Clinical Applications of Antimicrobial Peptides (AMPs): Where do we Stand Now?

In this era of multi-drug resistance (MDR), antimicrobial peptides (AMPs) are one of the most promising classes of potential drug candidates to combat communicable as well as noncommunicable diseases such as cancers and diabetes. AMPs show a wide spectrum of biological activities which include antiviral, antifungal, anti-mitogenic, anticancer, and anti-inflammatory properties. Apart from these prospective therapeutic potentials, the AMPs can act as food preservatives and immune modulators. Therefore, AMPs have the potential to replace conventional drugs and may gain a significant global drug market share. Although several AMPs have shown therapeutic potential in vitro or in vivo, in most cases they have failed the clinical trial owing to various issues. In this review, we discuss in brief (i) molecular mechanisms of AMPs in various diseases, (ii) importance of AMPs in pharmaceutical industries, (iii) the challenges in using AMPs as therapeutics and how to overcome, (iv) available AMP therapeutics in market, and (v) AMPs under clinical trials. Here, we specifically focus on the therapeutic AMPs in the areas of dermatology, surgery, oncology and metabolic diseases.

Synergistic chemopreventive effect of allyl isothiocyanate and sulforaphane on non-small cell lung carcinoma cells.

Isothiocyanates from cruciferous vegetables are known for their potential anti-carcinogenic activities. These isothiocyanates are frequently consumed together as part of a regular diet, but their combined effects on carcinogenesis have not been well studied. Herein, we tested the hypothesis that combination of two isothiocyanates, i.e. allyl isothiocyanate and sulforaphane, produced a synergy in inhibiting the growth of A549 lung cancer cells. Our results showed that the combination treatment led to a stronger growth inhibition than the singular treatment. Isobologram analysis proved that the enhanced inhibitory effect of the combination treatment was synergistic. Flow cytometry demonstrated that the combination treatment caused more extensive cell cycle arrest and apoptosis than the singular treatment with modified expression of key proteins regulating these cellular processes. The combined treatment resulted in the production of intracellular reactive oxygen species, which might contribute to the inhibitory effects on cancer cells. Moreover, a synergy between allyl isothiocyanate and sulforaphane was also observed in anti-cell migration. Collectively, our results have demonstrated the potential of different isothiocyanates used in combination to produce enhanced protective effects against carcinogenesis.

Glycine decarboxylase deficiency-induced motor dysfunction in zebrafish is rescued by counterbalancing glycine synaptic level.

Glycine encephalopathy (GE), or nonketotic hyperglycinemia (NKH), is a rare recessive genetic disease caused by defective glycine cleavage and characterized by increased accumulation of glycine in all tissues. Here, based on new case reports of GLDC loss-of-function mutations in GE patients, we aimed to generate a zebrafish model of severe GE in order to unravel the molecular mechanism of the disease. Using CRISPR/Cas9, we knocked out the gldc gene and showed that gldc-/- fish recapitulate GE on a molecular level and present a motor phenotype reminiscent of severe GE symptoms. The molecular characterization of gldc-/- mutants showed a broad metabolic disturbance affecting amino acids and neurotransmitters other than glycine, with lactic acidosis at stages preceding death. Although a transient imbalance was found in cell proliferation in the brain of gldc-/- zebrafish, the main brain networks were not affected, thus suggesting that GE pathogenicity is mainly due to metabolic defects. We confirmed that the gldc-/- hypotonic phenotype is due to NMDA and glycine receptor overactivation, and demonstrated that gldc-/- larvae depict exacerbated hyperglycinemia at these synapses. Remarkably, we were able to rescue the motor dysfunction of gldc-/- larvae by counterbalancing pharmacologically or genetically the level of glycine at the synapse.

Sodium Benzoate, a Food Additive and a Metabolite of Cinnamon, Enriches Regulatory T Cells via STAT6-Mediated Upregulation of TGF-ß.

Upregulation and/or maintenance of regulatory T cells (Tregs) during autoimmune insults may have therapeutic efficacy in autoimmune diseases. Earlier we have reported that sodium benzoate (NaB), a metabolite of cinnamon and a Food and Drug Administration-approved drug against urea cycle disorders, upregulates Tregs and protects mice from experimental allergic encephalomyelitis, an animal model of multiple sclerosis. However, mechanisms by which NaB increases Tregs are poorly understood. Because TGF-ß is an important inducer of Tregs, we examined the effect of NaB on the status of TGF-ß. In this study, we demonstrated that NaB induced the expression of TGF-ß mRNA and protein in normal as well as proteolipid protein-primed splenocytes. The presence of a consensus STAT6 binding site in the promoter of the TGF-ß gene, activation of STAT6 in splenocytes by NaB, recruitment of STAT6 to the TGF-ß promoter by NaB, and abrogation of NaB-induced expression of TGF-ß in splenocytes by small interfering RNA knockdown of STAT6 suggest that NaB induces the expression of TGF-ß via activation of STAT6. Furthermore, we demonstrated that blocking of TGF-ß by neutralizing Abs abrogated NaB-mediated protection of Tregs and experimental allergic encephalomyelitis. These studies identify a new function of NaB in upregulating TGF-ß via activation of STAT6, which may be beneficial in MS patients.

