Paraformaldehyde Research Articles

STUDIES ON FUNGAL ISOLATES INVOLVED IN BIODETERIORATION OF ANCIENT MANUSCRIPTS OF THE GENERAL EGYPTIAN BOOK ORGANIZATION (GEBO)

Microbiological contamination with fungi and bacteria can pose a significant destroy to oldmanuscripts or health hazard to those working in archives or library. Among 520 selected olddocuments, 162 manuscripts (31.15%) macroscopically showed fungal growth or damage andnumber of 75 documents (14.42%) were positive for presence of fungi but by culture fungalcontamination revealed in 30 manuscripts (5.77%). Also, about 199 representative fungalisolates developed on PDA medium were isolated from old manuscripts samples. Aspergillus,Fusarium and Penicillium spp. were the main contaminating mould genera of all testedmanuscripts and account over two third of contaminations. The fungal genera could bearranged on the basis of their frequent occurrence as follows: Aspergillus (45.57%), Fusarium(33.1%), Penicillium (8.6%), Alternaria (6.5%), Trichoderma (3.0%), Stymphylium (1.5%) andNigrospora (0.5%) of the total fungal count. The obtained data also showed that of the 53fungal isolates screened for cellulolytic activity on carboxy methyl cellulose (CMC) only31(58.49% ) had the ability to grow. Moreover, only 8 out off 31fungal isolates had a highability to decompose cellulose powder in Czepek's medium. The growth of A. flavus wascompletely inhibited (100% inhibition) at rhiozolex concentration of ≤ 200ppm and benlateconcentration of ≤ 400ppm. While, the growth of A. niger was also completely inhibited atrhizolex concentration of ≤ 400ppm and benlate at≤ 200ppm. The growth of F. oxysporumwas completely inhibited by rhizolex at 1600ppm and benlate at ≤ 100ppm. On the otherhand, fumigation with Para formaldehyde tablet for 9 days completely inhibited A. nigergrowth.

UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling.

SummaryNeural stem cell (NSC) proliferation and differentiation in the developing brain is a complex process precisely regulated by intrinsic and extrinsic signals. Although epigenetic modification has been reportedly involved in the regulation of the cerebral cortex, whether UTX, an H3K27me3 demethylase, regulates the development of cerebral cortex during the embryonic period is unclear. In this study, we demonstrate that Utx deficiency by knockdown and conditional knockout increases NSC proliferation and decreases terminal mitosis and neuronal differentiation. Furthermore, we find that impairment of cortical development caused by lack of Utx is less significant in males than in females. In addition, UTX demethylates H3K27me3 at the Pten promoter and promotes Pten expression. P-AKT and P-mTOR levels are increased in the Utx conditional knockout cortices. Finally, Utx or Pten overexpression can rescue the impairment of brain development caused by Utx loss. These findings may provide important clues toward deciphering brain diseases.

Immobilization of lipase on the surface of a micro tube and continuous transesterification

Candida antarctica lipase B (CALB) was cross-linked efficiently, using a reagent containing glutaraldehyde (GA), paraformaldehyde (PA), and a polylysine reagent. For the reaction under micro-flow conditions, CALB was immobilized on the inner surface of a polytetrafluoroethylene (PTFE) tube, by flowing the cross-linking reagents in an appropriate flow rate. The immobilized CALB formed a membrane-like structure on the inner surface of the tube and showed catalytic activity for transesterification. Although the enzyme activity was not high at the immobilized CALB, the productivity of micro-flow reaction using immobilized CALB was higher than that of batch-wise reaction using free CALB.

