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BackgroundDevelopment of new tuberculosis (TB) drugs and alternative treatment strategies are urgently required to control the global spread of TB. Previous results have shown that vitamin D3 (vitD3) and 4-phenyl butyrate (PBA) are potent inducers of the host defense peptide LL-37 that possess anti-mycobacterial effects.ObjectiveTo examine if oral adjunctive therapy with 5,000IU vitD3 or 2x500 mg PBA or PBA+vitD3 to standard chemotherapy would lead to enhanced recovery in sputum smear-positive pulmonary TB patients.MethodsAdult TB patients (n = 288) were enrolled in a randomized, double-blind, placebo-controlled trial conducted in Bangladesh. Primary endpoints included proportions of patients with a negative sputum culture at week 4 and reduction in clinical symptoms at week 8. Clinical assessments and sputum smear microscopy were performed weekly up to week 4, fortnightly up to week 12 and at week 24; TB culture was performed at week 0, 4 and 8; concentrations of LL-37 in cells, 25-hydroxyvitamin D3 (25(OH)D3) in plasma and ex vivo bactericidal function of monocyte-derived macrophages (MDM) were determined at week 0, 4, 8, 12 and additionally at week 24 for plasma 25(OH)D3.ResultsAt week 4, 71% (46/65) of the patients in the PBA+vitD3-group (p = 0.001) and 61.3% (38/62) in the vitD3-group (p = 0.032) were culture negative compared to 42.2% (27/64) in the placebo-group. The odds of sputum culture being negative at week 4 was 3.42 times higher in the PBA+vitD3-group (p = 0.001) and 2.2 times higher in vitD3-group (p = 0.032) compared to placebo. The concentration of LL-37 in MDM was significantly higher in the PBA-group compared to placebo at week 12 (p = 0.034). Decline in intracellular Mtb growth in MDM was earlier in the PBA-group compared to placebo (log rank 11.38, p = 0.01).ConclusionAdjunct therapy with PBA+vitD3 or vitD3 or PBA to standard short-course therapy demonstrated beneficial effects towards clinical recovery and holds potential for host-directed-therapy in the treatment of TB.Trial Registrationclinicaltrials.gov NCT01580007

BackgroundLow serum levels of 25-hydroxyvitamin D3 are associated with an increased risk of respiratory tract infections (RTIs). Clinical trials with vitamin D3 against various infections have been carried out but data are so far not conclusive. Thus, there is a need for additional randomised controlled trials of effects of vitamin D3 on infections.ObjectiveTo investigate if supplementation with vitamin D3 could reduce infectious symptoms and antibiotic consumption among patients with antibody deficiency or frequent RTIs.DesignA double-blind randomised controlled trial.SettingKarolinska University Hospital, Huddinge.Participants140 patients with antibody deficiency (selective IgA subclass deficiency, IgG subclass deficiency, common variable immune disorder) and patients with increased susceptibility to RTIs (>4 bacterial RTIs/year) but without immunological diagnosis.InterventionVitamin D3 (4000 IU) or placebo was given daily for 1 year.Primary and secondary outcome measuresThe primary endpoint was an infectious score based on five parameters: symptoms from respiratory tract, ears and sinuses, malaise and antibiotic consumption. Secondary endpoints were serum levels of 25-hydroxyvitamin D3, microbiological findings and levels of antimicrobial peptides (LL-37, HNP1–3) in nasal fluid.ResultsThe overall infectious score was significantly reduced for patients allocated to the vitamin D group (202 points) compared with the placebo group (249 points; adjusted relative score 0.771, 95% CI 0.604 to 0.985, p=0.04).LimitationsA single study centre, small sample size and a selected group of patients. The sample size calculation was performed using p=0.02 as the significance level whereas the primary and secondary endpoints were analysed using the conventional p=0.05 as the significance level.ConclusionsSupplementation with vitamin D3 may reduce disease burden in patients with frequent RTIs.

