Proliferation of Pneumocystis Trophozoites in Human Lymph Nodes and in Nude Mice Lungs

Pneumocystis infection in athymic nude mice lungs showed a particularly high trophozoite to cyst ratio. A similar observation was obtained from a study of a patient with lymph node infection with Pneumocystis . Eosinophilic foamy masses in these sites were observed by light microscopy. With the electron microscope, the masses were seen to be composed of large aggregates of trophozoites. Cystic forms (precysl, cyst and empty cyst) were extremely scarce in comparison with the huge numbers of trophozoites. These cystic forms were mostly undergoing degeneration. These observations indicate that the mode of proliferation in both situations was predominantly asexual, that is, proliferation by trophozoites, suggesting that certain conditions may enhance asexual reproduction or depress the fonnation of cysts.     

Intratracheal Instillation of High Dose Adenoviral Vectors Is Sufficient to Induce Lung Injury and Fibrosis in Mice

Rationale

Replication deficient adenoviruses (Ad) vectors are common tools in gene therapy. Since Ad vectors are known to activate innate and adaptive immunity, we investigated whether intratracheal administration of Ad vectors alone is sufficient to induce lung injury and pulmonary fibrosis.

Methods

We instilled Ad viruses ranging from 10⁷ to 1.625×10⁹ ifu/mouse as well as the same volume of PBS and bleomycin. 14 and 21 days after administration, we collected bronchoalveolar lavage fluid (BALF) and mouse lung tissues. We measured the protein concentration, total and differential cell counts, and TGF-β1 production, performed Trichrome staining and Sircol assay, determined gene and protein levels of profibrotic cytokines, MMPs, and Wnt signaling proteins, and conducted TUNEL staining and co-immunofluorescence for GFP and α-SMA staining.

Results

Instillation of high dose Ad vectors (1.625×10⁹ ifu/mouse) into mouse lungs induced high levels of protein content, inflammatory cells, and TGF-β1 in BALF, comparable to those in bleomycin-instilled lungs. The collagen content and mRNA levels of Col1a1, Col1a2, PCNA, and α-SMA were also increased in the lungs. Instillation of both bleomycin and Ad vectors increased expression levels of TNFα and IL-1β but not IL-10. Instillation of bleomycin but not Ad increased the expression of IL-1α, IL-13 and IL-16. Treatment with bleomycin or Ad vectors increased expression levels of integrin α1, α5, and αv, MMP9, whereas treatment with bleomycin but not Ad vectors induced MMP2 expression levels. Both bleomycin and Ad vectors induced mRNA levels of Wnt2, 2b, 5b, and Lrp6. Intratracheal instillation of Ad viruses also induced DNA damages and Ad viral infection-mediated fibrosis is not limited to the infection sites.

Conclusions

Our results suggest that administration of Ad vectors induces an inflammatory response, lung injury, and pulmonary fibrosis in a dose dependent manner.     

静脉注射和气管内滴入博莱霉素诱导小鼠肺纤维化的差异

目的 探讨静脉注射和气管内滴入博莱霉素(BLM)对小鼠肺纤维化形成的差异.方法 8周龄雌性C57BL/6小鼠40只,随机分为静脉注射组(V组)20只、气管内滴入组(I组)20只,分别经尾静脉一次性注射BLM 150 mg/kg和气管内滴入BLM 5 mg/kg.观察每组小鼠生存率、肺组织病理改变及肺组织羟脯胺酸的含量.结果 ① V组和I组小鼠的生存率分别为50%和75%,两者间统计学无显著性差异(P>0.05).②注射处置后28 d,两组小鼠均形成广泛、稳定的间质纤维化病理改变,但I组主要分布在肺门和支气管周围,而V组主要分布在胸膜下及血管周围.肺纤维化病理评分I组与V组之间无显著的统计学差异(P>0.05).③小鼠注射处置后28 d,V组与I组肺羟脯氨酸含量分别为634.4±67.1 μg/g和696.6±41.2 μg/g,两组间无显著的统计学差异(P>0.05).结论 利用BLM静脉注射和气管内滴入制备肺间质纤维化动物模型,纤维化形成的部位存在着一定的差异,而肺间质形成纤维化的程度和病理改变大致相同.     

