From bench to bedside: Improving the clinical safety of GalNAc-siRNA conjugates using seed-pairing destabilization.

Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc-siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2'-5'-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development.

Evaluation of RNAi therapeutics VIR-2218 and ALN-HBV for chronic hepatitis B: Results from randomized clinical trials.

BACKGROUND & AIMS: Current therapy for chronic hepatitis B virus (cHBV) infection involves lifelong treatment. New treatments that enable HBV functional cure would represent a clinically meaningful advance. ALN-HBV and VIR-2218 are investigational RNA interference therapeutics that target all major HBV transcripts. METHODS: We report on: i) the safety of single doses of VIR-2218 (modified from ALN-HBV by enhanced stabilization chemistry plus technology to reduce off-target, seed-mediated binding while maintaining on-target antiviral activity) and ALN-HBV in humanized mice; ii) a cross-study comparison of the safety of single doses of VIR-2218 and ALN-HBV in healthy human volunteers (n = 24 and n = 49, respectively); and iii) the antiviral activity of two doses of 20, 50, 100, 200 mg of VIR-2218 (total n = 24) vs. placebo (n = 8), given 4 weeks apart, in participants with cHBV infection. RESULTS: In humanized mice, alanine aminotransferase (ALT) levels were markedly lower following administration of VIR-2218 compared with ALN-HBV. In healthy volunteers, post-treatment ALT elevations occurred in 28% of participants receiving ALN-HBV compared with none in those receiving VIR-2218. In participants with cHBV infection, VIR-2218 was associated with dose-dependent reductions in hepatitis B surface antigen (HBsAg). The greatest mean reduction of HBsAg at Week 20 in participants receiving 200 mg was 1.65 log IU/ml. The HBsAg reduction was maintained at 0.87 log IU/ml at Week 48. No participants had serum HBsAg loss or hepatitis B surface antibody seroconversion. CONCLUSIONS: VIR-2218 demonstrated an encouraging hepatic safety profile in preclinical and clinical studies as well as dose-dependent HBsAg reductions in patients with cHBV infection. These data support future studies with VIR-2218 as part of combination regimens with a goal of HBV functional cure. TRIAL REGISTRATION: ClinicalTrials.gov identifiers: NCT02826018 and NCT03672188. IMPACT AND IMPLICATIONS: A significant unmet need exists for therapies for chronic HBV (cHBV) infection that achieve functional cure. We report clinical and non-clinical data on two investigational small-interfering RNAs that target HBx, ALN-HBV and VIR-2218, demonstrating that incorporation of enhanced stabilization chemistry plus technology in VIR-2218 reduces its propensity to cause ALT elevations relative to its parent compound, ALN-HBV. We also show that VIR-2218 reduces hepatitis B surface antigen levels in a dose-dependent manner in participants with cHBV infection. These studies support the continued development of VIR-2218 as part of therapeutic regimens for cHBV infection, with the goal of a functional cure, and are important for HBV researchers and physicians.

RNA Interference Targeting Liver Angiopoietin-Like Protein 3 Protects from Nephrotic Syndrome in a Rat Model Via Amelioration of Pathologic Hypertriglyceridemia.

