High Spatial Resolution and Temporally Resolved T2 * Mapping of Normal Human Myocardium at 7.0 Tesla: An Ultrahigh Field Magnetic Resonance Feasibility Study

Myocardial tissue characterization using T₂ ^* relaxation mapping techniques is an emerging application of (pre)clinical cardiovascular magnetic resonance imaging. The increase in microscopic susceptibility at higher magnetic field strengths renders myocardial T₂ ^* mapping at ultrahigh magnetic fields conceptually appealing. This work demonstrates the feasibility of myocardial T₂ ^* imaging at 7.0 T and examines the applicability of temporally-resolved and high spatial resolution myocardial T₂ ^* mapping. In phantom experiments single cardiac phase and dynamic (CINE) gradient echo imaging techniques provided similar T₂ ^* maps. In vivo studies showed that the peak-to-peak B₀ difference following volume selective shimming was reduced to approximately 80 Hz for the four chamber view and mid-ventricular short axis view of the heart and to 65 Hz for the left ventricle. No severe susceptibility artifacts were detected in the septum and in the lateral wall for T₂ ^* weighting ranging from TE = 2.04 ms to TE = 10.2 ms. For TE >7 ms, a susceptibility weighting induced signal void was observed within the anterior and inferior myocardial segments. The longest T₂ ^* values were found for anterior (T₂ ^* = 14.0 ms), anteroseptal (T₂ ^* = 17.2 ms) and inferoseptal (T₂ ^* = 16.5 ms) myocardial segments. Shorter T₂ ^* values were observed for inferior (T₂ ^* = 10.6 ms) and inferolateral (T₂ ^* = 11.4 ms) segments. A significant difference (p = 0.002) in T₂ ^* values was observed between end-diastole and end-systole with T₂ ^* changes of up to approximately 27% over the cardiac cycle which were pronounced in the septum. To conclude, these results underscore the challenges of myocardial T₂ ^* mapping at 7.0 T but demonstrate that these issues can be offset by using tailored shimming techniques and dedicated acquisition schemes.     

T7 Protein Synthesis in F-Factor-Containing Cells: Evidence for an Episomally Induced Impairment of Translation and Its Relation to an Alteration in Membrane Permeability

T7 infection of F-factor-containing PIFA⁺,B⁺ cells is abortive. In spite of the presence of mRNA for all three classes of T7 proteins, only the earliest of the T7 proteins are synthesized. A crucial question is whether the failure of T7 to develop in PIFA⁺,B⁺ cells is the result of an inability to translate the late classes of T7 mRNA or, as has been recently suggested (Britton, and Haselkorn, 1975; Condit, 1975), whether it is the result of a more generalized alteration in membrane permeability. We have examined the effects of the wild-type PIFA⁺,B⁺ episome and two episomal mutations (pifA⁻ and pifB⁻) on in vitro translation and membrane permeability. In vivo the episomal mutations allow partial or complete T7 development to occur. We demonstrate that cell-free protein-synthesizing systems from T7-infected PIFA⁺,B⁺ cells show a three- to fivefold decrease in the rate of translation of both natural and synthetic mRNA. In addition, ribosomes from T7-infected PIFA⁺,B⁺ cells are defective in their ability to bind Fmet tRNA_f in response to natural mRNA. By contrast, cell-free extracts from T7-infected pifA⁻ (PIFA⁻,B⁺) cells retain the ability to bind Fmet tRNA_f and to translate natural and synthetic mRNA at normal rates. The defective T7-infected PIFA⁺,B⁺ ribosomes can be restored to full activity by a trypsin-sensitive fraction from uninfected PIFA⁺,B⁺ or T7-infected PIFA⁻,B⁺ cells. Despite the differences in translational capacity of these extracts, both T7-infected PIFA⁺,B⁺ and PIFA⁻,B⁺ cells display the same permeability lesions as measured by the loss of ATP from the cells into the supernatant. Mutation of the episome to pfiB⁻ prevents the loss of ATP from the cells after T7 infection.     

Association between the IL1B (-511), IL1B (+3954), IL1RN (VNTR) Polymorphisms and Graves' Disease Risk: A Meta-Analysis of 11 Case-Control Studies

Background

Data on the association between the interleukin-1 (IL-1) gene polymorphisms and Graves'' disease (GD) risk were conflicting. A meta-analysis was undertaken to assess this association.

