Inhibition of Local Immune Responses by the Frog-Killing Fungus Batrachochytrium dendrobatidis

Amphibians are suffering unprecedented global declines. A leading cause is the infectious disease chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis. Chytridiomycosis is a skin disease which disrupts transport of essential ions leading to death. Soluble factors produced by B. dendrobatidis impair amphibian and mammalian lymphocytes in vitro, but previous studies have not shown the effects of these inhibitory factors in vivo. To demonstrate in vivo inhibition of immunity by B. dendrobatidis, a modified delayed-type-hypersensitivity (DTH) protocol was developed to induce innate and adaptive inflammatory swelling in the feet of Xenopus laevis by injection of killed bacteria or phytohemagglutinin (PHA). Compared to previous protocols for PHA injection in amphibians, this method induced up to 20-fold greater inflammatory swelling. Using this new protocol, we measured DTH responses induced by killed bacteria or PHA in the presence of B. dendrobatidis supernatants. Swelling induced by single injection of PHA or killed bacteria was not significantly affected by B. dendrobatidis supernatants. However, swelling caused by a secondary injection of PHA, was significantly reduced by B. dendrobatidis supernatants. As previously described in vitro, factors from B. dendrobatidis appear to inhibit lymphocyte-mediated inflammatory swelling but not swelling caused by an inducer of innate leukocytes. This suggests that B. dendrobatidis is capable of inhibiting lymphocytes in a localized response to prevent adaptive immune responses in the skin. The modified protocol used to induce inflammatory swelling in the present study may be more effective than previous methods to investigate amphibian immune competence, particularly in nonmodel species.     

Duplex Real-Time PCR for Rapid Simultaneous Detection of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in Amphibian Samples

Chytridiomycosis is a lethal fungal disease contributing to declines and extinctions of amphibian species worldwide. The currently used molecular screening tests for chytridiomycosis fail to detect the recently described species Batrachochytrium salamandrivorans. In this study, we present a duplex real-time PCR that allows the simultaneous detection of B. salamandrivorans and Batrachochytrium dendrobatidis. With B. dendrobatidis- and B. salamandrivorans-specific primers and probes, detection of the two pathogens in amphibian samples is possible, with a detection limit of 0.1 genomic equivalent of zoospores of both pathogens per PCR. The developed real-time PCR shows high degrees of specificity and sensitivity, high linear correlations (r² > 0.995), and high amplification efficiencies (>94%) for B. dendrobatidis and B. salamandrivorans. In conclusion, the described duplex real-time PCR can be used to detect DNA of B. dendrobatidis and B. salamandrivorans with highly reproducible and reliable results.     

Immune Defenses against Batrachochytrium dendrobatidis,a Fungus Linked to Global Amphibian Declines,in the South African Clawed Frog,Xenopus laevis

