B cell depletion in HIV-1 subtype A infected Ugandan adults: relationship to CD4 T cell count, viral load and humoral immune responses

To better understand the nature of B cell dysfunctions in subjects infected with HIV-1 subtype A, a rural cohort of 50 treatment-naïve Ugandan patients chronically infected with HIV-1 subtype A was studied, and the relationship between B cell depletion and HIV disease was assessed. B cell absolute counts were found to be significantly lower in HIV-1+ patients, when compared to community matched negative controls (p<0.0001). HIV-1-infected patients displayed variable functional and binding antibody titers that showed no correlation with viral load or CD4+ T cell count. However, B cell absolute counts were found to correlate inversely with neutralizing antibody (NAb) titers against subtype A (p = 0.05) and subtype CRF02_AG (p = 0.02) viruses. A positive correlation was observed between subtype A gp120 binding antibody titers and NAb breadth (p = 0.02) and mean titer against the 10 viruses (p = 0.0002). In addition, HIV-1 subtype A sera showed preferential neutralization of the 5 subtype A or CRF02_AG pseudoviruses, as compared with 5 pseudoviruses from subtypes B, C or D (p<0.001). These data demonstrate that in patients with chronic HIV-1 subtype A infection, significant B cell depletion can be observed, the degree of which does not appear to be associated with a decrease in functional antibodies. These findings also highlight the potential importance of subtype in the specificity of cross-clade neutralization in HIV-1 infection.     

MRI discriminates thrombus composition and ST resolution after percutaneous coronary intervention in patients with ST-elevation myocardial infarction

Histological composition of material obtained by thrombus aspiration during percutaneous coronary intervention (PCI) in patients with ST-segment elevation acute myocardial infarction (STEMI) is highly variable. We aimed to characterize this material using magnetic resonance imaging (MRI) and to correlate MRI findings with the success of PCI in terms of ST-segment resolution. Thrombus aspiration during primary or rescue PCI was attempted in 100 consecutive STEMI patients, of whom enough material for MRI was obtained in 59. MR images were obtained at 9.4T and T1 and T2 values were measured. Patients with (n = 31) and without (n = 28) adequate ST resolution 120 min after PCI (≥70% of pre-PCI value) had similar baseline characteristics except for a higher prevalence of diabetes mellitus in the latter (10 vs. 43%, p = 0.003). T1 values were similar in both groups (1248±112 vs. 1307±85 ms, respectively, p = 0.7). T2 values averaged 31.2±10.3 and 36.6±12.2 ms; in thrombus from patients with and without adequate ST resolution (p = 0.09). After adjusting for diabetes and other baseline characteristics, lower T2 values were significantly associated with inadequate ST resolution (odds ratio for 1 ms increase 1.08, CI 95% 1.01–1.16, p = 0.027). Histology classified thrombus in 3 groups: coagulated blood (n = 38), fibrin rich (n = 9) and lipid-rich (n = 3). Thrombi composed mostly of coagulated blood were characterized as being of short (n = 10), intermediate (n = 15) or long evolution (n = 13), T2 values being 34.0±13.2, 31.9±8.3 and 31.5±7.9 ms respectively (p = NS). In this subgroup, T2 was significantly higher in specimens from patients with inadequate perfusion (35.9±10.3 versus 28.6±6.7 ms, p = 0.02). This can be of clinical interest as it provides information on the probability of adequate ST resolution, a surrogate for effective myocardial reperfusion.     

Lack of evidence for changing virulence of HIV-1 in North America

Background

Several long-term cohort studies and in-vitro fitness assays have resulted in inconsistent reports on changes in HIV-1 virulence, including reports of decreasing, stable, and increasing virulence over the course of the AIDS pandemic. We tested the hypothesis of changing HIV-1 virulence by examining trends in prognostic clinical markers of disease progression from 1984 to 2005 among nearly 400 antiretroviral-naïve participants in the United States Multicenter AIDS Cohort Study (MACS), a longitudinal study of HIV infection in men who have sex with men (MSM).