Nephrotic syndrome (NS) is associated with metabolic perturbances including profound dyslipidemia characterized by hypercholesterolemia and hypertriglyceridemia. A major underlying mechanism of hypertriglyceridemia in NS is lipoprotein lipase (LPL) deficiency and dysfunction. There is emerging evidence that elevated angiopoietin-like protein 3 (ANGPTL3), an LPL inhibitor that is primarily expressed and secreted by hepatocytes, may be in part responsible for these findings. Furthermore, there is evidence pointing to the contribution of ANGPTL3 to the pathogenesis of proteinuria in NS. Therefore, we hypothesized that inhibition of hepatic ANGPTL3 by RNA interference will ameliorate dyslipidemia and other symptoms of NS and pave the way for a new therapeutic strategy. To this end, we used a subcutaneously delivered, GalNAc (N-Acetylgalactosamine)-conjugated small interfering RNA (siRNA) to selectively target and suppress liver Angptl3 in rats with puromycin-induced NS, which exhibits clinical features of NS including proteinuria, hypoalbuminemia, hyperlipidemia, and renal histologic abnormalities. The study demonstrated that siRNA-mediated knockdown of the liver Angptl3 relieved its inhibitory effect on LPL and significantly reduced hypertriglyceridemia in nephrotic rats. This was accompanied by diminished proteinuria and hypoalbuminemia, which are the hallmarks of NS, and significant attenuation of renal tissue inflammation and oxidative stress. Taken together, this study confirmed the hypothesis that suppression of Angptl3 is protective in NS and points to the possibility that the use of RNA interference to suppress hepatic Angptl3 can serve as a novel therapeutic strategy for NS. SIGNIFICANCE STATEMENT: The current standard of care for mitigating nephrotic dyslipidemia in nephrotic syndrome is statins therapy. However, the efficacy of statins and its safety in the context of impaired kidney function is not well established. Here, we present an alternate therapeutic approach by using siRNA targeting Angptl3 expressed in hepatocytes. As the liver is the major source of circulating Angptl3, siRNA treatment reduced the profound hypertriglyceridemia in a rat model of nephrotic syndrome and was also effective in improving kidney and cardiac function.

Observational Prospective Natural History of Patients with Sanfilippo Syndrome Type B.

OBJECTIVE: To evaluate the natural course of disease progression in patients with Sanfilippo syndrome type B (mucopolysaccharidosis type IIIB), identify potential end points for future therapy trials, and characterize biomarkers related to the disease. STUDY DESIGN: A prospective, multicenter study was conducted. Baseline, 6-month, and 12-month assessments included neurodevelopmental status (Bayley Scales of Infant Development, Third edition), adaptive status (Vineland Adaptive Behavior Scales, Second Edition), volumetric brain magnetic resonance imaging, cerebrospinal fluid heparan sulfate, and urine glycosaminoglycan (GAG) measurements. RESULTS: Nineteen patients aged 1.6-31.7 years were enrolled. Over 12 months, cognition, adaptive behavior, and cortical gray matter volume (GMV) declined in most patients. For patients diagnosed at <6 years, although there was no overall mean change over 12 months, there were 10%-48%, 3%-66%, and 1%-14% decreases in cognitive development quotient score, Vineland Adaptive Behavior Scales, Second Edition development quotient score, and cortical GMV in 8/12, 9/11, and 10/11 patients, respectively. Mean urine GAG and cerebrospinal fluid heparan sulfate levels were stable, but patients diagnosed at <6 years (n = 14) had higher levels than those ≥6 years at diagnosis (n = 4), which was likely associated with age as they also were generally younger. CONCLUSIONS: Cognition, adaptive behavior, and cortical GMV measures sensitively tracked deterioration in patients with mucopolysaccharidosis type IIIB aged ≤8.6 years. Biomarkers may have prognostic value, but their sensitivity to disease progression requires further investigation. These findings should help evaluate enzyme replacement and gene therapy agents for this rare, devastating, neurodegenerative disease. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01509768.

An Investigational RNAi Therapeutic Targeting Glycolate Oxidase Reduces Oxalate Production in Models of Primary Hyperoxaluria.