MICAN, a new fluorophore for vital and non-vital staining of human cells

Fluorescence time-lapse microscopy is in connection with the invasive properties of fluorochrome applied, and with the toxicity of the excitation energy and wavelength of the dye itself. Experiments with the newly synthesized fluorescent dye 1-N-methylamino-5-isocyanonaphthalene (MICAN) served to test its cytotoxicity on human HaCaT keratinocyte cell cultures. Experiments related to staining capability were performed with paraformaldehyde (PFA) fixed cells and observed with fluorescence microscope. It was assumed that the fluorophore 1-amino-5-isocyanonaphthalene (ICAN) and especially its N-methylamino derivative MICAN, containing condensed aromatic rings could serve as a nonselective fluorescent dye capable to stain cellular structures of fixed, living, damaged and dead cells. This notion was confirmed by the MICAN staining of cytoplasmic proteins primarily rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SEM) and less efficiently nuclear proteins suggesting the involvement of staining of subcellular structures involved in protein synthesis. MICAN was not only well tolerated by living cells but turned out to be a strong heterochromatin and RER staining agent. This led to the development of a MICAN staining protocol for native and living samples. Relative to other fluorescent dyes, MICAN is not only useful but also cost-effective. Toxicology tests were performed using 30, 10, 5, 0.5 μg/ml MICAN concentrations. Time-lapse videomicroscopy at near-infrared (NIR) illumination has been used for the examination of MICAN effect on cell division. It was found that MICAN as a vital stain had no significant harmful effect on HaCaT cells. MICAN turned out to be a non-toxic, highly quantum-efficient vital stain with minimal, or no photobleaching, and can be applied to co-stain with propidium-iodide due the strong spectral separation.

Frozen section with improved H&E staining for follicular morphometric analysis of mouse ovary in oestrus cycle.

Careful analysis of follicular morphology and numbers of different follicular maturation stages, in combination with the measurements of gonadotropic and sex hormone profiles, provide an accurate and rapid evaluation system of the ovarian function. The aim of this study is to improve the existing methods of ovarian tissue section preparation and staining methods, and to establish a fast and easy method to observe and evaluate follicular maturation stage and numbers using mouse ovary samples. Ovaries were collected at menstrual phases of proestrus, oestrus, metestrus and diestrus from C57BL/6J female mice. Then the ovaries were fixed in 4% paraformaldehyde, dehydrated with graded sucrose, embedded with OCT, frozen-sectioned at 7 μm thick, and subjected to quick haematoxylin & eosin staining for observation. The results showed that the present method was able to distinguish secondary, preantral, antral, and preovulatory follicles. Although our method was unable to discriminate and distinguish primordial follicles and primary follicles, the results were comparable to those from more complicated techniques. We conclude that this improved and quick method can be used in combination with hormone analysis to investigate ovarian development and function in different mouse models.

Simple and Rapid Tissue Clearing Method for Three-Dimensional Histology of the Pancreas.

Previously, high-resolution three-dimensional imaging of a whole and intact pancreas was not possible, since light is scattered when it passes through cell compartments with different refractive indices. CLARITY is one of the tissue clearing techniques that has yielded success with the central nervous system. To preserve tissue integrity after delipidation, conventional protocols embed tissue in an acrylamide-based hydrogel, which involves the use of specialized equipment. Recently, we determined that the hydrogel-embedding step could be simplified and replaced by passive tissue fixation in 4% paraformaldehyde (PFA). The whole procedure is less time-consuming and less error-prone, and can be completed within a week, compared to conventional CLARITY protocols that may take weeks to complete. Here, the detailed stepwise procedures involved in the simplified CLARITY workflow are applied to the pancreas of wild-type and gene-knockout 6-week old mice expressing green fluorescent protein (GFP) under the mouse insulin 1 promoter (MIP-GFP). This technique could facilitate high-resolution, three-dimensional imaging of pancreatic islets and comparison between different mouse genotypes under different disease and treatment conditions. © 2017 by John Wiley & Sons, Inc.