Methods

We searched for case-control studies investigating the association between the IL1B (-511), IL1B (+3954), IL1RN (VNTR) polymorphisms and GD risk. We extracted data using standardized forms and calculated odds ratios (OR) with 95% confidence intervals (CI).

Results

A total of 11 case-control studies were included in this meta-analysis. Available data indicated that the IL1B (-511) polymorphism was associated with GD risk in the overall populations (Caucasians and Asians) in homozygote model (TT vs. CC, OR = 0.86, 95% CI: 0.76–0.97, P_z = 0.015), but not in dominant and recessive models (TT+TC vs. CC: OR = 0.95, 95% CI: 0.81–1.12, P_z = 0.553 and TT vs. TC+CC: OR = 0.82, 95% CI: 0.60–1.12, P_z = 0.205, respectively). No association between the IL1B (+3954), IL1RN (VNTR) polymorphisms and GD risk was found in the overall populations in any of the genetic models. In subgroup analyses according to ethnicity, the IL1B (-511) polymorphism was associated with GD risk in Asians in recessive and homozygote models (TT vs. TC+CC: OR = 0.68, 95% CI: 0.55–0.84, P_z<0.001 and TT vs. CC: OR = 0.81, 95% CI: 0.70–0.93, P_z = 0.003, respectively), but not in dominant model (TT+TC vs. CC: OR = 0.92, 95% CI: 0.77–1.11, P_z = 0.389). No association between the IL1B (+3954), IL1RN (VNTR) polymorphisms and GD risk was indicated in Asians, and we found no association between the IL1B (-511), IL1B (+3954), IL1RN (VNTR) polymorphisms and GD risk in Caucasians in any of the genetic models.

Conclusion

The IL1B (-511) polymorphism, but not the IL1B (+3954) and IL1RN (VNTR) polymorphisms was associated with GD risk in Asians. There was no association between these polymorphisms and GD risk in Caucasians.     

A Novel Missense Mutation,I890T,in the Pore Region of Cardiac Sodium Channel Causes Brugada Syndrome

Brugada syndrome (BrS) is a life-threatening, inherited arrhythmogenic syndrome associated with autosomal dominant mutations in SCN5A, the gene encoding the cardiac Na⁺ channel alpha subunit (Na_v1.5). The aim of this work was to characterize the functional alterations caused by a novel SCN5A mutation, I890T, and thus establish whether this mutation is associated with BrS. The mutation was identified by direct sequencing of SCN5A from the proband’s DNA. Wild-type (WT) or I890T Na_v1.5 channels were heterologously expressed in human embryonic kidney cells. Sodium currents were studied using standard whole cell patch-clamp protocols and immunodetection experiments were performed using an antibody against human Na_v1.5 channel. A marked decrease in current density was observed in cells expressing the I890T channel (from −52.0±6.5 pA/pF, n = 15 to −35.9±3.4 pA/pF, n = 22, at −20 mV, WT and I890T, respectively). Moreover, a positive shift of the activation curve was identified (V _1/2 = −32.0±0.3 mV, n = 18, and −27.3±0.3 mV, n = 22, WT and I890T, respectively). No changes between WT and I890T currents were observed in steady-state inactivation, time course of inactivation, slow inactivation or recovery from inactivation parameters. Cell surface protein biotinylation analyses confirmed that Na_v1.5 channel membrane expression levels were similar in WT and I890T cells. In summary, our data reveal that the I890T mutation, located within the pore of Na_v1.5, causes an evident loss-of-function of the channel. Thus, the BrS phenotype observed in the proband is most likely due to this mutation.     

