Rapid and massive virus-specific plasmablast responses during acute dengue virus infection in humans.

Humoral immune responses are thought to play a major role in dengue virus-induced immunopathology; however, little is known about the plasmablasts producing these antibodies during an ongoing infection. Herein we present an analysis of plasmablast responses in patients with acute dengue virus infection. We found very potent plasmablast responses that often increased more than 1,000-fold over the baseline levels in healthy volunteers. In many patients, these responses made up as much 30% of the peripheral lymphocyte population. These responses were largely dengue virus specific and almost entirely made up of IgG-secreting cells, and plasmablasts reached very high numbers at a time after fever onset that generally coincided with the window where the most serious dengue virus-induced pathology is observed. The presence of these large, rapid, and virus-specific plasmablast responses raises the question as to whether these cells might have a role in dengue immunopathology during the ongoing infection. These findings clearly illustrate the need for a detailed understanding of the repertoire and specificity of the antibodies that these plasmablasts produce.

Platelet activation and platelet-leukocyte interaction in ß-thalassemia/hemoglobin E patients with marked nucleated erythrocytosis.

Patients with thalassemia, an inherited hemolytic anemia, have increased risk of hypercoagulable complications. A whole blood flow cytometric (FCM) method has been used for studies of platelet activation and platelet-leukocyte aggregation in these patients. However, this FCM method presents technical difficulties because of the high proportion of immature red blood cells (RBCs) in these patients. A protocol for the simultaneous measurement of platelet activation and their aggregation with leukocyte populations in whole blood using four-color FCM which excluded immature RBC was devised, and evaluated for the evaluation of platelet function in patients with ß-thalassemia/hemoglobin E (HbE). Whole blood from these patients and from healthy volunteers was stained for platelet activation and platelet-leukocyte aggregates using anti-CD42a, anti-CD62P, anti-CD45 and glycophorin A (GPA) conjugated with different fluorochromes. Our FCM method is simple, effective and based on the assumption that GPA is present on all immature RBCs, but is not expressed on CD45⁺ leukocytes. Results from the studies showed that blood samples from these patients contained a high frequency of circulating activated platelets (CD42a⁺/CD62P⁺) when compared to samples from healthy individuals. The percentage of platelet-neutrophil, platelet-monocyte-but not platelet-lymphocyte-aggregates were also elevated in both thalassemia genotypes with marked increase in patients who had undergone splenectomy. These findings suggest that platelets adhere to neutrophils and monocytes are activated which support the clinical observation that splenectomized thalassemia patients have an increased risk of arterial or venous thrombotic manifestations.

Kinetic Changes of Peripheral Blood Monocyte Subsets and Expression of Co-Stimulatory Molecules during Acute Dengue Virus Infection.

Monocytes, one of the main target cells for dengue virus (DENV) infection, contribute to the resolution of viremia and to pathogenesis. We performed a longitudinal study by a detailed phenotypic comparison of classical (CD14++CD16-, non-classical (CD14+CD16++) and intermediate (CD14++CD16+) monocyte subsets in blood samples from dengue fever (DF) to the severe dengue hemorrhagic fever (DHF) and healthy individuals. Various costimulatory molecules of CD40, CD80, CD86 and inducible costimulatory ligand (ICOSL) expressed on these three monocyte subsets were also analyzed. DENV-infected patients showed an increase in the frequency of intermediate monocytes and a decrease in the classical monocytes when compared to healthy individuals. Although these differences did not correlate with disease severity, changes during the early phase of infection gradually returned to normal in the defervescence phase. Moreover, decreased frequency of classical monocytes was associated with a significant up-regulation of co-stimulatory molecules CD40, CD86 and ICOSL. Kinetics of these co-stimulatory molecule-expressing classical monocytes showed different patterns throughout the sampling times of acute DENV infection. Different distribution of monocyte subsets and their co-stimulatory molecules in the peripheral blood during acute infection might exacerbate immune responses like cytokine storms and ADE, and future studies on intracellular molecular pathways utilized by these monocyte linages are warranted.

Febrile temperature but not proinflammatory cytokines promotes phosphatidylserine expression on Plasmodium falciparum malaria-infected red blood cells during parasite maturation.

Intraerythrocytic maturation of the malaria parasite Plasmodium falciparum is associated with profound changes in the asymmetry of phospholipids in the lipid bilayer of the parasitized red blood cells (pRBCs). These changes may contribute to adherence of pRBCs to endothelial cells. This study investigates the effect of febrile temperature and proinflammatory cytokines on phosphatidylserine (PS) expression on the exofacial surface of pRBCs during parasite maturation. The expression of PS on the pRBCs was determined by flow cytometry using fluorescein-labeled annexin V, which specifically binds to PS and a vital nucleic acid fluorochrome for parasite staining. The results showed that PS expression on the surface of pRBCs increased in association with parasite maturation, especially at the late parasite stage. Furthermore, the growth of P. falciparum also accelerated senescence of the uninfected RBCs in parasite cultures. Exposure to febrile temperature led to significant increases in the expression of PS on the surface of pRBCs, particularly at the late parasite stage associated with the virulence strain of the parasite. In contrast, proinflammatory cytokines had no detectable effect on PS expression on pRBCs. These data suggest that PS molecule expression is more dependent on fever, parasitemia, parasite strain, and virulence than on cytokine exposure. These findings contribute to our understanding of the factors that are involved in malaria pathogenesis.

External quality assessment program on CD4+ T-lymphocyte counts for persons with HIV/AIDS in Thailand: history and accomplishments.

