Ladawan Khowawisetsut

Characterizations and proteome analysis of platelet-free plasma-derived microparticles in β-thalassemia/hemoglobin E patients.

Aggregatability and oxidative damage of red blood cells (RBCs), platelet activation and increased amount of blood cells-derived microparticles (MPs) are thought to be the etiologies for the thrombotic risk in thalassemia, but with unclear mechanisms. Here we report cellular origins and increases in number, oxidative stress status, and procoagulant activity, as well as altered proteome of MPs isolated from β-thal/HbE patients. Flow cytometric analysis revealed that β-thal/HbE patients had significantly higher levels of phosphatidylserine (PS)-bearing MPs in platelet-free plasma (PFP) as compared to normal subjects. The high levels of MPs correlated with not only the increased procoagulant activity but also the increased platelet counts. Additionally, these PS-bearing MPs were originated mostly from platelets and RBCs, both of which had increased levels of reactive oxygen species. Proteome analysis of MPs by 2-DE followed by Q-TOF MS and MS/MS analyses identified 29 proteins with significantly altered levels in MPs derived from β-thal/HbE patients (e.g. the increased levels of peroxiredoxin 6, apolipoprotein E, cyclophilin A and heat shock protein 90). These findings suggest that the oxidative damage in platelets and RBCs potentially induces production of MPs with altered proteome that may, in turn, facilitate thromboembolic complications, which are commonly found in thalassemic patients. This article is part of a Special Issue entitled: Integrated omics.

In Vivo Administration of a JAK3 Inhibitor during Acute SIV Infection Leads to Significant Increases in Viral Load during Chronic Infection

The studies reported herein are the first to document the effect of the in vivo administration of a JAK3 inhibitor for defining the potential role of NK cells during acute SIV infection of a group of 15 rhesus macaques (RM). An additional group of 16 MHC/KIR typed RM was included as controls. The previously optimized in vivo dose regimen (20 mg/kg daily for 35 days) led to a marked depletion of each of the major NK cell subsets both in the blood and gastro-intestinal tissues (GIT) during acute infection. While such depletion had no detectable effects on plasma viral loads during acute infection, there was a significant sustained increase in plasma viral loads during chronic infection. While the potential mechanisms that lead to such increased plasma viral loads during chronic infection remain unclear, several correlates were documented. Thus, during acute infection, the administration of the JAK3 inhibitor besides depleting all NK cell subsets also decreased some CD8+ T cells and inhibited the mobilization of the plasmacytoid dendritic cells in the blood and their localization to the GIT. Of interest is the finding that the administration of the JAK3 inhibitor during acute infection also resulted in the sustained maintenance during chronic infection of a high number of naïve and central memory CD4+ T cells, increases in B cells in the blood, but decreases in the frequencies and function of NKG2a+ NK cells within the GIT and blood, respectively. These data identify a unique role for JAK3 inhibitor sensitive cells, that includes NK cells during acute infection that in concert lead to high viral loads in SIV infected RM during chronic infection without affecting detectable changes in antiviral humoral/cellular responses. Identifying the precise mechanisms by which JAK3 sensitive cells exert their influence is critical with important implications for vaccine design against lentiviruses.

Platelet activation and platelet–leukocyte interaction in β-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 β-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.

In Vivo Administration of a JAK3 Inhibitor to Chronically SIV Infected Rhesus Macaques Leads to NK Cell Depletion Associated with Transient Modest Increase in Viral Loads