Changes in the Bacterial Microbiota in Gut,Blood, and Lungs following Acute LPS Instillation into Mice Lungs

Introduction

Previous reports have shown that the gastrointestinal (GI) bacterial microbiota can have profound effects on the lungs, which has been described as the “gut-lung axis”. However, whether a “lung-gut” axis exists wherein acute lung inflammation perturbs the gut and blood microbiota is unknown.

Methods

Adult C57/Bl6 mice were exposed to one dose of LPS or PBS instillation (n = 3 for each group) directly into lungs. Bacterial microbiota of the bronchoalveolar lavage fluid, blood, and cecum were determined using 454 pyrotag sequencing and quantitative polymerase chain reaction (qPCR) at 4 through 168 hours post-instillation. We then investigated the effects of oral neomycin and streptomycin (n = 8) on the microbiota at 4 and 24 hours post LPS instillation versus control treatment (n = 5 at baseline and 4 hours, n = 7 at 24 hours).

Results

At 24 hours post LPS instillation, the total bacterial count was significantly increased in the cecum (P<0.05); whereas the total bacterial count in blood was increased at 4, 48, and 72 hours (P<0.05). Antibiotic treatment reduced the total bacteria in blood but not in the cecum. The increase in total bacteria in the blood correlated with Phyllobacteriaceae OTU 40 and was significantly reduced in the blood for both antibiotic groups (P<0.05).

Conclusion

LPS instillation in lungs leads to acute changes in the bacterial microbiota in the blood and cecum, which can be modulated with antibiotics.     

Repeated Intratracheal Instillation of PM10 Induces Lipid Reshaping in Lung Parenchyma and in Extra-Pulmonary Tissues

Adverse health effects of air pollution attributed mainly to airborne particulate matter have been well documented in the last couple of decades. Short term exposure, referring to a few hours exposure, to high ambient PM10 concentration is linked to increased hospitalization rates for cardiovascular events, typically 24 h after air pollution peaks. Particulate matter exposure is related to pulmonary and cardiovascular diseases, with increased oxidative stress and inflammatory status. Previously, we have demonstrated that repeated intratracheal instillation of PM10sum in BALB/c mice leads to respiratory tract inflammation, creating in lung a condition which could potentially evolve in a systemic toxic reaction. Additionally, plasma membrane and tissue lipids are easily affected by oxidative stress and directly correlated with inflammatory products. With this aim, in the present investigation using the same model, we analyzed the toxic potential of PM10sum exposure on lipid plasma membrane composition, lipid peroxidation and the mechanisms of cells protection in multiple organs such as lung, heart, liver and brain. Obtained results indicated that PM10 exposure led to lung lipid reshaping, in particular phospholipid and cholesterol content increases; concomitantly, the generation of oxidative stress caused lipid peroxidation. In liver we found significant changes in lipid content, mainly due to an increase of phosphatidylcholine, and in total fatty acid composition with a more pronounced level of docosahexaenoic acid; these changes were statistically correlated to lung molecular markers. Heart and brain were similarly affected; heart was significantly enriched in triglycerides in half of the PM10sum treated mice. These results demonstrated a direct involvement of PM10sum in affecting lipid metabolism and oxidative stress in peripheral tissues that might be related to the serious systemic air-pollution effects on human health.     

Plant Chromosome Analysis and Sorting by Flow Cytometry

During the past decade, significant progress has been made in the development of methods for the preparation of plant chromosome suspensions suitable for flow cytometric analysis. In addition to successful classification of chromosomes (flow karyotyping), sorting of single chromosome types with a high degree of purity was reported in several plant species. Sorted chromosomes were used for the establishment of chromosome-specific DNA libraries and for gene mapping. The results confirmed the potential of plant flow cytogenetics and form a solid basis for further progress in this area. This article reviews its current status, analyzes major problems, and assesses future directions.     