Primary hyperoxaluria type 1 (PH1), an inherited rare disease of glyoxylate metabolism, arises from mutations in the enzyme alanine-glyoxylate aminotransferase. The resulting deficiency in this enzyme leads to abnormally high oxalate production resulting in calcium oxalate crystal formation and deposition in the kidney and many other tissues, with systemic oxalosis and ESRD being a common outcome. Although a small subset of patients manages the disease with vitamin B6 treatments, the only effective treatment for most is a combined liver-kidney transplant, which requires life-long immune suppression and carries significant mortality risk. In this report, we discuss the development of ALN-GO1, an investigational RNA interference (RNAi) therapeutic targeting glycolate oxidase, to deplete the substrate for oxalate synthesis. Subcutaneous administration of ALN-GO1 resulted in potent, dose-dependent, and durable silencing of the mRNA encoding glycolate oxidase and increased serum glycolate concentrations in wild-type mice, rats, and nonhuman primates. ALN-GO1 also increased urinary glycolate concentrations in normal nonhuman primates and in a genetic mouse model of PH1. Notably, ALN-GO1 reduced urinary oxalate concentration up to 50% after a single dose in the genetic mouse model of PH1, and up to 98% after multiple doses in a rat model of hyperoxaluria. These data demonstrate the ability of ALN-GO1 to reduce oxalate production in preclinical models of PH1 across multiple species and provide a clear rationale for clinical trials with this compound.

A phase 1/2 study of intrathecal heparan-N-sulfatase in patients with mucopolysaccharidosis IIIA.

OBJECTIVE: This was an open-label, phase 1/2 dose-escalation, safety trial of intrathecal recombinant human heparan-N-sulfatase (rhHNS) administered via intrathecal drug delivery device (IDDD) for treating mucopolysaccharidosis IIIA (NCT01155778). STUDY DESIGN: Twelve patients received 10, 45, or 90mg of rhHNS via IDDD once monthly for a total of 6 doses. Primary endpoints included adverse events (AEs) and anti-rhHNS antibodies. Secondary endpoints included standardized neurocognitive assessments, cortical gray matter volume, and pharmacokinetic/pharmacodynamic analyses. RESULTS: All patients experienced treatment-emergent AEs; most of mild-to-moderate severity. Seven patients reported a total of 10 serious AEs (SAEs), all but one due to hospitalization to revise a nonfunctioning IDDD. No SAEs were considered related to rhHNS. Anti-rhHNS antibodies were detected in the serum of 6 patients and in the cerebrospinal fluid (CSF) of 2 of these. CSF heparan sulfate levels were elevated at baseline and there were sustained declines in all tested patients following the first rhHNS dose. No impact of anti-rhHNS antibodies on any pharmacodynamic or safety parameters was evident. 4 of 12 patients showed a decline in developmental quotient, 6 were stable, and 2 patients had only a single data point. No dose group showed a clearly different response pattern. CONCLUSIONS: rhHNS administration via IDDD appeared generally safe and well tolerated. Treatment resulted in consistent declines in CSF heparan sulfate, suggesting in vivo activity in the relevant anatomical compartment. Results of this small study should be interpreted with caution. Future studies are required to assess the potential clinical benefits of rhHNS and to test improved IDDD models.

A Prospective Natural History Study of Mucopolysaccharidosis Type IIIA.

OBJECTIVES: To characterize the clinical course of mucopolysaccharidosis type IIIA (MPS IIIA), and identify potential endpoints for future treatment trials. STUDY DESIGN: Children with a confirmed diagnosis of MPS IIIA, functioning above a developmental age of 1 year, were followed for up to 2 years. Cognitive status and brain atrophy were assessed by standardized tests and volumetric magnetic resonance imaging, respectively. Liver and spleen volumes and cerebrospinal fluid and urine biomarker levels were measured. RESULTS: Twenty-five children, from 1.1 to 18.4 years old, were enrolled, and 24 followed for at least 12 months. 19 exhibited a rapidly progressing (RP) form of MPS IIIA, and 5, a more slowly progressing form. Children with RP plateaued in development by 30 months, followed by rapid regression after 40-50 months. In patients with RP, cognitive developmental quotients showed consistent steep declines associated with progressive cortical gray matter atrophy. Children with slowly progressing had a similar but more prolonged course. Liver and spleen volumes were approximately double normal size, and cerebrospinal fluid and urine heparin sulfate levels were elevated and relatively constant over time. CONCLUSION: Developmental quotient and cortical gray matter volume are sensitive markers of disease progression in MPS IIIA, and may have utility as clinical endpoints in treatment trials. For optimal outcomes, treatment may need to be instituted in children before the onset of steep cognitive decline and brain atrophy. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01047306.