Angiotensin II Is a New Component Involved in Splenic T Lymphocyte Responses during Plasmodium berghei ANKA Infection

The contribution of T cells in severe malaria pathogenesis has been described. Here, we provide evidence for the potential role of angiotensin II (Ang II) in modulating splenic T cell responses in a rodent model of cerebral malaria. T cell activation induced by infection, determined by 3 to 4-fold enhancement in CD69 expression, was reduced to control levels when mice were treated with 20 mg/kg losartan (IC₅₀ = 0.966 mg/kg/d), an AT₁ receptor antagonist, or captopril (IC₅₀ = 1.940 mg/kg/d), an inhibitor of angiotensin-converting enzyme (ACE). Moreover, the production of interferon-γ and interleukin-17 by CD4⁺ T cells diminished 67% and 70%, respectively, by both treatments. Losartan reduced perforin expression in CD8⁺ T cells by 33% while captopril completely blocked it. The upregulation in chemokine receptor expression (CCR2 and CCR5) observed during infection was abolished and CD11a expression was partially reduced when mice were treated with drugs. T cells activated by Plasmodium berghei ANKA antigens showed 6-fold enhance in AT₁ levels in comparison with naive cells. The upregulation of AT₁ expression was reduced by losartan (80%) but not by captopril. Our results suggest that the AT₁/Ang II axis has a role in the establishment of an efficient T cell response in the spleen and therefore could participate in a misbalanced parasite-induced T cell immune response during P. berghei ANKA infection.     

A Subset of Circulating Blood Mycobacteria-Specific CD4 T Cells Can Predict the Time to Mycobacterium tuberculosis Sputum Culture Conversion

We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR⁺Ki67⁺ on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67⁺HLA-DR⁺ Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67⁺HLA-DR⁺ T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ⁺IL2⁺TNFα⁺ CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67⁺HLA-DR⁺) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment.     

Molecular Epidemiology of Clostridium difficile at a Medical Center in Taiwan: Persistence of Genetically Clustering of A?B+ Isolates and Increase of A+B+ Isolates

Introduction

We investigated the changing trend of various toxigenic Clostridium difficile isolates at a 3 500-bed hospital in Taiwan. Genetic relatedness and antimicrobial susceptibility of toxigenic C. difficile isolates were also examined.

Methods

A total of 110 non-repeat toxigenic C. difficile isolates from different patients were collected between 2002 and 2007. Characterization of the 110 toxigenic isolates was performed using agar dilution method, multilocus variable-number tandem-repeat analysis (MLVA) genotyping, tcdC genotyping, and toxinotyping.

Results

Among the 110 toxigenic isolates studied, 70 isolates harbored tcdA and tcdB (A⁺B⁺) and 40 isolates harbored tcdB only (A⁻B⁺). The annual number of A⁺B⁺ isolates considerably increased over the 6-year study (P = 0.055). A total of 109 different MLVA genotypes were identified, in which A⁺B⁺ isolates and A⁻B⁺ isolates were differentiated into two genetic clusters with similarity of 17.6%. Twenty-four (60%) of the 40 A⁻B⁺ isolates formed a major cluster, MLVA-group 1, with a similarity of 85%. Seven (6.4%) resistant isolates were identified, including two metronidazole-resistant and five vancomycin-resistant isolates.

Conclusions

This study indicated a persistence of a MLVA group 1 A⁻B⁺ isolates and an increase of A⁺B⁺ isolates with diverse MLVA types. Moreover, C. difficile isolates with antimicrobial resistance to metronidazole or vancomycin were found to have emerged. Continuous surveillance is warranted to understand the recent situation and control the further spread of the toxigenic C. difficile isolates, especially among hospitalized patients.     

Impact of Soluble CD26 on Treatment Outcome and Hepatitis C Virus-Specific T Cells in Chronic Hepatitis C Virus Genotype 1 Infection

Background

Interferon and ribavirin therapy for chronic hepatitis C virus (HCV) infection yields sustained virological response (SVR) rates of 50–80%. Several factors such as non-1 genotype, beneficial IL28B genetic variants, low baseline IP-10, and the functionality of HCV-specific T cells predict SVR. With the pending introduction of new therapies for HCV entailing very rapid clearance of plasma HCV RNA, the importance of baseline biomarkers likely will increase in order to tailor therapy. CD26 (DPPIV) truncates the chemokine IP-10 into a shorter antagonistic form, and this truncation of IP-10 has been suggested to influence treatment outcome in patients with chronic HCV infection patients. In addition, previous reports have shown CD26 to be a co-stimulator for T cells. The aim of the present study was to assess the utility of CD26 as a biomarker for treatment outcome in chronic hepatitis C and to define its association with HCV-specific T cells.

