The role of cell death in the pathogenesis of autoimmune disease: HMGB1 and microparticles as intercellular mediators of inflammation

Cell death is critical to normal homeostasis, although this process, when increased aberrantly, can lead to the production of pro-inflammatory mediators promoting autoimmunity. Two novel intercellular mediators of inflammation generated during cell death are high mobility group box 1 (HMGB1) protein and microparticles (MPs). HMGB1 is a nuclear protein that functions in transcription when inside the nucleus but takes on pro-inflammatory properties when released during cell death. Microparticles are small, membrane-bound structures that extrude from cells when they die and contain cell surface proteins and nuclear material from their parent cells. MPs circulate widely throughout the vasculature and mediate long-distance communication between cells. Both MPs and HMGB1 have been implicated in the pathogenesis of a broad spectrum of inflammatory diseases, including the prototypic autoimmune conditions systemic lupus erythematosus and rheumatoid arthritis. Given their range of activity and association with active disease, both structures may prove to be targets for effective therapy in these and other disorders

Icaritin Shows Potent Anti-Leukemia Activity on Chronic Myeloid Leukemia In Vitro and In Vivo by Regulating MAPK/ERK/JNK and JAK2/STAT3 /AKT Signalings

PURPOSE: To explore the effects of Icaritin on chronic myeloid leukemia (CML) cells and underlying mechanisms. METHOD: CML cells were incubated with various concentration of Icaritin for 48 hours, the cell proliferation was analyzed by MTT and the apoptosis was assessed with Annexin V and Hoechst 33258 staining. Cell hemoglobinization was determined. Western blotting was used to evaluate the expressions of MAPK/ERK/JNK signal pathway and Jak-2/Phorpho-Stat3/Phorsph-Akt network-related protein. NOD-SCID nude mice were applied to demonstrate the anti-leukemia effect of Icaritin in vivo. RESULTS: Icaritin potently inhibited proliferation of K562 cells (IC50 was 8 µM) and primary CML cells (IC50 was 13.4 µM for CML-CP and 18 µM for CML-BC), induced CML cells apoptosis and promoted the erythroid differentiation of K562 cells with time-dependent manner. Furthermore, Icaritin was able to suppress the growth of primary CD34+ leukemia cells (CML) and Imatinib-resistant cells, and to induce apoptosis. In mouse leukemia model, Icaritin could prolong lifespan of NOD-SCID nude mice inoculated with K562 cells as effective as Imatinib without suppression of bone marrow. Icaritin could up-regulate phospho-JNK or phospho-C-Jun and down-regulate phospho-ERK, phospho-P-38, Jak-2, phospho-Stat3 and phospho-Akt expression with dose- or time-dependent manner. Icaritin had no influence both on c-Abl and phospho-c-Abl protein expression and mRNA levels of Bcr/Abl. CONCLUSION: Icaritin from Chinese herb medicine may be a potential anti-CML agent with low adverse effect. The mechanism of anti-leukemia for Icaritin is involved in the regulation of Bcr/Abl downstream signaling. Icaritin may be useful for an alternative therapeutic choice of Imatinib-resistant forms of CML

Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins

Combination of Two but Not Three Current Targeted Drugs Can Improve Therapy of Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a cancer of the hematopoietic system and has been treated with the drug Imatinib relatively successfully. Drug resistance, acquired by mutations, is an obstacle to success. Two additional drugs are now considered and could be combined with Imatinib to prevent resistance, Dasatinib and Nilotinib. While most mutations conferring resistance to one drug do not confer resistance to the other drugs, there is one mutation (T315I) that induces resistance against all three drugs. Using computational methods, the combination of two drugs is found to increase the probability of treatment success despite this cross-resistance. Combining more than two drugs, however, does not provide further advantages. We also explore possible combination therapies using drugs currently under development. We conclude that among the targeted drugs currently available for the treamtent of CML, only the two most effective ones should be used in combination for the prevention of drug resistance

Molecular Epidemiology of HIV-1 Subtypes in India: Origin and Evolutionary History of the Predominant Subtype C