Innate immune responses are reasoned to play an important role during both acute and chronic SIV infection and play a deterministic role during the acute stages on the rate of infection and disease progression. NK cells are an integral part of the innate immune system but their role in influencing the course of SIV infection has been a subject of debate. As a means to delineate the effect of NK cells on SIV infection, use was made of a Janus kinase 3 (JAK3) inhibitor that has previously been shown to be effective in the depletion of NK cells in vivo in nonhuman primates (NHP). Extensive safety and in vitro/in vivo PK studies were conducted and an optimal dose that depletes NK cells and NK cell function in vivo identified. Six chronically SIV infected rhesus macaques, 3 with undetectable/low plasma viral loads and 3 with high plasma viral loads were administered a daily oral dose of 10 mg/kg for 35 days. Data obtained showed that, at the dose tested, the major cell lineage affected both in the blood and the GI tissues were the NK cells. Such depletion appeared to be associated with a transient increase in plasma and GI tissue viral loads. Whereas the number of NK cells returned to baseline values in the blood, the GI tissues remained depleted of NK cells for a prolonged period of time. Recent findings show that the JAK3 inhibitor utilized in the studies reported herein has a broader activity than previously reported with dose dependent effects on both JAK2 and JAK1 suggests that it is likely that multiple pathways are affected with the administration of this drug that needs to be taken into account. The findings reported herein are the first studies on the use of a JAK3 inhibitor in lentivirus infected NHP.

Differences in activation and tissue homing markers of natural killer cell subsets during acute dengue infection

Dengue virus (DENV) infection is considered one of the most important mosquito‐borne diseases. It causes a spectrum of illness that could be due to qualitative and/or quantitative difference(s) of the natural killer (NK) cell responses during acute DENV infection. This view prompted us to perform a detailed phenotypic comparative characterization of NK cell subsets from DENV‐infected patients with dengue fever (DF), patients with dengue haemorrhagic fever (DHF) and healthy controls. The activation/differentiation molecules, CD69 and CD57 and a variety of tissue homing molecules were analysed on the CD56hi CD16− and CD56lo CD16+ NK cells. Although there was no increase in the frequency of the total NK cells during DENV infection compared with the healthy individuals, there was a significant increase in the frequency of the CD56hi CD16− subset and the frequency of CD69 expression by both NK cell subsets during the febrile phase of infection. We also found an increase in the frequencies of cells expressing CD69 and CD57 in the CD56lo CD16+ subset compared with those in the CD56hi CD16− subset. Moreover, although the CD56lo CD16+ subset contained a high frequency of cells expressing skin‐homing markers, the CD56hi CD16− subset contained a high frequency of cells expressing bone marrow and lymph node trafficking markers. Interestingly, no differences of these NK cell subsets were noted in samples from patients with DF versus those with DHF. These findings suggest that activation and differentiation and the patterns of tissue homing molecules of the two major NK cell subsets are different and that these might play a critical role in the immune response against acute DENV infection.

Data analysis and presentation in flow cytometry

The advancement of flow cytometry technology together with a series of novel developments in hardware and software have facilitated both phenotypic and functional characterizations of different cell types. In addition, the availability of many monoclonal antibodies and an expanding range of dye-chemistry have made multi-parameter flow cytometry possible for simultaneous measurements of large numbers of cells with better information of complex cellular networks such as the immune system. Although it has the advantage of being a fast, objective and quantitative, but running polychromatic flow cytometry is a complex process with many challenges particularly in the data analysis. The purpose of this communication is to describe several types of presentation and analysis of both univariate and multivariate datasets.

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.

A closed-culture system using a GMP-grade culture bag and anti-CD3/28 coated bead stimulation for CD4+ T cell expansion from healthy and HIV-infected donors

CD4 immunotherapy is potentially useful in immune reconstitution of CD4+ T cells for HIV-infected patients. Transfusion of anti-CD3/28 expanded CD4+ T cells is also proved to be safe and effective in both SIV-infected macaques and HIV-infected patients. However, there is no such standardized and practical protocol available for cell production in order to use in clinics. This study thus aimed to develop a closed-culture system for in vitro CD4+ T lymphocyte expansion by using a commercially available GMP-grade culture bag and anti-CD3/28 activation. Freshly isolated CD4+ T cells by immunorosette formation from healthy donors and cryopreserved CD4+ T cells from HIV-infected patients with CD4 count over 500 cells/μL were stimulated with anti-CD3/28 coated beads. The activated cells were then expanded in conventional culture flasks and GMP-grade culture bags for three weeks. Fold expansion, cell viability, growth kinetic and phenotypic characters were observed. Results revealed that purified CD4+ T cells from healthy individuals cultured in flasks showed better expansion than those cultured in bags (797-fold and 331-fold, respectively), whereas, their cell viability, growth kinetic and expanded CD4+ T cell purity were almost similar. A large-scale production was also conducted and supported consistency of cell proliferation in the closed-culture system. Frozen CD4+ T lymphocytes from the patients were able to remain their growth function and well expanded with a good yield of 415-fold, 85% viability and 96% purity of CD4+ T cells at the end of a 3-week culture in bags. This developed closed-culture system using culture bags and anti-CD3/28 coated beads, therefore, can achieve a large number of expanded CD4+ T lymphocytes with good reproducibility, suggesting a promising protocol required for adoptive immunotherapy.