These authors contributed equally to this work.Keywords: antimicrobial peptides, cathelicidin, innate immunity, P2RX7, tuberculosis, vitamin D Abbreviations: 1,25(OH) 2 D 3 , 1,25-dihydroxy vitamin D 3 ; AMPK, adenosine monophosphate-activated protein kinase; AMPs, antimicrobial peptides; ATG, autophagy related; BECN1, Beclin 1, autophagy related; CAMP, cathelicidin antimicrobial peptide; CFUs, colony-forming units; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MDMs, monocyte-derived macrophages; Mtb, Mycobacterium tuberculosis; P2RX7, purinergic receptor P2X, ligand gated ion channel, 7; PBA, 4-phenylbutyrate; PBS, phosphate-buffered saline; PtdIns3K, phosphatidylinositol 3-kinase; RNA18S/18S rRNA, RNA 18S ribosomal; SQSTM1, sequestosome 1; TB, tuberculosis LL-37 is a human antimicrobial peptide (AMP) of the cathelicidin family with multiple activities including a mediator of vitamin D-induced autophagy in human macrophages, resulting in intracellular killing of Mycobacterium tuberculosis (Mtb). In a previous trial in healthy volunteers, we have shown that LL-37 expression and subsequent Mtb-killing can be further enhanced by 4-phenylbutyrate (PBA), also an inducer of LL-37 expression. Here, we explore a potential mechanism(s) behind PBA and LL-37-induced autophagy and intracellular killing of Mtb. Mtb infection of macrophages downregulated the expression of both the CAMP transcript and LL-37 peptide as well as certain autophagy-related genes (BECN1 and ATG5) at both the mRNA and protein levels. In addition, activation of LC3-II in primary macrophages and THP-1 cells was not detected. PBA and the active form of vitamin D 3 (1,25[OH] 2 D 3 ), separately or particularly in combination, were able to overcome Mtb-induced suppression of LL-37 expression. Notably, reactivation of autophagy occurred by stimulation of macrophages with PBA and promoted colocalization of LL-37 and LC3-II in autophagosomes. Importantly, PBA treatment failed to induce autophagy in Mtb-infected THP-1 cells, when the expression of LL-37 was silenced. However, PBA-induced autophagy was restored when the LL-37 knockdown cells were supplemented with synthetic LL-37. Interestingly, we have found that LL-37-induced autophagy was mediated via P2RX7 receptor followed by enhanced cytosolic free Ca 2C , and activation of AMPK and PtdIns3K pathways. Altogether, these results suggest a novel activity for PBA as an inducer of autophagy, which is LL-37-dependent and promotes intracellular killing of Mtb in human macrophages.

BackgroundArsenic (As) exposure during pregnancy induces oxidative stress and increases the risk of fetal loss and low birth weight.ObjectivesIn this study we aimed to elucidate the effects of As exposure on immune markers in the placenta and cord blood, and the involvement of oxidative stress.MethodsPregnant women were enrolled around gestational week (GW) 8 in our longitudinal, population-based, mother–child cohort in Matlab, an area in rural Bangladesh with large variations in As concentrations in well water. Women (n = 130) delivering at local clinics were included in the present study. We collected maternal urine twice during pregnancy (GW8 and GW30) for measurements of As, and placenta and cord blood at delivery for assessment of immune and inflammatory markers. Placental markers were measured by immunohistochemistry, and cord blood cytokines by multiplex cytokine assay.ResultsIn multivariable adjusted models, maternal urinary As (U-As) exposure both at GW8 and at GW30 was significantly positively associated with placental markers of 8-oxoguanine (8-oxoG) and interleukin-1β (IL-1β); U-As at GW8, with tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ); and U-As at GW30, with leptin; U-As at GW8 was inversely associated with CD3+ T cells in the placenta. Cord blood cytokines (IL-1β, IL-8, IFNγ, TNFα) showed a U-shaped association with U-As at GW30. Placental 8-oxoG was significantly positively associated with placental proinflammatory cytokines. Multivariable adjusted analyses suggested that enhanced placental cytokine expression (TNFα and IFNγ) was primarily influenced by oxidative stress, whereas leptin expression appeared to be mostly mediated by As, and IL-1β appeared to be influenced by both oxidative stress and As.ConclusionAs exposure during pregnancy appeared to enhance placental inflammatory responses (in part by increasing oxidative stress), reduce placental T cells, and alter cord blood cytokines. These findings suggest that effects of As on immune function may contribute to impaired fetal and infant health.

BackgroundCathelicidins and defensins are endogenous antimicrobial peptides (AMPs) that are downregulated in the mucosal epithelia of the large intestine in shigellosis. Oral treatment of Shigella infected rabbits with sodium butyrate (NaB) reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia.AimsTo develop novel regimen for treating infectious diseases by inducing innate immunity, we selected sodium 4-phenylbutyrate (PB), a registered drug for a metabolic disorder as a potential therapeutic candidate in a rabbit model of shigellosis. Since acute respiratory infections often cause secondary complications during shigellosis, the systemic effect of PB and NaB on CAP-18 expression in respiratory epithelia was also evaluated.MethodsThe readouts were clinical outcomes, CAP-18 expression in mucosa of colon, rectum, lung and trachea (immunohistochemistry and real-time PCR) and release of the CAP-18 peptide/protein in stool (Western blot).Principal findingsSignificant downregulation of CAP-18 expression in the epithelia of rectum and colon, the site of Shigella infection was confirmed. Interestingly, reduced expression of CAP-18 was also noticed in the epithelia of lung and trachea, indicating a systemic effect of the infection. This suggests a causative link to acute respiratory infections during shigellosis. Oral treatment with PB resulted in reduced clinical illness and upregulation of CAP-18 in the epithelium of rectum. Both PB and NaB counteracted the downregulation of CAP-18 in lung epithelium. The drug effect is suggested to be systemic as intravenous administration of NaB could also upregulate CAP-18 in the epithelia of lung, rectum and colon.ConclusionOur results suggest that PB has treatment potential in human shigellosis. Enhancement of CAP-18 in the mucosal epithelia of the respiratory tract by PB or NaB is a novel discovery. This could mediate protection from secondary respiratory infections that frequently are the lethal causes in dysentery.

