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Understanding the Mechanism of Action and Clinical Implications of Anti-Obesity Drugs Recently Approved in Korea

The Korean Ministry of Food and Drug Safety has approved three anti-obesity drugs for long-term management in the past decade. In addition, since 2019, bariatric surgery has been financially supported by National Health Insurance Service in Korea. In this review, the mechanisms of action and the clinical implications of the recently approved anti-obesity drugs, lorcaserin, naltrexone/bupropion, and liraglutide are explained. Lorcaserin stimulates proopiomelanocortin (POMC)/cocaine- and amphetamine-regulated transcript (CART) neurons and inhibits neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons, which results in the activation of melanocortin 3/4 receptors. Naltrexone/bupropion stimulates POMC neurons through bupropion; this stimulation is augmented by blocking the autoinhibitory mechanism of POMC with naltrexone. The hypophagic effect of liraglutide is mediated through the direct activation of POMC/CART neurons and the indirect suppression of NPY/AgRP neurons through γ-aminobutyric acid-dependent signaling, with adjunctive suppression of the mesolimbic dopamine reward system. In addition to liraglutide, another glucagon-like peptide-1 receptor agonist, semaglutide, is expected to be added to the list of anti-obesity drugs in the near future. In patients with obesity and high cardiovascular risk, lorcaserin was considered neutral and liraglutide was considered favorable, whereas inconclusive results were obtained for naltrexone/bupropion.

INTRODUCTION
There are five methods of obesity treatment: diet, exercise, behavior modification, drugs, and surgery. These treatment modalities can be divided into two categories, active and passive, based on the perspective of the patient. The active methods, including diet, exercise, and behavior modification, must be done by the patients themselves, whereas the passive methods of pharmacotherapy and bariatric surgery are performed by medical personnel.
A sufficient reduction in food intake and a radical increase in daily activity can reduce excess body weight and then maintain normal body weight in almost all patients with obesity. However, it is well established, through clinical experience and many previous clinical trials, 1) that the standard active treatments are not strong enough to induce clinically significant weight loss. Very few patients with obesity can resist hunger and select low-energy-density foods when facing the excess availability of high-energy-density foods. Moreover, very few patients with obesity can maintain an active daily life given the present social prevalence of sedentary lifestyles. Thus, obesity specialists are constantly seeking passive methods of weight reduction.
Over the past decade, there have been major changes in the global treatment of obesity, and in Korea. Between 1964 and 2009, 25 antiobesity drugs, most of which were monoamine neurotransmitter reuptake inhibitors or releasers, were withdrawn after marketing in several countries or worldwide due to cardiotoxicity or potential for drug abuse/dependency. 2) Consequently, in 2009, orlistat was the only antiobesity drug approved for long-term treatment in Korea. However, Korean Ministry of Food and Drug Safety has approved three more antiobesity drugs for long-term management in the past decade and another drug is under consideration for approval. The currently prescribed anti-obesity drugs are listed in Table 1, along with their status in the controlled substances schedule, by the US Drug Enforcement Administration. 3) In addition, since 2019, bariatric surgery has been supported financially by the National Health Insurance Service.
In this review paper, the mechanisms of action and the clinical implications of the recently approved anti-obesity drugs, lorcaserin, naltrexone/bupropion, and liraglutide, will be described.

MECHANISM OF ACTION OF CENTRALLY ACTING ANTI-OBESITY DRUGS
In the previous era of anti-obesity drugs, it was unclear how the centrally acting anti-obesity drugs suppressed appetite; however, the mechanism of action of orlistat, a gastric/pancreatic lipase inhibitor that inhibits the absorption of dietary fat in the small intestine, was well characterized. However, the appetite-suppressing mechanisms of lorcaserin, naltrexone/bupropion, and liraglutide are easy to understand. Before the mechanism of action of anti-obesity drugs is described, a brief overview of the mechanism of energy homeostasis in the brain will be provided.

Energy Homeostasis and the Arcuate Nucleus
For the maintenance of energy homeostasis, it is essential to sense the peripheral signals related to the amount and accessibility of energy.
The signals can be divided into two groups: the relatively stable signals that describe the amount of overall stored energy in the body; and the rapidly changing signals that describe the sudden energy consumption, the recent food intake, food availability, and anticipated energy consumption. These peripheral signals of body energy status are considered to be transferred to a specific region of the brain through the humoral and neural pathways and are integrated there. The signaling routes for the control of obesity have been described in detail previously. 4) The neurons in the arcuate nucleus of the hypothalamus, which control food intake and energy expenditure, can be grouped into two major populations: orexigenic neurons, which express neuropeptide Y (NPY) and/or agouti-related peptide (AgRP); and anorexigenic neurons, which express proopiomelanocortin (POMC) and/or cocaineand amphetamine-regulated transcript (CART). These two neuron populations gather and integrate the peripheral signals related to the current energy status of body, the presence of food, and the amount of food intake, and they activate the feeding center or transmit signs of satiety, which stop feeding behavior and suppress feeding actions for a certain period of time. Downstream of the POMC/CART neurons, the activation of melanocortin (MC) 3 and 4 receptors by α-melanocyte stimulating hormone occurs ( Figure 1).

Serotonergic Satiety in Brain
Serotonin (5-hydroxytryptophan, 5-HT) is a monoamine neurotransmitter that is been well conserved across species throughout evolution. 5) The 5-HT system and receptors are quite varied and the 5-HT receptors have been grouped into seven families, 5-HT1 to 5-HT7, which include 14 known subtypes. 5) 5-HT is involved in diverse physiology, including cognition, mood, sexual behavior, and feeding behavior. 5) Although 5-HT has been conserved well throughout evolution,

Interaction of Serotonin Satiety System with POMC/CART Neurons and NPY/AgRP Neurons
The anorexic effects of the serotonin receptor agonist (RA), fenfluramine, which was widely administered to patients with obesity in the early 1990s, were known to be mediated through the central melano-  hyperpolarize NPY neurons, whereas 5-HT1B receptor antagonists prevented the hyperpolarization induced by 5-HT1B RAs. 13) These findings suggested that 5-HT may directly inhibit NPY/AgRP neuron activity through the inhibitory 5-HT1B receptor.