用流式细胞仪检测大黄鱼三倍体

通过对大黄鱼二倍体和三倍体的倍性分析,建立流式细胞仪检测三倍体的方法。大黄鱼受精卵经三倍体诱导处理后,胚胎期进行染色体滴片证实在处理组中有三倍体细胞存在。接着对该组胚胎进行育苗,获得1￣3cm的鱼苗,用流式细胞仪进行检测。以二倍体大黄鱼的肌肉组织或血液细胞DNA含量的峰值道数作为对照,用同样的方法取样处理、上机、测定处理组样本个体细胞的DNA含量的峰值道数。如果处理组个体细胞的DNA含量的峰值道数是二倍体组的1.5±0.1倍,则认为该个体为三倍体。实验结果经冷休克或静水压诱导处理的样本共检测182个,三倍体检出率为12.09%,其中有一组检出率高达55.56%。     

Age-Dependent Cell Trafficking Defects in Draining Lymph Nodes Impair Adaptive Immunity and Control of West Nile Virus Infection

Impaired immune responses in the elderly lead to reduced vaccine efficacy and increased susceptibility to viral infections. Although several groups have documented age-dependent defects in adaptive immune priming, the deficits that occur prior to antigen encounter remain largely unexplored. Herein, we identify novel mechanisms for compromised adaptive immunity that occurs with aging in the context of infection with West Nile virus (WNV), an encephalitic flavivirus that preferentially causes disease in the elderly. An impaired IgM and IgG response and enhanced vulnerability to WNV infection during aging was linked to delayed germinal center formation in the draining lymph node (DLN). Adoptive transfer studies and two-photon intravital microscopy revealed a decreased trafficking capacity of donor naïve CD4⁺ T cells from old mice, which manifested as impaired T cell diapedesis at high endothelial venules and reduced cell motility within DLN prior to antigen encounter. Furthermore, leukocyte accumulation in the DLN within the first few days of WNV infection or antigen-adjuvant administration was diminished more generally in old mice and associated with a second aging-related defect in local cytokine and chemokine production. Thus, age-dependent cell-intrinsic and environmental defects in the DLN result in delayed immune cell recruitment and antigen recognition. These deficits compromise priming of early adaptive immune responses and likely contribute to the susceptibility of old animals to acute WNV infection.     

Distribution of Mast Cells in Mediastinal Lymph Nodes from Lung Cancer Patients

Background  

Mast cells have been documented to have several key functions with regards to malignant neoplasms. However, the functional significance of their accumulation is largely unknown. An analysis of the mast cell profile in mediastinal lymph nodes from lung cancer patients is reported here.     

Volumetric Instillation of Fixatives and Inert Substances Into Mouse Lungs

Mouse lungs were instilled with various fixatives to establish the optimum volume necessary to fix the lung without distortion and to compare the efficacy of the fixatives. Fixation with either Stieve's or Bouin's fluid was found preferable to 2.5% and 5% glutaraldehyde, 4% neutral buffered formalin, and to a mixture of formalin and Stieve's fixative. In addition, a comparison was made between diluted Ames O.C.T. Compound and 4% aq. gelatin as supportive substances for unfixed lungs in preparation of cryostat sections and for histochemistry. A 1:2 dilution of O.C.T. was found to be superior to 4% gelatin in preparative, cutting and adhesive properties. The optimal instilled volume for mouse lungs was found to be 0.1 ml for every 7 grams of body weight, introduced at a rate of 0.1 ml per 10 seconds.     

Dielectric Properties of Normal and Metastatic Lymph Nodes Ex Vivo From Lung Cancer Surgeries

The dielectric properties of normal and tumor human tissues have been widely reported in recent years. However, the dielectric properties of intrathoracic lymph nodes (LNs) have not been reported. In this communication, we measured the dielectric properties (i.e., permittivity and conductivity) of ex vivo intrathoracic LNs obtained from lung cancer surgeries. Results show that the permittivity and conductivity of metastatic LNs are higher than those of normal LNs over the frequency range of 1 MHz–4 GHz. Statistically significant differences are observed at single specific frequencies (64, 128, 298, 433, and 915 MHz and 2.45 GHz). Our study provides the basic data to support future-related research and fills the research gap on the dielectric properties of LNs in the lungs. Bioelectromagnetics. 2020;41:148–155. © 2020 Bioelectromagnetics Society.     