Rice hull smoke extract inactivates Salmonella Typhimurium in laboratory media and protects infected mice against mortality.

UNLABELLED: A previously characterized rice hull smoke extract (RHSE) was tested for bactericidal activity against Salmonella Typhimurium using the disc-diffusion method. The minimum inhibitory concentration (MIC) value of RHSE was 0.822% (v/v). The in vivo antibacterial activity of RHSE (1.0%, v/v) was also examined in a Salmonella-infected Balb/c mouse model. Mice infected with a sublethal dose of the pathogens were administered intraperitoneally a 1.0% solution of RHSE at four 12-h intervals during the 48-h experimental period. The results showed that RHSE inhibited bacterial growth by 59.4%, 51.4%, 39.6%, and 28.3% compared to 78.7%, 64.6%, 59.2%, and 43.2% inhibition with the medicinal antibiotic vancomycin (20 mg/mL). By contrast, 4 consecutive administrations at 12-h intervals elicited the most effective antibacterial effect of 75.0% and 85.5% growth reduction of the bacteria by RHSE and vancomycin, respectively. The combination of RHSE and vancomycin acted synergistically against the pathogen. The inclusion of RHSE (1.0% v/w) as part of a standard mouse diet fed for 2 wk decreased mortality of 10 mice infected with lethal doses of the Salmonella. Photomicrographs of histological changes in liver tissues show that RHSE also protected the liver against Salmonella-induced pathological necrosis lesions. These beneficial results suggest that the RHSE has the potential to complement wood-derived smokes as antimicrobial flavor formulations for application to human foods and animal feeds. PRACTICAL APPLICATION: The new antimicrobial and anti-inflammatory rice hull derived liquid smoke has the potential to complement widely used wood-derived liquid smokes as an antimicrobial flavor and health-promoting formulation for application to foods.

Immunomodulation of experimental allergic encephalomyelitis by cinnamon metabolite sodium benzoate.

Experimental allergic encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), the most common human demyelinating disease of the central nervous system. Sodium benzoate (NaB), a metabolite of cinnamon and a FDA-approved drug against urea cycle disorders in children, is a widely used food additive, which is long known for its microbicidal effect. However, recent studies reveal that apart from its microbicidal effects, NaB can also regulate many immune signaling pathways responsible for inflammation, glial cell activation, switching of T-helper cells, modulation of regulatory T cells, cell-to-cell contact, and migration. As a result, NaB alters the neuroimmunology of EAE and ameliorates the disease process of EAE. In this review, we have made an honest attempt to analyze these newly-discovered immunomodulatory activities of NaB and associated mechanisms that may help in considering this drug for various inflammatory human disorders including MS as primary or adjunct therapy.

Butylated hydroxytoluene chemoprevention of aflatoxicosis - effects on aflatoxin B(1) bioavailability, hepatic DNA adduct formation, and biliary excretion.

The extreme sensitivity of turkeys to aflatoxin B(1) (AFB(1)) is associated with efficient hepatic cytochrome P-450 (P450)-mediated bioactivation, and deficient glutathione S-transferase (GST) mediated detoxification. Butylated hydroxytoluene (BHT) protects against AFB(1) toxicity in turkeys through mechanisms that include competitive inhibition of P450-mediated AFB(1) bioactivation. To test whether dietary BHT alters hepatic AFB(1)-DNA adduct formation, excretion, and bioavailability of AFB(1)in vivo, turkeys were given diets with BHT (4000ppm) for 10 days, given a single oral dose of [(3)H]-AFB(1) (0.05microg/g; 0.02microCi/g), then sampled at intervals up to 24h. Radiolabel in serum, red blood cells, liver, and breast meat was frequently lower in BHT-treated compared to control. Hepatic AFB(1)-DNA adducts in BHT-treated turkeys were significantly lower at 12 and 24h. BHT-fed birds had significant higher bile efflux, though biliary radiolabel excretion was not different from control. The amount of aflatoxin M(1) (AFM(1)) excreted in the bile was lower than in control, but BHT had no effect on the biliary excretion of AFB(1), aflatoxin Q(1) or glucuronide and sulfate conjugates. Thus, the chemopreventive properties of BHT may also occur through a reduction in AFB(1) bioavailability in addition to inhibition of bioactivation.

