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Definition and Classification of PneumoniaWhen the word pneumonia is used in medical practice, it almost always refers to a syndrome caused by acute infection, usually bacterial, that is characterized by clinical and/or radiographic signs of consolidation of a part or parts of one or both lungs. The use of the term has however been greatly extended to include non-bacterial infection of the lungs caused by a wide variety of microorganisms. Pneumonitis is occasionally used as a synonym for pneumonia, particularly when inflammation of the lung has resulted from a non-infectious cause such as in chemical or radiation injury.For practical purposes the classification of pneumonia should be both anatomical, in which the descriptive terms used communicate the extent and distribution of the process in the lung or lungs, and causal, in which the responsible microorganism is named. When, as is often initially the case, the infectious cause is not known it is useful to consider whether the pneumonia is community-acquired or nosocomial (hospital-acquired). It is also useful to consider whether the pneumonia may have resulted from pharyngeal aspiration and whether or not it is occurring in an immunocompromised host.The anatomical terms used will indicate whether the pneumonia involves one or more entire lobes or whether the process is confined to a segment or segments. In its most confined form, pneumonia may be subsegmental. Such anatomical descriptions are in life entirely dependent upon the chest radiographic appeara nces which show the extent of pneumonia more accurately than can be gauged by physical examination. Early clinicians distinguished between bronchopneumonia and lobar pneumonia in pathological terms. Bronchopneumonia was regarded as a complication of bronchitis in which the inflammatory process was confined to the territory of a small or terminal bronchus and the lung lobule subtended by it, hence the alternative term lobular pneumonia. Lobar pneumonia, on the other hand, frequently occurred de novo and was characterized by an inflammatory out-pouring or exudation of fluid extending throughout most of a lobe or lobes.It is commonplace for the term lobar pneumonia to be used when there is clinical and radiographic evidence of confluent consolidation occupying the greater part of one or more lobes of one or both lungs. The term segmental pneumonia is used when such consolidation is not extensive enough to occupy most of a lobe but corresponds more closely to the anatomy of a bronchopulmonary segment in one or more lobes. Where the area of radiographic shadowing is even more confined, then subsegmental pneumonia is an appropriate descriptive term, although this still implies a confluent and localized process. Where subsegmental shadowing is patchy(non-confluent) and poorly localized, being scattered throughout part or the whole of one or both lungs, the term bronchopneumonia remains entirely acceptable.This anatomical classification is complementary but subservient to causal classification, being of only limited value in establishing the likely infective agent, for although lobar pneumonia is usually caused by Streptococcus pneumoniae it can be caused by many other microorganisms besides, as indeed can all other anatomical types. Every reasonable effort should therefore be made to establish the identity of the pathogenic organism responsible for pneumonia in each patient, in order that specific antimicrobial therapy can be directed against it, for without this information some patients will not recover who otherwise would have done so.The causal organism can only be guessed at when the patient is first seen and it is useful in this respect to classify the patient as having either community-acquired pneumonia or nosocomial pneumonia, as the spectrum of infecting organisms is different in each case. Nosocomial pneumonia is a particular problem in post-operative patients and in those treated in intensive care units, the latter group being highly susceptible to lower respiratory tract infection. The differing types of causal organisms found in the hospital situation are due in part to the altered immunocompetency of patients who are afflicted with other serious disease, in part to the alteration of bacterial flora that results from treatment with antibiotics and in part to the instrumentation or intubation of the upper airways of patients. Such hospital-acquired infections are more frequently due to aerobic gram-negative bacilli and Staphylococcus aureus than are those acquired in the community.。
COVID-19 could cut wildlife trade新冠肺炎肆虐让野生动物背锅Conservation experts are saying that the trade in wildlife could be greatly cut after the coronavirus pandemic has finished. They say that the virus probably started at a market selling wild animals in China. The virus came from either a bat or an animal called a pangolin. It then crossed over to infect humans. The conservation group Humane Society International said: "The consumption of wild animals which can carry diseases that can cross the species barrier poses a real threat to human health." The Wildlife Conservation Society called for a ban of animal markets that trade in wildlife. It said: "Not only will this help to prevent the spread of disease, it will address one of the major drivers of species extinction."A spokesman from the Zoological Society of London said animal markets could be "time bombs". He said the markets can provide perfect conditions for new viruses to start and grow. He added that if we treated animals like goods to buy and sell, we would be in trouble again in the future. Scientists say many new outbreaks of viruses start in animals. About 75 percent of new infectious diseases come from animals. Recent examples include SARS, MERS and Ebola. Infections from animals cause millions of illnesses and deaths worldwide every year. An expertsaid the world must act soon, because, "realistically, it's just a matter of time before the next zoonotic disease risk emerges".材料来源：https:///news/science-environmen生词表conservation （自然环境、文物）保护pandemic 大流行病pangolin 穿山甲consumption 消费species barrier 物种屏障poses a real threat 构成真正的威胁address 解决处理extinction 熄灭Zoological 动物的，动物学的infectious 传染的realistically 现实地zoonotic [医] 动物传染病的emerge 出现，浮现参考译文COVID-19可能会切断野生动物贸易保护专家说，在冠状病毒大流行结束后，野生动物贸易可能会大大减少。
2022考研英语阅读互根除脊髓灰质炎Eradicating polio,Late? Or never?根除脊髓灰质炎,为时已晚?或絕無可能?A plan to wipe out polio by the end of next year is in trouble明年底毁灭脊髓灰质炎的方案倍受挑战HUBRIS is always dangerous. In 1977 smallpox was eradicated andan accidentalinfection in a British laboratory a year later asidethat claim has stood the test of time.傲慢自满通常是危急的行为。
1977年，人类宣称毁灭了天花，这份声明经受了时间的考验。
Having eliminated one viral disease, the authorities decided they ought to be able to get ridof another: polio.在毁灭了一种病毒疾病后，权威专家们认为他们有力量再毁灭另一种病毒：脊髓灰质炎病毒。