Analysis of Coccidian Oocyst Populations by Means of Flow Cytometry

Flow cytometry was employed as a tool to analyze and characterize batches of oocysts from laboratory and field isolates of Eimeria spp. from chickens and to propagate sub-populations of batches of oocysts. Oocyst batches were cleaned of debris by a combination of salt flotation, washing and treatment with dilute sodium hypochlorite (1.5% aqueous). Oocyst size and shape were registered by forward-angle light scatter with the argon laser excitation set at 488 nm at 300 mW. Sub-populations of oocysts were collected by map gating and used for microscopy or for propagation. The profile of particle size was characteristic for each species. Propagation of sub-populations of oocysts of specified sizes resulted in cultures of coccidia that were pure species or nearly pure species. The small size of E. mills caused difficulty in separation from the remaining fine debris. This technique was useful for studying the variation in oocyst size within populations and characterization of field isolates of mixed species. Propagation of pure species from mixed isolates by bit-map gating had the same limitations as micromanipulation because of the overlapping size of Eimeria spp. Chancaerization is further limited by the lack of suitable size/shape standards for flow cytometry-Key words. Cell sorter. Eimeria spp., oocyst shape, oocyst size.     

Quantitative,Architectural Analysis of Immune Cell Subsets in Tumor-Draining Lymph Nodes from Breast Cancer Patients and Healthy Lymph Nodes

Background

To date, pathological examination of specimens remains largely qualitative. Quantitative measures of tissue spatial features are generally not captured. To gain additional mechanistic and prognostic insights, a need for quantitative architectural analysis arises in studying immune cell-cancer interactions within the tumor microenvironment and tumor-draining lymph nodes (TDLNs).

Methodology/Principal Findings

We present a novel, quantitative image analysis approach incorporating 1) multi-color tissue staining, 2) high-resolution, automated whole-section imaging, 3) custom image analysis software that identifies cell types and locations, and 4) spatial statistical analysis. As a proof of concept, we applied this approach to study the architectural patterns of T and B cells within tumor-draining lymph nodes from breast cancer patients versus healthy lymph nodes. We found that the spatial grouping patterns of T and B cells differed between healthy and breast cancer lymph nodes, and this could be attributed to the lack of B cell localization in the extrafollicular region of the TDLNs.

Conclusions/Significance

Our integrative approach has made quantitative analysis of complex visual data possible. Our results highlight spatial alterations of immune cells within lymph nodes from breast cancer patients as an independent variable from numerical changes. This opens up new areas of investigations in research and medicine. Future application of this approach will lead to a better understanding of immune changes in the tumor microenvironment and TDLNs, and how they affect clinical outcomes.     

Intramuscular Inoculation of Mice with the Live-Attenuated Recombinant Rabies Virus TriGAS Results in a Transient Infection of the Draining Lymph Nodes and a Robust,Long-Lasting Protective Immune Response against Rabies

A single intramuscular application of the live but not UV-inactivated recombinant rabies virus (RABV) variant TriGAS in mice induces the robust and sustained production of RABV-neutralizing antibodies that correlate with long-term protection against challenge with an otherwise lethal dose of the wild-type RABV. To obtain insight into the mechanism by which live TriGAS induces long-lasting protective immunity, quantitative PCR (qPCR) analysis of muscle tissue, draining lymph nodes, spleen, spinal cord, and brain at different times after TriGAS inoculation revealed the presence of significant copy numbers of RABV-specific RNA in muscle, lymph node, and to a lesser extent, spleen for several days postinfection. Notably, no significant amounts of RABV RNA were detected in brain or spinal cord at any time after TriGAS inoculation. Differential qPCR analysis revealed that the RABV-specific RNA detected in muscle is predominantly genomic RNA, whereas RABV RNA detected in draining lymph nodes is predominantly mRNA. Comparison of genomic RNA and mRNA obtained from isolated lymph node cells showed the highest mRNA-to-genomic-RNA ratios in B cells and dendritic cells (DCs), suggesting that these cells represent the major cell population that is infected in the lymph node. Since RABV RNA declined to undetectable levels by 14 days postinoculation of TriGAS, we speculate that a transient infection of DCs with TriGAS may be highly immunostimulatory through mechanisms that enhance antigen presentation. Our results support the superior efficacy and safety of TriGAS and advocate for its utility as a vaccine.     