Quantifying behaviors of children with Sanfilippo syndrome: the Sanfilippo Behavior Rating Scale.

The Sanfilippo Behavior Rating Scale (SBRS), a 68 item questionnaire, has been developed to assess the behavioral phenotype of children with Sanfilippo syndrome and its progression over time. Fifteen scales rate orality, movement/activity, attention/self-control, emotional function including anger and fear, and social interaction. Items within scales intercorrelate; measures of internal consistency are adequate. Twelve scales are grouped into 4 abnormality clusters: Movement, Lack of fear, Social/emotional and Executive Dysfunction. A Loess age-trajectory analysis showed that Lack of Fear, Social/Emotional and Executive Dysfunction increased steadily with age; Orality and Mood/Anger/Aggression leveled off. Movement peaked around 6years, then declined as children's excessive/purposeless actions stopped. Compared with standard scales, SBRS Movement was appropriately associated with the Vineland Motor scale; SBRS Lack of Fear had significant associations with the Autism Diagnostic Observation Schedule (ADOS), indicating a symptom overlap between Sanfilippo syndrome and autism. This suggests that reduced fearfulness may be the most salient/sensitive SBRS marker of disease progression. Volumetric MRI showed that increased Lack of Fear was significantly associated with reduced amygdala volume, consistent with our hypothesis that the behavior seen in Sanfilippo syndrome is a variant of Klüver-Bucy syndrome. Hippocampal volume loss had twice the effect on Social-Emotional Dysfunction as amygdala loss, consistent with a hippocampal role in attachment and social emotions. In conclusion, the SBRS assesses the Sanfilippo behavioral phenotype; it can measure behavior change that accompanies disease progression and/or results from treatment.

IL-17A is essential for cell activation and inflammatory gene circuits in subjects with psoriasis.

BACKGROUND: In subjects with psoriasis, inflammation and epidermal hyperplasia are thought to be controlled by T cell-derived cytokines. Evidence suggests that the T(H)17 cell cytokine IL-17A (IL-17) might play a role in disease pathogenesis. OBJECTIVE: We sought to understand the effect that neutralization of IL-17 has on the clinical features of psoriasis and to understand the role that IL-17 has in inflammatory pathways underlying psoriasis in human subjects. METHODS: We examined skin lesions obtained from 40 subjects participating in a phase I, randomized, double-blind, placebo-controlled trial of the anti-IL-17 mAb ixekizumab (previously LY2439821) in which subjects received 5, 15, 50, or 150 mg of subcutaneous ixekizumab or placebo at weeks 0, 2, and 4. RESULTS: There were significant dose-dependent reductions from baseline in keratinocyte proliferation, hyperplasia, epidermal thickness, infiltration into the dermis and epidermis by T cells and dendritic cells, and keratinocyte expression of innate defense peptides at 2 weeks. By week 6, the skin appeared normal. Quantitative RT-PCR and microarrays revealed an ablation of the disease-defining mRNA expression profile by 2 weeks after the first dose of study drug. The effect of IL-17 blockade on expression of genes synergistically regulated by IL-17 and TNF-α was of higher magnitude at 2 weeks than in prior studies with TNF-α antagonism. CONCLUSION: Our data suggest that IL-17 is a key "driver" cytokine that activates pathogenic inflammation in subjects with psoriasis. Neutralizing IL-17 with ixekizumab might be a successful therapeutic strategy in psoriasis.

Lower numbers of natural killer T cells in HIV-1 and Mycobacterium leprae co-infected patients.

