected RBCs predicted by the CG model at different stages of parasite maturation are in agreement with experimental results. In addition to providing a fundamental understanding of the stiffening mechanisms of Pf-infected RBCs, our simulation results suggest potential targets for antimalarial therapies.The most virulent human malaria parasite, Plasmodium falciparum (Pf), causes ∼700,000 deaths each year (1, 2). Following entry into red blood cells (RBCs), the parasite matures through the ring (0–24 h), trophozoite (24–36 h), and schizont stages (40–48 h). This intraerythrocyte maturation is accompanied by striking changes in the surface topography and membrane architecture of the infected RBC (3–5). A notable modification is the formation of nanoscale protrusions, commonly known as knobs, at the RBC surface during the second half (24–48 h) of the asexual cycle. These protrusions mainly comprise the knob-associated histidine-rich protein (KAHRP) and the membrane-embedded cytoadherence protein, Pf-erythrocyte membrane protein 1 (PfEMP1). KAHRP binds to the fourth repeat unit of the spectrin α-chain, to ankyrin, to spectrin–actin–protein 4.1 complexes, and to the cytoplasmic domain of PfEMP1 (6–9). These attachments enhance the vertical coupling between the lipid bilayer and the spectrin network. Another striking modification in the Pf-infected RBC membrane is the reorganization of the cytoskeletal network caused by parasite-induced actin remodeling (10). As a result of these molecular-level modifications, the Pf-infected RBC exhibits markedly increased stiffness [the shear modulus increases on average from ∼4−10 µN/m in normal/uninfected RBCs, to ∼40 µN/m at the trophozoite stage, and to as high as 90 µN/m at the schizont stage (11–13)] and cytoadherence to the vascular endothelium, which enable sequestration from circulation in vasculature, and evasion from the surveillance mechanisms of the spleen. Although in vitro experimental studies have revealed roles of particular parasite-encoded proteins in remodeling the host RBC (14–22), the mechanism by which Pf-infected RBCs gain dramatically increased stiffness has remained unclear. Indeed, uncertainty remains as to whether the loss of deformability arises from the structural reorganization of the host membrane components or from the deposition of parasite proteins. That is, it is not clear whether the stiffening is due to remodeling of the spectrin network, or to the formation of the knobs, or both. As experimental studies alone have heretofore not been able to determine the molecular details, numerical modeling, combined with a variety of experimental observations and measurements, offers an alternative approach to reveal the underlying mechanisms.We present here a coarse-grained (CG) molecular dynamics (MD) RBC membrane model to correlate structural modifications at the molecular ultrastructure level with the shear responses of the Pf-infected RBC membrane, focusing on the second half of the parasite’s intra-RBC asexual cycle (24–48 h), i.e., the trophozoite and schizont stages. The CG model is computationally efficient, and able to capture the molecular structures of the RBC membrane in both normal and infected states. CGMD simulations reveal that spectrin network remodeling accounts for a relatively small change in shear modulus. Instead, the knobs stiffen the membrane by multiple mechanisms, including composite strengthening, strain hardening, and knob density-dependent vertical coupling. Our findings provide molecular-level understanding of the stiffening mechanisms operating in Pf-infected RBCs and shed light on the pathogenesis of falciparum malaria.     

A randomized, double-blind trial of Lactobacillus GG versus placebo in addition to standard maintenance therapy for children with Crohn's disease

Probiotics are widely used by patients with Crohn's disease (CD) in an attempt to improve their health, but few controlled studies have been done to evaluate the efficacy of these therapies. We conducted a randomized, placebo-controlled trial of the probiotic Lactobacillus rhamnosus strain GG (LGG) to see if the addition of LGG to standard therapy prolonged remission in children with CD. Concomitant medications allowed in the study included aminosalicylates, 6-mercaptopurine, azathioprine, and low-dose alternate day corticosteroids. Seventy-five children (age range, 5-21 yr) with CD in remission were randomized to either LGG (n=39) or placebo (n=36) and followed for up to 2 years. The median time to relapse was 9.8 months in the LGG group and 11.0 months in the placebo group (P=0.24); 31% (12/39) of patients in the LGG group developed a relapse compared with 6/36 (17%) of the placebo group (P=0.18). The LGG was well tolerated, with a side effect profile comparable with placebo. This study suggests that LGG does not prolong time to relapse in children with CD when given as an adjunct to standard therapy.     