Methods

Baseline plasma from 153 genotype 1 and 58 genotype 2/3 infected patients enrolled in an international multicenter phase III trial (DITTO-HCV) and 36 genotype 1 infected patients participating in a Swedish trial (TTG1) were evaluated regarding baseline soluble CD26 (sCD26) and the functionality of HCV-specific CD8⁺ T cells.

Results

Genotype 1 infected patients achieving SVR in the DITTO (P = 0.002) and the TTG1 (P = 0.02) studies had lower pretreatment sCD26 concentrations compared with non-SVR patients. Sixty-five percent of patients with sCD26 concentrations below 600 ng/mL achieved SVR compared with 39% of the patients with sCD26 exceeding 600 ng/mL (P = 0.01). Patients with sCD26 concentrations below 600 ng/mL had significantly higher frequencies of HCV-specific CD8⁺ T cells (P = 0.02).

Conclusions

Low baseline systemic concentrations of sCD26 predict favorable treatment outcome in chronic HCV infection and may be associated with higher blood counts of HCV-specific CD8⁺ T cells.     

Fine Mapping and Functional Analysis of the Multiple Sclerosis Risk Gene CD6

CD6 has recently been identified and validated as risk gene for multiple sclerosis (MS), based on the association of a single nucleotide polymorphism (SNP), rs17824933, located in intron 1. CD6 is a cell surface scavenger receptor involved in T-cell activation and proliferation, as well as in thymocyte differentiation. In this study, we performed a haptag SNP screen of the CD6 gene locus using a total of thirteen tagging SNPs, of which three were non-synonymous SNPs, and replicated the recently reported GWAS SNP rs650258 in a Spanish-Basque collection of 814 controls and 823 cases. Validation of the six most strongly associated SNPs was performed in an independent collection of 2265 MS patients and 2600 healthy controls. We identified association of haplotypes composed of two non-synonymous SNPs [rs11230563 (R225W) and rs2074225 (A257V)] in the 2^nd SRCR domain with susceptibility to MS (P _{max(T) permutation} = 1×10⁻⁴). The effect of these haplotypes on CD6 surface expression and cytokine secretion was also tested. The analysis showed significantly different CD6 expression patterns in the distinct cell subsets, i.e. – CD4⁺ naïve cells, P = 0.0001; CD8⁺ naïve cells, P<0.0001; CD4⁺ and CD8⁺ central memory cells, P = 0.01 and 0.05, respectively; and natural killer T (NKT) cells, P = 0.02; with the protective haplotype (RA) showing higher expression of CD6. However, no significant changes were observed in natural killer (NK) cells, effector memory and terminally differentiated effector memory T cells. Our findings reveal that this new MS-associated CD6 risk haplotype significantly modifies expression of CD6 on CD4⁺ and CD8⁺ T cells.     

Higher CD27+CD8+ T Cells Percentages during Suppressive Antiretroviral Therapy Predict Greater Subsequent CD4+ T Cell Recovery in Treated HIV Infection

HIV-mediated immune dysfunction may influence CD4⁺ T cell recovery during suppressive antiretroviral therapy (ART). We analyzed cellular biomarkers of immunological inflammation, maturation, and senescence in HIV-infected subjects on early suppressive ART. We performed longitudinal analyses of peripheral immunological biomarkers of subjects on suppressive ART (n = 24) from early treatment (median 6.4 months, interquartile range [IQR] 4.8–13.9 months) to 1–2 years of follow-up (median 19.8 months, IQR 18.3–24.6 months). We performed multivariate regression to determine which biomarkers were associated with and/or predictive of CD4⁺ T cell recovery. After adjusting for the pre-ART CD4⁺ T cell count, age, proximal CD4⁺ T cell count, and length of ART medication, the percentage of CD27⁺CD8⁺ T cells remained significantly associated with the CD4⁺ T cell recovery rate (β = 0.092 cells/ul/month, P = 0.028). In HIV-infected subjects starting suppressive ART, patients with the highest percentage of CD8⁺ T cells expressing CD27 had the greatest rate of CD4⁺ T cell recovery.     

High Resolution T1ρ Mapping of In Vivo Human Knee Cartilage at 7T

Purpose

Spin lattice relaxation time in rotating frame (T1ρ) mapping of human knee cartilage has shown promise in detecting biochemical changes during osteoarthritis. Due to higher field strength, MRI at 7T has advantages in term of SNR compared to clinical MR scanners and this can be used to increase in image resolution. Objective of current study was to evaluate the feasibility of high resolution T1ρ mapping of in vivo human knee cartilage at 7T MR scanner.