Natural killer T (NKT) cells are a heterogeneous population of lymphocytes that recognize antigens presented by CD1d and have attracted attention because of their potential role linking innate and adaptive immune responses. Peripheral NKT cells display a memory-activated phenotype and can rapidly secrete large amounts of pro-inflammatory cytokines upon antigenic activation. In this study, we evaluated NKT cells in the context of patients co-infected with HIV-1 and Mycobacterium leprae. The volunteers were enrolled into four groups: 22 healthy controls, 23 HIV-1-infected patients, 20 patients with leprosy and 17 patients with leprosy and HIV-1-infection. Flow cytometry and ELISPOT assays were performed on peripheral blood mononuclear cells. We demonstrated that patients co-infected with HIV-1 and M. leprae have significantly lower NKT cell frequencies [median 0.022%, interquartile range (IQR): 0.007-0.051] in the peripheral blood when compared with healthy subjects (median 0.077%, IQR: 0.032-0.405, P < 0.01) or HIV-1 mono-infected patients (median 0.072%, IQR: 0.030-0.160, P < 0.05). Also, more NKT cells from co-infected patients secreted interferon-γ after stimulation with DimerX, when compared with leprosy mono-infected patients (P = 0.05). These results suggest that NKT cells are decreased in frequency in HIV-1 and M. leprae co-infected patients compared with HIV-1 mono-infected patients alone, but are at a more activated state. Innate immunity in human subjects is strongly influenced by their spectrum of chronic infections, and in HIV-1-infected subjects, a concurrent mycobacterial infection probably hyper-activates and lowers circulating NKT cell numbers.

Lessons of leprosy: the emergence of TH17 cytokines during type II reactions (ENL) is teaching us about T-cell plasticity.

BACKGROUND: Leprosy was the first disease classified according to the thymus derived T-cell in the 1960s and the first disease classified by the cytokine profile as intact interferon-γ (IFN-γ) and interleukin-2 (IL2) or TH1 (tuberculoid) and deficient IFN-γ and IL2 or TH2 (lepromatous), in the 1980s. OBJECTIVE: In the present study, we set out to explore the T helper 17 (TH17) lymphocyte subset, the hallmark of T-cell plasticity, in skin biopsies from patients with erythema nodosum leprosum (ENL) who were treated with thalidomide. METHOD: RNA was extracted from paraffin embedded tissue before and after thalidomide treatment of ENL and RT-PCR was performed. RESULTS: IL17A, the hallmark of TH17, was consistently seen before and after thalidomide treatment, confirming the TH17 subset to be involved in ENL and potentially up-regulated by thalidomide. CONCLUSION: A reduction in CD70, GARP, IDO, IL17B (IL-20), and IL17E (IL-25), coupled with increases in RORγT, ARNT, FoxP3, and IL17C (IL-21) following thalidomide treatment, opens the door to understanding the complexity of the immunomodulatory drug thalidomide, which can operate as an anti-inflammatory while simultaneously stimulating cell-mediated immunity (CMI). We conclude that TH17 is involved in the immunopathogenesis of ENL and that thalidomide suppresses inflammatory components of TH17, while enhancing other components of TH17 that are potentially involved in CMI.

GWAS of serum ALT and AST reveals an association of SLC30A10 Thr95Ile with hypermanganesemia symptoms.

Understanding mechanisms of hepatocellular damage may lead to new treatments for liver disease, and genome-wide association studies (GWAS) of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum activities have proven useful for investigating liver biology. Here we report 100 loci associating with both enzymes, using GWAS across 411,048 subjects in the UK Biobank. The rare missense variant SLC30A10 Thr95Ile (rs188273166) associates with the largest elevation of both enzymes, and this association replicates in the DiscovEHR study. SLC30A10 excretes manganese from the liver to the bile duct, and rare homozygous loss of function causes the syndrome hypermanganesemia with dystonia-1 (HMNDYT1) which involves cirrhosis. Consistent with hematological symptoms of hypermanganesemia, SLC30A10 Thr95Ile carriers have increased hematocrit and risk of iron deficiency anemia. Carriers also have increased risk of extrahepatic bile duct cancer. These results suggest that genetic variation in SLC30A10 adversely affects more individuals than patients with diagnosed HMNDYT1.