The regulation of preimaginal populations of Aedes aegypti L. (Diptera: Culicidae) on the Kenya coast. I. Preimaginal population dynamics and the role of human behaviour

On the Kenya coast Aedes aegypti breeds in pots maintained indoors for water storage. Pupal numbers vary greatly from one pot to another. The rhythm of water replenishment and the accidental introduction of food into the pots regulate pupal numbers. When water replenishment occurs more often than once per week, pupal numbers are small. When replenishment occurs less often, both low and high pupal numbers may be observed. The presence or absence of larval food distinguishes pots of high versus low productivity. A hypothesis is put forward that the introduction of food into the pots could be the result of human activity especially of young children when getting water for their meals.     

Mucosal delivery of a double-stapled RSV peptide prevents nasopulmonary infection

Respiratory syncytial virus (RSV) infection accounts for approximately 64 million cases of respiratory disease and 200,000 deaths worldwide each year, yet no broadly effective prophylactic or treatment regimen is available. RSV deploys paired, self-associating, heptad repeat domains of its fusion protein, RSV-F, to form a fusogenic 6-helix bundle that enables the virus to penetrate the host cell membrane. Here, we developed hydrocarbon double-stapled RSV fusion peptides that exhibit stabilized α-helical structure and striking proteolytic resistance. Pretreatment with double-stapled RSV peptides that specifically bound to the RSV fusion bundle inhibited infection by both laboratory and clinical RSV isolates in cells and murine infection models. Intranasal delivery of a lead double-stapled RSV peptide effectively prevented viral infection of the nares. A chitosan-based nanoparticle preparation markedly enhanced pulmonary delivery, further preventing progression of RSV infection to the lung. Thus, our results provide a strategy for inhibiting RSV infection by mucosal and endotracheal delivery of double-stapled RSV fusion peptides.     

Pratique de la biopsie rénale : résultat d’une enquête en France,revue de la littérature et recommandations

BackgroundAlthough several risk factors associated with complications after renal biopsy (RB) have been identified, recommendations for RB procedures are still lacking. Our working group, appointed by the scientific commission of the Société de néphrologie in France, aimed to depict the main aspects of the practice of RB in adults in France, before establishing some guidelines.MethodsMembers of the Société de néphrologie in France were asked to participate to a questionnaire survey on RB procedures.ResultsEighty-eight nephrologists from 74 units (27 in teaching hospitals, 35 in public general hospitals, and 12 in private centers) participated in our study. Native kidney and graft biopsies were performed in 73 and 35 units, respectively. RB activity was highly variable among units, ranging from several hundred to less than ten per year. Transjugular RB was judged to be smoothly accessible in 28 out of 73 units (38.4%). Significant variations in practices were observed regarding patient information before RB, assessment of hemorrhagic risk factors, care of patients with antiplatelet agents and hemorrhagic risk factors, and radiological guidance. Early discharge (< 12 hours) was the rule in three (4.1%) units for native kidney biopsies and in ten (28.6%) units for graft biopsies.ConclusionsOur study is the first to provide a representative picture of “everyday” RB practices in a country. Consensual recommendations on all points mentioned are provided here.     

Microfluidic study of retention and elimination of abnormal red blood cells by human spleen with implications for sickle cell disease

The spleen clears altered red blood cells (RBCs) from circulation, contributing to the balance between RBC formation (erythropoiesis) and removal. The splenic RBC retention and elimination occur predominantly in open circulation where RBCs flow through macrophages and inter-endothelial slits (IESs). The mechanisms underlying and interconnecting these processes significantly impact clinical outcomes. In sickle cell disease (SCD), blockage of intrasplenic sickled RBCs is observed in infants splenectomized due to acute splenic sequestration crisis (ASSC). This life-threatening RBC pooling and organ swelling event is plausibly triggered or enhanced by intra-tissular hypoxia. We present an oxygen-mediated spleen-on-a-chip platform for in vitro investigations of the homeostatic balance in the spleen. To demonstrate and validate the benefits of this general microfluidic platform, we focus on SCD and study the effects of hypoxia on splenic RBC retention and elimination. We observe that RBC retention by IESs and RBC–macrophage adhesion are faster in blood samples from SCD patients than those from healthy subjects. This difference is markedly exacerbated under hypoxia. Moreover, the sickled RBCs under hypoxia show distinctly different phagocytosis processes from those non-sickled RBCs under hypoxia or normoxia. We find that reoxygenation significantly alleviates RBC retention at IESs, and leads to rapid unsickling and fragmentation of the ingested sickled RBCs inside macrophages. These results provide unique mechanistic insights into how the spleen maintains its homeostatic balance between splenic RBC retention and elimination, and shed light on how disruptions in this balance could lead to anemia, splenomegaly, and ASSC in SCD and possible clinical manifestations in other hematologic diseases.