Materials and Methods

In this study we have used a T1ρ prepared GRE pulse sequence for obtaining high resolution (in plan resolution  = 0.2 mm²) T1ρ MRI of human knee cartilage at 7T. The effect of a global and localized reference frequency and reference voltage setting on B₀, B₁ and T1ρ maps in cartilage was evaluated. Test-retest reliability results of T1ρ values from asymptomatic subjects as well as T1ρ maps from abnormal cartilage of two human subjects are presented. These results are compared with T1ρ MRI data obtained from 3T.

Results

Our approach enabled acquisition of 3D-T1ρ data within allowed SAR limits at 7T. SNR of cartilage on T1ρ weighted images was greater than 90. Off-resonance effects present in the cartilage B₀, B₁ and T1ρ maps obtained using global shim and reference frequency and voltage setting, were reduced by the proposed localized reference frequency and voltage setting. T1ρ values of cartilage obtained with the localized approach were reproducible. Abnormal knee cartilage showed elevated T1ρ values in affected regions. T1ρ values at 7T were significantly lower (p<0.05) compared to those obtained at 3T.

Conclusion

In summary, by using proposed localized frequency and voltage setting approach, high-resolution 3D-T1ρ maps of in vivo human knee cartilage can be obtained in clinically acceptable scan times (<30 min) and SAR constraints, which provides the ability to characterize cartilage molecular integrity.     

IL-4 Haplotype -590T, -34T and Intron-3 VNTR R2 Is Associated with Reduced Malaria Risk among Ancestral Indian Tribal Populations

Background

Interleukin 4 (IL-4) is an anti-inflammatory cytokine, which regulates balance between T_H1 and T_H2 immune response, immunoglobulin class switching and humoral immunity. Polymorphisms in this gene have been reported to affect the risk of infectious and autoimmune diseases.

Methods

We have analyzed three regulatory IL-4 polymorphisms; -590C>T, -34C>T and 70 bp intron-3 VNTR, in 4216 individuals; including: (1) 430 ethnically matched case-control groups (173 severe malaria, 101 mild malaria and 156 asymptomatic); (2) 3452 individuals from 76 linguistically and geographically distinct endogamous populations of India, and (3) 334 individuals with different ancestry from outside India (84 Brazilian, 104 Syrian, and 146 Vietnamese).

Results

The -590T, -34T and intron-3 VNTR R2 alleles were found to be associated with reduced malaria risk (P<0.001 for -590C>T and -34C>T, and P = 0.003 for VNTR). These three alleles were in strong LD (r²>0.75) and the TTR2 (-590T, -34T and intron-3 VNTR R2) haplotype appeared to be a susceptibility factor for malaria (P = 0.009, OR = 0.552, 95% CI = 0.356 –0.854). Allele and genotype frequencies differ significantly between caste, nomadic, tribe and ancestral tribal populations (ATP). The distribution of protective haplotype TTR2 was found to be significant (χ² ₃ = 182.95, p-value <0.001), which is highest in ATP (40.5%); intermediate in tribes (33%); and lowest in caste (17.8%) and nomadic (21.6%).

Conclusions

Our study suggests that the IL-4 polymorphisms regulate host susceptibility to malaria and disease progression. TTR2 haplotype, which gives protection against malaria, is high among ATPs. Since they inhabited in isolation and mainly practice hunter-gatherer lifestyles and exposed to various parasites, IL-4 TTR2 haplotype might be under positive selection.     

Cortisol Patterns Are Associated with T Cell Activation in HIV

Objective

The level of T cell activation in untreated HIV disease is strongly and independently associated with risk of immunologic and clinical progression. The factors that influence the level of activation, however, are not fully defined. Since endogenous glucocorticoids are important in regulating inflammation, we sought to determine whether less optimal diurnal cortisol patterns are associated with greater T cell activation.

Methods

We studied 128 HIV-infected adults who were not on treatment and had a CD4⁺ T cell count above 250 cells/µl. We assessed T cell activation by CD38 expression using flow cytometry, and diurnal cortisol was assessed with salivary measurements.