Phase 1/2 Study of Lumasiran for Treatment of Primary Hyperoxaluria Type 1: A Placebo-Controlled Randomized Clinical Trial.

BACKGROUND AND OBJECTIVES: In the rare disease primary hyperoxaluria type 1, overproduction of oxalate by the liver causes kidney stones, nephrocalcinosis, kidney failure, and systemic oxalosis. Lumasiran, an RNA interference therapeutic, suppresses glycolate oxidase, reducing hepatic oxalate production. The objective of this first-in-human, randomized, placebo-controlled trial was to evaluate the safety, pharmacokinetic, and pharmacodynamic profiles of lumasiran in healthy participants and patients with primary hyperoxaluria type 1. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This phase 1/2 study was conducted in two parts. In part A, healthy adults randomized 3:1 received a single subcutaneous dose of lumasiran or placebo in ascending dose groups (0.3-6 mg/kg). In part B, patients with primary hyperoxaluria type 1 randomized 3:1 received up to three doses of lumasiran or placebo in cohorts of 1 or 3 mg/kg monthly or 3 mg/kg quarterly. Patients initially assigned to placebo crossed over to lumasiran on day 85. The primary outcome was incidence of adverse events. Secondary outcomes included pharmacokinetic and pharmacodynamic parameters, including measures of oxalate in patients with primary hyperoxaluria type 1. Data were analyzed using descriptive statistics. RESULTS: Thirty-two healthy participants and 20 adult and pediatric patients with primary hyperoxaluria type 1 were enrolled. Lumasiran had an acceptable safety profile, with no serious adverse events or study discontinuations attributed to treatment. In part A, increases in mean plasma glycolate concentration, a measure of target engagement, were observed in healthy participants. In part B, patients with primary hyperoxaluria type 1 had a mean maximal reduction from baseline of 75% across dosing cohorts in 24-hour urinary oxalate excretion. All patients achieved urinary oxalate levels ≤1.5 times the upper limit of normal. CONCLUSIONS: Lumasiran had an acceptable safety profile and reduced urinary oxalate excretion in all patients with primary hyperoxaluria type 1 to near-normal levels. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Study of Lumasiran in Healthy Adults and Patients with Primary Hyperoxaluria Type 1, NCT02706886.

A potential role for complement in immune evasion by Mycobacterium leprae.

Lepromatous leprosy is a model of immune evasion wherein pathogen-specific IL-10-secreting T cells and concomitant failure of Th-1 immunity permit uncontrolled proliferation of the intracellular pathogen, Mycobacterium leprae (M. leprae). The mechanism of this immune escape is unknown. Here, the authors report that phenolic glycolipid-1 (PGL-1), a major and distinguishing feature of the M. leprae cell wall, is expressed in the cell membrane of M. leprae-infected human dendritic cells, where it can activate complement in human serum. The authors demonstrate that PGL-1 and the C3 component of complement colocalize in lipid rafts in the dendritic cell membrane, and enter the immune synapse upon co-culture of M. leprae-infected DCs and T cells. Hence, activated C3 is strategically located to costimulate naïve T cells via the complement regulatory protein, CD46, a process known to stimulate the differentiation of IL-10-secreting regulatory T cells. These observations suggest a potential novel mechanism of immune evasion, wherein M. leprae may subvert host natural immunity to provoke an adaptive response that favors bacillary survival.

Activation of Protein Kinase A (PKA) signaling mitigates congenital hyperinsulinism associated hypoglycemia in the Sur1-/- mouse model.