Gut to Brain
Preproglucagon, coded by the proglucagon gene, is processed to glucagon-like peptide-1 (GLP-1), and is secreted from intestinal endocrine L-cells, which are located mainly in the distal ileum and colon, and preproglucagon-expressing neurons in the nucleus tractus solitarius (NTS) of the brain stem. 14,15) Although there are many GLP-1 binding sites in the brain, the half-life of endogenous GLP-1 secreted from the gut is too short to reach the brain owing to the rapid degradation; therefore, some GLP-1 RAs may reach the brain through the humoral pathway, cross the blood-brain barrier (BBB), and attach to GLP-1 binding sites in the brain. GLP-1 RAs that reach the inside of brain may act like brain-derived endogenous GLP-1.
GLP-1 can activate the hepatic vagal nerve and the vagal afferent fibers of nodose ganglion neurons. 16,17) In rats with subdiaphragmatic vagal deafferentation, the hypophagic effect of intraperitoneally injected GLP-1 was suppressed in the early stage (until 1 hour) of low-dose administration, whereas the hypophagic effect was observed in the later stages (≥2 hours) or by a high-dose administration. 18) Similarly, the hypophagic effect of liraglutide was sustained in rats with subdiaphragmatic vagal deafferentation. 19) According to these findings, putatively, the neural pathway through the vagus nerve is responsible for a large part of the influence of endogenous gut-derived GLP-1 on the brain, whereas peripherally administered long-acting GLP-1 RAs act directly on brain GLP-1 RAs through the humoral pathway.

Mechanisms of Action of Lorcaserin and Liraglutide
Considering the energy homeostasis mechanism described above, the action mechanism of lorcaserin, a  Values are presented as number or mean±standard deviation. The mean weight loss was calculated for the full analysis set, which comprised all randomized individuals exposed to trial drug with at least one post-randomization weight assessment. *The participants were patients with obesity and diabetes. † Treatment was combined with behavior modification. However, there are no GLP-1 receptors on NPY/AgRP neurons, and the inhibitory signal of liraglutide on orexigenic NPY neurons is transmitted via γ-aminobutyric acid (GABA)-dependent signaling. 19) In addition, the activation of GLP-1 receptors suppressed the reward system activity of mesolimbic dopamine neurons to highly palatable food intake, which may be a mechanism through which liraglutide demonstrates its hypophagic effect. 21) GLP-1 can induce direct thermogenesis. It was demonstrated that GLP-1 increased thermogenesis in rats, mediated by the lower brainstem and the sympathoadrenal system, with a larger increase observed from intravenous administration than from intracerebroventricular administration. 22) The thermogenic effect of GLP-1 is induced by the increase in interscapular brown adipose tissue activity, which depends on adrenergic signaling. 23) However, the increase in resting energy expenditure was not significant in patients with type 2 diabetes mellitus (T2DM). 24) In conclusion, the hypophagic effect of liraglutide depends on the direct activation of POMC/CART neurons and the indirect suppression of NPY/AgRP neurons through GABA-dependent signaling, and the suppression of mesolimbic dopamine reward system (Figure 1).

Combination of Naltrexone/Bupropion
The fixed dose drug combination of sustained-release (SR) naltrexone 32 mg and SR bupropion 360 mg (NB) is one of the approved antiobesity drugs for long-term treatment in Korea.
In the arcuate nucleus, dopamine receptor activation suppresses NPY neurons and stimulates POMC neurons, most likely through D2 receptors. 33,34) The intraperitoneal co-administration of a selective dopamine reuptake inhibitor with a selective norepinephrine reuptake inhibitor showed additive effects on negative energy balance in mice. 35) Although bupropion, a norepinephrine-dopamine reuptake inhibitor, is unique among anti-depressants owing to its association with weight loss due to its mechanism of action, the extent of weight reduction from bupropion is minimal in patients with obesity. In a randomized placebo-controlled trial, 24-week administration of SR bupropion 300 and 400 mg/d were associated with mean weight loss of 5.7% and 7.7%, respectively, compared with that of 4.0% in the placebo group. 36)  Values are presented as number or mean±standard deviation. The mean weight loss was calculated for the full analysis set, which comprised all randomized individuals exposed to trial drug with at least one post-randomization weight assessment. BMI, body mass index; FE, fast (2-weekly) dose escalation. *The participants were patients with obesity who lost ≥5% of their initial body weight during a variable-length (4-12 weeks) low-energy diet run-in period. † The participants were patients with obesity and diabetes (BMI ≥27 kg/m 2 with hemoglobin A1c 7.9%−10.0%).
Naltrexone, an opioid receptor antagonist used in the treatment of opioid or alcohol dependence, resulted in the dose-related suppression of eating and drinking in rats. 37) Consequently, naltrexone was administered to patients with obesity in several clinical trials in the 1980s to induce weight loss in humans, but the results were unsuccessful. 38,39) Although the weight loss effects of bupropion and naltrexone in humans were not satisfactory, in vivo electrophysiology studies by Greenway et al. 40) demonstrated that bupropion stimulated POMC neurons and the combination of bupropion and naltrexone showed greater effects on POMC neurons. Animal studies, preclinical, and clinical trials of NB showed that this combination had a greater weight loss effect compared with previous results for the monotherapy of each drug. [40][41][42][43][44] In conclusion, the mechanism of action of NB is the stimulation of POMC neurons with bupropion, with this stimulation augmented by blocking of the autoinhibitory mechanism of POMC by nal trexone (Figure 1). The weight loss results of clinical trials of NB are summarized in Table 2. [41][42][43][44]

GLP-1 RECEPTOR AGONISTS FOR THE TREATMENT OF OBESITY: LIRAGLUTIDE AND SEMAGLUTIDE
Presently, six GLP-1 RAs are used worldwide for the treatment of and long-acting (albiglutide, dulaglutide, exenatide long-acting release, liraglutide, and semaglutide). The main mechanism of the glucose-lowering effect is somewhat different between the two groups.
The short-acting GLP-1 RAs inhibit gastric emptying and mainly lower postprandial glucose levels, whereas the long-acting drugs stimulate postprandial insulin secretion and suppress fasting glucagon secretion, which can affect both fasting and postprandial blood glucose levels. 45,46) Endogenous GLP-1, liraglutide, and lixisenatide can cross the BBB and reach the hypothalamus. 19,47,48) However, there is no evidence that other GLP-1 RAs cross the BBB. The molecular size and structure of albiglutide and dulaglutide are completely different from endogenous GLP-1; therefore, it is difficult for them to cross BBB and act on the hypothalamic neurons through the humoral pathway.
Of the six GLP-1 RAs, only liraglutide and semaglutide were researched for long-term weight management; liraglutide is currently available in Korea. There have been several large phase 2 and 3 clinical trials for these drugs in the treatment of obesity and the participants and mean weight loss results are summarized in Table 3. [28][29][30][31][32] As shown in the clinical trials, the weight reduction effect of liraglutide was modest (mean weight loss from -5.7% to -8.0%) and that of semaglutide