Methodology/Principal Findings

Because clinical AIDS endpoints could not be used (due to antiretroviral therapies and prophylaxis), three prognostic markers of disease progression were used as proxies for HIV-1 virulence: plasma viral RNA load and CD4+ T cell count at “set point” (between ∼9 and ∼15 months after seroconversion), and rate of CD4 cell decline within three years after seroconversion. We performed multivariate analyses of the association between these markers and seroconversion year, with covariates including MACS site, race/ethnic group, seroconversion age, and CCR5Δ32 status. No statistically significant association was found between year of seroconversion and “set point” plasma viral load (at ∼9 months after seroconversion: slope = −0.004 log₁₀ copies/mL/year, p = 0.76; at ∼15 months: slope = −0.005 log₁₀ copies/mL/year, p = 0.71), CD4 cell count after seroconversion (at ∼9 months: slope = −0.112 cells/µL/year, p = 0.22; at ∼15 months: slope = −0.047 cells/µL/year, p = 0.64), or rate of CD4 cell decline over the first three years after seroconversion (slope = −0.010 cells/ul/yr², p = 0.88).

Conclusions/Significance

The lack of significant trends from 1984 to 2005 in these prognostic markers of HIV disease progression suggests no major change in HIV-1 virulence over the AIDS pandemic in MSM in the US.     

Chronic immune activation in HIV-1 infection contributes to reduced interferon alpha production via enhanced CD40:CD40 ligand interaction

Although a signature of increased interferon (IFN-)alpha production is observed in HIV-1 infection, the response of circulating plasmacytoid dendritic cells (PDC) to Toll-like receptor ligand stimulation is substantially impaired. This functional PDC deficit, which we specifically observed in HIV-1 infected individuals with less than 500 CD4+ T cells/µl, is not well understood. We provide evidence that the peripheral IFN-alpha production in HIV-1 infection is actively suppressed by the enhanced interaction of CD40 ligand (CD40L), a member of the tumor necrosis factor family, and its receptor CD40, which are both upregulated upon immune activation. Plasma levels of soluble CD40L were significantly higher in untreated HIV-1 infected individuals (n = 52) than in subjects on long-term antiretroviral therapy (n = 62, p<0.03) and in uninfected control donors (n = 16, p<0.001). Concomitantly, cell-associated CD40L and the expression of the receptor CD40 on the PDC were significantly upregulated in HIV-1 infection (p<0.05). Soluble and cell-associated CD40L inhibited the PDC-derived IFN-alpha production by CpG oligodeoxynucleotides dose-dependently. This suppressive effect was observed at much lower, physiological CD40L concentrations in peripheral blood mononuclear cells (PBMC) of HIV-1 infected individuals compared to controls (p<0.05). The CpG-induced IFN-alpha production in PBMC of HIV-1 infected donors was directly correlated with PDC and CD4+ T cell counts, and inversely correlated with the viral loads (p<0.001). In HIV-1 infected donors with less than 500 CD4+ T cells/µl, the CpG-induced IFN-alpha production was significantly correlated with the percentage of CD40-expressing PDC and the level of CD40 expression on these cells (p<0.05), whereas CD40L plasma levels played a minor role. In addition, low-dose CD40L contributed to the enhanced production of interleukin 6 and 8 in PBMC of HIV-1 infected donors compared to controls. Our data support the conclusion that the chronic immune activation in HIV-1 infection impairs peripheral PDC innate immune responses at least in part via enhanced CD40:CD40L interactions.     

HIV-1 disease progression is associated with bile-salt stimulated lipase (BSSL) gene polymorphism

Background

DC-SIGN expressed by dendritic cells captures HIV-1 resulting in trans-infection of CD4⁺ T-lymphocytes. However, BSSL (bile-salt stimulated lipase) binding to DC-SIGN interferes with HIV-1 capture. DC-SIGN binding properties of BSSL associate with the polymorphic repeated motif of BSSL exon 11. Furthermore, BSSL binds to HIV-1 co-receptor CXCR4. We hypothesized that BSSL modulates HIV-1 disease progression and emergence of CXCR4 using HIV-1 (X4) variants.

