Assessing the Vascular Deformability of Erythrocytes and Leukocytes: From Micropipettes to Microfluidics

Among the most crucial rheological characteristics of blood cells within the vasculature is their ability to undergo the shape change (i.e., deform). The significance of cellular deformability is readily apparent based solely on the disparate mean size of human erythrocytes (~8 μm) and leukocytes (10–25 μm) compared to the minimum luminal size of capillaries (4–5 μm) and splenic interendothelial clefts (0.5–1.0 μm) they must transit. Changes in the deformability of either cell will result in their premature mechanical clearance as well as an enhanced possibility of intravascular lysis. In this chapter, we will demonstrate how microfluidic devices can be used to examine the vascular deformability of erythrocytes and agranular leukocytes. Moreover, we will compare microfluidic assays with previous studies utilizing micropipettes, ektacytometry and micropore cell transit times. As will be discussed, microfluidics-based devices offer a low-cost, high throughput alternative to these previous, and now rather ancient, technologies

Altered ratio of IFN-γ/IL-10 in patients with drug resistant Mycobacterium tuberculosis and HIV-tuberculosis immune reconstitution inflammatory syndrome

We have described a clinical relationship between HIV-Tuberculosis Immune Reconstitution Inflammatory Syndrome (TB-IRIS) and anti-tubercular drug resistance. Here we studied the immune response of TB-IRIS patients from whom a drug-resistant (n = 11) or drug-susceptible (n = 25) Mycobacterium tuberculosis (MTB) strain was isolated after presenting with TB-IRIS. ELISpot analysis and multiplex cytokine analysis of the supernatant collected from peripheral blood mononuclear cells stimulated overnight with the heat-killed H37Rv MTB laboratory strain was used. Although there was no statistical difference in IFN-gamma ELISpot responses between the two groups, the results point towards higher bacterial load in the drug-resistant patients, possibly due to failed therapy. The ratio between secreted IFN-gamma/IL-10 and IL-2/IL-10 was significantly lower in TB-IRIS patients in whom the cause of TB was a drug-resistant strain compared to those with a fully sensitive strain (p = 0.02). Since host immune responses are dependent on the bacterial load, we hypothesise that the impaired cytokine balance is likely to be caused by the poorly controlled bacterial growth in these patients

HIV-1 infection alters CD4 1 memory T-cell phenotype at the site of disease in extrapulmonary tuberculosis

HIV-1-infected people have an increased risk of developing extrapulmonary tuberculosis (TB), the immunopathogenesis of which is poorly understood. Here, we conducted a detailed immunological analysis of human pericardial TB, to determine the effect of HIV-1 co-infection on the phenotype of Mycobacterium tuberculosis (MTB)-specific memory T cells and the role of polyfunctional T cells at the disease site, using cells from pericardial fluid and blood of 74 patients with (n 5 50) and without (n 5 24) HIV-1 co-infection. The MTB antigen-induced IFN-c response was elevated at the disease site, irrespective of HIV-1 status or antigenic stimulant. However, the IFN-c ELISpot showed no clear evidence of increased numbers of antigen-specific cells at the disease site except for ESAT-6 in HIV-1 uninfected individuals (p 5 0.009). Flow cytometric analysis showed that CD4 1 memory T cells in the pericardial fluid of HIV-1-infected patients were of a less differentiated phenotype, with the presence of polyfunctional CD4 1 T cells expressing TNF, IL-2 and IFN-c. These results indicate that HIV-1 infection results in altered phenotype and function of MTB-specific CD4 1 T cells at the disease site, which may contribute to the increased risk of developing TB at all stages of HIV-1 infection

Prevalence, hemodynamics, and cytokine profile of effusive-constrictive pericarditis in patients with tuberculous pericardial effusion

BACKGROUND: Effusive constrictive pericarditis (ECP) is visceral constriction in conjunction with compressive pericardial effusion. The prevalence of proven tuberculous ECP is unknown. Whilst ECP is distinguished from effusive disease on hemodynamic grounds, it is unknown whether effusive-constrictive physiology has a distinct cytokine profile. We conducted a prospective study of prevalence and cytokine profile of effusive-constrictive disease in patients with tuberculous pericardial effusion. METHODS: From July 2006 through July 2009, the prevalence of ECP and serum and pericardial levels of inflammatory cytokines were determined in adults with tuberculous pericardial effusion. The diagnosis of ECP was made by combined pericardiocentesis and cardiac catheterization. RESULTS: Of 91 patients evaluated, 68 had tuberculous pericarditis. The 36/68 patients (52.9%; 95% confidence interval [CI]: 41.2-65.4) with ECP were younger (29 versus 37 years, P=0.02), had a higher pre-pericardiocentesis right atrial pressure (17.0 versus 10.0 mmHg, P 15 mmHg (odds ratio [OR] = 48, 95%CI: 8.7-265; P 200 pg/ml (OR=10, 95%CI: 1.1, 93; P=0.04) were independently associated with ECP. CONCLUSION: Effusive-constrictive disease occurs in half of cases of tuberculous pericardial effusion, and is characterized by greater elevation in the pre-pericardiocentesis right atrial pressure and pericardial and serum IL-10 levels compared to patients with effusive non-constrictive tuberculous pericarditis