This thesis describes the translational genomics of HIV-1subtype C in India from its origin to therapeutic response with the aim to improve our knowledge for better therapeutic and preventive strategies to combat HIV/AIDS. In a systemic approach, we identified the molecular phylogeny of HIV-1 subtypes circulating in India and the time to most recent common ancestors (tMRCA) of predominant HIV-1 subtype C strains. Additionally, this thesis also studied drug resistance mutations in children, adolescents and adults, the role of host factors in evolution of drug resistance, and population dynamics of viremia and viral co-receptor tropism in perinatal transmission. Finally, the long term therapeutic responses on Indian national first-line antiretroviral therapy were also studied. In Paper I, we reported an increase in the HIV-1 recombinant forms in the HIV-1 epidemiology using a robust subtyping methodology. While the study confirmed HIV- 1 subtype C as a dominant subtype, its origin was dated back to the early 1970s from a single or few genetically related strains from South Africa, whereafter, it has evolved independently. In Paper II, the lethal hypermutations due to the activity of human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (hA3G) was significantly associated with antiretroviral therapy (ART) failure in Indian HIV-1 subtype C patients. The presence of M184I and M230I mutations were observed due to the editing of hA3G in the proviral compartment but stop codons were also found in the open reading frames and the same drug resistance mutations were absent in plasma virus. Therefore, it is unlikely that the viral variants which exhibit hypermutated sequences and M184I and/or M230I will mature and expand in vivo and hence are unlikely to have any clinical significance. The high concordance of drug resistance genotyping in the plasma and proviral compartments in therapy-naïve patients, gives weight to the idea of using whole blood for surveillance of drug resistance mutations which precludes logistic challenges of cold chain transport. In Papers III and IV, we identified a substantial proportion of HIV-1 subtype C perinatally-infected older children who had a high burden of plasma viremia but also had high CD4+ T-cell counts. In addition, older children with HIV-1 subtype C infection presented a high prevalence of predicted X4 and R5/X4 tropic strains which indicates that HIV-1 subtype C strains required longer duration of infection and greater disease progression to co-receptor transition from R5- to X4-tropic strains (IV). Our studies also indicate that transmitted drug resistance is low among Indian HIV-1 infected children, adolescents (III) and adults (II). In Paper V, in a longitudinal cohort study, a good long-term response to the Indian national first-line therapy for a median of nearly four years with 2.8% viral failure, indicating the overall success of the Indian ART program. Our study also showed that three immunologically well patients with virological rebound and major viral drug resistance mutations (M184V, K103N and Y181C) during one study visit had undetectable viral load at their next visit. These findings suggest that use of multiple parameters like patients’ immunological (CD4+ T-cell count), virological (viral load) and drug resistance data should all be used to optimize the treatment switch to second line therapy. In conclusion, this translational genomics study enhances our knowledge about the HIV-1 subtype C strains circulating in India which are genetically distinct from prototype African subtype C strains. Considerably more research using appropriate models need to be performed to understand the phenotypic and biological characteristics of these strains to guide efficient disease intervention and management strategies

Association of Coagulation Activation with Clinical Complications in Sickle Cell Disease

Background: The contribution of hypercoagulability to the pathophysiology of sickle cell disease (SCD) remains poorly defined. We sought to evaluate the association of markers of coagulation and platelet activation with specific clinical complications and laboratory variables in patients with SCD. Design and Methods: Plasma markers of coagulation activation (D-dimer and TAT), platelet activation (soluble CD40 ligand), microparticle-associated tissue factor (MPTF) procoagulant activity and other laboratory variables were obtained in a cohort of patients with SCD. Tricuspid regurgitant jet velocity was determined by Doppler echocardiography and the presence/history of clinical complications was ascertained at the time of evaluation, combined with a detailed review of the medical records. Results: No significant differences in the levels of D-dimer, TAT, soluble CD40 ligand, and MPTF procoagulant activity were observed between patients in the SS/SD/Sb 0 thalassemia and SC/Sb + thalassemia groups. Both TAT and D-dimer were significantly correlated with measures of hemolysis (lactate dehydrogenase, indirect bilirubin and hemoglobin) and soluble vascular cell adhesion molecule-1. In patients in the SS/SD/Sb 0 thalassemia group, D-dimer was associated with a history of stroke (p = 0.049), TAT was associated with a history of retinopathy (p = 0.0176), and CD40 ligand was associated with the frequency of pain episodes (p = 0.039). In multivariate analyses, D-dimer was associated with reticulocyte count, lactat

Anemia and growth failure among HIV-infected children in India: a retrospective analysis

<p>Abstract</p> <p>Background</p> <p>Anemia and poor nutrition have been previously described as independent risk factors for death among HIV-infected children. We sought to describe nutritional status, anemia burden and HIV disease correlates among infected children in India.</p> <p>Methods</p> <p>We analyzed retrospective data from 248 HIV-infected children aged 1–12 years attending three outpatient clinics in South India (2004–2006). Standard WHO definitions were used for anemia, HIV staging and growth parameters. Statistical analysis included chi square, t tests, univariate and multivariate logistic regression analyses.</p> <p>Results</p> <p>The overall prevalence of anemia (defined as hemoglobin < 11 gm/dL) was 66%, and 8% had severe anemia (Hb < 7 gm/dL). The proportion of underweight and stunted children in the population was 55% and 46% respectively. Independent risk factors of anemia by multivariate analysis included the pre-school age group (age younger than 6 years) (OR: 2.87; 95% CI: 1.45, 5.70; p < 0.01), rural residence (OR: 12.04; 95% CI: 5.64, 26.00; p < 0.01), advanced HIV disease stage (OR: 6.95; 95% CI: 3.06, 15.79; p < 0.01) and presence of stunting (Height-for-age Z Score < -2) (OR: 3.24; 95% CI: 1.65, 6.35; p < 0.01). Use of iron/multivitamin supplementation was protective against risk of anemia (OR: 0.44; 95% CI: 0.22, 0.90; p = 0.03). Pulmonary tuberculosis was an independent risk factor in multivariate analysis (OR: 3.36; 95% CI: 1.43, 7.89; p < 0.01) when correlated variables such as HIV disease stage and severe immunodeficiency, and nutritional supplement use were not included. Use of antiretroviral therapy (ART) was associated with a reduced risk of anemia (OR: 0.29; 95% CI: 0.16, 0.53; p < 0.01). No significant association was found between anemia and gender, cotrimoxazole, or ART type (zidovudine versus stavudine).</p> <p>Conclusion</p> <p>The high prevalence and strong interrelationship of anemia and poor nutrition among HIV-infected children in India, particularly those living in rural areas underscores the need for incorporating targeted nutritional interventions during national scale up of care, support and treatment among HIV-infected children.</p