CARDIOVASCULAR SAFETY OF ANTI-OBESITY DRUGS
Diabetes is diagnosed from an elevated blood glucose level and nonenzymatic glycation products; thus, the tight control of blood glucose should be the core of the prevention of cardiovascular disease, which is the most common cause of mortality, disability, and financial burden. However, intensive glycemic control was shown to fail in the reduction of macrovascular episodes, even though it reduced microvascular events in well-known large clinical observational or interventional studies. 57) It is certain that obesity increases cardiovascular risk and that weight reduction can ameliorate risk. However, similar to the glycemic control achieved with anti-diabetic drugs, there is little evidence on whether the weight reduction induced by anti-obesity drugs can decrease or postpone cardiovascular events in patients with obesity. Conversely, several anti-obesity drugs, including fenfluramine and sibutramine, were banned from market owing to their deleterious effects on the cardiovascular system. 2) Based on these historical events, newly developed anti-obesity drugs should have proven safety and/or efficacy on cardiovascular events in addition to its weight loss effect.
In the past several years, three clinical trials of the cardiovascular safety of anti-obesity drugs have been performed: the CAMELLIA-TIMI 61 58) trial of lorcaserin, the study by Nissen et al. 59) of NB, and the LEADER trial of liraglutide in patients with T2DM. 60) The results of the trials are summarized in   Values are presented as number (%), median (interquartile range), or mean±standard deviation, unless otherwise stated. HR, hazard ratio; NB, naltrexone 32 mg+bupropion 360 mg; AE, adverse event; MACE, major cardiovascular events (a composite of cardiovascular death, myocardial infarction, and stroke); NA, not applicable; CI, confidence interval. *The participants were people with obesity and pre-existing cardiovascular disease or with an increased cardiovascular risk. † The participants were patients with type 2 diabetes and high cardiovascular risk. ‡ Glycated hemoglobin level in patients with diabetes, median (interquartile range). § 99.7% CI.


extfieldsofstudy: Biology Medicine

text:
Fungal infection strategies

Fungi are increasingly recognized as the cause of not only superficial infections, which affect many people but are relatively easy to treat, but also invasive and disseminated infections. Difficulties in diagnosing fungal infections and the limited number of antifungal drugs available contribute to the high mortality rates observed in these infections. Furthermore, severe fungal infections mainly occur in immunosuppressed individuals, for example, patients undergoing solid organ or stem cell transplantations, a group that has been increasing with the advancements of modern medicine resulting in increased numbers of severe fungal infections [1]. Immunosuppression as a risk factor highlights the important role of the immune system in controlling opportunistic fungal pathogens; it also suggests supporting the host immune functions or targeting interactions between the host immune system and fungi as alternative therapeutic strategies that could be combined with antifungal treatment [2]. For such approaches, however, a thorough understanding of host–pathogen interactions is necessary. Human fungal pathogens are phylogenetically very diverse and belong to different phyla within the kingdom. The majority of severe infections, for example, are caused bymembers of the generaCandida andAspergillus, belonging to the Ascomycota, and Cryptococcus, a basidiomycete. More rare, but very severe, are infections caused by various species belonging to the phylum Mucoromycotina. This suggests that pathogenicity evolved independently across the fungal kingdom several times and raises two main questions: (i) in which context did pathogenicity evolve, and (ii) are there common and specific virulence traits in phylogenetically diverse fungal pathogens [3]. A common feature of dermatophytes is the ability to utilize keratin as a nutrient source. Keratins as structural proteins of hair, nails, and epidermal layers of the skin are found not only on animal and human hosts but also in the environment, as keratinous structures are constantly shed as part of the bodies’ renewal process. Dermatophyte species are classified as geophilic, zoophilic and anthropophilic, depending on their preferred niche; interestingly, clinical infection tends to be more severe, driven by both fungus-driven host tissue destruction and overt inflammation, if dermatophytes colonize a human or animal theyare not adapted to [4]. An example is the occurrence of certain zoophilic dermatophytes, such as Microsporium canis and Trichophytum benhamiae, on asymptomatic animal hosts, while the same strains cause inflammatory reactions in humans [5]. This suggests that specific adaptation of these fungi to natural host species is resulting in commensal behaviour while in untypical hosts the immune response and possibly altered fungal behaviour are driving inflammation resulting in clinical disease. Although dermatophyte infections are the most common superficial fungal infection in animals and humans, genetic features of dermatophytes and factors that drive pathogenesis are, however, not well understood yet, and the mechanisms underlying host adaptation remain to be elucidated. One explanation why deeply invasive or disseminated infections by dermatophytes are very rare is their limited tolerance to body core temperature, usually around 37.5°C in mammals. In contrast, other saprophytic environmental fungi, such as some Aspergillus species and several mucormycetes, are temperature-tolerant. This is likely the result of adaptation of the elevated temperatures found in decaying organic matter, such as compost heaps, which also represents the natural habitat of these fungi. Many Aspergillus and mucormycete species are found ubiquitously in the environment and have a versatile metabolism that allows them to thrive on various types of organic material. This Special Focus issue includes the paper of Lackner et al.[6] who investigated the correlation of temperature tolerance, resistance to oxidative stress, and growth rates of cryptic Aspergillus species of section Terrei for virulence in a wax moth larvae model.