That, though, proved a tougher opponent.但实践证明，脊髓灰质炎是个难以应付的对手。
The World Health Organisation s original target, set in 1988a polio-free world by 2000proved illusory.世界卫生组织于1988年定下的原始目标到2000年实现全世界无脊髓灰质炎被证明是天方夜谭。
The latest deadline set by the Global Polio EradicationInitiative , the multinationalbody charged with dealing with the disease, is next year.全球根除脊髓灰质炎行动小组是负责毁灭该疾病的跨国机构,它将毁灭脊髓灰质炎的最终期限定为明年。
抗病英语作文In the realm of modern medicine, the battle against diseasesis relentless and ever-evolving. As an English teacher, Ifind that the power of language can play a significant rolein this fight, not just in communicating scientific findings but also in raising awareness and inspiring action. Here'show we can harness the English language to combat diseases:1. Educational Awareness: Writing compositions on various diseases can educate students about their causes, symptoms, and prevention methods. This not only increases their knowledge but also empowers them to take preventive measures.2. Promoting Vaccination: English essays can be used todiscuss the importance of vaccination in controlling and eradicating diseases. By presenting facts and debunking myths, students can become advocates for vaccination within their communities.3. Highlighting Success Stories: Composing narratives about individuals or communities that have successfully overcome diseases can inspire hope and demonstrate the effectivenessof medical interventions.4. Encouraging Research: English compositions can be aplatform to explore and discuss the latest research indisease prevention and treatment. This can encourage students to consider careers in healthcare or scientific research.5. Cultural Sensitivity: Writing about diseases in different cultural contexts can promote understanding and respect for diverse health practices and beliefs, which is crucial in global health initiatives.6. Fundraising and Philanthropy: Composing persuasive essays can teach students how to effectively advocate for resources and support for disease prevention and treatment programs.7. Global Health Issues: By writing about diseases that are prevalent in different parts of the world, students can learn about the interconnectedness of global health and the importance of international cooperation.8. Personal Narratives: Encouraging students to write personal narratives about their experiences with diseases or those of their loved ones can humanize the statistics and foster empathy.9. Health Policies: English compositions can be used to analyze and critique health policies, teaching students the importance of civic engagement in shaping public health.10. The Role of Technology: Writing about how technology is used in disease detection, treatment, and management can highlight the importance of innovation in healthcare.By integrating these themes into English composition lessons, we not only improve students' language skills but alsocontribute to a more informed and proactive society that is better equipped to combat diseases.。
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SCI长难句呼吸内科第一章—社区获得性肺炎Community-acquired pneumonia is still a significant cause of morbidity and mortality and is often misdiagnosed and inappropriately treated.Although it can be caused by a wide variety of micro-organisms, the pneumococcus,atypicals,Staphylococcus aureus and certain Gram-negative rods are the usual pathogens encountered.Antimicrobial therapy should be started as soon as possible particularly in those requiring admission to hospita.社区获得性肺炎仍然具有很高的发病率和死亡率，且经常被误诊和不恰当地治疗。
虽然它可以由多种微生物引起，但涉及的常见病原体有肺炎球菌、非典型球菌、金黄色葡萄球菌和某些革兰氏阴性杆菌。
特别是（对于）那些需要住院的患者，抗菌治疗应尽快开始。
知识点总结：1pneum(o)-前缀，肺2pneumonia n.肺炎3pneumococcus n.肺炎球菌4atypical adj.非典型的5Staphylococcus aureus n.金黄色葡萄球菌6Gram-negative rods n.革兰氏阴性杆菌7pathogen n.病原体8antimicrobial adj.抗菌的Mandell munity-acquired pneumonia:An overview.Postgrad Med.2015 Aug;127(6):607-15.SCI长难句呼吸内科第二章—肺脓肿A lung abscess is an infectious pulmonary disease characterised by the presence of a pus-filled cavity within the lung parenchyma.The content of an abscess often drains into the airways spontaneously,leading to an air-fluid level visible on chest X-rays and CT scans. Primary lung abscesses occur in patients who are prone to aspiration or in otherwise healthy individuals;secondary lung abscesses typically develop in association with a stenosing lung neoplasm or a systemic disease that compromises immune defences,such as AIDS,or after organ transplantation.肺脓肿是一种感染性肺部疾病，其特征是肺实质内有充满脓的空洞。
2022高中英语精选外刊语法填空31 科学家发现蚂蚁能嗅出癌症比狗更胜一筹Ants have the ability to sniff out cancer in humans, study revealsAnts have the ability 1_____(sniff) out cancerous cells in humans, a new study has discovered, 2____(suggest) they could 3____(use) for cancer diagnosis in future.Researchers from the French National Centre for Scientific Research (CNRS) discovered that ant species Formica fusca (丝光褐林蚁) has a well 4____(develop) sense of smell.It was able to differentiate cancerous cells from healthy cells in humans, thanks 5_____their sense of smell, limited trials revealed. But more clinical tests must be carried out before they could be used in clinical settings like hospitals, theteam said.They suggest that in future, ants could turn out to be better at dogs when 6_____ comes to locating cancerous cells in humans.7_____(conduct) their research, the scientists performed tests with 36 ants, smelling cells under a laboratory setting.First, the specialists exposed the ants to the smell of a sample of cancerous human cells. This odor(气味) was then associated with a reward of sugar 8_____(solve).In a second step, the researchers exposed the ants to two different odors. One was a new smell and the second was the smell of the cancerous cells.Once this test was 9____(success), the researchers exposed the ants to different cancerous cells.As such, the scientists found that 'ants discriminate between cancerous and healthy cells and between two cancerous lines.'After training, Formica fusca ants are able to detect volatile organic compounds emitted by cancerous cells.'This first study shows that ants have high potential, are capable of learning very quickly, at lower cost, and areefficient,' points out CNRS in a news release.This isn't the first time that scientists 10____(use) the animal sense of smell to locate cancerous cells.'Dogs' noses are well suited for medical diagnosis and used for the detection of cancer-specific volatile organic compounds],' the researchers explained.However, training them to do so requires several months to a year.On the other hand, 'insects can be easily reared(养殖) in controlled conditions, they are inexpensive, they have a very well-developed olfactory system and hundreds of individuals can be conditioned with very few trials,' the researchers point out. keys:1 to sniff2 suggesting3 be used4 developed5 to6 it7 To conduct8 solution9 successful 10 have used2 为什么流行歌曲通常都是3分钟左右？