Lymphoid tissues induce NGF-dependent and NGF-independent neurite outgrowth from rat superior cervical ganglia explants in culture

Induction of neurite outgrowth from superior cervical ganglia (SCG) by rat lymphoid tissues was studied using a tissue culture model. Neonatal rat SCG were cultured with 6–12-week-old rat thymus, spleen, or mesenteric lymph node (MLN) explants in a Martrigel layer, in defined culture medium without exogenous nerve growth factor (NGF). SCG were also co-cultured with neonatal rat heart (as positive control) or spinal cord (SC; as negative control). To determine whether inflammation affects the ability of lymphoid tissues to induce neurite outgrowth, we also examined MLN at various times after infecting rats with Nippostrongylus brasiliensis (Nb-MLN). In one series of experiments, a single lymphoid tissue explant was surrounded by four SCG at a distance of 1 mm. The extent of neurite outgrowth was determinded by counting the number of neurites 0.5 mm away from each ganglion at several time points. Adult thymus and, to a lesser extent, spleen had strong stimulatory effects on neurite outgrowth from SCG after 12 hr or more in culture. For thymus tissue, this was similar to the positive control heart explants. MLN from normal rats had minimal effect on neurite outgrowth; however, Nb-MLN showed a time-dependent enhancement of the neurite outgrowth, maximal at 3 weeks after infection. The relative efficacy of neurite outgrowth induction (heart ≥ thymus ≥ Nb-MLN ≥ spleen ≥ MLN ≥ SC) was confirmed in a second series of experiments where one SCG was surrounded by three different tissue explants. We then examined the role of 2.5S NGF, a well-known trophic factor for sympathetic nerves, in the lymphoid tissue-induced neurite outgrowth. Anti-NGF treatment of co-cultures of SCG and heart almost completely blocked the neurite outgrowth. Anti-NGF also significantly inhibited thymus- and spleen-induced neurite outgrowth, but not as effectively as heart-induced neuritogenesis (93,80, and 77% inhibition at 24 hr; 86,70, and 68% inhibition at 48 hr for heart, thymus, and spleen, respectively). On the other hand, anti-NGF inhibited only 8% of neurite outgrowth induced by 3-week post-infection Nb-MLN at 24 hr, and 41% at 48 hr. These data show that several adult rat lymphoid tissues exert neurotrophic/tropic effects. The predominant growth factor in thymus and spleen is NGF, while Nb-MLN produces factor(s) which is (are) immunologically distinguishable from NGF. These neurotrophic/tropic factors are produced during the reactive lymphoid hyperplasia that forms part of the inflammatory response against the nematode, N. brasiliensis. This suggests the possibility that cytokines produced by lymphocytes or other inflammatory cells may stimulate sympathetic neurite outgrowth in vivo. © 1994 Wiley-Liss, Inc.     

Anorexia Induced by the Parasitic Nematode, Nippostrongylus brasiliensis: Effects on NPY and CRF Gene Expression in the Rat Hypothalamus

Infections of the gastrointestinal nematode, Nippostrongylus brasiliensis, in the laboratory rat result in a characteristic biphasic anorexia which is followed by hyperphagia once the worm burden has been cleared. Despite the importance of parasite-induced anorexia, relatively little is known of the underlying mechanisms. We have investigated the involvement of the central appetite drive in this anorexia by studying the gene expression of two neuropeptides with opposing actions on energy balance, NPY and CRF. Gene expression was assessed by in situ hybridization at 2, 8 and 16 days post-infection (p.i.) in infected rats, in uninfected controls, and in a group with food intake restricted to match that taken voluntarily by the parasitized animals. The sampling intervals corresponded to each of the two phases of maximum anorexia and the period of compensatory hyperphagia. Surprisingly, we found that increases in NPY gene expression in the hypothalamic arcuate nucleus (ARC) accompany anorexia in rats infected with N. brasiliensis; there was a significant relationship between degree of anorexia and induction of NPY mRNA after 8 days of infection. Furthermore, ARC NPY mRNA levels in parasitized animals were similar to those in pair-fed individuals with food intake restricted to match the infected rats. The number of larvae used to establish the infection affected both the degree of anorexia and the level of NPY mRNA at 8 days p.i. in a dose-dependent manner. NPY gene expression remained elevated in infected rats during at least the initial stages of compensatory hyperphagia. This suggests that animals detect a state of energy deficit during the early stages of the infection, yet do not feed, but become hyperphagic coincident with worm loss. The failure of anorectic parasitized animals to feed in response to activation of the NPYergic system makes this a novel system in which to study the regulation of hypothalamic NPY by physiological challenge. There were no significant differences in CRF gene expression between the groups at any of the sampling intervals.     