Results

The relation between BSSL genotype and HIV-1 disease progression and emergence of X4 variants was studied using Kaplan Meier and multivariate Cox proportional hazard analysis in a cohort of HIV-1 infected men having sex with men (n = 334, with n = 130 seroconverters). We analyzed the association of BSSL genotype with set-point viral load and CD4 cell count, both pre-infection and post-infection at viral set-point. The number of repeats in BSSL exon 11 were highly variable ranging from 10 to 18 in seropositive individuals and from 5–17 in HRSN with 16 repeats being dominant (>80% carry at least one allele with 16 repeats). We defined 16 to 18 repeats as high (H) and less than 16 repeats as low (L) repeat numbers. Homozygosity for the high (H) repeat number BSSL genotype (HH) correlated with high CD4 cell numbers prior to infection (p = 0.007). In HIV-1 patients, delayed disease progression was linked to the HH BSSL genotype (RH = 0.462 CI = 0.282–0.757, p = 0.002) as was delayed emergence of X4 variants (RH = 0.525, 95% CI = 0.290–0.953, p = 0.034). The LH BSSL genotype, previously found to be associated with enhanced DC-SIGN binding of human milk, was identified to correlate with accelerated disease progression in our cohort of HIV-1 infected MSM (RH = 0.517, 95% CI = 0.328–0.818, p = 0.005).

Conclusion

We identify BSSL as a marker for HIV-1 disease progression and emergence of X4 variants. Additionally, we identified a relation between BSSL genotype and CD4 cell counts prior to infection.     

A plasma biomarker signature of immune activation in HIV patients on antiretroviral therapy

Background

Immune activation is a strong predictor of disease progression in HIV infection. Combinatorial plasma biomarker signatures that represent surrogate markers of immune activation in both viremic and aviremic HIV patients on combination antiretroviral therapy (cART) have not been defined. Here, we identify a plasma inflammatory biomarker signature that distinguishes between both viremic and aviremic HIV patients on cART and healthy controls and examine relationships of this signature to markers of disease progression.

Methods

Multiplex profiling and ELISA were used to detect 15 cytokines/chemokines, soluble IL-2R (sIL-2R), and soluble CD14 (sCD14) in plasma from 57 HIV patients with CD4 nadir <300 cells/µl and 29 healthy controls. Supervised and unsupervised analyses were used to identify biomarkers explaining variance between groups defined by HIV status or drug abuse. Relationships between biomarkers and disease markers were examined by Spearman correlation.

Results

The majority (91%) of HIV subjects were on cART, with 38% having undetectable viral loads (VL). Hierarchical clustering identified a biomarker cluster in plasma consisting of two interferon-stimulated gene products (CXCL9 and CXCL10), T cell activation marker (sIL-2R), and monocyte activation marker (sCD14) that distinguished both viremic and aviremic HIV patients on cART from controls (p<0.0001) and were top-ranked in variables important in projection plots. IL-12 and CCL4 were also elevated in viremic and aviremic patients compared to controls (p<0.05). IL-12 correlated with IFNα, IFNγ, CXCL9, and sIL-2R (p<0.05). CXCL10 correlated positively with plasma VL and percentage of CD16+ monocytes, and inversely with CD4 count (p = 0.001, <0.0001, and 0.04, respectively).

Conclusion

A plasma inflammatory biomarker signature consisting of CXCL9, CXCL10, sIL-2R, and sCD14 may be useful as a surrogate marker to monitor immune activation in both viremic and aviremic HIV patients on cART during disease progression and therapeutic responses.     

Interferon-alpha administration enhances CD8+ T cell activation in HIV infection

Background

Type I interferons play important roles in innate immune defense. In HIV infection, type I interferons may delay disease progression by inhibiting viral replication while at the same time accelerating disease progression by contributing to chronic immune activation.

Methods

To investigate the effects of type I interferons in HIV-infection, we obtained cryopreserved peripheral blood mononuclear cell samples from 10 subjects who participated in AIDS Clinical Trials Group Study 5192, a trial investigating the activity of systemic administration of IFNα for twelve weeks to patients with untreated HIV infection. Using flow cytometry, we examined changes in cell cycle status and expression of activation antigens by circulating T cells and their maturation subsets before, during and after IFNα treatment.