Why are so many pop songs roughly three minutes long?No matter where you usually hear today’s top hits—the radio, Spotify(流媒体服务平台), a mixtape on your Sony Walkman—you’ve probably noticed that they tend 1____(be ) around three minutes long.As Vox explains, the custom dates back 2_____ the early 20th century, when shellac(虫蛟的) records first appeared in the market. The rates at 3____ these records spun(旋转) varied, but 78 revolutions per minute (RPM) quickly became the norm. The most popular record sizes were 10 inches—which could hold about three minutes of music per side—and 12 inches, which held 4____(rough) four to five minutes per side. To get radio stations to broadcast their music and get people to buy it, musicians pretty much had to accommodate those time limits.The late 1940s saw the 5____(bear) of the 45 RPM record(45转的唱片): a smaller, cheaper disc 6____(make) of vinyl (乙烯基)that couldn’t manage much more than three minutes of music per side. Long-playing records (LPs) were introduced around the same time, but it was much easier for radio stations to play single tracks from 45s—which hordes of listeners then went out and bought. While 10-inch 78s hadoriginated the three-minute trend, 45s really helped make it a necessity for radio singles throughout the mid-20th century.As music technology evolved （进步）over the years, from records to cassette tapes to CDs, three minutes didn’t fall out of fashion as the de facto estimate for pop songs. According to Classic FM, this is partly 7_____ radio stations could fit more commercials into a program if the songs stayed relatively short. Record labels may have favored shorter singles, too, since longer songs didn’t mean fatter royalty checks. It’s also likely that pop music listeners just preferred concision; after all, it’s 8____the last several decades had taught them 9____(expect). As Thomas Tierney, director of the Sony Music Archives Library, told Mashable, “it’s embedded(根植) in our DNA.”In other 10____(word), success is now less about sales and more about number of streams. Today’s pop stars are making music that reflects the shift—not unlike how 20th-century musicians learned to work within the confines of the original three-minute limit.keys:1 to be2 to3 which4 roughly5 birth6 made7 because8 what9 to expect 10 words3庆祝大熊猫抵美50周年美国动物园纪念活动丰富多彩50 years of giant pandas' presence in US celebratedThe Smithsonian National Zoo in Washington kicked off a six-month celebration on Wednesday of the 50th anniversary of the 1_____(arrive) of two giant pandas, 2____(celebrate) the close cooperation between China and the United States in panda exchanges and preservation.The first pandas from China in the US－Ling Ling and Hsing Hsing－arrived at the Smithsonian National Zoo and Conservation Biology Institute on April 16, 1972."We are going to have six months of celebrations both online and at the zoo. People can come 3____(see) the cubs and the mom and dad," Brandie Smith, director of the national zoo, told China Daily.Annalisa Meyer, the zoo's deputy director of communications, said the celebrations will run through Aug 27.The zoo will hold online events, as well as on-site events on April 8 and 21.Smith said one of the 4_____(highlight) will be a "screening of the film, The Miracle Panda, 5_____ is our story of the giant pandas at the national zoo. And we also are going to have 6_____event with the Chinese embassy. The embassy is going to come and provide treats for people as they see the pandas."The documentary The Miracle Panda, by the Smithsonian Channel, will be screened for a limited time at the zoo's Visitor Center Theater on April 16 and 17.Also on April 16, from 9 am to 2 pm, visitors can enjoy lion dance performances, panda-shaped Bao buns(面点) and calligraphy demonstrations. They also can speak with the zoo's scientists 7____ study giant panda biology and ecology, and they can watch the pandas receive special treats.In 1972, then-US president Richard Nixon and first lady Pat Nixon made a historic visit to China. The first lady went to the Beijing Zoo to visit pandas on 8____(they) second day in China.At a dinner before they left China, Premier Zhou Enlai told Pat Nixon that China 9____(present) the US with two pandas,which surprised and delighted her.The two pandas, Ling Ling and Hsing Hsing, lived at the national zoo for more than 20 years. Their arrival not only brought warmth and joy to tens of millions of Americans but also launched the US-China giant panda program.Now the panda couple, Mei Xiang and Tian Tian, and their cub Xiao Qi Ji ("little miracle"), 10____(bear) in August 2020, live at the national zoo.Mei Xiang and Tian Tian's other three cubs, Tai Shan, born in 2005; Bao Bao, born in 2013; and Bei Bei, born in 2015, were returned to China.keys:1 arrival2 celebrating3 to see4 highlights5 which6 an7 who8 their9 would present 10 born。
英文外刊，抗击疟疾的科学家们，陷入了生物伦理学的争论Scientists at this lab in Burkina Faso have deployed gene warfare against the parasite carrying mosquitoes that spread malaria.布基纳法索一个实验室的科学家已经对传播疟疾同时携带寄生虫的蚊子进行了基因改造。
The conventional tools at our disposal today have reached a ceiling and can't become more efficient than they are right now.我们现在使用的传统工具已经达到了极限，不能比现在的效率更高。
We have no choice but to look at complementary methods.我们别无选择，只能寻找辅助性疗法。
That is why we're using genetically modified mosquitoes.这就是我们对蚊子进行转基因的原因。
Professor Diabate runs the experiment for target malaria, a research consortium backed by the Bill and Melinda Gates Foundation.迪亚巴特教授为目标疟疾组织(比尔和梅琳达.盖茨基金会支持的研究联盟)开展了这项实验。
The group developed an enzyme that sterilizes male mosquitoes.研究小组研发出一种可以使雄蚊绝育的酶，可以使雄蚊绝育。
The action of the enzyme continues after fertilization which means if the male copulates with a female, the embryo is dead and the female can no longer have offspring.这种酶在雌蚊子受精后继续发挥作用，这意味着如果雄蚊子与雌蚊子交配，胚胎就会死亡，雌蚊子就不能再生育后代。
