Th1-skewed tissue responses to a mycolyl glycolipid in mycobacteria-infected rhesus macaques

Trehalose 6,6′-dimycolate (TDM) is a major glycolipid of the cell wall of mycobacteria with remarkable adjuvant functions. To avoid detection by the host innate immune system, invading mycobacteria down-regulate the expression of TDM by utilizing host-derived glucose as a competitive substrate for their mycolyltransferases; however, this enzymatic reaction results in the concomitant biosynthesis of glucose monomycolate (GMM) which is recognized by the acquired immune system. GMM-specific, CD1-restricted T cell responses have been detected in the peripheral blood of infected human subjects and monkeys as well as in secondary lymphoid organs of small animals, such as guinea pigs and human CD1-transgenic mice. Nevertheless, it remains to be determined how tissues respond at the site where GMM is produced. Here we found that rhesus macaques vaccinated with Mycobacterium bovis bacillus Calmette–Guerin mounted a chemokine response in GMM-challenged skin that was favorable for recruiting T helper (Th)1 T cells. Indeed, the expression of interferon-γ, but not Th2 or Th17 cytokines, was prominent in the GMM-injected tissue. The GMM-elicited tissue response was also associated with the expression of monocyte/macrophage-attracting CC chemokines, such as CCL2, CCL4 and CCL8. Furthermore, the skin response to GMM involved the up-regulated expression of granulysin and perforin. Given that GMM is produced primarily by pathogenic mycobacteria proliferating within the host, the Th1-skewed tissue response to GMM may function efficiently at the site of infection.     

Effect of oral exposure ofMycobacterium avium intracellular on the protective immunity induced by BCG

The relative protective efficacy of oral administration of mycobacteria as compared to the conventional intradermal route of vaccination has been assessed in guinea pigs. Skin test reactivity to partially purified protein derivative and protective immunity to challenge with virulentMycobacterium tuberculosis were used as parameters of protective immunity. Oral immunisation of guinea pigs either with BCG or withMycobacterium avium intracellulare induces skin test reactivity and protective immunity comparable to that induced by intradermal route of vaccination. Oral exposure ofMycobacterium avium intracellulare prior to oral or intradermal dose of BCG did not interfere with the protective immunity induced by BCG in guinea pigs challenged withMycobacterium tuberculosis H₃₇Rv.     

Different Leishmania species determine distinct profiles of immune and histopathological responses in CBA mice

Most experimental studies on leishmaniasis compare two different inbred strains of mice that are resistant or susceptible to one species of Leishmania. In the present study we characterized some cytokines and nitric oxide production as well as histological changes related to resistance and susceptibility in isogenic CBA mice infected with Leishmania major or Leishmania amazonensis. CBA mice are capable of controlling infection with L.  major, but they succumb to infection with L. amazonensis. Cells from susceptible L. amazonensis-infected CBA mice produced interleukin (IL)-4 and IL-10 but no interferon (IFN)-γ. On the other hand, resistant L. major-infected CBA mice produced IFN-γ and IL-10, but IL-4 was detected only in the first week of infection. Histopathological studies showed patterns of tissue responses at the site of the infection and in the draining lymph nodes that correlated with resistance or susceptibility. Resistant mice showed a mixed inflammatory cell infiltration and granulomas in the lesions, whereas in susceptible mice only heavily parasitized macrophages were seen. Our results indicate an important role of the parasite species in determining the pattern of immune response. L. amazonensis induces a Th2-type immune response, whereas L.  major induces a Th1-type response. These factors must be identified and taken into account in the strategies for the development of vaccines against leishmaniasis. The model presented here will be useful for the study of such factors.     

Glycerol Monomycolate Is a Novel Ligand for the Human,but Not Mouse Macrophage Inducible C-type Lectin,Mincle