Results

The proportion of CD38+HLA-DR+CD8+ T cells increased from a mean of 11.7% at baseline to 24.1% after twelve weeks of interferon treatment (p = 0.006). These frequencies dropped to an average of 20.1% six weeks after the end of treatment. In contrast to CD8+ T cells, the frequencies of activated CD4+ T cells did not change with administration of type I interferon (mean percentage of CD38+DR+ cells = 2.62% at baseline and 2.17% after 12 weeks of interferon therapy). As plasma HIV levels fell with interferon therapy, this was correlated with a “paradoxical” increase in CD8+ T cell activation (p<0.001).

Conclusion

Administration of type I interferon increased expression of the activation markers CD38 and HLA DR on CD8+ T cells but not on CD4+ T cells of HIV+ persons. These observations suggest that type I interferons may contribute to the high levels of CD8+ T cell activation that occur during HIV infection.     

Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Background

With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.

Methodology and Principal Findings

We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC₅₀±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC₅₀ = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC₅₀ = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC₅₀ = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC₅₀ = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC₅₀ = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC₅₀ = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC₅₀ = 7.05±2.24 µM and 6.18±1.51 µM.

Conclusion

The in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.     

Urinary podocyte-associated mRNA profile in various stages of diabetic nephropathy

Background

Podocyte injury and subsequent excretion in urine play a crucial role in the pathogenesis and progression of diabetic nephropathy (DN). Quantification of messenger RNA (mRNA) expression in urinary sediment by real-time PCR is emerging as a noninvasive method of screening DN-associated biomarkers. We hypothesized that the urinary mRNA profile of podocyte-associated molecules may provide important clinical insight into the different stages of diabetic nephropathy.

Methods

DN patients (N = 51) and healthy controls (N = 13) were enrolled in this study. DN patients were divided into a normoalbuminuria group (UAE<30 mg/g, n = 17), a microalbuminuria group (UAE 30∼300 mg/g, n = 15), and a macroalbuminuria group (UAE>300 mg/g, n = 19), according to their urinary albumin excretion (UAE). Relative mRNA abundance of synaptopodin, podocalyxin, CD2-AP, α-actin4, and podocin were quantified, and correlations between target mRNAs and clinical parameters were examined.

Results

The urinary mRNA levels of all genes studied were significantly higher in the DN group compared with controls (p<0.05), and mRNA levels increased with DN progression. Urinary mRNA levels of all target genes positively correlated with both UAE and BUN. The expression of podocalyxin, CD2-AP, α-actin4, and podocin mRNA correlated with serum creatinine (r = 0.457, p = 0.001; r = 0.329, p = 0.01; r = 0.286, p = 0.021; r = 0.357, p = 0.006, respectively). Furthermore, podocalyxin mRNA was found to negatively correlate with eGFR (r = −0.349, p = 0.01).

Conclusion

The urinary mRNA profiles of synaptopodin, podocalyxin, CD2-AP, α-actin4, and podocin were found to increase with the progression of DN, which suggested that quantification of podocyte-associated molecules will be useful biomarkers of DN.     

The dynamics of naturally acquired immune responses to Plasmodium falciparum sexual stage antigens Pfs230 & Pfs48/45 in a low endemic area in Tanzania

Background

Naturally acquired immune responses against sexual stages of P. falciparum can reduce the transmission of malaria from humans to mosquitoes. These antigens are candidate transmission-blocking vaccines but little is known about the acquisition of sexual stage immunity after exposure to gametocytes, or their longevity and functionality. We conducted a longitudinal study on functional sexual stage immune responses.