Fungi are increasingly recognized as the cause of not only superficial infections, which affect many people but are relatively easy to treat, but also invasive and disseminated infections. Difficulties in diagnosing fungal infections and the limited number of antifungal drugs available contribute to the high mortality rates observed in these infections. Furthermore, severe fungal infections mainly occur in immunosuppressed individuals, for example, patients undergoing solid organ or stem cell transplantations, a group that has been increasing with the advancements of modern medicine resulting in increased numbers of severe fungal infections [1]. Immunosuppression as a risk factor highlights the important role of the immune system in controlling opportunistic fungal pathogens; it also suggests supporting the host immune functions or targeting interactions between the host immune system and fungi as alternative therapeutic strategies that could be combined with antifungal treatment [2]. For such approaches, however, a thorough understanding of host-pathogen interactions is necessary.
Human fungal pathogens are phylogenetically very diverse and belong to different phyla within the kingdom. The majority of severe infections, for example, are caused by members of the genera Candida and Aspergillus, belonging to the Ascomycota, and Cryptococcus, a basidiomycete. More rare, but very severe, are infections caused by various species belonging to the phylum Mucoromycotina. This suggests that pathogenicity evolved independently across the fungal kingdom several times and raises two main questions: (i) in which context did pathogenicity evolve, and (ii) are there common and specific virulence traits in phylogenetically diverse fungal pathogens [3].
A common feature of dermatophytes is the ability to utilize keratin as a nutrient source. Keratins as structural proteins of hair, nails, and epidermal layers of the skin are found not only on animal and human hosts but also in the environment, as keratinous structures are constantly shed as part of the bodies' renewal process. Dermatophyte species are classified as geophilic, zoophilic and anthropophilic, depending on their preferred niche; interestingly, clinical infection tends to be more severe, driven by both fungus-driven host tissue destruction and overt inflammation, if dermatophytes colonize a human or animal theyare not adapted to [4]. An example is the occurrence of certain zoophilic dermatophytes, such as Microsporium canis and Trichophytum benhamiae, on asymptomatic animal hosts, while the same strains cause inflammatory reactions in humans [5]. This suggests that specific adaptation of these fungi to natural host species is resulting in commensal behaviour while in untypical hosts the immune response and possibly altered fungal behaviour are driving inflammation resulting in clinical disease. Although dermatophyte infections are the most common superficial fungal infection in animals and humans, genetic features of dermatophytes and factors that drive pathogenesis are, however, not well understood yet, and the mechanisms underlying host adaptation remain to be elucidated.
One explanation why deeply invasive or disseminated infections by dermatophytes are very rare is their limited tolerance to body core temperature, usually around 37.5°C in mammals. In contrast, other saprophytic environmental fungi, such as some Aspergillus species and several mucormycetes, are temperature-tolerant. This is likely the result of adaptation of the elevated temperatures found in decaying organic matter, such as compost heaps, which also represents the natural habitat of these fungi. Many Aspergillus and mucormycete species are found ubiquitously in the environment and have a versatile metabolism that allows them to thrive on various types of organic material. This Special Focus issue includes the paper of Lackner et al. [6] who investigated the correlation of temperature tolerance, resistance to oxidative stress, and growth rates of cryptic Aspergillus species of section Terrei for virulence in a wax moth larvae model.
Using 73 isolates of six clinically relevant species they show that different strains of all species were well adapted to growth in this insect host and that virulence mainly correlated with growth rates at 37°C. Their data furthermore suggest that in this group of fungi virulence is a strainrather than a species-specific feature.
In the environment, fungi however not only have to compete with other fungi, bacteria, and protozoa for nutrients but are also facing predators such as nematodes and amoeba. In their ability to take up and digest microorganisms, amoebas resemble mammalian phagocytes, and some of the mechanisms mediating prey recognition and killing by amoeba are also found in phagocytes. It thus appears likely that counterstrategies evolved in fungi in response to amoeba also enable them to prevent recognition or killing by mammalian phagocytes, and thus promote virulence, a concept also called "amoeboid predator-fungal animal virulence hypothesis" [7].
Not surprisingly, the fungal cell surface plays a key role in recognition by amoeba and phagocytes alike; on Aspergillus fumigatus spores, specific proteins (rodlets) and pigmentation convey protection from environmental stresses such as UV, but also hamper recognition by phagocytes [8,9]. Germination and growth of vegetative hyphae, however, results in the production or exposure of cell wall components such as galactosaminogalactan (GAG) and β-1,3-glucan [10]. In this special focus issue, Speth et al. review the impact of GAG on Aspergillus pathogenesis [11]. GAG is a heteropolysaccharide secreted by actively growing hyphae and binding to the surface of these hyphae. It mediates interaction with host factors, such as surfactant D, shields the underlying β-1,3 glucan from recognition by immune cells, interferes with neutrophil activity against A. fumigatus, and modulates cytokine and cell responses. Overall, GAG mediates immunosuppressive effects, which has led to an interest in the use of soluble GAG in the treatment of inflammatory diseases [11]. In contrast to GAG, β-1,3 glucan is a well-known ligand of Dectin-1, a C-type lectin pattern recognition receptor (PRR) known to be involved in antifungal immunity [12]. While the role of PRRs in antifungal defense has been extensively studied, the role of opsonization by complement is less well understood. This topic has been addressed by Steger et al. in work included in this Special Focus issue [13]. They demonstrate that complement deposition enhances recognition and phagocytosis of A. fumigatus conidia by dendritic cells. Not only melanin, the pigment contributing to immune evasion of A. fumigatus spores, interferes with complement deposition but also β-1,3 glucan, highlighting the complexity of interactions of the immune system with the fungal cell wall [13].
Pigmentation is also an important feature of Cryptococcus neoformans and Cryptococcus gattii, dimorphic basidiomycetes that grow as filaments in the environment but switch to yeast growth in warmblooded hosts. Like Aspergillus and molds, Cryptococcus likely acquired its virulence traits in the environment as a mean to withstand adverse environmental conditions and predators [14]. It has not only become the fungal pathogen causing the highest number of severe human infections as a result of the HIV pandemic but is also known to infect a wide range of animals. In a review in this Special Focus issue, Oscar Zaragoza summarizes the principles of Cryptococcus virulence which, in addition to general adaptation to the host environment and melanin production, includes the production of a capsule and specific types of polysaccharides [15]. Another interesting feature of Cryptococcus is the formation of giant yeast cells, termed Titan cells that due to their size cannot be phagocytosed, and are more resistant to stressors [16]. Further examples presented in this Special Focus issue of opportunistic environmental fungal pathogens which produce pigments are Exophiala dermatitidis, reviewed by Kirchhoff et al. [17], and Coccidioides. E. dermatitidis is a black yeast, a term originating from the dark pigmentation of this group of fungi. It can cause phaeohyphomycosis in immunosuppressed patients, but also fatal infections in healthy patients. It is also regularly isolated from cystic fibrosis patients, but its role in disease in this patient group is unclear. Besides pigmentation, the secretion of extracellular polysaccharides (acid mucopolysaccharides), adherence and biofilm formation, and the ability to form invasive filaments contribute to virulence of this fungus [17]. Interestingly, E. dermatitidis is most commonly isolated from indoor man-made habitats, and although it is considered a ubiquitous fungus, isolation from the environment is rare. Its main environmental habitat is assumed to be tropical rainforests, but little is known about the natural habitat and the transmission routes. One can thus only speculate on the context in which the virulence traits of E. dermatitidis evolved, but it is likely the environmental niche, rather than an occasional association with humans. This is also the case for Coccidioides immitis and C. posadasii, the causative agents of coccidioidomycosis, a disease commonly known as valley fever [18].
Kollath et al. review our current understanding of both the disease and the pathogens' biology which remains largely enigmatic to date [19]. Coccidioides infects immunocompetent mammals including humans, and although infection is commonly asymptomatic or results only in mild, self-limiting pneumonia, disseminated infections occur that require medical intervention.
Like Cryptococcus, Coccidioides is dimorphic with filamentous growth in the environment while in the host spherules that produce endospores are formed. C. immitis and C. posadasii are endemic to areas with arid to semi-arid alkaline thermic soils throughout western North America and into Central and South America, and it appears that sufficiently high soil temperatures in combination with low water-holding capacity are required for these fungi. Although Coccidioides has been thought to be a saprotrophic soil-dwelling fungi, the exact conditions required for growth and maturation in the environment are unknown [19]. Recent evidence suggests that its natural life requires close association with small desert mammals such as rodents, and utilization of animal-derived substrates. Coccidioides was isolated from deer mice, pocket mice, ground squirrels, grasshopper mice, kangaroo rats, and pack rats; it thus appears possible that virulence in these fungi not only evolved in the environment as a consequence of encounters with predators like amoeba, but as a result of adaptation to rodents, with the close proximity facilitating infections [13].
From the examples described above, it appears that pigmentation is a common feature of environmental opportunistic fungal pathogens, mediating both protection in the environment and immune evasion in the host. Human pathogenic fungi of the genus Candida, however, do not produce any pigments. Furthermore, while some Candida species are found in the environment, such as C. tropicalis and C. krusei, others like C. glabrata and C. albicans have been isolated only or predominantly from skin or mucosal surfaces of warm-blooded animals, including humans. It thus appears that these fungi are evolutionarily adapted to a commensal lifestyle [20], albeit this is challenged by recent whole-genome analyses [21]. Adaptation to a host however clearly occurred in Pneumocystis, a group of fungi that are extremely difficult to grow outside a warm-blooded host and have evolved to host-specific species [22]. Humans are probably infected with Pneumocystis early in life, but clinical infections usually occur only if the host is severely immunocompromised [23].
Virulence of C. albicans is associated not only with metabolic adaptation and resistance to potential stressors in the host [24,25] but also the ability to switch between yeast and invasive filamentous growth forms associated with production of the peptide toxin Candidalysin [26,27]. The "classical" fitness properties and virulence factors of Candida have been reviewed extensively elsewhere [26,28,29]; in this Special Focus, we included a review by Chakraborty et al. on a topic less studied: Eicosanoid production by pathogenic yeasts [30]. Eicosanoids are bioactive lipid mediators generated in many mammalian cells from the oxidation of arachidonic acid and regulate various physiological functions, including cellular homoeostasis and modulation of immune responses. Various human pathogenic fungi are also able to produce eicosanoids, to some extent even the very same molecules as their mammalian hosts [30]. There is some evidence that fungi-derived lipid mediators affect the host immune response and thereby pathogenesis, albeit the exact function is yet to be unravelled. As eicosanoids are not only found in fungi associated with mammals but also environmental fungi and plant pathogens, it is likely that they did not primarily evolve as virulence traits but rather serve yet to be discovered functions in fungi themselves.
For Candida species occurring as commensals, it appears essential that virulence factors are regulated as damage associated with virulence factors might trigger the immune system and subsequent elimination of the fungus [31]. In plant pathogenic fungi factors have been identified that trigger a hypersensitive immune response in the infected plant, which promotes plant resistance and renders the pathogen avirulent [32]. Such factors have been termed "antivirulence factors" and the respective genes "avirulence genes" [32,33]. In the final paper of this Special Focus, Siskar-Lewin et al. discuss how this concept could be transferred to human pathogenic fungi and extend our understanding of host-fungal interaction [34].