IntroductionImmunocytology, a specialized branch of cellular biology, delves into the intricate world of immune cells, their structure, function, and interactions within the complex network of the immune system. These cells, often referred to as leukocytes or white blood cells, play a pivotal role in defending our bodies against a myriad of pathogens, foreign substances, and even aberrant cells that arise from within. This essay provides a comprehensive, high-quality analysis of immunocytology, examining various aspects of immune cells, including their classification, development, activation mechanisms, effector functions, and the emerging therapeutic applications that harness their power.Classification and Development of Immune CellsThe immune system is composed of a diverse array of cell types, each with distinct roles and characteristics. Broadly, immune cells can be classified into two main categories: innate immune cells and adaptive immune cells. Innate immune cells, such as neutrophils, monocytes/macrophages, dendritic cells (DCs), natural killer (NK) cells, and mast cells, provide the first line of defense against invading pathogens. They recognize conserved pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) and respond rapidly but non-specifically.In contrast, adaptive immune cells, comprising B cells and T cells, offer a highly specific, long-lasting defense. B cells produce antibodies, while T cells execute cytotoxic or helper functions depending on their subsets (CD4+ T helper cells, CD8+ cytotoxic T cells, regulatory T cells, etc.). The development of these immune cells occurs primarily in the bone marrow (for B cells and myeloid cells) and the thymus (for T cells). A tightly regulated process involving hematopoietic stem cell (HSC) differentiation, gene rearrangements, positive and negative selection, and maturation ensures the generation of a diverse and self-tolerant immune repertoire.Activation Mechanisms and Signal TransductionThe activation of immune cells is a finely orchestrated process triggeredby the recognition of antigens or danger signals. For innate immune cells, PRR engagement initiates signaling cascades involving adaptor proteins like MyD88 and TRIF, leading to the activation of transcription factors such as NF-κB and IRF3/7, which drive the expression of pro-inflammatory cytokines, chemokines, and antimicrobial peptides.Adaptive immune cells, particularly T and B cells, require antigen recognition through their unique antigen receptors (TCR for T cells, BCR for B cells). This interaction, when accompanied by appropriate co-stimulatory signals, activates intracellular signaling pathways involving kinases such as Lck, Zap70, and PI3K, ultimately leading to the activation of transcription factors like NF-κB, AP-1, and NFAT. These transcription factors orchestrate the expression of genes involved in cell proliferation, differentiation, and effector function.Effector Functions of Immune CellsInnate immune cells execute various effector functions to combat infections. Neutrophils phagocytose and kill pathogens through the release of reactive oxygen species (ROS) and granule contents. Monocytes/macrophages display similar phagocytic abilities and also present antigens to T cells, produce inflammatory cytokines, and participate in tissue repair. DCs are professional antigen-presenting cells (APCs) that capture, process, and present antigens to naïve T cells, initiating adaptive immune responses. NK cells directly eliminate virus-infected or transformed cells without prior sensitization, relying on the balance of activating and inhibitory receptors interacting with cell surface ligands.Adaptive immune cells contribute to immunity through antibody production and cell-mediated responses. B cells differentiate into plasma cells that secrete antibodies, which neutralize pathogens, opsonize them for enhanced phagocytosis, or activate complement. T cells, upon activation, differentiate into effector subsets: CD4+ T helper cells (Th1, Th2, Th17, Tfh, etc.) that provide help to other immune cells, and CD8+ cytotoxic T cells that directlykill infected or transformed cells. Regulatory T cells (Tregs) maintain immune homeostasis by suppressing excessive immune responses and preventing autoimmunity.Emerging Therapeutic ApplicationsRecent advances in immunocytology have paved the way for innovative therapeutic strategies targeting immune cells. Cancer immunotherapy, for instance, has revolutionized cancer treatment, with approaches such as immune checkpoint inhibitors (e.g., anti-PD-1, anti-CTLA-4 antibodies) that unleash the cytotoxic potential of T cells suppressed by tumor microenvironment. Chimeric antigen receptor (CAR)-T cell therapy involves engineering patient's T cells to express CARs, enabling targeted recognition and destruction of tumor cells. Additionally, adoptive transfer of ex vivo expanded or genetically modified NK cells is being explored for cancer therapy due to their inherent ability to recognize and kill malignant cells.In autoimmune diseases and transplant rejection, therapies targeting immune cells aim to suppress pathogenic immune responses. These include the use of monoclonal antibodies against pro-inflammatory cytokines or their receptors, T cell-depleting agents, and Treg-based therapies. Moreover, modulation of innate immune cells, particularly DCs, through targeted delivery of antigens or immunomodulatory molecules, holds promise for the induction of tolerance in autoimmune and allergic disorders.ConclusionImmunocytology offers a rich tapestry of knowledge, elucidating the complexities of immune cells and their integral role in maintaining host defense. From the classification and development of these cells to the intricate mechanisms governing their activation and effector functions, understanding immunocytology is crucial for both fundamental biological insights and translational applications. The ongoing advancements in this field continue to fuel the development of novel therapeutic strategies that harness the power of immune cells, transforming the landscape of modern medicine in the fight againstinfectious diseases, cancer, and autoimmune disorders.。
2022年考研考博-考博英语-中国人民大学考试全真模拟易错、难点剖析B卷（带答案）一.综合题(共15题)1.单选题British Prime Minister Tony Blair promised the electorate that guns would not be fired without an attempt to win a further U. N. sanction.问题1选项A.alliesB.delegatesC.votersD.juries【答案】C【解析】名词词义辨析。
electorate的前缀为elect-（选-），可以推测出electorate意为“选民”，所以选项C正确。
2.单选题For the last 20 years or so the subject of global warming has（）heated debate among the world’s brightest minds.问题1选项A.spawnedB.injectedC.modeledD.moved【答案】A【解析】动词词义辨析。
句意：在过去20年左右的时间里，全球变暖这个话题在全球最聪明的头脑中引发了激烈的辩论。
只有选项A有引起的意思，所以正确。
3.单选题Tony has not the least （） of giving up his research work.问题1选项A.intentionB.interestC.wishD.desire【答案】A【解析】名词词义辨析。
句意：Tony丝毫没有放弃其研究工作的打算。
选项A符合语境。
4.单选题The gang derived their nickname from their dark clothing and blacked up faces for nocturnal raids in the forest.问题1选项A.illegalB.night-timeC.brutalD.abusive【答案】B【解析】形容词词义辨析。