Effect of Matrigel on the establishment of human osteosarcoma animal model

Objective To investigate the effects of cell suspension including Matrigel, normal culture medium and phosphate buffer saline (PBS) on the xenograft model establishment of human osteosarcoma. The function of Matrigel on regulating human osteosarcoma cell differentiation and proliferation was analyzed. Methods Twenty-four BALB/c-nu/nu nude mice were randomly divided into three groups with 8 animals in every group: Matrigel and RPMI 1640 suspension (group M), RPMI 1640 culture medium (group R), PBS (group P). Human osteosarcoma cell-SaOS-2 was suspended in the three groups respectively. 1×106/ml equivalent cell counts were injected into the back of each anesthetized nude mouse (400 μl per mouse). Xenograft tumors were measured at regular intervals and the tumor volume was calculated. After 5 weeks of inoculation, the tumor parts were dissected. Paraffin-embedded sections from xenograft tumor tissues were fixed in 4 % paraformaldehyde and pathological study was made after paraffin embedding and cutting under microscope by HE stains. Results Eight nude mice formed tumor lumps at 0 day in group M and were gradually increased over time. Xenograft tumors of group R and group P disappeared in 2-4 days and some appeared again after 1 week with an increase of tumor size. After 5 weeks, the tumor volume in the group M was significantly larger than that in the group P and group R [(3 185 ± 488), (598 ± 189), (512 ± 109) mm3 respectively, F= 85.7, P 0.05). Histopathological analysis showed that cells in the group M could keep original degrees of pathological differentiation in osteosarcoma cells. Besides, cells suspension of culture medium or PBS in the group P and group R were poorly differentiated. Conclusions Matrigel can promote high tumor growth rate and good uniformity of human osteosarcoma cells in experimental animals. The histological state is similar to original structure, which conforms to the occurrence and development of human osteosarcoma. Key words: Osteosarcoma; Models, animal; Transplantation; Matrigel

Preservation of three-dimensional spatial structure in the gut microbiome.

Preservation of three-dimensional structure in the gut is necessary in order to analyze the spatial organization of the gut microbiota and gut luminal contents. In this study, we evaluated preparation methods for mouse gut with the goal of preserving micron-scale spatial structure while performing fluorescence imaging assays. Our evaluation of embedding methods showed that commonly used media such as Tissue-Tek Optimal Cutting Temperature (OCT) compound, paraffin, and polyester waxes resulted in redistribution of luminal contents. By contrast, a hydrophilic methacrylate resin, Technovit H8100, preserved three-dimensional organization. Our mouse intestinal preparation protocol optimized using the Technovit H8100 embedding method was compatible with microbial fluorescence in situ hybridization (FISH) and other labeling techniques, including immunostaining and staining with both wheat germ agglutinin (WGA) and 4', 6-diamidino-2-phenylindole (DAPI). Mucus could be visualized whether the sample was fixed with paraformaldehyde (PFA) or with Carnoy’s fixative. The protocol optimized in this study enabled simultaneous visualization of micron-scale spatial patterns formed by microbial cells in the mouse intestines along with biogeographical landmarks such as host-derived mucus and food particles.

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA‐based protocols. Glyoxal acted faster than PFA, cross‐linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA‐based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.

Clinical-Scale Cell-Surface-Marker Independent Acoustic Microfluidic Enrichment of Tumor Cells from Blood.

Enumeration of circulating tumor cells (CTCs) predicts overall survival and treatment response in metastatic cancer, but as many commercialized assays isolate CTCs positive for epithelial cell markers alone, CTCs with little or no epithelial cell adhesion molecule (EpCAM) expression stay undetected. Therefore, CTC enrichment and isolation by label-free methods based on biophysical rather than biochemical properties could provide a more representative spectrum of CTCs. Here, we report on a clinical-scale automated acoustic microfluidic platform processing 5 mL of erythrocyte-depleted paraformaldehyde (PFA)-fixed blood (diluted 1:2) at a flow rate of 75 μL/min, recovering 43/50 (86 ± 2.3%) breast cancer cell line cells (MCF7), with 0.11% cancer cell purity and 162-fold enrichment in close to 2 h based on intrinsic biophysical cell properties. Adjustments of the voltage settings aimed at higher cancer cell purity in the central outlet provided 0.72% cancer cell purity and 1445-fold enrichment that resulted in 62 ± 8.7% cancer cell recovery. Similar rates of cancer-cell recovery, cancer-cell purity, and fold-enrichment were seen with both prostate cancer (DU145, PC3) and breast cancer (MCF7) cell line cells. We identified eosinophil granulocytes as the predominant white blood cell (WBC) contaminant (85%) in the enriched cancer-cell fraction. Processing of viable cancer cells in erythrocyte-depleted blood provided slightly reduced results as to fixed cells (77% cancer cells in the enriched cancer cell fraction, with 0.2% WBC contamination). We demonstrate feasibility of enriching either PFA-fixed or viable cancer cells with a clinical-scale acoustic microfluidic platform that can be adjusted to meet requirements for either high cancer-cell recovery or higher purity and can process 5 mL blood samples in close to 2 h.