Disclosure statement
No potential conflict of interest was reported by the author.


PREVALENCE OF INTESTINAL PROTOZOA AND HELMINTHIC PA RASITIC INFECTIONS IN ADULT POPULATION OF PUDUCHERRY

INFECTIONS IN ADULT POPULATION OF PUDUCHERRY NAMRATA K. BHOSALE , MUNUSWAMY SANGEETHA 2 & JOSHY M. EASOW 3 Assistant Professor, Department of Microbiology, Ma hatma Gandhi Medical College & Research Institute,  Sri Balaji Vidyapeeth (Deemed-to-be-University), Po ndicherry, India MD Microbiology, Mahatma Gandhi Medical College & R esearch Institute, Sri Balaji Vidyapeeth (Deemed-to-be-University), Po ndicherry, India 3 Professor & Head, Microbiology, Mahatma Gandhi Medi cal College & Research Institute, Sri Balaji Vidyapeeth (Deemed-to-be-University), Po ndicherry, India ABSTRACT

INTRODUCTION
In India, one third of the populations were mostly affected by the intestinal parasites viz., Entamoeba histolytica, Entamoeba dispar, Ascaris, Trichuris trichiura, and Hook worm (Kang et al. 1988:6). Increasing burden of helminthic infection in India due to the unhygienic personnel, overcrowding and water scarcity. Infection may pose major health problem among humans and induces severity of the disease ( Bartelt et al., 2013:1). In the late 19 th century, the true prevalence of pathogenic intestinal parasites was recorded among South Indian population (Kang et al. 1998:6). Prevalence of intestinal parasitic infections in different states which ranges from 5.56% to 90%and most of them were filed high in rural areas than the urban (Mathuria et al., 2017:4;Mareeswaran et al., 2018:5). Parasitic infection is almost exists in every states of India in which it has been documented by several studies (Rituparna, et al. 2017 Helminths and Protozoal parasites such as Ascaris lumbricoides, hookworm, Enterobius vermicularis, Entamoeba histolytica and Giardia lamblia are commonly found in the intestines of humans, which yielded high morbidity mostly in child population (Kaur et al. 2002:5). Our main objective of the study is to determine the prevalence of intestinal parasites among clinically suspected patients who are attending our tertiary care teaching hospital.