Peptides 56(2014)132–140Contents lists available at ScienceDirectPeptidesj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /p e p t i d esA family of antimicrobial and immunomodulatory peptides related to the frenatins from skin secretions of the Orinoco lime frog Sphaenorhynchus lacteus (Hylidae)J.Michael Conlon a ,∗,Milena Mechkarska a ,Gordana Radosavljevic b ,Samir Attoub c ,Jay D.King d ,Miodrag L.Lukic b ,Stephen McClean eaDepartment of Biochemistry,College of Medicine and Health Sciences,United Arab Emirates University,17666Al Ain,United Arab Emirates bCenter for Molecular Medicine,Faculty of Medicine,University of Kragujevac,Kragujevac,Serbia cDepartment of Pharmacology,College of Medicine and Health Sciences,United Arab Emirates University,17666Al Ain,United Arab Emirates dRare Species Conservatory Foundation,St.Louis,MO 63110,USA eSchool of Biomedical Sciences,University of Ulster,Cromore Road,Coleraine,Northern Ireland BT521SA,UKa r t i c l ei n f oArticle history:Received 23February 2014Received in revised form 25March 2014Accepted 25March 2014Available online 4April 2014Keywords:Frog skin Frenatin HylidaeAntimicrobialImmunomodulatory Cytokinea b s t r a c tPeptidomic analysis of norepinephrine-stimulated skin secretions of the Orinoco lime tree frog Sphaenorhynchus lacteus (Hylidae,Hylinae)revealed the presence of three structurally related host-defense peptides with limited sequence similarity to frenatin 2from Litoria infrafre-nata (Hylidae,Pelodryadinae)and frenatin 2D from Discoglossus sardus (Alytidae).Frenatin 2.1S (GLVGTLLGHIGKAILG.NH 2)and frenatin 2.2S (GLVGTLLGHIGKAILS.NH 2)are C-terminally ␣-amidated but frenatin 2.3S (GLVGTLLGHIGKAILG)is not.Frenatin 2.1S and 2.2S show potent bactericidal activity against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA)and Staphylococcus epidermidis (MIC ≤16M)but are less active against a range of Gram-negative bacteria.Frenatin 2.1S (LC 50=80±6M)and 2.2S (LC 50=75±5M)are cytotoxic against non-small cell lung adenocarcinoma A549cells but are less hemolytic against human erythrocytes (LC 50=167±8M for frenatin 2.1S and 169±7M for 2.2S).Weak antimicrobial and cytotoxic potencies of frenatin 2.3S demonstrate the importance of C-terminal ␣-amidation for activity.Frenatin 2.1S and 2.2S significantly (P <0.05)increased production of proinflammatory cytokines IL-1and IL-23by lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and frenatin 2.1S also enhanced production of TNF-␣.Effects on IL-6production were not significant.Frenatin 2.2S significantly downregulated production of the anti-inflammatory cytokine IL-10by LPS-stimulated cells.The data support speculation that frenatins act on skin macrophages to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms.They may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.©2014Elsevier Inc.All rights reserved.1.IntroductionSkin secretions of frogs belonging to certain families contain high concentrations of cytotoxic host-defense peptides with the ability to inhibit the growth of bacteria and fungi that may also show anti-tumor and anti-viral activity [12,14].With few excep-tions,these peptides are cationic (charge between +1and +6at pH 7),contain between 40and 70%hydrophobic amino acids,∗Corresponding author.E-mail address:jmconlon@uaeu.ac.ae (J.M.Conlon).and adopt an amphipathic ␣-helical conformation in a membrane-mimetic environment [40].At the time of writing,peptides with antimicrobial activity have been identified in the skins of frogs from species belonging to the Alytidae,Bombinatoridae,Hylidae,Hyper-oliidae,Leiopelmatidae,Leptodactylidae,Myobatrachidae,Pipidae,and Ranidae families [12]and the Antimicrobial Peptide Database (/AP )lists 929amphibian host-defense pep-tides [40].Although considered to be a component of the animal’s system of innate immunity,the sporadic species distribution of these peptides suggests that their production in the skin may confer some evolutionary advantage to the organism but is not neces-sary for survival.It has been suggested that cutaneous symbiotic/10.1016/j.peptides.2014.03.020J.M.Conlon et al./Peptides56(2014)132–140133bacteria may provide the major system of defense against pathogenic microorganisms in the environment with antimicrobial peptides assuming a supplementary role in some species[11].According to currently accepted taxonomic classifications,the extensive and widely distributed family Hylidae is divided into the subfamilies Phyllomedusinae(currently59species in5genera), Pelodryadinae(currently203species in the single genus Litoria), and Hylinae(currently674species in43genera)[18].Skin secre-tions from the Central and South American tree frogs,particularly species in the genera Agalychnis,Hylomantis,Pachymedusa,and Phyllomedusa in the subfamily Phyllomedusinae,have proved to be rich source of host-defense peptides with antimicrobial activity (reviewed in[30]).These include the dermaseptins,dermatox-ins,hyposins,phylloseptins,phylloxins,plasticins,and a number of“orphan”peptides not included in these groups.Similarly,the skin secretions from the tree frogs of the Australo-Papuan region belonging to the genus Litoria in the subfamily Pelodryadinae have provided multiple antimicrobial peptides that may be arranged in at least six groups on the basis of structural similarity.These are the aureins,caerins,citropins,dahleins,frenatins,and maculatins (reviewed in[5]).Synthesis of dermal host-defense peptides among species in the subfamily Hylinae is more restricted.Four structurally related pep-tides,termed pseudin1–4,were isolated from an extract of the skin of the South American paradoxical frog Pseudis paradoxa,although these peptides were not detected in norepinephrine-stimulated skin secretions[31].Skin secretions from several North American species belonging to the genera Hyla,Hypsiboas,Osteopilus,and Pseudacris in the sub-family Hylinae have been shown not to con-tain host-defence peptides[12]but peptides with broad-spectrum antimicrobial activity and low hemolytic activity have been iden-tified in skin secretions from the tree frogs Hyla punctata[34], Hypsiboas albopunctatus[7],and Hypsiboas raniceps[25]from Brazil and Hyla eximia from Mexico[39].The Orinoco lime tree frog Sphaenorhynchus lacteus(Daudin, 1802)in the subfamily Hylinae is a moderately sized(snout-vent length2.6–4.2cm in males,3.8–4.6cm in females)frog that is widely distributed in the Amazon basin of Columbia,Venezuela, Peru,Brazil and Ecuador and also in the Guianas and on the islands of Trinidad and Tobago[18].Its preferred habitat is wetland,such as flooded plains,ponds and lagoons withfloating vegetation,and sea-sonallyflooded agricultural land and it is common throughout its range.It is listed as a Species of Least Concern by the International Union for Conservation of Nature(IUCN)Red List of Threatened Species in view of its relatively wide distribution and tolerance of a broad range of habitats but numbers have declined in some areas due to deforestation and water pollution[23].The present study describes the purification of three structurally related peptides from norepinephrine-stimulated skin secretions from cteus and presents their antimicrobial potencies against a range of reference strains and clinical isolates of Gram-positive and Gram-negative bacteria and cytotoxic activities against human erythrocytes