Methodology/Principal Findings

Parasitaemic individuals (n = 116) were recruited at a health centre in Lower Moshi, Tanzania. Patients presented with gametocytes (n = 16), developed circulating gametocytes by day 7 (n = 69) or between day 7 and 14 (n = 10) after treatment or did not develop gametocytes (n = 21). Serum samples were collected on the first day of gametocytaemia and 28 and 84 days post-enrolment (or d7, 28, 84 after enrolment from gametocyte-negative individuals). Antibody responses to sexual stage antigens Pfs230 and Pfs48/45 were detected in 20.7% (72/348) and 15.2% (53/348) of the samples, respectively, and were less prevalent than antibodies against asexual stage antigens MSP-1₁₉ (48.1%; 137/285) and AMA-1 (52.4%; 129/246)(p<0.001). The prevalence of anti-Pfs230 (p = 0.026) and anti-Pfs48/45 antibodies (p = 0.017) increased with longer duration of gametocyte exposure and had an estimated half-life of approximately 3 months. Membrane feeding experiments demonstrated a strong association between the prevalence and concentration of Pfs230 and Pfs48/45 antibodies and transmission reducing activity (TRA, p<0.01).

Conclusions/Significance

In a longitudinal study, anti-Pfs230 and Pf48/45 antibodies developed rapidly after exposure to gametocytes and were strongly associated with transmission-reducing activity. Our data indicate that the extent of antigen exposure is important in eliciting functional transmission-reducing immune responses.     

MRKAd5 HIV-1 Gag/Pol/Nef Vaccine-Induced T-Cell Responses Inadequately Predict Distance of Breakthrough HIV-1 Sequences to the Vaccine or Viral Load

Background

The sieve analysis for the Step trial found evidence that breakthrough HIV-1 sequences for MRKAd5/HIV-1 Gag/Pol/Nef vaccine recipients were more divergent from the vaccine insert than placebo sequences in regions with predicted epitopes. We linked the viral sequence data with immune response and acute viral load data to explore mechanisms for and consequences of the observed sieve effect.

Methods

Ninety-one male participants (37 placebo and 54 vaccine recipients) were included; viral sequences were obtained at the time of HIV-1 diagnosis. T-cell responses were measured 4 weeks post-second vaccination and at the first or second week post-diagnosis. Acute viral load was obtained at RNA-positive and antibody-negative visits.

Findings

Vaccine recipients had a greater magnitude of post-infection CD8+ T cell response than placebo recipients (median 1.68% vs 1.18%; p = 0·04) and greater breadth of post-infection response (median 4.5 vs 2; p = 0·06). Viral sequences for vaccine recipients were marginally more divergent from the insert than placebo sequences in regions of Nef targeted by pre-infection immune responses (p = 0·04; Pol p = 0·13; Gag p = 0·89). Magnitude and breadth of pre-infection responses did not correlate with distance of the viral sequence to the insert (p>0·50). Acute log viral load trended lower in vaccine versus placebo recipients (estimated mean 4·7 vs 5·1) but the difference was not significant (p = 0·27). Neither was acute viral load associated with distance of the viral sequence to the insert (p>0·30).

Interpretation

Despite evidence of anamnestic responses, the sieve effect was not well explained by available measures of T-cell immunogenicity. Sequence divergence from the vaccine was not significantly associated with acute viral load. While point estimates suggested weak vaccine suppression of viral load, the result was not significant and more viral load data would be needed to detect suppression.     

Risk of all-cause mortality in HIV infected patients is associated with clinical, immunologic predictors and the CCR5 Δ32 deletion

Objective

Investigation of the interplay between the CCR5 Δ32/wt genotype and demographic, epidemiological, clinical and immunological factors associated with mortality in the cART era.

Design

Longitudinal data from 507 HIV-infected patients following the Δ32 allele detection were analyzed.

Methods

Cumulative 15 years mortality was calculated using Kaplan-Meyer methodology. Hazard ratios were estimated using univariate Cox models. Basing on Akakie information criteria and statistical significance multivariate Cox model was constructed and effect plots presenting adjusted hazard ratio time-dependency were drawn. Analysis of the association of all-cause mortality and CCR5 Δ32/wt genotype prior to the antiretroviral treatment (cART) initiation (n = 507) and on the therapy (n = 422) was also performed.