B cell subset alteration and the expression of tissue homing molecules in dengue infected patients

BackgroundB cells play an essential role during dengue viral infection. While a major expansion of antibody secreting cells (ASCs) was observed, the importance of these increased frequencies of ASCs remains unclear. The alteration of B cell subsets may result from the expression of tissue specific homing molecules leading to their mobilization and distribution to different target organs during acute dengue viral infection.MethodsIn this study, whole blood samples were obtained from thirty pediatric dengue-infected patients and ten healthy children and then stained with fluorochrome-conjugated monoclonal antibodies against CD3, CD14, CD19, CD20, CD21, CD27, CD38, CD45, CD138 and homing molecules of interest before analyzed by polychromatic flow cytometry. B cell subsets were characterized throughout acute infection period.ResultsData shows that there were no detectable differences in frequencies of resting, activated and tissue memory cells, whereas the frequency of ASCs was significantly increased and associated with the lower frequency of naïve cells. These results were found from patients with both dengue fever and dengue hemorrhagic fever, suggesting that such change or alteration of B cells was not associated with disease severity. Moreover, several homing molecules (e.g., CXCR3 and CCR2) were found in ASCs, indicating that ASCs may distribute to inflamed tissues and various organs.ConclusionsFindings from this study provide insight into B cell subset distribution. Furthermore, organ mobilization according to homing molecule expression on different B cell subsets during the course of dengue viral infection also suggests they are distributed to inflamed tissues and various organs.

Therapeutic trial of doxycyclin plus ivermectin for the treatment of Brugia malayi naturally infected cats

Lymphatic filariasis (LF) is one of the neglected tropical diseases which causes permanent and long term disability worldwide. LF is caused by filarial nematode parasites, i.e. Wuchereria bancrofti, Brugia malayi, and B. timori. All available antifilarial drugs currently being used have shown a limited adulticidal activity. Discoveries of endosymbiont rickettsia-like bacterium, Wolbachia in filarial nematodes provided a novel approach for antibiotic use in eradication of filarial diseases. The earlier studies revealed the macrofilaricidal efficacy of doxycycline against filarial nematodes. Chemotherapeutic efficiency of doxycycline has been studied against many filarial parasites, but there are still no therapeutic trials of the drug regimens for B. malayi treatment in naturally infected cats. Thus, this study would be the first attempt to study the efficiency of doxycycline (DOXY) alone or in combination with ivermectin (IVM) for treatment of B. malayi in naturally infected cats. A total of 26 B. malayi-infected cats in the endemic areas were recruited and divided into 3 groups, receiving different treatment regimens; a single dose of ivermectin only (IVM), doxycycline only (DOXY) and a combination of ivermectin and doxycycline (DOXY-IVM). The efficacy of each therapatic regimen was evaluated by detecting the presence of microfilaria using parasitological and molecular techniques monthly up to 2 years after starting the treatment. The IVM treated group had a significant rapid reduction of microfilariae in the first month; however, recurrence of microfilaraemia was observed in some cats. By contrast, the DOXY and DOXY-IVM groups showed a better result with a gradual decrease in microfilariae with no recurrence. These 2 groups were not only virtually deprived of infection but also sustained the sterility of infection through the course of study. These results revealed the advantages of using in B. malayi treatment in cats. Doxycycline showed to have both microfilaricidal and adulticidal effects on lymphatic filariae which maintained the long-term response to control of B. malayi infection in cats.