Batrachochytrium dendrobatidis is a chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians. It is regarded as an emerging infectious disease affecting diverse amphibian populations in many parts of the world. Because there are few model amphibian species for immunological studies, little is known about immune defenses against B. dendrobatidis. We show here that the South African clawed frog, Xenopus laevis, is a suitable model for investigating immunity to this pathogen. After an experimental exposure, a mild infection developed over 20 to 30 days and declined by 45 days postexposure. Either purified antimicrobial peptides or mixtures of peptides in the skin mucus inhibited B. dendrobatidis growth in vitro. Skin peptide secretion was maximally induced by injection of norepinephrine, and this treatment resulted in sustained skin peptide depletion and increased susceptibility to infection. Sublethal X-irradiation of frogs decreased leukocyte numbers in the spleen and resulted in greater susceptibility to infection. Immunization against B. dendrobatidis induced elevated pathogen-specific IgM and IgY serum antibodies. Mucus secretions from X. laevis previously exposed to B. dendrobatidis contained significant amounts of IgM, IgY, and IgX antibodies that bind to B. dendrobatidis. These data strongly suggest that both innate and adaptive immune defenses are involved in the resistance of X. laevis to lethal B. dendrobatidis infections.Batrachochytrium dendrobatidis is a newly described chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians (29). Growing evidence links amphibian declines in Australia, Central America, the western United States, Europe, and Africa to this emerging infectious disease (4, 9, 12, 26, 29, 34-36, 45, 65). B. dendrobatidis colonizes skin cells of adults and the keratinized mouth parts of tadpoles (3, 4, 29, 34) but does not invade other tissues. It is spread by waterborne zoospores that attach to the skin and migrate to the basal layer of the epidermis (3). The pathogen replicates within the epidermal cells and moves to the surface as the cells mature. Emerging zoospores may infect the same host or another nearby host (3, 4, 29, 34). Recent evidence supports the hypothesis that death results from impaired retention of essential ions by the skin resulting in eventual cardiac arrest (63, 64). Some species of amphibians are very resistant to lethal infections of B. dendrobatidis, whereas others are more susceptible (4, 26, 27, 38, 66-68), and the factors that determine resistance or susceptibility are not well understood. Although much is known about amphibian immunity in general (9, 14, 41), there is limited information about specific immune responses against B. dendrobatidis.We hypothesized that resistant species have antimicrobial peptides or antibodies in the mucus that limit initial infections by B. dendrobatidis zoospores and prevent the further colonization of the same host by zoospores emerging from the skin. Previous work has shown that individual purified antimicrobial peptides (11, 44-50, 52, 68) and enriched skin peptides (48, 52, 66-68) from many species can inhibit the growth of B. dendrobatidis zoospores and mature sporangia in vitro. The skin of amphibians is also protected by the adaptive immune system. Antigens in the skin can be transported to the spleen, where an immune response involving both T cells and B cells can occur (9, 14, 41). In mammals and in fish, antibodies are present in mucosal secretions (10, 28, 31, 53), but there have been no previous studies of antibodies in amphibian mucus.X. laevis was chosen as the species to investigate immunity to B. dendrobatidis because this species has been widely used as a model for studies of amphibian immunity since the 1960s (9, 14, 41). X. laevis is quite resistant to the lethal effects of infection with B. dendrobatidis in nature. Infections were detected in archived specimens of X. laevis as early as 1938, and the incidence of infected individuals appears to be constant (∼3%) over the last 60 years (1941 to 2001) (65).We show here that after exposure to B. dendrobatidis, immunocompetent frogs developed a mild infection that is almost completely cleared by 45 days. Antimicrobial skin peptides inhibited B. dendrobatidis growth, were present at effective concentrations in resting frogs, and increased in number when frogs were exposed to an “alarm” stress. Treatment with norepinephrine depleted skin peptide stores and increased host susceptibility to infection. X-irradiation depleted leukocytes in the spleen without altering the capacity to secrete skin peptides, and the infection intensity was significantly greater in the irradiated frogs. Immunization with heat-killed B. dendrobatidis induced significantly elevated pathogen-specific IgM and IgY in the serum detectable for at least 1 month after the last immunization. In addition to antimicrobial peptides, skin mucus samples from frogs exposed to B. dendrobatidis 5 months earlier contained antibodies of all three immunoglobulin classes that bind B. dendrobatidis. Whether the mucosal antibodies are protective will be determined in ongoing studies. Collectively, these data demonstrate that X. laevis is a good model species to study immune defenses against B. dendrobatidis, and both innate and adaptive immune mechanisms appear to be involved in the resistance to lethal infections.     

Activity of antimicrobial skin peptides from ranid frogs against Batrachochytrium dendrobatidis, the chytrid fungus associated with global amphibian declines

Accumulating evidence suggests that a chytrid fungus, Batrachochytrium dendrobatidis, is responsible for recent declines in amphibian populations in Australia, Central America, Europe, and North America. Because the chytrid infects the keratinized epithelium of the skin, we investigated the possible role of antimicrobial peptides produced in the skin as inhibitors of infection and growth. We show here that 10 peptides representing eight families of peptides derived from North American ranid frogs can effectively inhibit growth of this chytrid. The peptides are members of the ranatuerin-1, ranatuerin-2, esculentin-1, esculentin-2, brevinin-2, temporin, palustrin-3, and ranalexin families. All the tested peptides inhibit growth of mature fungal cells at concentrations above 25 microM, and some of them inhibit at concentrations as low as 2 microM. A comparison of the sensitivity of infectious zoospores with that of mature cells showed that the zoospores are inhibited at significantly lower concentrations of peptides. To determine whether cold temperature interferes with the inhibitory effects of these peptides, we tested their effectiveness at both 22 and 10 degrees C. Although the peptides inhibit at both temperatures, they appear to be more effective against zoospores at the lower temperature. These results suggest that the ranid frogs have, within their repertoire of antimicrobial substances, a number of skin peptides that should be a deterrent to chytrid infection. This may provide some natural resistance to infection, but if environmental factors inhibit the synthesis and release of the skin peptides, the pathogen could gain the advantage.     