Results

Lower waking cortisol levels correlated with greater T cell immune activation, measured by CD38 mean fluorescent intensity, on CD4⁺ T cells (r = −0.26, p = 0.006). Participants with lower waking cortisol also showed a trend toward greater activation on CD8⁺ T cells (r = −0.17, p = 0.08). A greater diurnal decline in cortisol, usually considered a healthy pattern, correlated with less CD4⁺ (r = 0.24, p = 0.018) and CD8⁺ (r = 0.24, p = 0.017) activation.

Conclusions

These data suggest that the hypothalamic-pituitary-adrenal (HPA) axis contributes to the regulation of T cell activation in HIV. This may represent an important pathway through which psychological states and the HPA axis influence progression of HIV.     

A Functional Variant of PTPN22 Confers Risk for Vogt-Koyanagi-Harada Syndrome but Not for Ankylosing Spondylitis

Background

Protein tyrosine phosphatase non-receptor 22 (PTPN22) is a key negative regulator of T lymphocytes and has emerged as an important candidate susceptibility factor for a number of immune-related diseases. This study aimed to examine the predisposition of PTPN22 SNPs to Vogt-Koyanagi-Harada (VKH) syndrome and acute anterior uveitis (AAU) associated with ankylosing spondylitis (AS).

Methods

A total of 1005 VKH syndrome, 302 AAU⁺AS⁺ patients and 2010 normal controls among the Chinese Han population were enrolled in the study. Genotyping, PTPN22 expression, cell proliferation, cytokine production and cell activation were examined by PCR-RFLP, Real-time PCR, CCK8, ELISA and Flow cytometry.

Results

The results showed significantly increased frequencies of the rs2488457 CC genotype and C allele but a decreased frequency of the GG genotype in VKH syndrome patients (P_{Bonferroni correction} (P_c) = 3.47×10⁻⁷, OR = 1.54; P_c = 3.83×10⁻⁸, OR = 1.40; P_c = 6.35×10⁻⁴, OR = 0.62; respectively). No significant association of the tested SNPs with AAU⁺AS⁺ patients was observed. Functional studies showed a decreased PTPN22 expression, impaired cell proliferation and lower production of IL-10 in rs2488457 CC cases compared to GG cases (P_c = 0.009, P_c = 0.015 and P_c = 0.048 respectively). No significant association was observed concerning T cell activation and rs2488457 genotype.

Conclusions

The study showed that a functional variant of PTPN22 confers risk for VKH syndrome but not for AAU⁺AS⁺ in a Chinese Han population, which may be due to a modulation of the PTPN22 expression, PBMC proliferation and IL-10 production.     

Association of Cytokines in Individuals Sensitive and Insensitive to Dust Mites in a Brazilian Population

Introduction

Allergic reaction to dust mites is a relatively common condition among children, triggering cutaneous and respiratory responses that have a great impact on the health of this population. Anaphylactic hypersensitivity is characterized by an exacerbated response involving the production of regulatory cytokines responsible for stimulating the production of IgE antibodies.

Objective

To investigate an association of variants in cytokine genes (IL1A ⁻⁸⁸⁹, IL1B ^{−511, +3962}, IL1R ¹⁹⁷⁰, IL1RA ¹¹¹⁰⁰, IL4RA ⁺¹⁹⁰², IL12 ⁻¹¹⁸⁸, IFNG ⁺⁸⁷⁴, TGFB1 ^{codon 10, codon 25}, TNFA ^{−308, −238}, IL2 ^{−330, +166}, IL4 ^{−1098, −590, −33}, IL6 ^{−174, nt565}, and IL10 ^{−1082, −819, −592}) between patients sensitive to dust mites and a control group.

Methods

A total of 254 patients were grouped as atopic and non-atopic according to sensitivity as evaluated by the Prick Test and to cytokine genotyping by the polymerase chain reaction-sequence specific primers (PCR-SSP) method using the Cytokine Genotyping Kit.