and non-small cell lung adenocarcinoma A549cells. Several frog peptides that werefirst identified on the basis of their abilities to inhibit growth of bacteria have been shown to pos-sess cytokine-mediated immunomodulatory properties and effects upon production of both pro-and anti-inflammatory cytokines have been described[1,16,27,32,33,37].Consequently,the action of the synthetic cteus peptides on the production of proinflam-matory tumor necrosis factor-␣(TNF-␣),interleukin IL-1(IL-1), interleukin-6(IL-6),and interleukin-23(IL-23)and immunosup-presive interleukin-10(IL-10)by mouse peritoneal macrophages was investigated.The peptides show limited structural similarity to previously isolated frenatin2from Litoria infrafrenata(Hylidae,2.Experimental2.1.Collection of skin secretionsAll experiments with live animals were approved by the Ani-mal Research Ethics committees of U.A.E.University(Protocol No. A21-09)and the University of Kragujevac Animal Ethics Committee and were carried out by authorized investigators.Adult specimens (n=2;weights7.0g and8.1g;sex not determined)of cteus were collected in north central Guyana near the Essequibo river (6◦28 N,58◦35 W).Each animal was injected at two sites into the dorsal lymphatic sac with norepinephrine bitartrate(40nmol per g body weight)and immersed in water(50ml)for15min.The solu-tion containing the secretions was acidified with trifluoroacetic acid(TFA)(final concentration1%,v/v)and stored at−20◦C.After collection of the secretions,the animals were released into their environment unharmed.2.2.Peptide purificationThe pooled skin secretions were passed at aflow rate of2ml/min through6Sep-Pak C-18cartridges(Waters Associates,Milford, MA,USA)connected in series.Bound material was eluted with acetonitrile/water/TFA(70.0/29.9/0.1,v/v/v)and freeze-dried.The lyophilized skin secretions were redissolved in0.1%(v/v)TFA/water (4ml)and injected onto a(2.2cm×25cm)Vydac218TP1022(C-18) reversed-phase HPLC column(Grace,Deerfield,IL,USA)equili-brated with0.1%(v/v)TFA/water at aflow rate of6.0ml/min.The concentration of acetonitrile in the eluting solvent was raised to 21%(v/v)over10min and to63%(v/v)over60min using linear gra-dients.Absorbance was monitored at214nm and280nm and peaks were collected by hand.Aliquots(50l)of each major peak were analysed by MALDI-TOF mass spectrometry.The major peaks in the chromatogram were sequentially injected onto(1cm x25cm) Vydac214TP510(C-4)and(1cm×25cm)Vydac208TP510(C-8) columns.The concentration of acetonitrile in the eluting solvent was raised from21%to56%over50min and theflow rate was 2.0ml/min.2.3.Structural characterizationMALDI-TOF mass spectrometry was carried out using a Voyager DE-PRO instrument(Applied Biosystems,Foster City,CA,USA)that was operated in reflector mode as previously described[16].The instrument was calibrated with peptides of known molecular mass in the1–4kDa range.The accuracy of mass determinations was ±0.02%.The primary structures of the peptides were determined by automated Edman degradation using a model491Procise sequena-tor(Applied Biosystems).MS/MS sequencing was performed using a Q-ToF Ultima API mass spectrometer(Micromass,Manchester, UK)using collision-induced dissociation(CID)-based fragmenta-tion.Ions are fragmented by subjecting them to collisions with the background gas,argon as previously described[38].2.4.Peptide synthesisFrenatin 2.1S(GLVGTLLGHIGKAILG.NH2),frenatin 2.2S (GLVGTLLGHIGKAILS.NH2),and frenatin 2.3S(GLVGTLL-GHIGKAILG)were supplied in crude form by GL Biochem Ltd(Shanghai,China).The peptides were purified to near homogeneity by reversed-phase HPLC on a(2.2cm×25cm) Vydac218TP1022(C-18)column equilibrated with acetoni-trile/water/TFA(21.0/78.9/0.1,v/v/v)at aflow rate of6ml/min. The concentration of acetonitrile was raised to56%(v/v)over134J.M.Conlon et al./Peptides56(2014)132–140was collected manually.Thefinal purity of all peptides tested was >98%.The purities and identities of the synthetic peptides were confirmed by electrospray mass spectrometry.2.5.Verification of the frenatin structures by chemical synthesisSynthetic frenatin2.1S,frenatin2.2S,and frenatin2.3S(approx. 2nmol)were individually mixed with the corresponding naturally occurring cteus peptides(approx.2nmol)and each mixture chromatographed on(0.46cm×25cm)Vydac218TP54(C-18)col-umn equilibrated with acetonitrile/water/TFA(21.0/78.9/0.1,v/v/v) at aflow rate of1.5ml/min.The concentration of acetonitrile was raised to56%(v/v)over60min using a linear gradient.Absorbance was measured at214nm.2.6.Antimicrobial assaysThe following reference strains were purchased from the American Type Culture Collection(ATCC;Rockville,MD,USA): Staphylococcus aureus(ATCC25923),Escherichia coli(ATCC25726), Klebsiella pneumoniae(ATCC700603),Pseudomonas aeruginosa (ATCC27853),and Candida albicans(ATCC90028).The biofilm-producing Staphylococcus epidermidis RP62A strain produces polysaccharide intercellular adhesin that protects the bacteria against the components of the human innate immune system.Its full genome sequence is in the GenBank:NC002976.S.epidermidis RP62A/1is a stable biofilm non-producer phase variant of RP62A [43].Six clinical sporadic isolates of MRSA(127/08,145/08,274/08, V4180,S908,and T4/6)and two isolates of S.epidermidis(T6/19and T37/8)were obtained from wounds of patients admitted to Tawam hospital(Al Ain,UAE).Characterization of the MRSA strains by multilocus sequence typing(MLST),staphylococcal cassette chro-mosome(SCC mec)typing,accessory gene regulator(agr)typing, Staphylococcus protein A(spa)typing,and toxin gene carriage has been described previously[36].The strains were resistant to all-lactam antibiotics tested and to a range of non--lactam antibiotics.Five independent well-characterized multidrug-resistant Acine-tobacter baumannii strains(NM8,NM35,NM75,NM109,and NM124)andfive Stenotrophomonas maltophilia strains(B32/1, B32/4,B5/5,B6/2,and U8708)were included in the study.These strains were isolated at four different hospitals in Abu Dhabi Emirate,UAE and their clonal lineages and antibiotic suscepti-bilities have been described previously[13,22].The abilities of the synthetic frenatin2S peptides to inhibit the growth of these microorganisms were measured in the concentration range of 4–250M by standard double dilution methods[9,10].In order to determine the rate at which frenatin2.2S caused cell death,log-phase cultures of S.aureus(ATCC25923)at a concentra-tion of0.5×106colony forming units(CFU)/ml in Mueller-Hinton broth(1ml)were incubated in polypropylene tubes at37◦C with the peptide at2x minimum inhibitory concentration(MIC).A con-trol incubation in the absence of peptide was carried out.Aliquots (25l)were removed at0,0.5,1,2,3,4and24h and the CFU of the serially diluted samples was determined on Mueller-Hinton agar