MATERIALS & METHODS
This prospective and laboratory investigative study was carried out in the Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Puducherry. This study was conducted during the period of January 2018-January 2019. Totally 571 stool samples were collected from the patients who had suspicion of parasitic infections and they sent to the Parasitology laboratory for routine diagnostic testing like saline and iodine wet mount followed by formal ether concentration technique. Patients who are attending our hospital are from rural and urban areas.
Inclusion Criteria: patients with diarrhoea and other gastrointestinal tract infections like nausea, vomiting etc.
Exclusion Criteria: Improperly labelled samples, Samples with incomplete requisition forms and previously treated with antihelminthic drugs.

Processing of Stool Samples
Patients was educated to collect the samples in clean, wide, leak proof labelled containers provided by the laboratory with pinch of stool sample and asked the patients to avoid to full fill the containers till the rim. After collecting the samples, they were screened with Microscopic examination of Lugol'siodine and saline wet mounts for the presence of ova, larvae and cysts of parasites. Formal-Ether concentration technique will be performed for cases which had high clinical suspicion but were negative by the above mentioned tests. Modified acid fast staining will then be performed for all the samples for detection of intestinal coccidian parasites on methanol fixed stool smears.

STATISTICAL ANALYSIS
Percentages were calculated for Categorical variables. Mean and Standard deviation with 95% confidence interval was calculated for the patients' age. The fisher's exact test will be used to determine the statistical significance between rural and urban patients who had positive for intestinal parasitic infections.    (Saurabh K, et al. 2017:4), Chandigarh (Sehgal R, et al. 2010:4), Bihar (Rituparna B, et al. 2017:5) and Puducherry (Manochitra K, et al. 2016:8). But low prevalent in Uttarkhand (Kotian S, et al. 2014:4) and New

Mean
Delhi (Kaur R, et al. 2002:5). But in the contrast, the present study shows hookworm infections are more prevalent in the region of Puducherry and neighbouring Tamil Nadu state. Primarily an retrospective study was carried out in Puducherry with overall positivity is about 22.21% (Manochitra K, et al. 2016:8)but in our study projected a low prevalence 7.70% when compared to the above study. Coccidian parasites are absent in our stool samples. According to Kotian et al., G. lamblia and Hymenolepis nana were the most common parasites found in the targeted population. There was increase of intestinal parasitic infections among rural than urban population in two health centres of Anakaputhur and Padappai of Kancheepuram district (Mareeswaran et al., 2018:5). But in contrast, the present study shows that urban patients were mostly affected than the rural patients.

CONCLUSIONS
Microscopy is highly helpful and cost effective for diagnosing intestinal parasitic infections from stool samples.
The study which mainly focuses on adult population of Puducherry and yielded that the urban populations are mostly infected with intestinal parasites. This may be due to improper hygienic conditions and large number of populations in a


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Anti-Inflammatory and Immunomodulatory Effects of Barberry (Berberis vulgaris) and Its Main Compounds

Berberis vulgaris is a well-known herb in Iran that is widely used as a medicinal plant and a food additive. The aim of this study was to investigate the anti-inflammatory and immunomodulatory effects of Barberry and its main compounds. This narrative review was conducted by searching keywords such as B. vulgaris, Barberry, immunomodulatory, anti-inflammatory, medicinal herbs, plants, and extract, separately or combined in various databases, such as Web of Sciences, PubMed, and Scopus. According to the inclusion and exclusion criteria, just English language articles, which reported effective whole plants or herbal compounds, were included. 21 articles were reviewed in this study. In the in vivo models (mice, rats, and human cells) and in the in vitro models (some organ cells such as the spleen, kidney, blood, and brain), B. vulgaris and its main components showed anti-inflammatory effects in both models. The main mechanisms were the shift of cell immune response to Th2, T reg induction, inhibition of inflammatory cytokines (IL-1, TNF, and IFN-γ), and stimulation of IL-4 and IL-10. The induction of apoptosis in APCs and other effector cells was another important mechanism.