Results

A comparison between individuals allergic to Dermatophagoides farinae, Dermatophagoides pteronyssinus, and Blomia tropicalis and a non-atopic control group showed significant differences between allele and genotype frequencies in the regulatory regions of cytokine genes, with important evidence for IL4 ⁻⁵⁹⁰ in T/C (10.2% vs. 43.1%, odd ratio [OR] = 0.15, p = 5.2 10⁻⁸, pc = 0.0000011, and 95% confidence interval [95%CI] = 0.07–0.32) and T/T genotypes (42.9% vs. 13.8%, OR = 4.69, p = 2.5 10⁻⁶, pc = 0.000055, and 95%CI = 2.42–9.09). Other associations were observed in the pro-inflammatory cytokines IL1A ⁻⁸⁸⁹ (T/T, C, and T) and IL2 ⁻³³⁰ (G/T and T/T) and the anti-inflammatory cytokines IL4RA ⁺¹⁹⁰² (A and G), IL4 ⁻⁵⁹⁰ (T/C, T/T, C, and T), and IL10 ⁻⁵⁹² (A/A, C/A, A, and C).

Conclusion

Our results suggest a possible association between single nucleotide polymorphisms (SNPs) in cytokine genes and hypersensitivity to dust mites.     

A Correlate of HIV-1 Control Consisting of Both Innate and Adaptive Immune Parameters Best Predicts Viral Load by Multivariable Analysis in HIV-1 Infected Viremic Controllers and Chronically-Infected Non-Controllers

HIV-1 infected viremic controllers maintain durable viral suppression below 2000 copies viral RNA/ml without anti-retroviral therapy (ART), and the immunological factor(s) associated with host control in presence of low but detectable viral replication are of considerable interest. Here, we utilized a multivariable analysis to identify which innate and adaptive immune parameters best correlated with viral control utilizing a cohort of viremic controllers (median 704 viral RNA/ml) and non-controllers (median 21,932 viral RNA/ml) that were matched for similar CD4⁺ T cell counts in the absence of ART. We observed that HIV-1 Gag-specific CD8⁺ T cell responses were preferentially targeted over Pol-specific responses in viremic controllers (p = 0.0137), while Pol-specific responses were positively associated with viral load (rho = 0.7753, p = 0.0001, n = 23). Viremic controllers exhibited significantly higher NK and plasmacytoid dendritic cells (pDC) frequency as well as retained expression of the NK CD16 receptor and strong target cell-induced NK cell IFN-gamma production compared to non-controllers (p<0.05). Despite differences in innate and adaptive immune function however, both viremic controllers (p<0.05) and non-controller subjects (p<0.001) exhibited significantly increased CD8⁺ T cell activation and spontaneous NK cell degranulation compared to uninfected donors. Overall, we identified that a combination of innate (pDC frequency) and adaptive (Pol-specific CD8⁺ T cell responses) immune parameters best predicted viral load (R² = 0.5864, p = 0.0021, n = 17) by a multivariable analysis. Together, this data indicates that preferential Gag-specific over Pol-specific CD8⁺ T cell responses along with a retention of functional innate subsets best predict host control over viral replication in HIV-1 infected viremic controllers compared to chronically-infected non-controllers.     

DNA Methylation Changes Separate Allergic Patients from Healthy Controls and May Reflect Altered CD4+ T-Cell Population Structure

Altered DNA methylation patterns in CD4⁺ T-cells indicate the importance of epigenetic mechanisms in inflammatory diseases. However, the identification of these alterations is complicated by the heterogeneity of most inflammatory diseases. Seasonal allergic rhinitis (SAR) is an optimal disease model for the study of DNA methylation because of its well-defined phenotype and etiology. We generated genome-wide DNA methylation (N_patients = 8, N_controls = 8) and gene expression (N_patients = 9, N_controls = 10) profiles of CD4⁺ T-cells from SAR patients and healthy controls using Illumina''s HumanMethylation450 and HT-12 microarrays, respectively. DNA methylation profiles clearly and robustly distinguished SAR patients from controls, during and outside the pollen season. In agreement with previously published studies, gene expression profiles of the same samples failed to separate patients and controls. Separation by methylation (N_patients = 12, N_controls = 12), but not by gene expression (N_patients = 21, N_controls = 21) was also observed in an in vitro model system in which purified PBMCs from patients and healthy controls were challenged with allergen. We observed changes in the proportions of memory T-cell populations between patients (N_patients = 35) and controls (N_controls = 12), which could explain the observed difference in DNA methylation. Our data highlight the potential of epigenomics in the stratification of immune disease and represents the first successful molecular classification of SAR using CD4⁺ T cells.     

