Milk-Based Nutraceutical for Treating Autoimmune Arthritis via the Stimulation of IL-10- and TGF-β-producing CD39+ Regulatory T Cells

Autoimmune diseases arise from the loss of tolerance to self, and because the etiologies of such diseases are largely unknown, symptomatic treatments rely on anti-inflammatory and analgesic agents. Tolerogenic treatments that can reverse disease are preferred, but again, often thwarted by not knowing the responsible auto-antigens (auto-Ags). Hence, a viable alternative to stimulating regulatory T cells (Tregs) is to induce bystander tolerance. Colonization factor antigen I (CFA/I) has been shown to evoke bystander immunity and to hasten Ag-specific Treg development independent of auto-Ag. To translate in treating human autoimmune diseases, the food-based Lactococcus was engineered to express CFA/I fimbriae, and Lactococcus-CFA/I fermented milk fed to arthritic mice proved highly efficacious. Protection occurred via CD39⁺ Tregs producing TGF-β and IL-10 to potently suppress TNF-α production and neutrophil influx into the joints. Thus, these data demonstrate the feasibility of oral nutraceuticals for treating arthritis, and potency of protection against arthritis was improved relative to that obtained with Salmonella-CFA/I.     

Lrp4 Domains Differentially Regulate Limb/Brain Development and Synaptic Plasticity

Apolipoprotein E (ApoE) genotype is the strongest predictor of Alzheimer’s Disease (AD) risk. ApoE is a cholesterol transport protein that binds to members of the Low-Density Lipoprotein (LDL) Receptor family, which includes LDL Receptor Related Protein 4 (Lrp4). Lrp4, together with one of its ligands Agrin and its co-receptors Muscle Specific Kinase (MuSK) and Amyloid Precursor Protein (APP), regulates neuromuscular junction (NMJ) formation. All four proteins are also expressed in the adult brain, and APP, MuSK, and Agrin are required for normal synapse function in the CNS. Here, we show that Lrp4 is also required for normal hippocampal plasticity. In contrast to the closely related Lrp8/Apoer2, the intracellular domain of Lrp4 does not appear to be necessary for normal expression and maintenance of long-term potentiation at central synapses or for the formation and maintenance of peripheral NMJs. However, it does play a role in limb development.     

Placental Fatty Acid Ethyl Esters Are Elevated with Maternal Alcohol Use in Pregnancies Complicated by Prematurity

The accumulation of fatty acid ethyl esters (FAEEs) in meconium of term newborns has been described as one potential biomarker of maternal alcohol use during pregnancy. FAEEs accumulate in multiple alcohol-exposed fetal tissues and in the placenta. Limited research has focused on the identification of the premature newborn exposed to alcohol in utero. We hypothesized that maternal alcohol use occurs in a significant proportion of premature deliveries and that this exposure can be detected as elevated placental FAEEs. The goals of this study were to 1) determine the prevalence of maternal alcohol use in the premature newborn and 2) investigate whether placental FAEEs could identify those newborns with fetal alcohol exposure. This prospective observational study evaluated 80 placentas from 80 women after premature delivery. Subjects were interviewed for alcohol intake and placental FAEEs were quantified via GC/MS. Receiver Operator Characteristic (ROC) Curves were generated to evaluate the ability of placental FAEEs to predict maternal drinking during pregnancy. Adjusted ROC curves were generated to adjust for gestational age, maternal smoking, and illicit drug use. 30% of the subjects admitted to drinking alcohol during pregnancy and approximately 14% answered questions indicative of problem drinking (designated AUDIT+). The specific FAEEs ethyl stearate and linoleate, as well as combinations of oleate + linoleate + linolenate (OLL) and of OLL + stearate, were significantly (p<0.05) elevated in placentas from AUDIT+ pregnancies. Adjusted ROC Curves generated areas under the curve ranging from 88–93% with negative predictive values of 97% for AUDIT+ pregnancies. We conclude that nearly one third of premature pregnancies were alcohol-exposed, and that elevated placental FAEEs hold great promise to accurately determine maternal alcohol use, particularly heavy use, in pregnancies complicated by premature delivery.     

SIRT5 regulation of ammonia-induced autophagy and mitophagy

In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.     

Nitrogenous Nutrients Promote the Growth and Toxicity of Dinophysis acuminata during Estuarine Bloom Events

Diarrhetic Shellfish Poisoning (DSP) is a globally significant human health syndrome most commonly caused by dinoflagellates within the genus Dinophysis. While blooms of harmful algae have frequently been linked to excessive nutrient loading, Dinophysis is a mixotrophic alga whose growth is typically associated with prey availability. Consequently, field studies of Dinophysis and nutrients have been rare. Here, the temporal dynamics of Dinophysis acuminata blooms, DSP toxins, and nutrients (nitrate, ammonium, phosphate, silicate, organic compounds) were examined over four years within two New York estuaries (Meetinghouse Creek and Northport Bay). Further, changes in the abundance and toxicity of D. acuminata were assessed during a series of nutrient amendment experiments performed over a three year period. During the study, Dinophysis acuminata blooms exceeding one million cells L-1 were observed in both estuaries. Highly significant (p<0.001) forward stepwise multivariate regression models of ecosystem observations demonstrated that D. acuminata abundances were positively dependent on multiple environmental parameters including ammonium (p = 0.007) while cellular toxin content was positively dependent on ammonium (p = 0.002) but negatively dependent on nitrate (p<0.001). Nitrogen- (N) and phosphorus- (P) containing inorganic and organic nutrients significantly enhanced D. acuminata densities in nearly all (13 of 14) experiments performed. Ammonium significantly increased cell densities in 10 of 11 experiments, while glutamine significantly enhanced cellular DSP content in 4 of 5 experiments examining this compound. Nutrients may have directly or indirectly enhanced D. acuminata abundances as densities of this mixotroph during experiments were significantly correlated with multiple members of the planktonic community (phytoflagellates and Mesodinium). Collectively, this study demonstrates that nutrient loading and more specifically N-loading promotes the growth and toxicity of D. acuminata populations in coastal zones.     