Reparative and Immunomodulatory Potential of Low-Molecular-Weight Fractions of Secondary Metabolites of Bacillus sp.

Bulletin of Experimental Biology and Medicine - The experiment was performed on male BALB/c mice with modeled skin wound. Two chromatographic fractions of secondary metabolites of permafrost...     

Clonotype Patterns of Antibodies Released by Single Lymph Nodes

Experimental primary, secondary and tertiary stimulation with streptococcal vaccine of isolated lymph nodes in vivo in the sheep model induces largely persistence of anti-group polysaccharide antibody clonotype patterns with the rare occurrence of additional clonotypes demonstrable after secondary stimulation persisting during the tertiary stimulus. It is not clear whether these additional clonotypes are the products of mutants or whether they pre-existed and were demonstrable only after a secondary stimulus, because of threshold concentrations required for identification. Further, isolated contralateral popliteal and prescapular lymph nodes of individual sheep share completely overlapping clonotype patterns during experimental primary anti-polysaccharide antibody responses indicating an identical repertoire of specific clonotypes under this condition of responsiveness.     

Bone Regeneration by Lactoferrin Released from a Gelatin Hydrogel

The objective of this study is to evaluate the potential of lactoferrin (LF), an iron-binding glycoprotein, to induce bone regeneration. A biodegradable gelatin hydrogel was prepared to allow LF release in vivo in a sustained fashion. When subcutaneously implanted into the back of mice, the gelatin hydrogel incorporating LF showed a longer LF retention period at the site of implantation than that of LF solution injection. An in vitro culture experiment with 3T3E1 cells (mouse-derived osteoblasts) revealed that the cells were proliferated to a significantly greater extent by the repeated addition of LF compared with a single addition of LF at the same dose. Following the implantation of gelatin hydrogels incorporating LF into a skull bone defect of rats, a significantly stronger bone regeneration at the defect was observed than in LF-free- or low-LF-treated rats. It is concluded that the sustained release from the gelatin hydrogels enables LF to enhance the in vivo activity of bone regeneration.     

Immunomodulatory role of the corticotropin-releasing factor

Corticotropin-releasing factor (CRF) was originally identified as a hypothalamic peptide which stimulates secretion of the hypophyseal adrenocorticotropin hormone. CRF exhibits its actions through G protein-dependent seven membrane domain receptors. Two subtypes of CRF receptors (CRF-R1 and CRF-R2) have been characterized thus far. CRF and its receptors were found in a number of brain regions, where they function by neuromodulation and also in several peripheral organs. Besides CRF, another naturally occurring CRF-like peptide, urocortin, has been characterized. In the immune system, CRF and CRF-R1 were so far detected at both mRNA and protein levels in several lymphoid organs and at sites of inflammation. Locally injected CRF was shown to modulate the severity of inflammation. This effect was not only a result of hemodynamic changes known to be induced by CRF or by activation of the hypothalamo-pituitary-adrenal axis, as CRF-binding sites were also found on immune cells. CRF was shown to directly modulate secretion of cytokines and neuropeptides, proliferation, chemotaxis and degranulation of purified macrophage and lymphocyte populations in vitro. Functional CRF-R was more recently demonstrated also on polymorphonuclear cells and significant amounts of CRF were shown to be produced in lymphoid organs, or delivered to lymphoid organs by peripheral nerves. Taken together, the experimental results obtained so far strongly point to the importance of CRF as a signaling molecule in lymphoid tissues and at the sites of inflammation.     

Cytotoxic Factors Released by Dengue Virus-Infected Human Blood Monocytes

Human monocyte-derived cytotoxic factors (CF) induced by dengue virus were studied. Using several human leukemia cell lines as precursors, the biological activities of CF in conditioned medium from dengue virus-infected monocytes were demonstrated through the measurement of tumor cell growth inhibition. The conditioned medium from dengue virus infected monocytes suppressed significantly growth of CEM, HL60, K562, and U937 cells. In the presence of 10% conditioned medium (v/v) from dengue virus infected monocytes, DNA synthesis of U937 cells, as measured by [³H]thymidine incorporation, decreased by 99% in contrast to their synthesis in conditioned medium from noninfected control monocytes, which did not have any suppressive effect. Partial characterization of CF showed that it is a proteinase-K-sensitive and heat-labile protein with a molecular mass over 100 kDa. Employing a flow cytometric analysis of the cell cycle, it was found that U937 cells, treated either with conditioned medium from dengue virus infected monocytes or with CF, but not treated with conditioned medium from noninfected monocytes, showed cell-cycle arrest in G1 phase by 48 hr. This suppressive effect of CF on U937 growth was dose- and time-dependent. These results suggest that dengue virus-infected monocytes may produce CF to target myeloid cells, resulting in the hematological changes observed in patients with dengue fever. © 1995 Wiley-Liss, Inc.     