An array of lipidic compounds that constitute the cell wall of mycobacteria is recognized by host receptors. Examples include trehalose dimycolate (TDM), which is a major surface-exposed glycolipid of mycobacteria, that interacts with the macrophage inducible C-type lectin, Mincle, and exerts its highly potent adjuvant functions. Recent evidence has suggested that glycerol monomycolate (GroMM), another mycolate-containing lipid species produced by mycobacteria, can stimulate innate immune cells; however, its specific host receptors have yet to be identified. We here demonstrated that cell transfectants expressing human Mincle (hMincle) reacted to both TDM and GroMM, while those expressing mouse Mincle (mMincle) only reacted to TDM and failed to recognize GroMM. Studies using domain swap chimeras confirmed that the ectodomain of hMincle, but not that of mMincle, interacted with GroMM, and site-directed mutagenesis analyses revealed that short stretches of amino acid residues at positions 174–176 and 195–196 were involved in GroMM recognition. To further substantiate the differential recognition of GroMM by hMincle and mMincle, hMincle transgenic/mMincle knock-out mice (i.e. hMincle⁺ mice) were established and compared with non-transgenic mice (i.e. mMincle⁺ mice). We showed that macrophages derived from hMincle⁺ mice were activated by GroMM and produced inflammatory cytokines, whereas those derived from mMincle⁺ mice did not exhibit any reactivity to GroMM. Furthermore, local inflammatory responses were elicited in the GroMM-injected skin of hMincle⁺, but not mMincle⁺ mice. These results demonstrated that GroMM is a unique ligand for hMincle that is not recognized by mMincle.     

禽分枝杆菌对豚鼠的致病性分析

【背景】我国禽型结核菌素(avian tuberculin)的制造用菌株为CVCC 68201、CVCC 68202和CVCC 68203株,但目前仍未明确这3株菌的生物学特性及对豚鼠致病性的情况。【目的】探究禽分枝杆菌(Mycobacterium avium)的生物学特性及对动物机体的致病性,为禽结核病和牛结核病的防控工作提供技术支撑。【方法】对3株禽分枝杆菌基因组进行鉴定分析及核酸相似度分析;用3株禽分枝杆菌分别感染豚鼠,观察感染后的临床症状、病理学变化、体重增重情况分析、皮内变态反应结果、脏器系数变化等,进而分析3株禽分枝杆菌对豚鼠的致病力。【结果】种型鉴定和进化分析结果表明,CVCC 68201、CVCC 68202和CVCC 68203均为禽分枝杆菌,基因组与Mycobacterium avium subsp. avium FDAARGOS_1608最为相近;在感染前期、中期、后期对3株禽分枝杆菌感染豚鼠的体重增重情况分析发现,感染禽分枝杆菌影响豚鼠增重,主要表现为生长迟缓,感染第5周时,CVCC 68201、CVCC 68202组豚鼠的平均体重明显轻于未感染组;皮内变态反应试验结果显示,感染CVCC 68201组豚鼠的皮肤红肿面积明显大于其他2个感染组,CVCC 68201可引起机体更为强烈的迟发型变态反应;3株禽分枝杆菌感染后,豚鼠脾脏和肺脏存在不同程度的肿大与出血,其中感染CVCC 68201豚鼠的肺脏系数与未感染组相比差异显著(P<0.01);病理学观察结果显示,豚鼠肺脏可见不同程度病变,其中CVCC 68201组更为严重,表现为肿大和轻微出血。各感染组豚鼠肺脏和脾脏组织切片抗酸染色均可见红色的分枝杆菌散在浸润。【结论】3株禽分枝杆菌对豚鼠均有一定程度的致病性,可引发局部病变。本研究为禽分枝杆菌的制备和鉴定提供依据,也为牛结核病的鉴别诊断方法研究提供参考。     

Response of Hypersensitive Mice to the Footpad Injection of Living Homologous or Heterologous Mycobacteria: Preliminary Report

Mice sensitized by the injection of viable mycobacteria into one of the hind footpads responded to a second injection of mycobacteria (3 to 4 weeks later), introduced into the contralateral foot, with a degree of footpad swelling that was both accelerated and exaggerated beyond that observed after the first inoculation. The degree of specificity of this reaction (i.e., response to homologous versus heterologous mycobacteria) was comparable to that previously reported for dermal reactions of hypersensitive guinea pigs to tuberculin or tuberculin-like antigens from mycobacteria. In preliminary studies it was impossible to achieve this state of specific sensitization by vaccinating mice subcutaneously with water-in-oil emulsions of heat-killed mycobacteria; reasons for the failure are discussed. It is suggested that this tool could prove useful in both taxonomic and immunological investigations. Advantages and disadvantages of the mouse footpad test in relation to the dermal skin test in guinea pigs are discussed.     