Synergistic effect of Brønsted and Lewis acid sites for the synthesis of polyoxymethylene dimethyl ethers over highly efficient SO42−/TiO2 catalysts

Polyoxymethylene dimethyl ethers (PODEn, CH3O(CH2O)nCH3, where n≥1) are receiving much attention as ideal diesel fuel additives because they can significantly reduce the emission of smoke and engine exhaust during combustion. PODEn are synthesized over acid catalysts. In this work, highly efficient sulfate titania catalysts were prepared by impregnation for the synthesis of PODEn from dimethoxymethane (DMM) and paraformaldehyde (PF). XPS and XRF were used to determine the spatial distribution of S, and NH3-TPD and Pyridine-IR were used to measure acid properties. The concentration of the precursor solution had an important effect on the catalyst activity. The spatial distribution of S influenced the acid properties and catalyst activity. A catalytic mechanism based on Brønsted and Lewis acid sites was proposed, which explained the synergistic effect of Brønsted and Lewis acid sites in the reaction for the first time.

The investigation of the distribution of nerves, blood vessels and immune cells on the fresh human corneal surface using optimized protocols for immunostaining of flat mounted whole cornea

PurposeTo present a reliable protocol for the immunostaining of flat mounted corneas and provide a map of the distribution of nerves, blood vessels and immune cells on fresh human corneal epithelium and conjunctiva.MethodsOrgan‐cultured human corneas were used for our technique development. 15 fixatives and 5 antigen retrieval (AR) methods were assessed using 4 ubiquitous proteins with different, well‐characterized subcellular localization: ZO‐1 (membrane), actin (cytoplasm), hnRNP L (nucleosol) and histone H3 (chromatin). The distribution of nerves (Neurofilaments and tubulin β3), blood vessels (CD31 and α‐SMA) and immune cells (CD45, CD4, CD8, CD68) in fresh human corneas (without any preservation in culture medium) was then investigated using these optimized protocols.Results0.5% paraformaldehyde (PFA) and pure methanol were selected as the most efficient fixatives. Using our optimised protocol, we revealed not only superficial cells (cytokeratin 3, E‐cadherin), but also basal cells (ΔNp63, PAX6). Nerve fibers were mainly stained by tubulin β3 on epithelium and by Neurofilament on conjunctiva. CD31 stained only the capillaries situated at the limbus, whereas α‐SMA stained all types of vessels on the corneal surface. Numerous immune cells were detected on conjunctival and capillary loops below the limbus. A few immune cells were observed on central epithelium.ConclusionsTissue fixation with 0.5% PFA or pure methanol, without any subsequent AR techniques, were found to produce the best results. Compared to IHC on cross sections, immunostaining of flat mounted cornea allows a precise localization of targeted cells through the full thickness of the corneal epithelium and conjunctiva. The distribution of nerves, blood vessels and immune cells on the corneal surface has therefore been mapped.

A facile one-pot Mannich reaction for the construction of fluorescent polymeric nanoparticles with aggregation-induced emission feature and their biological imaging

Multicomponent reactions (MCRs) have recently attracted great attention as one of the most important tools for the construction of various organic compounds in modern organic chemistry. In this work, we introduced an efficient one-pot strategy to successfully fabricate the fluorescent polymeric nanoparticles (FPNs) with aggregation-induced emission (AIE) characteristic via the conjugation of hyperbranched polyamino compound polyethyleneimine (PEI), AIE dye (named as PhE-OH) and paraformaldehyde (PF) through a Mannich reaction. The final amphiphilies (PEI-PF-PhE) can self-assemble into micelles in aqueous solution. We demonstrated PEI-PF-PhE FPNs showed high water dispersity, intense orange-yellow fluorescence, excellent photostability, low toxicity and high cell imaging performance. As compared with other construction strategies, the one-pot Mannich reaction possesses a number of advantages, such as simplicity, atom economy, high-efficiency and multifunctional potential. Combined with the remarkable properties of the AIE-active FPNs and the one-pot Mannich reaction, we could expect that the strategy developed in this work should be a useful tool for construction of various AIE-active functional materials for biomedical applications.