The human spleen is a unique organ that plays an important role in our immune and circulatory systems. The spleen is composed primarily of two distinct functional regions, the red pulp and the white pulp, which are intermingled by the marginal/perifollicular zone (1). It contains complex vascular pathways involving direct and indirect connections. Direct connections exist between the fast perifollicular microcirculation and venous sinuses drained in splenic veins (“closed circulation”), whereas indirect connections exist between red pulp arterioles and veins through the reticular meshwork (“open circulation”) and across the wall of sinuses (1). As much as about 80% of the spleen parenchyma is populated with the red pulp, which mainly comprises the vascular sinuses and cords of Billroth (1, 2). Approximately 3 to 10% of blood from cardiac output flows through the spleen, and about 10% of splenic inflow passes through slow open circulation in the red pulp (1, 3–5).Splenic filtration of abnormal red blood cells (RBCs) is predominately performed in the open circulation, through macrophage-rich zones (M-filter) and across splenic inter-endothelial slits (IESs) in the wall of sinuses (S-filter), as shown in Fig. 1A. The specialized elongated shape of littoral cells in the splenic sinuses and their three-dimensional (3D) barrel-like structure impose sub-micrometer scale physical barriers or constraints on RBCs navigating the open circulation (6). Prior to returning to the systemic circulation through the IESs across the spleen, circulating RBCs are checked for surface integrity by a scattered collection of resident macrophages (7). These two structural and functional spleen filters, S-filter and M-filter (Fig. 1B), sustain the remarkable capacity of the spleen to retain and destroy abnormal RBCs. Consequently, they contribute to the fine balance between RBC production in the bone marrow and the removal of abnormal RBCs from the blood circulation (1).Open in a separate windowFig. 1.Splenic filtration of altered RBCs. (A) Schematic diagram of blood circulation through splenic red pulp, including closed circulation and open circulation. The splenic filtration of altered RBCs is achieved in the open circulation in the red pulp, through macrophages (M-filter) and the splenic IESs (S-filter). (B) Schematic diagram of the oxygen gradient near the sinus. The two structural and functional spleen filters, the S-filter and M-filter, respectively, are modeled in vitro using the S-Chip and M-Chip, respectively. Created with BioRender.com.Healthy human RBCs (AA RBCs) have an average life span of 100 to 120 d (8), indicating that about 1% of the RBCs are recycled each day by the human body (9). Senescent RBCs typically have reduced deformability (10) and send signals to macrophages by expressing higher phosphatidylserine (PS), higher band-3, and reduced CD47 levels (11, 12), on their external cell surface. Senescent RBCs could exhibit a higher propensity to be sequestered at the IESs due to their reduced deformability and then cleared by macrophages, or trapped by adhesion and phagocytosed in the meshwork (13). Here, a balance between RBC retention rate and RBC elimination rate (by post-retention processing) should be dynamically maintained to ascertain homeostasis. Such homeostatic balance, however, can be severely disrupted due to hemolytic disorders, resulting in serious, and sometimes life-threatening, complications such as splenomegaly and/or hypersplenism (14). As a result, the RBC retention rate can significantly surpass the RBC post-retention elimination rate in the spleen. It is, therefore, important to investigate systematically and quantitatively both the RBC retention rate and the elimination rate in the spleen, and to compare their relative changes, as a function of disease state, with the baseline condition of healthy hemostasis.The spleen is generally in a hypoxic (low oxygen level) condition owing to its slow and open blood circulation in the red pulp (see the brief review of splenic oxygen level and transit time/velocity of RBCs in SI Appendix, Table S2). An oxygen gradient exists in the spleen from locations nearest to the arteriolar end of capillaries to the distal locations in the proximity of the IESs, as shown schematically in Fig. 1B (15). Under normal circumstances, hypoxia is mild due to continuous oxygen delivery by the RBCs through splenic circulation. However, it deepens with the reduction in oxygen delivery arising from the obstruction of RBC flow or anemia in many hemolytic blood disorders. For instance, in sickle cell disease (SCD), such obstruction/anemia-induced local hypoxia