炎症因子干预影响间充质干细胞的免疫调控特性

文题释义： 免疫调节的可塑性：患者免疫系统对炎症反应的应答程度,局部微环境炎症递质的种类、浓度及参与的免疫细胞都会发生不同程度的改变,此时输注间充质干细胞既可以促进免疫应答也可以抑制免疫反应。 炎症因子影响间充质干细胞免疫调节特性研究的意义：间充质干细胞的免疫调节特性是其发挥疾病治疗作用的重要机制之一,在自身免疫性疾病和多种炎症损伤性疾病的治疗中显示了良好疗效。不同炎症环境对间充质干细胞功能、特性的改变也直接影响间充质干细胞的免疫调控作用,阐明间充质干细胞、免疫细胞和炎症环境3者之间的相互作用和变化,不但有利于明确间充质干细胞的作用机制,更有利于针对不同疾病病理机制精确提供疾病治疗的新策略,尤其是有的放矢地研发免疫增强型间充质干细胞制剂,进一步提高疗效,恢复免疫平衡,提供治疗学理论基础和实验依据。 背景：间充质干细胞拥有强大的免疫调节能力,主要表现在调控免疫细胞的增殖、分化和功能状态,调节炎症因子的分泌等。间充质干细胞的免疫调节能力受炎症因子调控,依据微环境中炎症因子的种类、水平,间充质干细胞的免疫调节功能也会发生变化。 目的：综述炎症因子影响间充质干细胞免疫调控特性的研究进展。 方法：应用计算机检索PubMed数据库、Elsevier数据库、CNKI数据库,中文检索关键词或主题词为“间充质干细胞,免疫调节,可塑性,干扰素γ,转化生长因子β,白细胞介素17,白细胞介素35,前列腺素E2”,英文检索关键词为“mesenchymal stem cells,immune regulation,plasticity,interferon gamma,transforming growth factor beta,interleukin17,interleukin 35,prostaglandin E2”。纳入炎症因子干预影响间充质干细胞免疫调控特性的相关文献。 结果与结论：采用干扰素γ、转化生长因子β、白细胞介素17、白细胞介素35和前列腺素E2干预或预处理后,间充质干细胞的生物学特性随之改变,不但可重塑组织微环境、影响炎症反应,而且可重建免疫平衡,进而治疗疾病或缓解疾病进展。针对炎症、免疫反应的不同,在疾病的不同阶段进行合理的个体化、差异化治疗,都有望进一步提高间充质干细胞对免疫炎症性疾病的治疗效果。 ORCID: 0000-0003-4724-0848(王婕) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Some Characteristics of the Outer Membrane Material Released by Growing Enterotoxigenic Escherichia coli

The high-molecular-weight material released into the medium by Escherichia coli AP1, an enterotoxigenic strain of porcine origin, has been isolated and resolved into two clearly distinct fractions, based on sucrose density gradient and differential centrifugation, chemical analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and freeze-fracture electron microscopy. These two fractions, referred to as “medium vesicles” and “medium lipopolysaccharides”, were compared with the cellular outer and cytoplasmic membranes, the periplasmic fraction, and the cytoplasmic fraction. The medium vesicles closely resembled outer membrane and accounted for 3 to 5% of the total cellular outer membrane. They contained most of the heat-labile enterotoxin (LT) activity released into the medium by E. coli AP1. The medium lipopolysaccharide consisted mostly of lipopolysaccharide and a small amount of outer membrane and contained relatively little LT activity. Based on experiments with E. coli K-12 strains, in which about 5% of the newly synthesized outer membrane is lost from areas of outer membrane synthesis, it is proposed that enterotoxigenic E. coli strains release LT as part of such newly synthesized outer membrane fragments and that released outer membrane fragments may function as physiologically significant LT carriers.