Immunological analysis of pericardial tuberculosis

Includes bibliographical references (leaves 297-327).Pericardial tuberculosis (TB) offers a relevant human model to study TB at the site of disease and to determine the effect of HIV-1 infection. 96 Patients with pericardial TB were recruited into this study, 68 of whom were HIV-1 infected. Where clinically indicated, pericardiocentesis was performed to drain pericardial fluid and blood was drawn. The data derived from the study provide novel insight into the immune response in the pericardium to TB infection. Furthermore, HIV-1 infection caused a dysregulation of the immune response

IF device (JRSi)

Human leukocytes processed by fast-rate inertial microfluidics retain conventional functional characteristic

Degradation of red blood cell deformability during cold storage in blood bags

Abstract Red blood cells (RBCs) stored in blood bags develop a storage lesion that include structural, metabolic, and morphologic transformations resulting in a progressive loss of RBC deformability. The speed of RBC deformability loss is donor‐dependent, which if properly characterized, could be used as a biomarker to select high‐quality RBC units for sensitive recipients or to provide customized storage timelines depending on the donor. We used the microfluidic ratchet device to measure the deformability of red blood cells stored in blood bags every 14 days over a span of 56 days. We observed that storage in blood bags generally prevented RBC deformability loss over the current standard 42‐day storage window. However, between 42 and 56 days, the deformability loss profile varied dramatically between donors. In particular, we observed accelerated RBC deformability loss for a majority of male donors, but for none of the female donors. Together, our results suggest that RBC deformability loss could be used to screen for donors who can provide stable RBCs for sensitive transfusion recipients or to identify donors capable of providing RBCs that could be stored for longer than the current 42‐day expiration window

REDUCED DEFORMABILITY OF PARASITIZED RED BLOOD CELLS AS A BIOMARKER FOR ANTIMALARIAL DRUG EFFICACY

Background Malaria remains a challenging and fatal infectious disease throughout the developing world. Malaria progressively induces structural and functional changes causing rigidification (loss of deformability) of infected red blood cells. Antimalarials may accelerate this process, thereby allowing the infected erythrocyte to be removed from the circulation earlier. The rapid spread of antimalarial drug resistance increases the urgency for the development of new drugs. Many biomarkers, including malaria specific genes and parasite synthesized proteins and metabolites, have been developed to evaluate drug efficacy as well as to screen new drugs. Methods Recently, we developed a microfluidic mechanism, called the multiplexed fluidic plunger that provides sensitive and rapid measurement of single red blood cell deformability. Here, we systematically evaluated the deformability changes of late stage trophozoite infected-RBCs after treatment with various well known as well as unknown antimalarials. Results We show a concentration and time - dependent response relationship that exists between chloroquine treatment and iRBCs deformability change. We determined that rigidification of trophozoite-infected RBCs is a universal property of almost all clinical antimalarial drug treatments. Spiroindolone compounds (+)-SJ733 and NITD246, inhibitors to a Plasmodium falciparum cation-transporting ATPase ATP4, induced the highest rigidified trophozoite-infected RBCs. Collectively, these results suggest that changes in the deformability of iRBCs could be used as a biomarker for antimalarial drug treatments. Therefore, as a proof-of-principle, we tested a group of bisindole alkaloids. The results revealed that cladoniamide A, which has a rare scaffold and lower IC50 value than that of chloroquine may be a promising antimalarial drug candidate. Conclusions Our results demonstrate that rigidification of infected-RBCs may be used as a biomarker for antimalarial drug efficacy as well as for new drug screening. As a proof of principle, we successfully discovered a potential antimalarial drug

Cell-­phoresis o fRed Blood Cells Revealing Biophysical Signatures in Falciparum Malaria

We describe the cell-phoresis mechanism for massively parallel analysis of red blood cell (RBC) deformability by transporting single cells through microstructures to measure their spatial dispersion. Analogous to gel electrophoresis, which transport molecules through nanostructures to measure their length, the spatial dispersion of RBCs within microstructures indicate their deformability. Similar to gel electrophoresis, cell-phoresis require minimal instrumentation, provide a simple image-based readout, and could be performed simultaneously on multiple samples as part of a biophysical assay. We applied the cell-phoresis mechanism to study the biophysical signatures of falciparum malaria where we demonstrate label-­‐free and calibration-­‐free detection of ring-­‐stage infection, as well as in vitro assessment of antimalarial drug efficacy. We show that all clinical antimalarial drugs rigidify RBCs infected P. falciparum and that recently discovered PfATP4 inhibitors show a distinct biophysical signature. We anticipate cell-phoresis to be a functional assay for screening new antimalarials and adjunctive agents, as well as for validating their mechanisms of action