Delayed-type Hypersensitivity Response to Crude and Fractionated Antigens from Fonsecaea pedrosoi CMMI 1 Grown in Different Culture Media

Chromoblastomycosis is a subcutaneous fungal disease caused by dematiaceous fungi, especially by Fonsecaea pedrosoi, regarded as its major causative agent in Brazil. In recent years there has been a decline in the use of skin testing for delayed-type hypersensitivity (DTH) in epidemiological surveys of fungal infections, mainly because of the unpredictability of positive reactions and lack of specificity of the antigens used. The aim of the present study was to assess delayed-type skin tests in guinea pigs experimentally infected with F. pedrosoi using exoantigens prepared from two culture filtrates. Sixteen adult male guinea pigs were inoculated intratesticularly with fungal cells and submitted to sensitivity assays 4 weeks after inoculation. They received an intradermal injection with crude and fractionated antigens from Alviano’s and Smith’s cultures, and were assessed 24 and 48 h thereafter. Except for one animal, all of them had positive indurations after 48 h. There were no statistical differences between the measurements at 24 and 48 h for each exoantigen used, neither among the induration measurements at 48 h when different preparations were compared. Our results suggest that a delayed-type skin test using antigens produced in synthetic media may be useful for the assessment of primary exposure to chromoblastomycosis.     

The formation of delayed hypersensitivity to mycobacterial antigens

In experiments on guinea pigs and BALB/c mice delayed hypersensitivity to mycobacterial antigens was induced by the sensitization of the animals with live BCG or killed Mycobacterium bovis or M. avium in incomplete Freund's adjuvant. In the study of the dynamics of the development of skin reactivity to tuberculin some advantages of the sensitization of guinea pigs with live mycobacteria were revealed, while after the revaccination of the animals no development of secondary cell-mediated immune response was observed. The immunization of guinea pigs with atypical mycobacteria prior to their sensitization with BCG was found to lead to the development of higher skin reactivity to allergen prepared from atypical mycobacteria than skin reactivity to tuberculin.     

Life cycle and host specificity of Amblyomma triste (Acari: Ixodidae) under laboratory conditions

We report biological data of two generations of Amblyomma triste in laboratory and compared the suitability of different host species. Infestations by larval and nymphal stages were performed on guinea pigs (Cavia porcellus), chickens (Gallus gallus), rats (Rattus norvegicus), rabbits (Oryctolagus cuniculus), wild mice (Calomys callosus), dogs (Canis familiaris) and capybaras (Hydrochaeris hydrochaeris). Infestations by adult ticks were performed on dogs, capybaras and rabbits. Tick developmental periods were observed in an incubator at 27 °C and RH 90%. Guinea pigs were the most suitable hosts for larvae and nymphs, followed by chickens. The remaining host species were less suitable for immature ticks as fewer engorged ticks were recovered from them. Mean larval feeding periods varied from 3.8 to 4.7 d between different host species. Mean larval premolt periods ranged from 8.9 to 10.4 d. Nymphal mean feeding periods varied from 4.2 to 6.2 d for ticks fed on different host species. Premolt period of male nymphs (mean: 15.4 d) was significantly longer than that of female nymphs (14.7 d). Female nymphs were significantly heavier than male nymphs. The overall sex ratio of the adult ticks emerged from nymphs was 0.9:1 (M:F). Capybaras were the most suitable host for the tick adult stage as significantly more engorged females were recovered from them and these females were significantly heavier than those recovered from dogs or rabbits. The life cycle of A. triste in laboratory could be completed in an average period of 155 d. The potential role of guinea pigs, birds and capybaras, as hosts for A. triste in nature, is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Plasma membrane cholesterol plays a critical role in the Salmonella-induced anti-inflammatory response in intestinal epithelial cells

Our recent study demonstrated that a phosphatidylinositol-3 kinase (PI3K)/Akt-dependent anti-inflammatory pathway was activated by Salmonella in intestinal epithelial cells. Salmonella virulence is dependent on the ability of the bacterium to invade nonphagocytic host cells and then survive and replicate within modified Salmonella-containing vacuoles where cholesterol accumulates. In addition, cholesterol in membrane lipid rafts is frequently a platform for the activation of downstream signaling pathways, including the PI3K/Akt pathway. However, the role of plasma membrane cholesterol in the Salmonella-induced anti-inflammatory response in intestinal epithelial cells has not been elucidated. Here, we show that the effect of plasma membrane cholesterol depletion on the inhibition of Akt activation allows sustained ERK activation and the subsequent upregulation of IL-8 expression. These results demonstrate that plasma membrane cholesterol plays a critical role in the PI3K-dependent anti-inflammatory pathway activated by Salmonella in intestinal epithelial cells.     