BackgroundWe earlier showed that 4-phenylbutyrate (PB) can induce cathelicidin LL-37 expression synergistically with 1,25-dihydroxyvitamin D3 in a lung epithelial cell line. We aimed to evaluate a therapeutic dose of PB alone or in combination with vitamin D3 for induction of LL-37 expression in immune cells and enhancement of antimycobacterial activity in monocyte-derived macrophages (MDM).MethodsHealthy volunteers were enrolled in an 8-days open trial with three doses of PB [250 mg (Group-I), 500 mg (Group-II) or 1000 mg (Group-III)] twice daily (b.d.) together with vitamin D3 {5000 IU once daily (o.d.)}, PB (500 mg b.d.) (Group-IV) or vitamin D3 (5000 IU o.d.) (Group-V), given orally for 4 days. Blood was collected on day-0, day-4 and day-8; plasma was separated, peripheral blood mononuclear cells (PBMC), non-adherent lymphocytes (NAL) and MDM were cultured. LL-37 transcript in cells and peptide concentrations in supernatant were determined by qPCR and ELISA, respectively. In plasma, 25-hydorxyvitamin D3 levels were determined by ELISA. MDM-mediated killing of Mycobacterium tuberculosis (Mtb) (H37Rv) was performed by conventional culture method.ResultsMDM from Group-II had increased concentration of LL-37 peptide and transcript at day-4, while Group-I showed increased transcript at day-4 and day-8 compared to day-0 (p < 0.05). Both Group-I and -II exhibited higher levels of transcript on day-4 compared to Group-III and Group-V (p < 0.035). Increased induction of peptide was observed in lymphocytes from Group-II on day-4 compared to Group-I and Group-IV (p < 0.05), while Group-IV showed increased levels on day-8 compared to Group-I and Group-III (p < 0.04). Intracellular killing of Mtb on day-4 was significantly increased compared to day-0 in Group-I, -II and -V (p < 0.05).ConclusionThe results demonstrate that 500 mg b.d. PB with 5000 IU o.d. vitamin D3 is the optimal dose for the induction of LL-37 in macrophages and lymphocytes and intracellular killing of Mtb by macrophages. Hence, this dose has potential application in the treatment of TB and is now being used in a clinical trial of adults with active pulmonary TB (NCT01580007).

BackgroundTreatment of shigellosis in rabbits with butyrate reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia. Here, we aimed to evaluate whether butyrate can be used as an adjunct to antibiotics in the treatment of shigellosis in patients.MethodsA randomized, double-blind, placebo-controlled, parallel-group designed clinical trial was conducted. Eighty adult patients with shigellosis were randomized to either the Intervention group (butyrate, n = 40) or the Placebo group (normal saline, n = 40). The Intervention group was given an enema containing sodium butyrate (80 mM), twice daily for 3 days, while the Placebo group received the same dose of normal saline. The primary endpoint of the trial was to assess the efficacy of butyrate in improving clinical, endoscopic and histological features of shigellosis. The secondary endpoint was to study the effect of butyrate on the induction of antimicrobial peptides in the rectum. Clinical outcomes were assessed and concentrations of antimicrobial peptides (LL-37, human beta defensin1 [HBD-1] and human beta defensin 3 [HBD-3]) and pro-inflammatory cytokines (interleukin-1β [IL-1β] and interleukin-8 [IL-8]) were measured in the stool. Sigmoidoscopic and histopathological analyses, and immunostaining of LL-37 in the rectal mucosa were performed in a subgroup of patients.ResultsCompared with placebo, butyrate therapy led to the early reduction of macrophages, pus cells, IL-8 and IL-1β in the stool and improvement in rectal histopathology. Butyrate treatment induced LL-37 expression in the rectal epithelia. Stool concentration of LL-37 remained significantly higher in the Intervention group on days 4 and 7.ConclusionAdjunct therapy with butyrate during shigellosis led to early reduction of inflammation and enhanced LL-37 expression in the rectal epithelia with prolonged release of LL-37 in the stool.Trial RegistrationClinicalTrials.gov, NCT00800930.