There is a significant unmet need for a safe and effective therapy for the treatment of children with congenital hyperinsulinism. We hypothesized that amplification of the glucagon signaling pathway could ameliorate hyperinsulinism associated hypoglycemia. In order to test this we evaluated the effects of loss of Prkar1a, a negative regulator of Protein Kinase A in the context of hyperinsulinemic conditions. With reduction of Prkar1a expression, we observed a significant upregulation of hepatic gluconeogenic genes. In wild type mice receiving a continuous infusion of insulin by mini-osmotic pump, we observed a 2-fold increase in the level of circulating ketones and a more than 40-fold increase in Kiss1 expression with reduction of Prkar1a. Loss of Prkar1a in the Sur1-/- mouse model of KATP hyperinsulinism significantly attenuated fasting induced hypoglycemia, decreased the insulin/glucose ratio, and also increased the hepatic expression of Kiss1 by more than 10-fold. Together these data demonstrate that amplification of the hepatic glucagon signaling pathway is able to rescue hypoglycemia caused by hyperinsulinism.

Increased Plasma Levels of the TH2 chemokine CCL18 associated with low CD4+ T cell counts in HIV-1-infected Patients with a Suppressed Viral Load.

The chemokine (C-C motif) chemokine ligand 18 (CCL18) is a structural homolog of CCL3 primarily produced by monocyte-derived cells with an M2 phenotype. Elevated levels of CCL18 have been observed in several diseases associated with malignancies and chronic inflammation. The role of CCL18 in Human Immunodeficiency Virus (HIV-1) infection remains unknown. We analyzed expression levels of T helper cell-mediated (TH2) chemokines CCL18, CCL17, and CCL22 by ELISA in plasma collected from HIV-1-infected and healthy donors. In HIV-1-infected individuals, plasma viral loads were monitored by NucliSense HIV-1 QT assay and T cell counts and expression of the activation marker CD38 were determined by flow cytometry. Our data showed a significant increase in plasma levels of CCL18 in HIV-1-infected individuals compared to uninfected controls (p < 0.001) and a significant correlation between CCL18 levels and viral load in untreated patients. No significant difference of CCL18 levels was detected among the HIV-1-infected patients treated with combined antiretroviral therapy (cART) and HIV-1-untreated patients.CCL18 values are negatively correlated with CD4+CD38+ cell numbers and total CD4+ T cell counts in patients with a suppressed viral load. Notably, plasma levels of the TH2 chemokines CCL17 and CCL22 are also elevated during HIV-1 infection. However, no correlation of CCL17 and CCL22 production with CD4+ T cell counts was detected. Presented data shows that the chemokines, CCL17, CCL18, and CCL22 are increased during HIV-1 infection. However, only increased levels of CCL18, a marker of M2 macrophages, correlate with low CD4+ T cell counts in patients with suppressed viral load, raising the possibility that CCL18 and/or CCL18-producing cells may interfere with their reconstitution in HIV-1-infected patients on cART.

Improving Drug Discovery by Nucleic Acid Delivery in Engineered Human Microlivers.

The liver plays a central role in metabolism; however, xenobiotic metabolism variations between human hepatocytes and those in model organisms create challenges in establishing functional test beds to detect the potential drug toxicity and efficacy of candidate small molecules. In the emerging areas of RNA interference, viral gene therapy, and genome editing, more robust, long-lasting, and predictive human liver models may accelerate progress. Here, we apply a new modality to a previously established, functionally stable, multi-well bioengineered microliver-fabricated from primary human hepatocytes and supportive stromal cells-in order to advance both small molecule and nucleic acid therapeutic pipelines. Specifically, we achieve robust and durable gene silencing in vitro to tune the human metabolism of small molecules, and demonstrate its capacity to query the potential efficacy and/or toxicity of candidate therapeutics. Additionally, we apply this engineered platform to test siRNAs designed to target hepatocytes and impact human liver genetic and infectious diseases.