Increased Frequency of CD4 and CD8 Regulatory T Cells in Individuals under 15 Years with Multibacillary Leprosy

Background

Leprosy is a chronic disease, caused by Mycobacterium leprae, which poses a serious public health problem worldwide. Its high incidence in people under 15 years old in Ceará state, Brazil, reflects the difficulty of its control. The spectrum of clinical manifestations is associated with the immune response developed, with the Th1 and Th2 responses being related to the paucibacillary and multibacillary forms, respectively. Regulatory T cells (Treg), which can suppress Th1 and Th2 response, have received special attention in the literature and have been associated with development of chronic infections. However, their role in leprosy in individuals under 15 years old has not yet been elucidated. We evaluated the frequency of CD4⁺/CD8⁺CD25^highFOXP3⁺ and CD4⁺/CD8⁺CD25^highFOXP3^high cells in leprosy patients and household contacts, in both cases under 15 years old.

Methodology/Principal Findings

PBMC from 12 patients and 17 contacts were cultured for 72 hours with anti-CD3 and anti-CD28 (activators) or with activators associated with total sonicated fraction of M. leprae. After culture, the frequency of CD4⁺/CD8⁺ Treg was identified by flow cytometry. Cells stimulated by activators and antigen from multibacillary patients showed Treg frequencies almost two times that of the contacts: CD4⁺FOXP3⁺ (21.93±8.43 vs. 13.79±8.19%, p = 0.0500), CD4⁺FOXP3^high (10.33±5.69 vs. 5.57±4.03%, p = 0.0362), CD8⁺FOXP3⁺ (13.88±9.19 vs. 6.18±5.56%, p = 0.0230) and CD8⁺FOXP3^high (5.36±4.17 vs. 2.23±2.68%, p = 0.0461). Furthermore, the mean fluorescence intensity of FOXP3 in Treg was higher in multibacillary patients than in the contacts. Interestingly, there was a positive correlation of the bacillary index and number of lesions with the frequency of all Treg evaluated in patients.

Conclusions/Significance

We have demonstrated for the first time that multibacillary leprosy patients under 15 years old have greater CD4⁺ and CD8⁺ Treg frequencies and these correlate with clinical and laboratorial aspects of disease. These findings suggest the involvement of these cells in the perpetuation of M. leprae infection.     

Mycobacterium tuberculosis Specific CD8+ T Cells Rapidly Decline with Antituberculosis Treatment

Rationale

Biomarkers associated with response to therapy in tuberculosis could have broad clinical utility. We postulated that the frequency of Mycobacterium tuberculosis (Mtb) specific CD8⁺ T cells, by virtue of detecting intracellular infection, could be a surrogate marker of response to therapy and would decrease during effective antituberculosis treatment.Objectives: We sought to determine the relationship of Mtb specific CD4⁺ T cells and CD8⁺ T cells with duration of antituberculosis treatment.

Materials and Methods

We performed a prospective cohort study, enrolling between June 2008 and August 2010, of HIV-uninfected Ugandan adults (n = 50) with acid-fast bacillus smear-positive, culture confirmed pulmonary TB at the onset of antituberculosis treatment and the Mtb specific CD4⁺ and CD8⁺ T cell responses to ESAT-6 and CFP-10 were measured by IFN-γ ELISPOT at enrollment, week 8 and 24.

Results

There was a significant difference in the Mtb specific CD8⁺ T response, but not the CD4⁺ T cell response, over 24 weeks of antituberculosis treatment (p<0.0001), with an early difference observed at 8 weeks of therapy (p = 0.023). At 24 weeks, the estimated Mtb specific CD8⁺ T cell response decreased by 58%. In contrast, there was no significant difference in the Mtb specific CD4⁺ T cell during the treatment. The Mtb specific CD4⁺ T cell response, but not the CD8⁺ response, was negatively impacted by the body mass index.

Conclusions

Our data provide evidence that the Mtb specific CD8⁺ T cell response declines with antituberculosis treatment and could be a surrogate marker of response to therapy. Additional research is needed to determine if the Mtb specific CD8⁺ T cell response can detect early treatment failure, relapse, or to predict disease progression.