Results

A mortality rate of 2.66 (CI 2.57–3.19) per 100 person-years was observed. Univariate analysis factors modifying the risk of death included the CCR5 genotype, gender, history of cART, AIDS diagnosis and also CD4 lymphocyte nadir, zenith, the latest CD4 count and stable levels >500 cells/µl. For multivariate analysis the following predictors were selected: CCR5 genotype (HR for wt/wt 2.53, CI 1.16–5.53, p = 0.02), gender (HR for males 1.91, 95%CI 1.1–3.36, p = 0.023), introduction of combined antiretroviral treatment (HR 4.85, CI 3.0–7.89, if untreated or treated <1 month, p<0.0001) CD4 count of 500 cells/µl for six months or more (HR 4.16, CI 1.95–8.88 if not achieved, p = 0.028), the latest CD4 count (HR 5.44, CI 3.39–8.74 for <100 cells/µl, p<0.0001) and history of AIDS (HR 1.69, CI 1.03–2.79, p = 0.039). Among untreated individuals the Δ32/wt genotype was associated with notably better survival (p = 0.026), while among cART treated individuals the Δ32 mutation did not correlate significantly with higher survival rates (p = 0.23).

Conclusions

The Δ32 CCR5 allele is associated with a reduction of the risk of all-cause mortality in HIV (+) patients alongside clinical and immunologic predictors such as AIDS, history of cART, lymphocyte CD4 cell count and gender.     

Functional energetics of CD4+-cellular immunity in monoclonal antibody-associated progressive multifocal leukoencephalopathy in autoimmune disorders

Background

Progressive multifocal leukoencephalopathy (PML) is an opportunistic central nervous system- (CNS-) infection that typically occurs in a subset of immunocompromised individuals. An increasing incidence of PML has recently been reported in patients receiving monoclonal antibody (mAb) therapy for the treatment of autoimmune diseases, particularly those treated with natalizumab, efalizumab and rituximab. Intracellular CD4⁺-ATP-concentration (iATP) functionally reflects cellular immunocompetence and inversely correlates with risk of infections during immunosuppressive therapy. We investigated whether iATP may assist in individualized risk stratification for opportunistic infections during mAb-treatment.

Methodology/Principal Findings

iATP in PHA-stimulated, immunoselected CD4⁺-cells was analyzed using an FDA-approved assay. iATP of mAb-associated PML (natalizumab (n = 8), rituximab (n = 2), efalizumab (n = 1)), or other cases of opportunistic CNS-infections (HIV-associated PML (n = 2), spontaneous PML, PML in a psoriasis patient under fumaric acids, natalizumab-associated herpes simplex encephalitis (n = 1 each)) was reduced by 59% (194.5±29 ng/ml, mean±SEM) in comparison to healthy controls (HC, 479.9±19.8 ng/ml, p<0.0001). iATP in 14 of these 16 patients was at or below 3^rd percentile of healthy controls, similar to HIV-patients (n = 18). In contrast, CD4⁺-cell numbers were reduced in only 7 of 15 patients, for whom cell counts were available. iATP correlated with mitochondrial transmembrane potential (ΔΨ_m) (iATP/ΔΨ_m−correlation:tau = 0.49, p = 0.03). Whereas mean iATP of cross-sectionally analysed natalizumab-treated patients was unaltered (448.7±12 ng/ml, n = 150), iATP was moderately decreased (316.2±26.1 ng/ml, p = 0.04) in patients (n = 7) who had been treated already during the pivotal phase III trials and had received natalizumab for more than 6 years. 2/92 (2%) patients with less than 24 months natalizumab treatment revealed very low iATP at or below the 3^rd percentile of HC, whereas 10/58 (17%) of the patients treated for more than 24 months had such low iATP-concentrations.

Conclusion

Our results suggest that bioenergetic parameters such as iATP may assist in risk stratification under mAb-immunotherapy of autoimmune disorders.     