Dissociation of specific and total IgE antibody responses following repeated low-level infections with Nippostrongylus brasiliensis in rats.

IgE, IgG and mast cell responses were studied in rats infected weekly with 10 larvae of Nippostrongylus brasiliensis (NB). Worm recovery at 8 weeks of repeated infections was six-fold greater than that of a single infection with 10 larvae, suggesting the accumulation of worms during the repeated infections. Total serum IgE was increased after 2 weeks of infection, and further increased after repeated infections: at 6 weeks of infection the level was four to six times higher than that after a single infection. Anti-NB IgG1 levels were also significantly higher after repeated infections than after a single infection. On the other hand, there was no significant difference in the level of anti-NB IgE between single and repeated infections, as determined by ELISA, as well as by passive cutaneous anaphylaxis (PCA) reaction. Mastocytosis was induced in the small intestine after both single and repeated infections, but the levels did not differ between the two. These results indicate that total IgE and specific IgG1 production are augmented by repeated helminth infections, but specific IgE and mast cell responses are not. This pattern of response may minimize the development of IgE-dependent hypersensitivity reactions with repeated helminth infections.     

Effects of latex membrane on guided regeneration of long bones

Abstract

Natural latex extracted from Hevea brasiliensis is one of the materials pointed out as potential tissue regenerators. The use of latex-based membranes in bone regeneration might be an alternative to stimulate bone formation. The aim of this study was to evaluate the effects of latex membranes in guided bone regeneration of defects produced in long bones of rats. Sixty rats were equally divided into latex and control groups, and each group was subdivided into two subgroups according to treatment duration of 1 and 4 weeks. Bone defects with 2.5?mm in diameter were surgically made in the left tibia. In the animals of the latex group, a latex membrane was placed over the bone defect. The samples underwent quantitative histological analysis of bone formation and collagen matrix, immunohistochemical analysis of osteogenic protein markers, assessment of bone mechanical properties and bone densitometry, and radiological assessment. The osteocalcin immunostaining data were submitted to the generalized linear model test with two independent factors. For the other data, the multivariate ANOVA with two independent factors was performed. The use of the latex membrane significantly improved (p?<?0.005) the volume of newly formed bone, collagen type I matrix, expression of osteopontin, and bone stiffness, both in the early and late stages of regeneration. In conclusion, the latex membrane was able to promote bone regeneration in long bones.     

The detection and measurement of coproantibodies to Nippostrongylus brasiliensis in rats following a primary infection

Summary Investigations were initiated to study the possible detection and measurement of coproantibodies in animals infected with a gastrointestinal nematode parasite. Faecal extracts, extracts of small intestinal mucosa and sera of rats infected with intestinal nematode Nippostrongylus brasiliensis were examined for total IgA, IgM and IgG levels and haemagglutinating and precipitating antibodies specific to parasite antigens over a 30-day-period following infection. It was found that in both faecal and mucosal extracts immunoglobulin concentrations increased after a primary infection. In faecal extracts there was a seven-fold increase of IgA, a three to six-fold increase of IgG and about a fifty-fold increase of IgM. Haemagglutinins in faecal extracts detected by adult worm excretory-secretory (ES) products and adult worm and infective larvae somatic extracts were observed from 3 days after infection (DAI). Haemagglutinins detected by ES products reached their highest titres on 11–12 DAI while those reacting with adult worm somatic extracts showed the highest level between IS and 19 DAI. A similar pattern of response was found in the antibody levels of the intestinal mucosa. Haemagglutinins detected in faeces during the first 12 DAI reacted with the same antigens as antibodies present in the sera at that time but coproantibodies from 18, 24 and 30 DAI were different from those circulating in sera at that stage of the infection. The results suggest that measurement of coproantibody levels may provide a convenient and useful index of local immune responses to gastrointestinal helminths.     