plates.The time required for99.9%cell death was determined.2.7.Cytotoxicity assaysHuman non-small cell lung adenocarcinoma A549cells were maintained at37◦C in RPMI1640medium containing2mM l-glutamine and supplemented with10%fetal calf serum(FCS; Biowest,Nouaille,France),and antibiotics(penicillin50U/ml; streptomycin50g/ml)as previously described[1,2,15].In all density of5×103cells/well.After24h,all cells were treated for 24h with increasing concentrations of frenatin2.1S,2.2S,and2.3S (1–100M)in triplicate.The effect of the peptides on cell viabil-ity was determined by measurement of ATP concentrations using a CellTiter-Glo Luminescent Cell Viability assay(Promega Corpora-tion,Madison,WI,USA).Luminescent signals were measured using a GLOMAX Luminometer system.The LC50value,calculated by non-linear regression analysis using commercially available software (SPSS version17.0;SPSS Inc.,Chicago,IL,USA),was taken as the mean concentration of peptide producing50%cell death in three independent experiments.Hemolytic activities of synthetic frenatin2.1S,2.2S,and2.3S (16–500M)against human erythrocytes taken from a healthy donor were measured as previously described[1,27].The LC50value was calculated using commercially available software(SPSS ver-sion17.0)and was taken as the mean concentration of peptide producing50%hemolysis in three independent experiments.2.8.Effects on cytokine production by mouse peritoneal cellsAll chemicals were supplied by Sigma(Germany).Effects of fre-natin2.1S,2.2S,and2.3S on cytokine production by peritoneal cells from C57BL/6mice were determined using previously described methodologies[27,32,37].The peritoneal cavity was washed with 5ml of ice cold phosphate buffered saline,pH7.4.Cells were placed on glass Petri dishes and non-adherent cells were dis-carded after two hours incubation at37◦C.Adherent cells were recovered by vigorous washing with ice-cold medium.This pop-ulation contained more than95%of F4/80positive macrophages. Cell number and viability were determined using trypan blue and acridine orange/ethidium bromide staining.The peptides(5,10 and20g/ml;approx.2.5,5,and10M)were incubated with peritoneal cells(2×105cell/well),with and without lipopolysac-charide from E.coli055:B5(0.5g/ml).Cells were cultured for24h at37◦C in supplemented culture medium(RPMI1640containing 10%autologous serum,2mM l-glutamine,100IU/ml penicillin G and100g/ml streptomycin)in an humidified atmosphere of5% CO2–95%air.After incubation cells were centrifuged,supernatants collected and kept at-20◦C until time of assay.Concentrations of TNF-␣,IL-1,IL-6,IL-10,and IL-23and were determined in tripli-cate using ELISA assay kits from R&D Systems(Minneapolis,USA) according to manufacturer’s recommended protocols.Data are pre-sented as mean±SEM for two series of independent experiments with5mice per group.2.9.Statistical analysisStatistical analyses were performed using commercially avail-able software(SPSS version13.0).The distributions of data were evaluated for normality using Kolmogorov–Smirnov test and then retested with Chi-Square parison of quantitative para-metric data between two study groups was done by application of unpaired t-test.Differences between the paired data were eval-uated using the paired t-test.A P-value of<0.05,from two-tailed tests,was considered statistically significant.3.Results3.1.Purification of the peptidesThe pooled skin secretions from cteus,after partial purifi-cation on Sep-Pak C-18cartridges,were chromatographed on a Vydac C-18preparative reversed-phase HPLC column(Fig.1).J.M.Conlon et al./Peptides56(2014)132–140135Fig.1.Reversed-phase HPLC on a preparative Vydac C-18column of skin secretions from cteus,after partial purification on Sep-Pak cartridges.The peaks designated 1(containing frenatin2.3S),2(containing frenatin2.1S),and3(containing frenatin 2.2S)were purified further.The dashed line shows the concentration of acetonitrile in the eluting solvent.1in Fig.1contained a component with monoisotopic mass(M+H+) 1518.9,subsequently shown to contain frenatin2.3S(calculated mass1519.0).Peak2contained a component with mass(M+H+) 1518.0,subsequently shown to contain frenatin2.1S(calculated mass1518.0).Peak3contained a component with mass(M+H+) 1547.9subsequently shown to be frenatin2.2S(calculated mass 1548.0).The peptides were purified to near homogeneity,as assessed by mass spectrometry and symmetrical peak shape,by sequen-tial chromatography on Vydac C-4and Vydac C-8semipreparative reversed-phase HPLC columns.The methodology is illustrated in Fig.2by the purification of frenatin2.1S.The approximatefinal yields of purified peptides(nmol)were frenatin2.1S13,frenatin2.2S17,and frenatin2.3S55.3.2.Structural characterizationThe C-terminal regions of the frenatin peptides are strongly hydrophobic so that it was not possible to establish unambigu-ously their complete amino acid sequence by automated Edman degradation due to wash-out from the sequencer ing an Applied Biosystems Procise sequencer,the common amino acid sequence at the N-terminus of the each peptide was established as Gly-Leu-Val-Gly-Thr-Leu-Leu-Gly-His-Ile-Gly.CID-based MS/MS sequencing of frenatin 2.1S produced b-ions with m/z1444.0, 1330.9,1217.7and1018.6indicating the probable structure Lys-Ala-Ile/Leu-Ile-Leu-Gly.NH2at the C-terminus of the peptide. While this work was in progress,the nucleotide sequence of a cloned cDNA from cteus was deposited in the National Center for Biotechnology Information(NCBI)database by Munoz-Camargo C,Correa M.Salazar VA,Moscoso JM,Mitrani E,and Groot,H.(Accession no.AGB51284.1)as unpublished data.The predicted amino acid sequence corresponding to the nucleotide sequence is AFLKKSLFLVLFLGLVSLSMGEREKREEEEEEEEN KEEEANEEGKGESEEKRGLVGTLLGHIGKAILGG.The residues under-lined correspond to the sequence of frenatin 2.1S with the C-terminal glycine in the precursor acting as nitrogen donor to produce a C-terminally␣-amidated peptide.The difference of+1 Dalton in frenatin2.3S compared with frenatin2.1S suggests that frenatin-2.3S represents the non-amidated form of the peptideFig.2.Purification to near homogeneity on semipreparative(A)Vydac C-4and(B) Vydac C-8columns of frenatin2.1S.The arrowheads show where peak collection began and ended.The peak denoted by the asterisk contains frenatin2.1S.The dashed line shows the concentration of acetonitrile in the eluting solvent.The proposed primary structures of the peptides were con-firmed by chemical synthesis.In each case,the synthetic replicates of frenatin2.1S,2.2S,and2.3S with the amino acid sequences shown in Fig.4co-eluted with corresponding endogenous peptide as single sharp peaks on reversed-phase HPLC(data not shown).3.3.Antimicrobial activities of the peptidesThe antimicrobial potencies of the cteus frenatin peptides against reference strains