A CD4 count External Quality Assessment (EQA) program is important for the clinical monitoring of persons infected with HIV/AIDS. The purpose of the present study was to evaluate the CD4 EQA performance program of the flow cytometer laboratories that perform routine CD4 counts for these patients in Thailand. Stabilized whole blood samples were sent to participating laboratories to determine the percentage and absolute counts of CD4+ T-lymphocytes using their routine procedures. The data were analyzed and reports sent to the participants within one month. Most participating laboratories produced results that were within two standard deviations (SD) of the mean, while the average inter-laboratory coefficients of variation were less than 8% for CD4+ T-lymphocytes. This program was found to improve the reliability of CD4+ T-lymphocyte determinations. This test is becoming increasingly important as Thailand and other Southeast Asian countries scale up their national programs that provide access to antiretroviral therapy for persons living with HIV/AIDS.

Identification of changes in dendritic cell subsets that correlate with disease severity in dengue infection.

Dengue virus (DENV) is the most prevalent arthropod-borne viral disease in humans. DENV causes a spectrum of illness ranging from mild to potentially severe complications. Dendritic cells (DCs) play a critical role in initiating and regulating highly effective antiviral immune response that include linking innate and adaptive immune responses. This study was conducted to comparatively characterize in detail the relative proportion, phenotypic changes, and maturation profile of subsets of both myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in children with dengue fever (DF), dengue hemorrhagic fever (DHF) and for purposes of control healthy individuals. The mDCs (Lin-CD11c+CD123lo), the pDCs (Lin-CD11c-CD123+) and the double negative (DN) subset (Lin-/HLA-DR+/CD11c-CD123-) were analyzed by polychromatic flow cytometry. The data were first analyzed on blood samples collected from DENV-infected patients at various times post-infection. Results showed that the relative proportion of mDCs were significantly decreased which was associated with an increase in disease severity in samples from DENV-infected patients. While there was no significant difference in the relative proportion of pDCs between healthy and DENV-infected patients, there was a marked increase in the DN subset. Analysis of the kinetics of changes of pDCs showed that there was an increase but only during the early febrile phase. Additionally, samples from patients during acute disease showed marked decreases in the relative proportion of CD141+ and CD16+ mDC subsets that were the major mDC subsets in healthy individuals. In addition, there was a significant decrease in the level of CD33-expressing mDCs in DENV patients. While the pDCs showed an up-regulation of maturation profile during acute DENV infection, the mDCs showed an alteration of maturation status. This study suggests that different relative proportion and phenotypic changes as well as alteration of maturation profile of DC subsets may play a critical role in the dengue pathogenesis and disease outcome.

Alteration of CD8+ T cell effector diversity during HIV-1 infection with discordant normalization in effective antiretroviral therapy.

BACKGROUND: Although the impairment of HIV-specific T lymphocytes is evident during chronic HIV-infection, it is unclear whether the increased CD8+ T cells associates with either selective or overall change of effector functional phenotype. Instead of study on HIV-specific T cells only, analyzing bulk T cell populations represent a neglected area of T cell impairment, which go far beyond HIV-specific T cells. METHODS: In this study, we determined the diversity of CD8+ T cells in term of cytolytic molecule expression (perforin, granzyme A, and granzyme B) and cytokine production ability (IFN-gamma, TNF-alpha, and IL-2) using intracellular staining and flow cytometry technique. The results were compared between healthy individuals, untreated, and antiretroviral therapy (ART) treated HIV infected patients. RESULTS: We demonstrated the presence of four different subsets of CD8+ T cells that expressed different combinations of cytolytic molecules. We also identified seven different subsets of cytokine producing cells based on different combination of IFN-gamma, TNF-alpha, and IL-2. Results showed significant alterations of these cell subsets that expressed different combination of cytolytic effector molecules or cytokines in HIV infected patients. Furthermore, cytolytic molecule expressing cell subsets are not normalized in effective ART treated patients, whereas the selective population of cytokine producing cells returned to normal value. CONCLUSIONS: The effector diversity of CD8+ T cells changed in HIV infected patients. Although effective ART altered functional diversity of these cells, long-term suppression of viral replication may be required to normalize the selected CD8+ T cell effector phenotype in HIV infected patients.

Performance evaluation of the Alere PIMA CD4 test for monitoring HIV-infected individuals in resource-constrained settings.

BACKGROUND: Enumeration of CD4+ T-lymphocytes is important in the management of HIV. However, standard laboratory systems based on flow cytometry are expensive, complicated, and thus unavailable to most resource-limited settings where a low-cost and fully automated point-of-care CD4 testing system is required. In attempts to address this issue, a study was conducted to validate the Alere PIMA point-of-care CD4 test. METHOD: Duplicate values of the absolute number of CD4+ T-lymphocytes in 203 HIV-infected blood samples obtained using the PIMA system were compared with the two predicate single-platform FACSCount and the dual-platform FACSCan (Becton Dickinson Biosciences). RESULTS: The overall absolute CD4+ T-lymphocyte count obtained using the PIMA system correlated highly with the FACSCount (r = 0.957; mean bias, -54.2 cells/µL; limit of agreement, -190.9 to +82.5 cells/µL) and the FACSCan (r = 0.957; mean bias -44.0 cells/µL; limit of agreement, -179.7 to +91.6 cells/µL). Good correlation and low biases were also observed for samples with CD4+ T-lymphocyte count ranges of 0 to 200 and 0 to 350 cells/µL. Additionally, there was no significant difference in absolute CD4+ T-lymphocyte counts noted between the duplicate samples using the PIMA system. CONCLUSIONS: This new point-of-care product is a simple and reliable system and should contribute significantly to the simplification of performing CD4 testing and thus increase access for patients in resource-limited settings. The inability to obtain values for the frequency (%) of CD4+ T-lymphocyte count is one limitation of the PIMA system, the addition of which would be of value for clinical staging or monitoring in HIV-infected pediatric patients.