Introduction
Medicinal plants can change the body's physiological or pathological mechanisms to prevent or treat diseases. These plants have been used widely from ancient ages for medical purposes.
Nowadays, easy access, low cost, and the prevailing belief in the low side effects have led to a dramatic increase in the use of these plants compared to chemical medications [1,2]. In Iran, the use of medicinal plants is also significantly increased [3,4]. Medicinal plants can be used for a variety of purposes, including gastroenterology [5], headache and migraine [6], reproductive disorders [7], diabetes [8,9], hyperlipidemia [10], stress and depression [11,12], liver disorders [13,14], and nervous system disorders [15]. Of course, the use of these drugs also has disadvantages, including deficiencies and lack of experiments for purity, health effects, or teratogenicity [16].
Berberis vulgaris is a common herb in Iran (called Barberry) and other countries that widely used it as a medicinal plant and its fruit as a food additive. The scientific classification of B. vulgaris is given in Table 1 [17]. This shrub plant with a height of 1 to 3 meters is barbed with yellow wood and ovoid leaves and has hanging yellow flowers that ultimately turn to pulled reddish fruits with a length of 7 to 10 mm and a width of 3 to 5 mm. These fruits come ripening in the end of summer and autumn and are edible. They also have a sour taste and are rich in vitamin C [18][19][20]. This plant is found in regions such as north of America and central and southern Europe, and south of Asia [21,22]. The various parts of this plant, including stems, roots, fruits and leaveshave been used in Iran and other countries as traditional medicine [23]. In traditional medicine, this plant is used in several purposes, including cleansing of renal stones and urinary tract diseases and gastrointestinal, liver, and gallbladder diseases as well as a circulatory system stimuli [24][25][26]. Some traditional uses of Barberry are listed in Table 2.
About 22 alkaloid compounds have been identified in the roots, the leaves, and the fruits of Barberry [27]. Studies about the chemical components of the extract of this plant show that alkaloids with an isoquinoline core such as protoberberine, berberamine, tetrandrine, chondocurine, and palmatine are among the important contents of Barberry. In the quantitative HPLC (high-performance liquid chromatography) analysis of the principal alkaloids of the root, shell, and stem of B. vulgaris, berberine and barbramine were reported 1.24% and 2.5%, respectively. The anti-inflammatory and immunosuppressive properties of these compounds are very important [28][29][30][31]. The fruits, flowers, and seeds of this plant contain significant amounts of phenolic compounds (including anthocyanin and carotenoid pigments), pectin, oleoresin, vitamin C, and organic acids such as chelidonic acid, resin, and tannin [18,20,32,33]. The fruits of this plant also have a sour taste and contain dextrose, malic acid, tartaric acid, and citric acid. Barberry extract contains flavonoids such as quercetin, chrysanthamine, hyperoside, dolphinidin-3-O-beta-D-glucoside, pelargonin, petunidin-3-O-beta-D-glucoside, alpha tocopherol, and beta-carotene ( Figure 1) [34,35]. Some of the important compounds found in Barberry are listed in Table 3. For Barberry, many properties are mentioned. In various studies, some pharmacological effects, such as antioxidant and cytoprotective properties [36], inhibition of vascular permeability [37] and epidermal growth factor (EGF) [38], and anticholinergic and antihistaminergic properties [18], for anthocyanins and Barberry fruits are mentioned. The antioxidant activity of Barberry is such that it reduces the survival of cancer cells, and this property is probably due to phenolic compounds and flavonols in the Barberry plant [39,40].
Studies have shown that alkaloids in this plant increase immunity through T cells [41,42]. Berberine in the root of Barberry has anticonvulsant, sedative, and diuretic effects [43]. Also, the berberine in Barberry shows the elimination activity of the ONOO(-) and NOO(2)(-) radicals that can contribute to oxidative damage reduction [44]. The extract of this plant is effective in inhibiting activating protein 1 (AP1) of human hepatoma cells [45]. However, the consumption of high levels of alkaloid in this plant (berberine) can cause respiratory paralysis, but death from the high consumption of this plant has not been reported so far. Barberry, by controlling the ACE enzyme (angiotensin converting enzyme) in the brain or by antioxidant property, can reduce the symptoms of Parkinson's disease. Barberry extract inhibits monoamine oxidase A (MAO-A), thus increasing the level of monoamines such as epinephrine and dopamine in the brain, which has an antidepressant effect [28,43]. Some of the important compounds of this plant are mentioned in Table 3.
Since access to and use of medicinal plants are very easy today, extensive studies on these compounds are important in order to understand their benefits and side effects and can be used to better understanding and explanation of the status of these medications in the community's health. The aim of this study was to investigate the anti-inflammatory and immunomodulatory effects of Barberry and its main compounds.

Methodology
This narrative review studied the main research performed on anti-inflammatory and immunomodulatory effects of B. vulgaris. Keywords such as B. vulgaris, Barberry, immunomodulatory, anti-inflammatory, medicinal herbs, plants, and extract were searched separately or combined in various databases, such as Web of Sciences, PubMed, and Scopus.
According to the inclusion and exclusion criteria, just English language articles, which reported effective whole plants or herbal compounds that performed standard laboratory tests, were included. Unrelated articles that studied other plants were excluded. The titles and abstracts of the studies were evaluated independently by two authors according to the inclusion and exclusion criteria. Data extracted from various articles were included in the study and entered into a check list that included some information: Publication year, authors' name, model type, concentration or dose of extract, and mechanisms of action (if reported).

Results
21 articles were reviewed in this study. Details of the studies are presented in Table 4. In the following, we will refer to the most important observations of these studies: In the study of W-C. Lin and J-Y. Lin, anti-inflammatory effects of berberine were investigated in nonobese diabetic (NOD) rats with spontaneously developed type 1 diabetes. The results showed that type 1 diabetes alone causes inflammation in some visceral organs. The administration of berberine reduced spontaneous thymus and spleen inflammation in mice. The secretion of IL-6 by splenocytes in the presence of Con A in NOD mice was significantly higher than that of ICR mice. Berberine administration (especially at high doses of 500 mg/kg BW; 20 g mouse/day) reduced ratios of the Th1 (IL-2)/Th2 (IL-4) cytokines expression in NOD mice splenocytes in the absence/presence of LPS in a preventive way [46]. In the study of Lee et al., the pulmonary inflammation process was simulated by stimulating pulmonary factors. Increased levels of inflammatory cytokines such as IL-1b and TNF-α are associated with pulmonary inflammation. This study showed that different inflammatory factors such as lipopolysaccharide, 12-o-tetradecanoylphorbol-13-acetate, hydrogen peroxide, okadaic acid, and ceramide induced the production of IL-1b and TNF-α in human pulmonary epithelial cells (A-549), fibroblast (HFL1), and lymphoma cells. However, the result showed that Berberine suppressed inflammatory factors produced due to the production of TNF-α and IL-1b in human pulmonary cells [47]. In the study of W-C. Lin and J-Y. Lin, the effects of berberine on the expression of the inflammatory cytokines gene in the initial splenocyte of mice, in the presence and absence of lipopolysaccharide (LPS), were investigated in four experimental models under laboratory conditions. In the initial splenocyte of mice, in the absence and presence of LPS, the anti-inflammatory potential of berberine was reported. A study conducted by Marinova et al. on tubulointerstitial nephritis (TIN) model rats showed that the severity of pathologic damage in TIN rats treated by berberine was significantly lower than that of the control group. In these rats, berberine reduced the number of TCD8+ and TCD4+ lymphocytes. The number of these cells in the peripheral blood is reduced by treatment with berberine and also reduces the infiltrated cells in the kidney [48].
Yan et al. investigated berberine effects on intestinal injury induced by DSS (dextran sulfate sodium) as well as on rat colitis and showed that berberine improved weight loss in rats induced by DSS, as well as the activity of myeloperoxidase, inflammatory scars, and colon injury. In addition, berberine inhibited the production of proinflammatory cytokines such as TNF-α, IFN-γ, and IL-17 in colon macrophages and epithelial cells in DSS mice [49]. A study by Jiang et al. was conducted to evaluate the protective effects of berberine on neural cells of the brain parenchyma in EAE mice, and the results showed that berberine can reduce MMP9 activity in the brain of EAE mice and, by reducing the expression of MMP9, causes reducing laminin decomposition and thereby preserving brain blood barrier (BBB) that results in a decrease in the number of inflammatory cells infiltrated in the brain and a reduction in the expression of cytokines and chemokines in the brain [50]. In another study, Hu et al. showed that treatment with berberine suppressed the pathological changes caused by the CIA. In the Aziz study,     was found that NO levels in the treated B. vulgaris cells were 5% lower than the control group (no treatment was applied in this group). In the study of Hu et al., berberine treatment improved EAN symptoms in affected rats [51].