Immunomodulatory Effects of an Enzymatic Extract from Ecklonia cava on Murine Splenocytes

We investigated whether the brown seaweed Alariaceae Ecklonia cava (E. cava) has immunological effects on splenocytes in vitro. For that purpose, we prepared an enzymatic extract from E. cava (ECK) by using the protease, Kojizyme. Here, ECK administered to ICR mice dramatically enhanced the proliferation of their splenocytes and increased the number of their lymphocytes, monocytes and granulocytes. In flow cytometry assays performed to identify in detail the specific phenotypes of these proliferating cells after ECK treatment, the numbers of CD4⁺ T cells, CD8⁺ T cells and CD45R/B220⁺ B cells increased significantly compared to those in untreated controls. In addition, the mRNA expression and production level of Th1-type cytokines, i.e., TNF-α and IFN-γ, were down-regulated, whereas those of Th2-type cytokines, i.e., IL-4 and IL-10, were up-regulated by ECK. Overall, this dramatic increase in numbers of splenocytes indicated that ECK could induce these cells to proliferate and could regulate the production of Th1- as well as Th2-type cytokines in immune cells. These results suggest that ECK has the immunomodulatory ability to activate the anti-inflammatory response and/or suppress the proinflammatory response, thereby endorsing its usefulness as therapy for diseases of the immune system. Y-J. Jeon and Y. Jee contributed equally to this study.     

Mycobacterium tuberculosislpdC, Rv0462, induces dendritic cell maturation and Th1 polarization

Mycobacterium tuberculosis, the etiological factor of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). In this study, we demonstrated that the gene encoding lipoamide dehydrogenase C (lpdC) from M. tuberculosis, Rv0462, induce maturation and activation of DCs involved in the MAPKs signaling pathway. Moreover, Rv0462-treated DCs activated naïve T cells, polarized CD4⁺ and CD8⁺ T cells to secrete IFN-γ in syngeneic mixed lymphocyte reactions, which would be expected to contribute to Th1 polarization of the immune response. Our results suggest that Rv0462 can contribute to the innate and adaptive immune responses during tuberculosis infection, and thus modulate the clinical course of tuberculosis.     

Interactions between the tomato spotted wilt virus movement protein and plant proteins showing homologies to myosin, kinesin and DnaJ-like chaperones

The non-structural protein encoded by the M RNA segment (NSm) of tomato spotted wilt virus (TSWV) has been implicated in cell-to-cell movement of nucleocapsids through modified plasmodesmata. Recently, DnaJ-like proteins from Nicotiana tabacum (tobacco) and Arabidopsis thaliana have been identified as NSm interacting host proteins, implying an involvement of molecular chaperones during systemic spread of the virus or other, presently unknown NSm-mediated virus functions. Examination of additional TSWV host plants and improvement of yeast two-hybrid interaction trap experiments led to the isolation of a DnaJ-like protein from Lycopersicon esculentum (tomato) and the identification of a protein from A. thaliana sharing some homologies with myosin and kinesin-like polypeptides. Sequence alignments of the tomato DnaJ-like protein unveiled the corresponding gene as an orthologue to the tobacco and A. thaliana DnaJ genes, substantiating that NSm interacting DnaJ-like polypeptides, identified from three different TSWV host species, apparently form a subgroup distinct from archetypical DnaJ chaperones. Increased levels of DnaJ-like proteins could be detected in TSWV systemically infected leaves and in plants exposed to heat shock, showing that the NSm interacting DnaJ-like chaperones are inducible upon biotic and abiotic stress. All together, the identification of DnaJ-like proteins and a protein resembling myosin and kinesin as NSm interacting plant proteins is in accordance with results accomplished for movement proteins from other plant attacking viruses showing an involvement of molecular chaperones and the cytoskeleton in at least intracellular trafficking.     