Antitumor Activity of T Cells Generated from Lymph Nodes Draining the SEA-expressing Murine B16 Melanoma and Secondarily Activated with Dendritic Cells

The successful use of tumor-draining lymph nodes (TDLN) as a source of effector cells for cancer immunotherapy depends largely on the immunogenicity of the tumor drained by the lymph nodes as well as the methods for secondary in vitro T cell activation and expansion. We transferred the bacterial superantigen staphylococcal enterotoxin A (SEA) gene into B16 murine melanoma tumor cells, and used them to induce TDLN (SEA TDLN) in syngeneic hosts. Wild-type (wt) TDLN induced by parental B16 tumor was used as a control. In vitro, SEA TDLN cells proliferated more vigorously, produced more IFNγ and demonstrated higher CTL activity than wt TDLN cells when activated with anti-CD3/anti-CD28/IL-2. In vivo, SEA TDLN cells mediated tumor eradication more effectively than similarly activated wt TDLN cells (p<0.01). Furthermore, use of dendritic cells (DC) plus tumor antigen in vitro in addition to anti-CD3/anti-CD28/IL-2 stimulation further amplified the immune function and therapeutic efficacy of SEA TDLN cells. DC-stimulated SEA TDLN cells eliminated nearly 90% of the pulmonary metastasis in mice bearing established B16 melanoma micrometastases. These results indicate that enforced expression of superantigen SEA in poorly immunogenic tumor cells can enhance their immunogenicity as a vaccine in vivo. The combined use of genetically modified tumor cells as vaccine to induce TDLN followed by secondary stimulation using antigen-presenting cells and tumor antigen in a sequential immunization/activation procedure may represent a unique method to generate more potent effector T cells for adoptive immunotherapy of cancer.     

In Situ Analysis of Lymphocyte Migration to Lymph Nodes

Blood-borne lymphocytes migrate continuously to peripheral lymph nodes (PLN) and other organized lymphoid tissues where they are most likely to encounter their cognate antigen. Lymphocyte homing to PLN is a highly regulated process that occurs exclusively in specialized high endothelial venules (HEV) in the nodal paracortex. Recently, it has become possible to explore this vital aspect of peripheral immune surveillance by intravital microscopy of the subiliac lymph node microcirculation in anesthetized mice. This paper reviews technical and experimental aspects of the new model and summarizes recent advances in our understanding of the molecular mechanisms of lymphocyte homing to PLN which were derived from its use. Both lymphocytes and granulocytes initiate rolling interactions via L-selectin binding to the peripheral node addressin (PNAd) in PLN HEV. Subsequently, a G protein-coupled chemoattractant stimulus activates LEA-1 on rolling lymphocytes, but not on granulocytes. Thus. granulocytes continue to roll through the PLN, whereas LEA-I activation allows lymphocytes to arrest and emigrate into the extravascular compartment. We have also identified a second homing pathway that allows L-selectin low/(activated/memory) lymphocytes to home to PLN. P-selectin on circulating activated platelets can mediate simultaneous platelet adhesion to PNAd in HEV and to P-selectin glycoprotein ligand (PSGL)-l on lymphocytes. Through this mechanism, platelets can form a cellular bridge which can effectively substitute for the loss of L-selectin on memory cell subsets.     

Isolation of Chromosomes from Picea abies and their Analysis by Flow Cytometry

A high-yield method for preparation of suspensions of intact Norway spruce [Picea abies (L.) Karst.] chromosomes was developed for the first time. To accumulate meristem root tip cells at metaphase, actively growing roots were subjected to subsequent treatments with 0.625 mM hydroxyurea for 18 h and after 8 h recovery in distilled water with 0.05 % (m/v) colchicine for 8 h. These treatments resulted in 50 % metaphase indices. Synchronized root tips were fixed in 2 % formaldehyde for 10 min and chromosomes were released into a lysis buffer by mechanical homogenisation, producing 5 × 10⁵ chromosomes from 50 root tips, at average. The isolated chromosomes were morphologically intact and suitable for flow cytometric analysis. Flow karyotypes obtained after the analysis of DAPI-stained chromosomes indicated a possibility to sort at least three different chromosome types. This revised version was published online in July 2006 with corrections to the Cover Date.