may in turn trigger sickling of homozygous sickle cell disease (HbSS) RBCs (SS RBCs) and subsequently lead to further reduction in deformability and increase in the expression of adhesion molecules (16, 17). Therefore, sickled RBCs have a higher propensity to be retained by the venous sinuses as well as cords of Billroth, thereby contributing further to a reduction in the oxygen level in the spleen (18). The splenic retention of SS RBCs depends on a trade-off between the local oxygen level (which determines the sickling kinetics) and the transit time of RBCs through the spleen (19). Excessive retention of stiff and sickled SS RBCs in the patient’s spleen has been considered a dominant cause of acute splenic sequestration crisis (ASSC), a life-threatening complication, in SCD (17, 20, 21). This process might involve a vicious cycle: the more RBCs the spleen traps, the larger the spleen grows, and the larger the spleen grows, the deeper the hypoxia is, resulting in more and more sickled SS RBCs that are consequently being trapped and destroyed. Indeed, following splenectomy in young SCD children with still functional spleen, sickled RBCs have been found retained and congested upstream of IESs during ASSC (17, 22, 23). On the other hand, surface modulations such as PS externalization (24), decreased levels of CD47 (25), and elevated binding of autologous immunoglobulin (26), as well as increased membrane rigidity of sickled RBCs may also promote the retention and elimination of SS RBCs by the splenic macrophages (27–29). From these considerations, we postulate that both increased mechanical retention and hyperactive phagocytosis elimination of abnormal RBCs can exacerbate significantly under some extreme conditions such as hypoxia in SCD. These factors could, in turn, play a key role in disrupting the homeostatic balance thereby causing spleen dysfunction in hemolytic disorders.Recent in vitro studies based on microfluidic spleen-on-a-chip platforms, which simulate the micro-constrictions of IESs and hydrodynamic conditions, have advanced the functional study of RBC filtration in the spleen (30–33). However, to our knowledge, no prior in vitro assays have effectively integrated a controlled gaseous microenvironment within a microfluidic system to enable the quantitative investigation of the hypoxic effect on splenic retention and post-retention elimination of RBCs, especially for SS RBCs. Moreover, there is a compelling need for an in vitro assay that elucidates the mechanisms underlying the interaction of RBCs with splenic phagocytes during the low-velocity microcirculation through the red pulp. Most existing erythrophagocytosis (RBC elimination) assays measure phagocytic activity in a static condition, which does not faithfully replicate in vivo conditions (27, 28). To this end, the development and validation of an oxygen-mediated in vitro assay for investigating the kinetics of both splenic retention of RBCs and erythrophagocytosis under hypoxia are critically needed for a better understanding of the mechanisms responsible for splenic functions in physiology and disease.Here we present a general microfluidic platform to systematically probe the retention and elimination functions undertaken by IESs and macrophages in the human spleen, by developing and validating two functional modules of an oxygen-mediated spleen-on-a-chip. This platform entails the S-Chip and the M-Chip, which model the S-filter for RBC retention through splenic IESs and the M-filter for RBC adhesion and elimination by splenic resident macrophages, respectively. While the microfluidic platform and assays presented in this work can, by design, potentially provide mechanistic insights into a wide spectrum of hereditary and acquired human diseases, we focus particular attention here on the study of homeostatic processes in SCD. We make comparisons with healthy subjects as a negative control group. We additionally use heated AA RBCs as a positive control, while considering it as a generic model for exploring different controlled concentrations of altered RBCs in hemolytic disorders. We demonstrate that our microfluidic platform can also be used to mimic in vitro the two major components of the spleen filtering unit, namely surface sensing by macrophages and mechanical sensing by splenic IESs under controlled oxygen pressure. We further show that this approach enables systematic investigations of the cellular mechanisms underlying anemia and ASSC in SCD, while also providing potential pathways to explore, with appropriate modifications, splenomegaly and hypersplenism in other diseases such as Plasmodium falciparum malaria.