杜鹃花属映山红亚属植物叶表皮显微形态特征及其分类学意义

为澄清映山红亚属(Rhododendron subg. Tsutsusi)内的系统关系问题,该研究选择杜鹃花属映山红亚属37种植物作为对象,其中29种为首次报道,通过扫描电镜观察其叶表皮显微特征。结果表明:(1)气孔器均为无规则型且均在远轴面。(2)根据叶片微形态特征将映山红亚属的种类分为杜鹃型(R. simisii-type)、岭南杜鹃型(R. mariae-type)、皋月杜鹃型(R. indicum-type)、崖壁杜鹃型(R. saxatile-type)及丁香杜鹃型(R. farrerae-type)5种类型。(3)杜鹃型植物的气孔器周边无或具间断的条形突起。(4)岭南杜鹃型植物的气孔器周围有多层条形突起环绕,保卫细胞两极不具T型加厚。(5)皋月杜鹃型植物的叶表皮保卫细胞两极具有T型加厚,与叶状苞亚属(Subg. Therorhodion)叶状苞杜鹃(R. redowskianum)的气孔器特征相似,推测其与叶状苞亚属具有一定的亲缘关系。(6)崖壁杜鹃型植物的叶表皮毛单一,多呈卷曲状,与其他类型有所不同且未见腺体。(7)丁香杜鹃型植物的叶表皮仅有腺体。(8)依据叶表皮微形态特征讨论了一些近缘种类的关系,如倾向于将背绒杜鹃(R. hypoblematosum)和千针叶杜鹃(R. polyraphidoideum)处理为独立的种,支持保留紫薇春(R. naamkwanense var. cryptonerve)作为南昆杜鹃(R. naamkwanense)的变种地位,不支持将腺花杜鹃(R. adenanthum)并入细瘦杜鹃(R. tenue)作异名处理等。该研究结果表明叶表皮显微特征在映山红亚属内物种划分上具有重要价值。     

Chrysosporium tropicum as a probable cause of mycosis of poultry in India

Chrysosporium tropicum was isolated from comb lesions in two different breeds of chickens in India and subcultures were shown to be pathogenic when inoculated onto prepared skin of guinea pigs. This report provides additional evidence to considerCh. tropicum as a pathogenic fungus and a probable cause of a dermatomycosis in chickens.     

Immunogenicity,Protective Efficacy,and Non-Replicative Status of the HSV-2 Vaccine Candidate HSV529 in Mice and Guinea Pigs

HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine.     

Induced sensitization to nickel in guinea pigs immunized with mycobacteria by injection of purified protein derivative with nickel

Nickel has been reported to be one of the most common causes of allergic contact dermatitis. Despite the fact that nickel is a frequent sensitizer in humans, establishing animal models for nickel allergy has met with considerable difficulties. In clinical cases, allergic contact hypersensitivity to nickel develops much more readily in inflamed skin than normal skin. In this study, we tried to induce nickel sensitization when inflammation has been evoked in guinea pigs immunized with mycobacteria followed by co-administration of a mycobacterial component with nickel. We first examined the delayed-type hypersensitivity (DTH) reaction of mycobacterial components such as the cell wall, cell membrane, 70S ribosomal fraction, cytoplasm, tuberculin purified protein derivative (PPD), RNA and DNA from Mycobacterium bovis BCG in guinea pigs immunized with live M. bovis BCG or heat killed M. tuberculosis. When PPD was used, the hypersensitivity reaction was strongest. Next, we tested whether PPD with nickel could induce nickel sensitivity in guinea pigs immunized with mycobacteria. Strong sensitization to nickel was achieved by injecting PPD with nickel. However, if too large an amount of PPD or nickel salts was used, sensitization to nickel decreased. In this way, sensitization of nickel developed much more easily in guinea pigs immunized with mycobacteria by injection of an appropriate amount of nickel at the inflammation site induced by a suitable amount of PPD.     

Mycobacterial induction of autophagy varies by species and occurs independently of mammalian target of rapamycin inhibition

The interaction of host cells with mycobacteria is complex and can lead to multiple outcomes ranging from bacterial clearance to latent infection. Although many factors are involved, the mammalian autophagy pathway is recognized as a determinant that can influence the course of infection. Intervention aimed at utilizing autophagy to clear infection requires an examination of the autophagy and signal transduction induced by mycobacteria under native conditions. With both pathogenic and non-pathogenic mycobacteria, we show that infection correlates with an increase in the mammalian target of rapamycin (mTOR) activity indicating that autophagy induction by mycobacteria occurs in an mTOR-independent manner. Analysis of Mycobacterium smegmatis and Mycobacterium bovis bacille Calmette-Guérin (BCG), which respectively induce high and low autophagy responses, indicates that lipid material is capable of inducing both autophagy and mTOR signaling. Although mycobacterial infection potently induces mTOR activity, we confirm that bacterial viability can be reduced by rapamycin treatment. In addition, our work demonstrates that BCG can reduce autophagy responses to M. smegmatis suggesting that specific mechanisms are used by BCG to minimize host cell autophagy. We conclude that autophagy induction and mTOR signaling take place concurrently during mycobacterial infection and that host autophagy responses to any given mycobacterium stem from multiple factors, including the presence of activating macromolecules and inhibitory mechanisms.