Effect of Cellular Quiescence on the Success of Targeted CML Therapy

Similar to tissue stem cells, primitive tumor cells in chronic myelogenous leukemia have been observed to undergo quiescence; that is, the cells can temporarily stop dividing. Using mathematical models, we investigate the effect of cellular quiescence on the outcome of therapy with targeted small molecule inhibitors.According to the models, the initiation of treatment can result in different patterns of tumor cell decline: a biphasic decline, a one-phase decline, and a reverse biphasic decline. A biphasic decline involves a fast initial phase (which roughly corresponds to the eradication of cycling cells by the drug), followed by a second and slower phase of exponential decline (corresponding to awakening and death of quiescent cells), which helps explain clinical data. We define the time when the switch to the second phase occurs, and identify parameters that determine whether therapy can drive the tumor extinct in a reasonable period of time or not. We further ask how cellular quiescence affects the evolution of drug resistance. We find that it has no effect on the probability that resistant mutants exist before therapy if treatment occurs with a single drug, but that quiescence increases the probability of having resistant mutants if patients are treated with a combination of two or more drugs with different targets. Interestingly, while quiescence prolongs the time until therapy reduces the number of cells to low levels or extinction, the therapy phase is irrelevant for the evolution of drug resistant mutants. If treatment fails as a result of resistance, the mutants will have evolved during the tumor growth phase, before the start of therapy. Thus, prevention of resistance is not promoted by reducing the quiescent cell population during therapy (e.g., by a combination of cell activation and drug-mediated killing).The mathematical models provide insights into the effect of quiescence on the basic kinetics of the response to targeted treatment of CML. They identify determinants of success in the absence of drug resistant mutants, and elucidate how quiescence influences the emergence of drug resistant mutants

Prevalence of Transmitted Drug Resistance and Impact of Transmitted Resistance on Treatment Success in the German HIV-1 Seroconverter Cohort

BACKGROUND: The aim of this study is to analyse the prevalence of transmitted drug resistance, TDR, and the impact of TDR on treatment success in the German HIV-1 Seroconverter Cohort. METHODS: Genotypic resistance analysis was performed in treatment-naïve study patients whose sample was available 1,312/1,564 (83.9% October 2008). A genotypic resistance result was obtained for 1,276/1,312 (97.3%). The resistance associated mutations were identified according to the surveillance drug resistance mutations list recommended for drug-naïve patients. Treatment success was determined as viral suppression below 500 copies/ml. RESULTS: Prevalence of TDR was stable at a high level between 1996 and 2007 in the German HIV-1 Seroconverter Cohort (N = 158/1,276; 12.4%; CI(wilson) 10.7-14.3; p(for trend) = 0.25). NRTI resistance was predominant (7.5%) but decreased significantly over time (CI(Wilson): 6.2-9.1, p(for trend) = 0.02). NNRTI resistance tended to increase over time (NNRTI: 3.5%; CI(Wilson): 2.6-4.6; p(for trend)= 0.07), whereas PI resistance remained stable (PI: 3.0%; CI(Wilson): 2.1-4.0; p(for trend) = 0.24). Resistance to all drug classes was frequently caused by singleton resistance mutations (NRTI 55.6%, PI 68.4%, NNRTI 99.1%). The majority of NRTI-resistant strains (79.8%) carried resistance-associated mutations selected by the thymidine analogues zidovudine and stavudine. Preferably 2NRTI/1PIr combinations were prescribed as first line regimen in patients with resistant HIV as well as in patients with susceptible strains (susceptible 45.3%; 173/382 vs. resistant 65.5%; 40/61). The majority of patients in both groups were treated successfully within the first year after ART-initiation (susceptible: 89.9%; 62/69; resistant: 7/9; 77.8%). CONCLUSION: Overall prevalence of TDR remained stable at a high level but trends of resistance against drug classes differed over time. The significant decrease of NRTI-resistance in patients newly infected with HIV might be related to the introduction of novel antiretroviral drugs and a wider use of genotypic resistance analysis prior to treatment initiation