Persistent infection of Strongyloides venezuelensis and normal expulsion of Nippostrongylus brasiliensis in Mongolian gerbils, Meriones unguiculatus, with reference to the cellular responses in the intestinal mucosa

The kinetics of daily faecal egg count, worm burdens, and intestinal cellular responses were examined in Mongolian gerbils after infection with either Strongyloides venezuelensis or Nippostrongylus brasiliensis alone, or concurrently with both parasites. The results show that, both in individual and concurrent infections, S. venezuelensis infection persisted for over 10 weeks and elicited a gradual increase in number of mast cells in the jejunal mucosa. On the other hand, N. brasiliensis worms were expelled by 3 weeks in association with goblet cell hyperplasia. These results suggest that effector/regulator cells involved in worm expulsion are different and highly selective depending on the genus of intestinal helminths.     

Histopathological observations in new and classic models of experimental Schistosoma haematobium infections

The authors present marsh rat Holochilus brasiliensis, jirds Meriones shawi and M. unguiculatus as new models of Schistosoma haematobium infection. Histological findings were compared with those of classic models mouse Mus and hamster Mesocricetus. In new models, embryonated eggs were seen in the stool from 90 days post infestation (DPI) and active disease developed from 117 to 175 DPI. Seven out of 10 rodents presented granulomatous and/or chronic cystitis, fibrosis, polyps and urothelial changes: squamous metaplasia, precancerous dysplasia and squamous cell carcinoma of the urinary bladder. In the digestive tract of all new models, granulomas eroded the mucosa, formed inflammatory polyps, infiltrated the wall and accumulated into bilharziomas. In the liver, granulomatous hepatitis surrounded by bilharzial pigment deposit was apparent. Pipe-stem fibrosis involved 4 rodents with precirrhotic changes in 1 and portal hypertension in 2. One female Meriones suffered from granulomatous endometritis and salpingitis. All new models developed pulmonary granulomatosis with associated vascular lesions: giant cell arteritis in 1 rodent, thromboses in 3 and pulmonary hypertension in 4 others. In classic models, 1 Mus presented a squamous cell carcinoma of the urinary bladder while Mesocricetus displayed diverse lesions in digestive and genital tracts, liver and lungs. All tissue lesions, resembling those seen in humans in all points, were far more frequent and severe in new models than in classic ones. Those involving the urinary bladder have never been reported in other models such as monkeys: Pan troglodytes, Cercopithecus aethiops and Cebus apella. A comparison was carried out between different models on the basis of experimental conditions: definitive hosts, number of cercariae used, type and duration of infection. This study clearly demonstrated that Holochilus brasiliensis, Meriones shawi and M. unguiculatus are perfectly adequate models in terms of laboratory facilities. They are helpful in investigating the pathogenic mechanism of some disorders in S. haematobium infection, particularly tumours of the urinary bladder, and this may enhance therapeutic assays.     

IL‐25 exhibits disparate roles during Th2‐cell differentiation versus effector function

A keenly sought therapeutic approach for the treatment of allergic disease is the identification and neutralization of the cytokine that regulates the differentiation of T helper 2 (Th2) cells. Th2 cells are exciting targets for asthma therapies. Recently, the cytokine IL‐25 has been shown to enhance Th2‐type immune activity and play important roles in mediating allergic inflammatory responses. To investigate this further, we crossed IL‐25^?/? C57BL/6 mice with G4 IL‐4 C57BL/6 reporter mice and developed an assay for in vitro and in vivo IL‐4‐independent Th2‐cell differentiation. These assays were used to determine whether IL‐25 was critical for the formation of Th2 cells. We found there was no physiological role for IL‐25 in either the differentiation of Th2 cells or their development to effector or memory Th2‐cell subsets. Importantly, this data challenges the newly found and growing status of the cytokine IL‐25 and its proposed role in promoting Th2‐cell responses.     