Sodium benzoate, a food additive and a metabolite of cinnamon, modifies T cells at multiple steps and inhibits adoptive transfer of experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is the animal model for multiple sclerosis. This study explores a novel use of sodium benzoate (NaB), a commonly used food additive and a Food and Drug Administration-approved nontoxic drug for urea cycle disorders, in treating the disease process of relapsing-remitting EAE in female SJL/J mice. NaB, administered through drinking water at physiologically tolerable doses, ameliorated clinical symptoms and disease progression of EAE in recipient mice and suppressed the generation of encephalitogenic T cells in donor mice. Histological studies reveal that NaB effectively inhibited infiltration of mononuclear cells and demyelination in the spinal cord of EAE mice. Consequently, NaB also suppressed the expression of proinflammatory molecules and normalized myelin gene expression in the CNS of EAE mice. Furthermore, we observed that NaB switched the differentiation of myelin basic protein-primed T cells from Th1 to Th2 mode, enriched regulatory T cell population, and down-regulated the expression of various contact molecules in T cells. Taken together, our results suggest that NaB modifies encephalitogenic T cells at multiple steps and that NaB may have therapeutic importance in multiple sclerosis.

Bactericidal activity of wasabi (Wasabia japonica) against Helicobacter pylori.

In this study, the bactericidal activity of Korean and Japanese wasabi roots, stems and leaves against Helicobacter pylori were examined. Allyl isothiocyanate (AIT) in roots, stems and leaves of Korean wasabi were 0.75, 0.18 and 0.32 mg/g, respectively. AIT in roots, stems and leaves of Japanese wasabi were 1.18, 0.41 and 0.38 mg/g, respectively. All parts of wasabi showed bactericidal activities against H. pylori strain NCTC 11637, YS 27 and YS 50. The leaves of both wasabi showed the highest bactericidal activities with the minimum bactericidal concentration of 1.05-1.31 mg of dry weight/ml against three strains of H. pylori. The roots showed a little lower bactericidal activity with 2.09-4.17 mg of dry weight/ml against them. The main component related to antimicrobial activity in wasabi is well known to be AIT. In this study, the bactericidal activity of leaves was higher than that of roots, although AIT amount of leaves was lower than that of roots. These results suggest that certain components besides AIT in wasabi are effective in killing H. pylori.

Antioxidant properties of extracts from Alchornea laxiflora (Benth) Pax and Hoffman.

The antioxidant activities of the leaf and root extracts of Alchornea laxiflora, a plant used locally for the preservation of food items in Nigeria, were evaluated using the ferric thiocyanate method, horseradish peroxidase catalysed oxidation of 2,2 azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), beta-carotene linoleate model system and Fe(2+)/ascorbate/H(2)O(2)-induced rat liver microsomal lipid peroxidation. The crude hexane root (HR), methanol root (MR), methanol leaf (ML) and hexane leaf (HL) extracts from A. laxiflora were tested for antioxidant activities. Antioxidant activity decreased in the following order: HR (76.4%), MR (63%), ML (40%) and HL (38%) at a concentration of 0.05% v/v. The antioxidant activity of HR compared to that of butylated hydroxyanisole (BHA) (80%), a standard antioxidant. The total antioxidant activity (TAA) of the crude extracts suggests that activity is highest in the HR compared with the others. The TAA value was estimated to be 8.0 measured as mm of vitamin C equivalent. Six column chromatographic fractions (FI-FVI) from HR showed antioxidant activity to varying extents in the beta-carotene model system in the order of FII > FI > FVI > FIII > FIV > FV. FII exhibited the highest antioxidant activity in all model systems utilized, it recorded a higher antioxidant activity than BHA and quercetin in the beta-carotene linoleate and Fe(2+)/ascorbate/H(2)O(2). TLC analysis of fraction II revealed the presence of terpenoid compounds (radiant green coloration with 2,4 dinitrophenylhydrazine). Our results suggest that A. laxiflora contains potent natural antioxidants and may therefore be relevant in the preservation of lipid food products, which are prone to oxidation and rancidity.

Ornithine transcarbamylase deficiency: a urea cycle defect.