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus.

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3'-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

A new method for gene expression analysis of pure lymphocytes recovered from heterogeneous fixed mouse tissue.

e23089 Background: Tumor infiltrating lymphocytes (TILs) are biomarkers that play a critical role in cancer diseases, including differential diagnosis, determination of prognosis, prediction of response to treatment, and evaluation of disease progression. Gene expression analysis in TILs derived from fresh tissue may not accurately depict the gene profile of the tissue microenvironment as it can change aggressively during lymphocyte isolation and RNA extraction. In addition, tissue sample size can limit the isolation of TILs with current technologies. In this study, we demonstrate the use of the DEPArray™platform to isolate pure populations of lymphocytes from a fixed mouse tissue for RNA analysi. Methods: Mouse splenocytes were activated in vitro with anti-CD3 and -CD28 for 72hs. Cells were harvested, fixed with 2% paraformaldehyde (PFA) for 20 min at RT, and stained for either CD4 or CD8 expression. Gene expression analysis of CD45, ADORA2A, GLS and GAPDH was performed in CD4+ and CD8+ DEPArray™sorted cells using the TaqMan PreAmp Cells-to-Ct kit. Results: The table below summarizes the Ct values for CD45, ADORA2A, GLS and GAPDH expression in 300 fixed unsorted control and DEPArray™sorted lymphocytes. Conclusions: We have demonstrated the feasibility of gene expression analysis on pure populations of CD4+ and CD8+ cells isolated from a fixed tissue using the DEPArray™ platform. The advantage of this approach is the DEPArray’s ability to identify and isolate subpopulations of cells from complex heterogeneous samples and/or specimens that are limited by size or content. This methodology will be applied for isolation of TILs in syngeneic and xenograft models of cancers for downstream RNA applications. [Table: see text]

Mechanical properties of paraformaldehyde-treated individual cells investigated by atomic force microscopy and scanning ion conductance microscopy

BackgroundCell fixation is an essential step to preserve cell samples for a wide range of biological assays involving histochemical and cytochemical analysis. Paraformaldehyde (PFA) has been widely used as a cross-linking fixation agent. It has been empirically recognized in a gold standard protocol that the PFA concentration for cell fixation, CPFA, is 4%. However, it is still not quantitatively clear how the conventional protocol of CPFA is optimized.MethodsHere, we investigated the mechanical properties of cell fixation as a function of CPFA by using atomic force microscopy and scanning ion conductance microscopy. The goal of this study is to investigate the effect of CPFA (0–10 wt%) on the morphological and mechanical properties of live and fixed mouse fibroblast cells.ResultsWe found that both Young’s modulus, E, and the fluctuation amplitude of apical cell membrane, am, were almost constant in a lower CPFA (<10−4%). Interestingly, in an intermediate CPFA between 10−1 and 4%, E dramatically increased whereas am abruptly decreased, indicating that entire cells begin to fix at CPFA = ca. 10−1%. Moreover, these quantities were unchanged in a higher CPFA (>4%), indicating that the cell fixation is stabilized at CPFA = ca. 4%, which is consistent with the empirical concentration of cell fixation optimized in biological protocols.ConclusionsTaken together, these findings offer a deeper understanding of how varying PFA concentrations influence the mechanical properties of cells and suggest new avenues for establishing refined cell fixation protocols.