Interactions between macrophages and helminths

Macrophages, the major population of tissue-resident mononuclear phagocytes, contribute significantly to the immune response during helminth infection. Alternatively activated macrophages (AAM) are induced early in the anti-helminth response following tissue insult and parasite recognition, amplifying the early type 2 immune cascade initiated by epithelial cells and ILC2s, and subsequently driving parasite expulsion. AAM also contribute to functional alterations in tissues infiltrated with helminth larvae, mediating both tissue repair and inflammation. Their activation is amplified and occurs more rapidly following reinfection, where they can play a dual role in trapping tissue migratory larvae and preventing or resolving the associated inflammation and damage. In this review, we will address both the known and emerging roles of tissue macrophages during helminth infection, in addition to considering both outstanding research questions and new therapeutic strategies.     

巴西日圆线虫虫体蛋白体外诱导THP-1来源巨噬细胞极化的研究

目的　分析巴西日圆线虫虫体蛋白体外诱导人单核细胞白血病THP?1细胞来源巨噬细胞的极化方向,探索机体对巴西日圆线虫感染的免疫应答机制。方法　建立并维持巴西日圆线虫培养的实验室循环,无菌条件下收集L3和L5虫体,分别制备虫体蛋白;建立THP?1细胞体外培养体系,经500 ng/mL佛波酯（PMA）刺激培养后呈贴壁状态的M0型细胞分别采用500 ng/mL脂多糖（LPS）+ 100 ng/mL γ?干扰素（IFN?γ）、白细胞介素4（IL?4）+ IL?13（浓度均为100 ng/mL）、L3及L5虫体蛋白（浓度均为5 mg/mL）进行刺激,同时设不加任何刺激的阴性对照组。通过显微镜镜检观察刺激后细胞形态、实时荧光定量PCR（qPCR）检测M1/M2型巨噬细胞特异性基因mRNA表达水平、流式细胞术检测巨噬细胞表面标志物及酶联免疫吸附试验（ELISA）检测M1/M2型巨噬细胞分泌的细胞因子含量,观察巴西日圆线虫虫体蛋白体外诱导THP?1来源巨噬细胞的极化方向。结果　THP?1细胞经PMA刺激培养呈贴壁的M0型细胞后,经LPS + IFN?γ刺激培养后呈特征性M1型极化;经IL?4 + IL?13刺激培养后呈特征性M2型极化;经L3和L5蛋白刺激培养后均呈特征性M2型极化。阴性对照组、LPS + IFN?γ刺激组、IL?4 + IL?13刺激组、L3蛋白刺激组、L5蛋白刺激组间M1型巨噬细胞比例差异有统计学意义（[χ2] = 3 721.00,P < 0.001）,其中LPS + IFN?γ刺激组M1型巨噬细胞比例最高;各组间M2型巨噬细胞比例差异有统计学意义（[χ2] = 105.43,P < 0.001）。各组间C?C基序趋化因子配体2（CCL2）、肿瘤坏死因子α（TNF?α）、IL?12b、过氧化物酶体增殖物激活受体γ（PPARγ）、IL?10、甘露糖受体C型1（Mrc1）基因mRNA表达水平差异均有统计学意义（F = 191.95、129.95、82.89、11.30、9.51、12.35,P均< 0.001）,各组间CD86、CD206阳性率差异均有统计学意义（[χ2] = 24 004.33、832.50,P均< 0.001）。LPS + IFN?γ刺激组IL?1β、TNF?α表达水平均显著高于IL?4 + IL?13刺激组、L3蛋白刺激组及L5蛋白刺激组（P均< 0.001）;IL?4 + IL?13刺激组、L3蛋白刺激组和L5蛋白刺激组TGF?β1、IL?10表达水平均显著高于阴性对照组和LPS + IFN?γ刺激组（P均< 0.05）。结论　巴西日圆线虫L3和L5虫体蛋白体外均可诱导THP?1来源巨噬细胞向M2型极化。