The symptoms and signs of ornithine transcarbamylase deficiency are discussed. When the condition occurs among males in the neonatal period it is likely to be lethal. Pathological findings are non-specific. The diagnosis should be considered if coma with cerebral oedema and respiratory alkalosis occurs for no obvious reason. When hyperammonaemia is found, enzyme assay on a liver biopsy should be considered. A useful clue in an asymptomatic patient is a voluntary adoption of a vegetarian diet. Provocative tests, such as the allopurinol test can be used, but the method most frequently applied is mutation analysis. In the case of prenatal diagnosis this is possible on a chorionic villus sample. The prognosis of ornithine transcarbamylase deficiency is better for those with an onset after infancy, but morbidity from brain damage does not appear to be linked to the number of episodes of hyperammonaemia that have occurred. The syndrome results from a deficiency of the mitochondrial enzyme ornithine transcarbamylase which catalyses the conversion of ornithine and carbamoyl phosphate to citrulline. The gene responsible for this enzyme is located on Xp21.1, and is expressed in the liver and gut. Mutations can be divided into two groups: those with neonatal onset with all enzyme activity abolished, and those with later onset with partial and varying enzyme deficiency. There can be a variety of precipitating causes, for example sodium valproate. Treatment can be given with a low protein diet, and with alternate pathway drugs such as sodium benzoate and phenylbutyrate. Liver transplant can be considered when symptoms are life-threatening, although there may be severe complications.Gene replacement therapy is the hope of the future.

The effects of benzoate and fluoride on dental caries in intact and desalivated rats.

The decline in prevalence of dental caries in some segments of the population has been attributed mainly to extensive exposure to fluoride. Over the past decades, the use of fluoridated products has increased. During the same period, the consumption of food preservatives such as benzoates and sorbates has also increased substantially. Benzoates, in vitro, possess antibacterial properties similar to those of fluoride and in combination with fluoride could affect caries development. In the present study we explored the effects of sodium benzoate and fluoride in combination and alone on dental caries in our animal model. The results showed a combination of benzoate and fluoride reduced caries activity more effectively in rodents fed a cariogenic diet ad libitum than fluoride alone (p = 0.038).

Citrullinemia. Clinical experience with 23 cases.

A retrospective study was performed on the clinical outcome of 23 patients with citrullinemia diagnosed during the last 20 years in our clinic. The study group consisted of 13 patients with the neonatal form of the disease, four patients with the subacute form, five patients with the late-onset form and one with the asymptomatic form. All patients were treated with natural protein restriction, sodium benzoate and arginine administration. Almost all of the neonatal-onset patients were treated with exchange transfusions and/or peritoneal dialysis. Fourteen patients died: 11 with the neonatal form, one with the subacute form, and two with the late-onset form. The general neurological outcome of the patients who were alive was not satisfactory. Despite these results, it was concluded that the prognosis and quality of life of patients with citrullinemia might be improved with early diagnosis and appropriate therapy.

Vida de anaquel y evaluación sensorial en tortillas de maíz elaboradas con conservadores y mejoradores / Shelf life and sensory evaluation of corn tortillas, made with preservatives and texture conditioners

Con el propósito de elaborar tortillas empacadas, con una vida de anaquel prolongada y que a su vez tenga la calidad de un producto fresco, se planteó determinar el efecto conservadores y mejoradores sobre las características reológicas, sensoriales y vida de anaquel en tortillas de harina de maíz nistamalizado. Los conservadores evaluados fueron; sorbado de potasio (SK) y propionato de calcio (PCa). Como mejoradores de la textura se usaron: carboximetilcelulosa (CMC) y estearil-2 lactilato de sodio (SS2L). En las tortillas frescas se evaluó pH, rendimiento, textura, elasticidad y evaluación sensorial. Las tortillas empacadas en bolsas de polietileno se almacenaron a 4ºC. Las tortillas que tenían SK (0,1 por ciento), PCa (0,1 por ciento) y SS2L (0,25 por ciento), fueron las más ácidas (pH=5,12), las de mayor vida de anaquel (53 días sin desarrollo de microorganismos), y la menor cuenta total de mesófilos aerobicos (23 unidades formadoras de colonias). Los mayores rendimientos correspondieron a los tratamientos con 0,5 por ciento de CMC, siendo las tortillas más elásticas, pero con menor vida de anaquel (18 y 24 días). Diez días después de empacar las tortillas, fueron recalentadas y evaluadas sensorialmente por 5 panelistas entrenados, quienes juzgaron que el mejoramiento fue el que contenía SK (0,1 por ciento), PCa (0,1 por ciento) y CMC (0,5 por ciento), por ser las tortillas más suaves y elásticas; en el resto de los atributos las mejores tortillas fueron las hechas con el método tradicional (frescas). En la prueba de nivel de agrado, los consumidores señalaron que la tortilla de mejor sabor era la tradicional. no obstante, los tratamientos con mezcla de conservadores (SK y PCa) y mejoradores, obtuvieron calificaciones semejantes a las obtenidas por dos marcas comerciales de tortillas empacadas

Absence of acidosis in the initial presentation of propionic acidaemia.

The clinical presentation and results of the initial biochemical and haematological investigations in 11 newborn term infants with propionic acidaemia are described. All patients had neurological symptoms. Only four had clinically important acidosis, but all had a raised blood ammonia. A diagnosis of propionic acidaemia should be considered in all newborn infants with unexplained neurological deterioration even in the absence of a metabolic acidosis.