Immune cellular parameters of leprosy and human immunodeficiency virus-1 co-infected subjects.

Leprosy and human immunodeficiency virus-1 (HIV-1) are examples of human infections where interactions between the pathogen and the host cellular immunity determine the clinical manifestations of disease. Hence, a significant immunopathological interaction between HIV-1 and leprosy might be expected. In the present study we explored several aspects of cellular immunity in patients co-infected with HIV-1 and Mycobacterium leprae. Twenty-eight individuals were studied, comprising four groups: healthy controls, HIV-1 and M. leprae co-infection, HIV-1 mono-infection, and M. leprae mono-infection. Subjects in the mono-infection and co-infection groups were matched as far as possible for bacillary load and HIV disease status, as appropriate. Peripheral blood mononuclear cells (PBMC) were analysed using six- and seven-colour flow cytometry to evaluate T-cell subpopulations and their activation status, dendritic cell (DC) distribution phenotypes and expression of IL-4 by T cells. The co-infected group exhibited lower CD4 : CD8 ratios, higher levels of CD8(+) T-cell activation, increased V delta : V delta 2 T cell ratios and decreased percentages of plasmacytoid DC, compared with HIV-1 mono-infected subjects. Across infected groups, IL-4 production by CD4(+) T lymphocytes was positively correlated with the percentage of effector memory CD4(+) T cells, suggesting antigenically driven differentiation of this population of T cells in both HIV-1 and M. leprae infections. Co-infection with M. leprae may exacerbate the immunopathology of HIV-1 disease. A T helper 2 (Th2) bias in the CD4(+) T-cell response was evident in both HIV-1 infection and leprosy, but no additive effect was apparent in co-infected patients.

Selective defect in plasmacyoid dendritic cell function in a patient with AIDS-associated atypical genital herpes simplex vegetans treated with imiquimod.

We report a case of acquired immunodeficiency syndrome (AIDS)-associated, acyclovir-refractory genital herpes infection treated with topical imidazoquinoline therapy. The patient's plasmacytoid dendritic cells made a robust interferon- alpha response following in vitro stimulation with imidazoquinoline but not with herpes simplex virus. We hypothesize that disease resulting from defective herpes simplex virus-stimulated interferon- alpha may be overcome by stimulating intact alternative pathways.

A whole blood assay to assess peripheral blood dendritic cell function in response to Toll-like receptor stimulation.

We report a practical technique to assess peripheral blood dendritic cell (DC) maturation and function in whole blood (WB), which requires minimal blood volumes and minimizes ex vivo manipulations of clinical specimens. We determined optimal conditions for flow cytometric analysis of markers of DC maturation, including CCR7, CD25, CD80 and CD83, and of the intracellular cytokines, tumor necrosis factor-alpha (TNF-alpha) and interferon-alpha (IFN-alpha), in both myeloid (mDC) and plasmacytoid (pDC) lineages. We demonstrate concentration-dependent production of these cytokines by DC following short-term stimulation with ligands to Toll-like receptors (TLRs) 2/1, 3, 4, 7, 8 and 9. Kinetic studies revealed maximal TNF-alpha and IFN-alpha protein expression at 2 to 3 h after stimulation with certain TLR ligands. Finally, utilizing cells from a cohort of eight healthy donors, we compared DC responses to TLR activation in WB, freshly isolated peripheral blood mononuclear cells (PBMC) and cryopreserved PBMC. We found that TNF-alpha responses were essentially preserved, but IFN-alpha responses were profoundly diminished or entirely abrogated following cryopreservation. In conclusion, we propose that WB analysis of peripheral DC function is a rapid, reliable and simple method to evaluate TLR function in clinical specimens, which obviates artifact-prone cell purification. The major impact of cryopreservation on some DC responses further strengthens the case for a rapid method that uses fresh blood.