Genetic association study identifies HSPB7 as a risk gene for idiopathic dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a structural heart disease with strong genetic background. Monogenic forms of DCM are observed in families with mutations located mostly in genes encoding structural and sarcomeric proteins. However, strong evidence suggests that genetic factors also affect the susceptibility to idiopathic DCM. To identify risk alleles for non-familial forms of DCM, we carried out a case-control association study, genotyping 664 DCM cases and 1,874 population-based healthy controls from Germany using a 50K human cardiovascular disease bead chip covering more than 2,000 genes pre-selected for cardiovascular relevance. After quality control, 30,920 single nucleotide polymorphisms (SNP) were tested for association with the disease by logistic regression adjusted for gender, and results were genomic-control corrected. The analysis revealed a significant association between a SNP in HSPB7 gene (rs1739843, minor allele frequency 39%) and idiopathic DCM (p = 1.06×10⁻⁶, OR = 0.67 [95% CI 0.57–0.79] for the minor allele T). Three more SNPs showed p < 2.21×10⁻⁵. De novo genotyping of these four SNPs was done in three independent case-control studies of idiopathic DCM. Association between SNP rs1739843 and DCM was significant in all replication samples: Germany (n = 564, n = 981 controls, p = 2.07×10⁻³, OR = 0.79 [95% CI 0.67–0.92]), France 1 (n = 433 cases, n = 395 controls, p = 3.73×10⁻³, OR = 0.74 [95% CI 0.60–0.91]), and France 2 (n = 249 cases, n = 380 controls, p = 2.26×10⁻⁴, OR = 0.63 [95% CI 0.50–0.81]). The combined analysis of all four studies including a total of n = 1,910 cases and n = 3,630 controls showed highly significant evidence for association between rs1739843 and idiopathic DCM (p = 5.28×10⁻¹³, OR = 0.72 [95% CI 0.65–0.78]). None of the other three SNPs showed significant results in the replication stage.This finding of the HSPB7 gene from a genetic search for idiopathic DCM using a large SNP panel underscores the influence of common polymorphisms on DCM susceptibility.     

Hypertension is associated with marked alterations in sphingolipid biology: a potential role for ceramide

Background

Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function.

Methods and Findings

In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p<0.05). Imaging mass spectrometry and immunohistochemistry indicated that these contractions were most likely mediated by ceramide and dependent on iPLA₂, cyclooxygenase-1 and thromboxane synthase. Expression levels of these enzymes were higher in SHR vessels. In concurrence, infusion of dimethylsphingosine caused a marked rise in blood pressure in anesthetized SHR (42±4%; n = 7), but not in WKY (−12±10%; n = 6). Lipidomics analysis by mass spectrometry, revealed elevated levels of ceramide in arterial tissue of SHR compared to WKY (691±42 vs. 419±27 pmol, n = 3–5 respectively, p<0.05). These pronounced alterations in SHR sphingolipid biology are also reflected in increased plasma ceramide levels (513±19 pmol WKY vs. 645±25 pmol SHR, n = 6–12, p<0.05). Interestingly, we observed similar increases in ceramide levels (correlating with hypertension grade) in plasma from humans with essential hypertension (185±8 pmol vs. 252±23 pmol; n = 18 normotensive vs. n = 19 hypertensive patients, p<0.05).

Conclusions

Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone.     

Elevated cerebral spinal fluid cytokine levels in boys with cerebral adrenoleukodystrophy correlates with MRI severity

Background

X-linked adrenoleukodystrophy (ALD) is a metabolic, peroxisomal disease that results from a mutation in the ABCD1 gene. The most severe course of ALD progression is the cerebral inflammatory and demyelinating form of the disease, cALD. To date there is very little information on the cytokine mediators in the cerebral spinal fluid (CSF) of these boys.

Methodology/Principal Findings

Measurement of 23 different cytokines was performed on CSF and serum of boys with cerebral ALD and patients without ALD. Significant elevations in CSF IL-8 (29.3±2.2 vs 12.8±1.1 pg/ml, p = 0.0001), IL-1ra (166±30 vs 8.6±6.5 pg/ml, p = 0.005), MCP-1 (610±47 vs 328±34 pg/ml, p = 0.002), and MIP-1b (14.2±1.3 vs 2.0±1.4 pg/ml, p<0.0001) were found in boys with cALD versus the control group. The only serum cytokine showing an elevation in the ALD group was SDF-1 (2124±155 vs 1175±125 pg/ml, p = 0.0001). The CSF cytokines of IL-8 and MCP-1b correlated with the Loes MRI severity score (p = 0.04 and p = 0.008 respectively), as well as the serum SDF-1 level (p = 0.002). Finally, CSF total protein was also significantly elevated in boys with cALD and correlated with both IL-8, MCP-1b (p = 0.0001 for both), as well as Loes MRI severity score (p = 0.0007).