of the Gram-positive bacteria S.aureus and S.epidermidis,clinical isolates of MRSA and S.epidermidis,and the biofilm-producing strain RP62A of S.epidermidis are shown in Table1.The abilities of the peptides to inhibit the growth of ref-erence strains of the Gram-negative bacteria E.coli,K.pneumoniae, P.aeruginosa,and the opportunistic yeast pathogen C.albicans as well as multidrug-resistant clinical isolates of Acinetobacter bau-mannii,and S.maltophilia are compared in Table2.Overall,the C-terminally␣-amidated frenatin2.1S and2.2S are appreciably more active against all strains tested than the non-amidated fre-natin2.3S.The growth inhibitory potencies of frenatin2.1S and2.2S were greatest against Gram-positive bacteria,including multidrug-136J.M.Conlon et al./Peptides 56(2014)132–140Table 1Minimum inhibitory concentrations of synthetic frenatins from cteus against reference strains and clinical isolates of Gram-positive bacteria.S.aureusATCC 25923127/08145/08274/08S908T4/6V4180Frenatin 2.1S 881616161616Frenatin 2.2S 1681616161616Frenatin 2.3S125125250250125250250S.epidermidisRP62ARP62A/1T6/19T37/8Frenatin 2.1S 88816Frenatin 2.2S 168816Frenatin 2.3S125125125250Data are expressed in M.Table 2Minimum inhibitory concentrations of synthetic frenatins from cteus against reference strains and isolates of clinically relevant bacteria and the opportunistic yeast pathogen C.albicans .Reference strainsE.coliK.pneumoniaeP.aeruginosaE.faecalisC.albicansFrenatin 2.1S 12525025062.562.5Frenatin 2.2S 12525025062.562.5Frenatin 2.3S250>250>250250>250A.baumannii clinical isolatesNM8NM35NM75NM109NM124Frenatin 2.1S 62.562.5125125125Frenatin 2.2S 12512562.5125125Frenatin 2.3S250250250250250S.maltophilia clinical isolatesB32/1B32/4U8908B5/5B6/2Frenatin 2.1S 62.562.562.562.531.3Frenatin 2.2S 62.562.562.562.531.3Frenatin 2.3S>250250250>250250Data are expressed in M.Fig.3.Survival of S.aureus (ATCC 25923)in Mueller-Hinton broth after addition of 2x MIC frenatin 2.2S.Control represents incubation in the absence of peptide.CFU:colony forming units.cteus 2.1S GLVGTLLGHIGKAILG a cteus 2.2S GLVGTLLGHIGKAILS acteus 2.3S GLVGTLLGHIGKAILG L. infrafrenata 2 GLLGTLGNLLNGLGL a D. sardus 2D DLLGTLGNLPLPFIaFig.4.A comparison of the primary structures of the frenatin 2-related peptides from cteus with frenatin 2from L.infrafrenata and frenatin 2D from D.sardus .Table 3Cytotoxic activities (LC 50)of synthetic frenatins from cteus against non-small cell lung adenocarcinoma A549cells and human red blood cells (RBC).A549RBC Frenatin 2.1S 80±6167±8Frenatin 2.2S 65±5169±7Frenatin 2.3S>100404±11Data are expressed in M.25923)with frenatin 2.2S at a concentration of 2×MIC resulted in killing of >99.9%of the bacteria within 180min.3.4.Cytotoxic activities of the peptidesFrenatin 2.1S (LC 50=80±6M)and frenatin 2.2S (LC 50=75±5M)are cytotoxic against human non-small cell lung adenocarcinoma A549cells whereas frenatin 2.3S did not produce significant cell death at concentrations up to 100M (Table 3).The peptides were significantly less cytotoxic to human erythrocytes giving therapeutic indicies (defined as the ratio of LC 50for erythrocytes to the LC 50for tumor cells)of 2.4for frenatin 2.1S and 2.8for frenatin 2.2S.3.5.Effects of the peptides on cytokine productionThe effects of the frenatin 2S peptides,alone or together with a sub-maximal stimulatory dose of LPS (0.5g/ml),on the production of proinflammatory TNF-␣,IL-1,IL-6,and IL-23and downregulatory IL-10by mouse peritoneal macrophages are shown in Figs.5–8.Both frenatin 2.1S and 2.2S,at a dose of 20g/ml produce significant (P <0.05)enhancement of IL-1production alone or in the presence of LPS but frenatin 2.3S was without signif-icant effect (Fig.5).Frenatin 2.1S (20g/ml)also increases TNF-␣production but only in the presence of LPS,while frenatin 2.2S and frenatin 2.3S do not have significant effects (Fig.6).Frenatin 2.1S,2.2S,and 2.3S had a stimulatory effect on IL-23production in the presence of LPS (Fig.7).The increase in IL-23production in both unstimulated and LPS-stimulated macrophages by frenatin 2.2S was significant at lower doses (5and 10g/ml).Frenatin 2.2S (20g/ml)significantly downregulated production of the anti-inflammatory cytokine IL-10when incubated together with LPS (Fig.8).There was no significant effect on IL-6production by any of the peptides studied (data not shown).4.DiscussionThis study has identified three cytotoxic peptides present in skin secretions of cteus that show limited structural similarity to frenatin 2previously isolated from the Australopapuan frog L.infrafrenata (Hylidae,Pelodryadinae)[35]and frenatin 2D from the Tyrrhenian painted frog D.sardus (Alytidae)that is largely restricted to the islands of Corsica and Sardinia [16](Fig.4).As neither frenatin 2nor frenatin 2D contain a basic residue,their lack of antimicrobial activity was not unexpected [17].Frenatin 2D was inactive against S.aureus ,E.coli ,A.baumannii ,and S.maltophilia [16].Frenatin 2from L.infrafrenata did show growth inhibitory activity against Micrococcus luteus but was inactive against all other microorgan-isms tested [35].In contrast,the C-terminally ␣-amidated frenatin 2.1S and 2.2S show relatively high potency (MIC ≤16M)against the Gram-positive bacteria S.aureus and S.epidermidis (Table 1)and are also active against the Gram-negative bacteria A.bauman-nii ,E.coli ,K.pneumoniae ,P.aeruginosa ,and S.maltophilia albeit with lower potency.The peptides are also active against the opportunis-J.M.Conlon et al./Peptides56(2014)132–140137Fig.5.Effects of cteus frenatins on the production of IL-1by unstimulated and lipopolysaccharide(LPS)-stimulated mouse peritoneal macrophages.(*)P<0.05vs. medium only.peptide is bactericidal not bacteriostatic(Fig.3).The rate at which the peptide kills bacteria is less than that observed for some other frog skin peptides.For examples,99.9%of a clinical isolate of A.bau-mannii was killed by2×MIC of the[E4K]analog of alyteserin-1c within30min[13].The emergence in all regions of the world of strains of pathogenic bacteria and fungi with resistance to commonly used antibiotics constitutes a serious threat to public health.The appear-ance of strains of the Gram-positive bacterium S.aureus that are resistant to all-lactam antibiotics,generally referred to as MRSA, are of particular concern.These strains often exhibit multi-drug resistance,including non-susceptibility to quinolones,macrolides and sulfonamides.Although originally nosocomial,community-acquired MRSA infections are becoming increasingly common[21]. In particular,MRSA is a serious problem in infections of the super-ficial epidermis,such as impetigo in infants and children[20]and in colonization of surface lesions such as the foot ulcers of diabetic patients[6].Frog skin host-defense peptides have excitedinterest。