High Rate of Subclinical Chikungunya Virus Infection and Association of Neutralizing Antibody with Protection in a Prospective Cohort in The Philippines

Background

Chikungunya virus (CHIKV) is a globally re-emerging arbovirus for which previous studies have indicated the majority of infections result in symptomatic febrile illness. We sought to characterize the proportion of subclinical and symptomatic CHIKV infections in a prospective cohort study in a country with known CHIKV circulation.

Methods/Findings

A prospective longitudinal cohort of subjects ≥6 months old underwent community-based active surveillance for acute febrile illness in Cebu City, Philippines from 2012-13. Subjects with fever history were clinically evaluated at acute, 2, 5, and 8 day visits, and at a 3-week convalescent visit. Blood was collected at the acute and 3-week convalescent visits. Symptomatic CHIKV infections were identified by positive CHIKV PCR in acute blood samples and/or CHIKV IgM/IgG ELISA seroconversion in paired acute/convalescent samples. Enrollment and 12-month blood samples underwent plaque reduction neutralization test (PRNT) using CHIKV attenuated strain 181/clone25. Subclinical CHIKV infections were identified by ≥8-fold rise from a baseline enrollment PRNT titer <10 without symptomatic infection detected during the intervening surveillance period. Selected CHIKV PCR-positive samples underwent viral isolation and envelope protein-1 gene sequencing. Of 853 subjects who completed all study procedures at 12 months, 19 symptomatic infections (2.19 per 100 person-years) and 87 subclinical infections (10.03 per 100 person-years) occurred. The ratio of subclinical-to-symptomatic infections was 4.6:1 varying with age from 2:1 in 6 month-5 year olds to 12:1 in those >50 years old. Baseline CHIKV PRNT titer ≥10 was associated with 100% (95%CI: 46.1, 100.0) protection from symptomatic CHIKV infection. Phylogenetic analysis demonstrated Asian genotype closely related to strains from Asia and the Caribbean.

Conclusions

Subclinical infections accounted for a majority of total CHIKV infections. A positive baseline CHIKV PRNT titer was associated with protection from symptomatic CHIKV infection. These findings have implications for assessing disease burden, understanding virus transmission, and supporting vaccine development.     

Evaluation of Integrated Community Case Management in Eight Districts of Central Uganda

Objective

Evidence is limited on whether Integrated Community Case Management (iCCM) improves treatment coverage of the top causes of childhood mortality (acute respiratory illnesses (ARI), diarrhoea and malaria). The coverage impact of iCCM in Central Uganda was evaluated.

Methods

Between July 2010 and December 2012 a pre-post quasi-experimental study in eight districts with iCCM was conducted; 3 districts without iCCM served as controls. A two-stage household cluster survey at baseline (n = 1036 and 1042) and end line (n = 3890 and 3844) was done in the intervention and comparison groups respectively. Changes in treatment coverage and timeliness were assessed using difference in differences analysis (DID). Mortality impact was modelled using the Lives Saved Tool.

Findings

5,586 Village Health Team members delivered 1,907,746 treatments to children under age five. Use of oral rehydration solution (ORS) and zinc treatment of diarrhoea increased in the intervention area, while there was a decrease in the comparison area (DID = 22.9, p = 0.001). Due to national stock-outs of amoxicillin, there was a decrease in antibiotic treatment for ARI in both areas; however, the decrease was significantly greater in the comparison area (DID = 5.18; p<0.001). There was a greater increase in Artemisinin Combination Therapy treatment for fever in the intervention areas than in the comparison area but this was not significant (DID = 1.57, p = 0.105). In the intervention area, timeliness of treatments for fever and ARI increased significantly higher in the intervention area than in the comparison area (DID = 2.12, p = 0.029 and 7.95, p<0.001, respectively). An estimated 106 lives were saved in the intervention area while 611 lives were lost in the comparison area.

Conclusion

iCCM significantly increased treatment coverage for diarrhoea and fever, mitigated the effect of national stock outs of amoxicillin on ARI treatment, improved timeliness of treatments for fever and ARI and saved lives.     

Evaluation of Borrelia burgdorferi BbHtrA Protease as a Vaccine Candidate for Lyme Borreliosis in Mice

Borrelia burgdorferi synthesizes an HtrA protease (BbHtrA) which is a surface-exposed, conserved protein within Lyme disease spirochetes with activity toward CheX and BmpD of Borrelia spp, as well as aggrecan, fibronectin and proteoglycans found in skin, joints and neural tissues of vertebrates. An antibody response against BbHtrA is observed in Lyme disease patients and in experimentally infected laboratory mice and rabbits. Given the surface location of BbHtrA on B. burgdorferi and its ability to elicit an antibody response in infected hosts, we explored recombinant BbHtrA as a potential vaccine candidate in a mouse model of tick-transmitted Lyme disease. We immunized mice with two forms of BbHtrA: the proteolytically active native form and BbHtrA ablated of activity by a serine to alanine mutation at amino acid 226 (BbHtrA^S226A). Although inoculation with either BbHtrA or BbHtrA^S226A produced high-titer antibody responses in C3H/HeJ mice, neither antigen was successful in protecting mice from B. burgdorferi challenge. These results indicate that the search for novel vaccine candidates against Lyme borreliosis remains a challenge.