Conclusions/Significance

IL-8, IL-1ra, MCP-1, MIP-1b and CSF total protein were significantly elevated in patients with cALD; IL-8, MCP-1b, and CSF total protein levels correlated with disease severity determined by MRI. This is the largest report of CSF cytokine levels in cALD to date, and identification of these key cytokines will provide further insight into disease progression and perhaps lead to improved targeted therapies.     

Increased prevalence of albuminuria in HIV-infected adults with diabetes

Objective

HIV and type 2 diabetes are known risk factors for albuminuria, but no previous reports have characterized albuminuria in HIV-infected patients with diabetes.

Research Design and Methods

We performed a cross-sectional study including 73 HIV-infected adults with type 2 diabetes, 82 HIV-infected non-diabetics, and 61 diabetic control subjects without HIV. Serum creatinine >1.5 mg/dL was exclusionary. Albuminuria was defined as urinary albumin/creatinine ratio >30 mg/g.

Results

The prevalence of albuminuria was significantly increased among HIV-infected diabetics (34% vs. 13% of HIV non-diabetic vs. 16% diabetic control, p = 0.005). HIV status and diabetes remained significant predictors of albuminuria after adjusting for age, race, BMI, and blood pressure. Albumin/creatinine ratio correlated significantly with HIV viral load (r = 0.28, p = 0.0005) and HIV-infected subjects with albuminuria had significantly greater cumulative exposure to abacavir (p = 0.01). In an adjusted multivariate regression analysis of HIV-infected subjects, the diagnosis of diabetes (p = 0.003), higher HIV viral load (p = 0.03) and cumulative exposure to abacavir (p = 0.0009) were significant independent predictors of albuminuria.

Conclusions

HIV and diabetes appear to have additive effects on albuminuria which is also independently associated with increased exposure to abacavir and HIV viral load. Future research on the persistence, progression and management of albuminuria in this unique at-risk population is needed.     

Prominent plasmacytosis following intravenous immunoglobulin correlates with clinical improvement in Guillain-Barré syndrome

Background

High doses of pooled polyclonal IgG are commonly used to treat numerous autoimmune diseases. Their mode of action nevertheless remains only partially explained. At the same time, until now, no early biological marker has been able to predict their efficacy.

Methodology/Principal Findings

In a first pilot retrospective analysis, we reviewed white blood cell counts and blood smears in consecutive patients with autoimmune disease (n = 202) and non-autoimmune disease (n = 104). Autoimmune patients received either intravenous immunoglobulin (IVIg, n = 103), plasma exchange (n = 78) or no specific treatment (n = 21). We then prospectively monitored consecutive autoimmune patients with IVIg injection (n = 67), or without any specific treatment (n = 10) using the same routine laboratory tests, as well as flow cytometry. Both retrospective and prospective analyses identified large plasma-cell mobilization exclusively in IVIg-treated autoimmune patients 7 days after initiation of treatment. The majority of IVIg-mobilized plasma cells were immature HLA-DR^high/CD138^low/CXCR4^low plasma cells expressing intracellular immunoglobulin G which were neither IVIg- nor human IgG-specific. Importantly, we found a strong negative correlation between the absolute number of IVIg-mobilized plasma cells and time to improve neurological function in both retrospective and prospective studies of Guillain-Barré syndrome (GBS), (r = −0.52, p = 0.0031, n = 30, r = −0.47, p = 0.0028, n = 40, respectively).

Conclusions/Significance

IVIg promotes immature plasma-cell mobilization in patients with GBS, chronic inflammatory demyelinating polyneuropathy, myasthenia gravis and inflammatory myopathy. Prominent day 7 plasma-cell mobilization is a favourable prognostic marker in patients with GBS receiving IVIg treatment.