Discussion
In this study, 21 articles were reviewed. In the in vivo models, mice, rat, and human cells, and in the in vitro models, some organ cells such as the spleen, kidney, blood, and brain were used. In this section, we mention the most important mechanisms of the effect of B. vulgaris and its important compounds, including berberine.
In the study of W-C Lin and J-Y Lin, the antiinflammatory effects of berberine were attributed to a reduction in TNF-α, IL-6, and IL-1b cytokines. The use of berberine in NOD mice was positively correlated with LPS-stimulated IL-10/IL-1b and Con A-stimulated IL-10/TNF-α. This effect was particularly higher at high doses (500 mg/kg BW; 20 g mouse/day). In the confirmation of this finding, Lee et al. showed that berberine reduces the inflammatory factors induced by the production of TNF-α and IL-1b in human pulmonary cells. Berberine inhibited this mechanism by inhibiting Ik-Ba phosphorylation. Berberine also inhibited the expression of cyclooxygenase 2 (COX2) by the regulation of AP-1 [47]. In the study of W-C Lin and J-Y Lin, change in the expression of Th1 (TNF-α) and Th2 (IL-4) cytokines and the shift of Th1/Th2 to Th2 and further increase in the expression of IL-10 and IL-4 were known as the effective processes in the immunomodulatory activity of berberine [46]. Yan et al. showed that berberine improved myeloperoxidase activity, inflammatory scars, and colon damage. In addition, berberine inhibited the production of proinflammatory cytokines such as TNF-α, IFN-γ, and IL-17 in colon macrophages and epithelial cells in DSS-treated mice [49]. In the study of Ren et al., berbamine altered the response of autoreactive T cells by two ways: on one hand, by inhibiting the nuclear factor of activated T cells (NFAT), it reduced the T cells proliferation; on the other hand, it changed the cytochrome profile of encephalitogenic T cells. By selective inhibition of IFN-γ through the Jak/stat pathway and by increasing the SLIM, which is ubiquitin E3 ligase for STAT4, it boosts STAT4 protease degradation and reduces IFN-γ production [58]. In study Cui et al., it was found that berberine reduced the differentiation of Th1 and Th17 cells by reducing the expression of lineage markers. The berberine inhibits the differentiation to Th17 by activating ERK1/2. ERK plays this role by decreasing the STAT3 phosphorylation and expressing RORγt. Berberine also inhibits the differentiation of Th1 by inhibiting the activity of p38 MAPK and JNK. Berberine reduces STAT1 and STAT4 activities by suppressing p38 MAPK and JNK activity and controls the stability of STAT4 through the ubiquitin proteasome pathway [42].
However, the response shift mechanism of Th2 in the study of Marinova et al. was rejected because in their study berberine reduced the number of TCD8+ and TCD4+ lymphocytes in mice, which showed suppression of both Th1 and Th2 cells. The suppression of Th1 response in this study was confirmed by inhibiting the delayed type hypersensitivity in TIN+BB mice and suppression of the Th2 response by decreasing the amount of IL-6 in the studied animals [48].
Another mechanism that is effective in berberine's immunomodulatory effect is the involvement of APCs. By reducing the activity of NF-κB and thus reducing the supply of stimulatory stimulant molecules such as CD80 and CD86 on APCs, berberine reduces antigen delivery performance in these cells. Berberine also suppresses the production of IL-6, IL-12p40, and IL-23p19 in APCs. In confirmation of these findings, Hu et al. showed that berberine can limit the maturity of DCs and reduce their longevity by inducing selective apoptosis. Berberine can lead to oxygen free radical production, activation of caspase-3, and polarization loss in the mitochondrial membrane of DCs, which together result in the apoptosis of these cells [51].
But Jiang et al. presented another mechanism for the anti-inflammatory and immunosuppressive properties of berberine. The purpose of these researchers was to evaluate the protective effects of berberine on neural cells of the brain parenchyma in EAE mice, and they reported that berberine can reduce MMP9 activity in EAE mice, and by decreasing the expression of MMP9, it decreases laminin decomposition and thereby preserves the blood brain barrier, resulting in a reduction in the number of inflammatory cells infiltrated in the brain and reduced expression of inflammatory cytokines in the brain [50].

Conclusion
It seems that B. vulgaris and its most important component berberine play their anti-inflammatory and immunomodulatory effect through the shift of cell immune response to Th2, T reg induction, inhibition of inflammatory cytokines (IL-1, TNF, and IFN-γ), and stimulation of IL-4 and IL-10. The induction of apoptosis in APCs and other effector cells was another important mechanism. Berberine has showed to have several pharmacological properties, including antimicrobial, anticancer, and immunomodulatory activities. However, hypocholesterolemic and hypoglycaemic activities of berberine have come to light recently. Furthermore, berberine seems to have useful effects on the cardiovascular system, owing to its vase-relaxing and hypotensive activities, as well as the potency of prevention of congestive heart failure, cardiac hypertrophy, and arrhythmia. The experimental and clinical evidence available to date in the literature shows interesting employment prospects of berberine in the treatment of hypercholesterolemia and diabetes. This could open the way not only to a new therapeutic possibility effective in the control of hypercholesterolemia in patients who do not tolerate statins but also to new forms of diabetes therapy and all those situations characterized by evident signs of metabolic syndrome, with possible reduction of a cardiovascular risk. In the end, it is recommended to test and apply these effective compounds in the pharmaceutical industry to control both inflammatory and autoimmune and metabolic diseases.

Conflicts of Interest
The authors declare that they have no conflict of interest.