Development of an anti-ferret CD4 monoclonal antibody for the characterisation of ferret T lymphocytes

The ferret is an established animal model for a number of human respiratory viral infections, such as influenza virus and more recently, Ebola virus. However, a paucity of immunological reagents has hampered the study of cellular immune responses. Here we describe the development and characterisation of a novel monoclonal antibody (mAb) against the ferret CD4 antigen and the characterisation of ferret CD4 T lymphocytes. Recombinant production and purification of the ferret CD4 ectodomain soluble protein allowed hybridoma generation and the generation of a mAb (FeCD4) showing strong binding to ferret CD4 protein and lymphoid cells by flow cytometry. FeCD4 bound to its cognate antigen post-fixation with paraformaldehyde (PFA) which is routinely used to inactivate highly pathogenic viruses. We have also used FeCD4 in conjunction with other immune cell markers to characterise ferret T cells in both primary and secondary lymphoid organs. In summary, we have developed an important reagent for the study of cellular immunological responses in the ferret model of infectious disease.

Pattern and distribution of extracellular matrix proteins in human reparative dentin by an immunohistochemical approach

Dentin is a large and complex component of the tooth synthesized by odontoblasts during the process of dentinogenesis. Dentin formed, before the completion of root formation, is define primary dentin (PD), while dentin formed after and associated with the normal aging process is designated secondary dentin (SD). Tertiary dentin (TD) is produced in reaction to external noxious stimulus/injury, such as attrition or dental caries, adjacent to the preexisting dentin layer and further classified reparative dentin (RD) (1, 2). Aim this study was to compare pattern and distribution of extracellular matrix proteins, produced by odontoblast cells during dentin mineralization and during reparative process, in response to stimulus in human sound dentin vs human reparative dentin matrix. Sixteen sound carious human molars were selected, demineralized, fixed in paraformaldehyde and then processed for immunohistochemical approach to detect extracellular matrix proteins. In particular specimens were submitted to an immunolabeling technique by using primary antibodies anti dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), osteoponti (OPN). Results indicate that the region of the exposed pulp, formed a layer of reparative dentin bridge sealing the communication between the cavity and pulp chamber. In addition results indicate that in RD is present a lower levels of DMP1 and DSP than PD layer, while BSP and OPN are present in RD but absent in PD layer. The expression of BSP and OPN in RD indicates that the odontoblast-like cells were attempting to produce a hard tissue at a very rapid process. In according with previous scientific literature, our results suggested that the deposition of OPN and BSP at the calcification front is essential for the type I collagen secretion by newly differentiated odontoblast-like cells to form reparative dentin during pulpal healing following cavity preparation.

Histochemical Staining of Collagen and Identification of Its Subtypes by Picrosirius Red Dye in Mouse Reproductive Tissues.

Collagen is one of the foremost components of tissue extracellular matrix (ECM). It provides strength, elasticity and architecture to the tissue enabling it to bear the wear and tear from external factors like physical stress as well as internal stress factors like inflammation or other pathological conditions. During normal pregnancy or pregnancy related pathological conditions like preterm premature rupture of membranes (PPROM), collagen of the fetal membrane undergoes dynamic remodeling defining biochemical properties of the fetal membrane. The protocol in this article describes the histochemical method to stain total collagen by Picrosirius red stain which is a simple, quick and reliable method. This protocol can be used on paraformaldehyde (PFA) and formaldehyde fixed paraffin embedded tissue sections. We further describe the staining and distribution of collagen in different mouse reproductive tissues and also demonstrate how this technique in combination with polarization microscopy is useful to detect the distribution of different subtypes of collagen.

Interleukin-1 receptor type 1 signaling induces excessive inflammatory responses in H1N1 influenza virus infection

Objective To investigate the role of interleukin-1 receptor type 1 (IL-1R1) signaling in H1N1 influenza virus infection. Methods IL-1R1 knockout (IL-1R1-/-) mice and wild type (WT) mice were infected intranasally with 2×104 TCID50 (50% tissue culture infective dose) of influenza virus H1N1 PR8. Changes in clinical signs, survivals and bodyweights of those mice were monitored daily for 14 consecutive days. Three mice from each group were sacrificed at 3, 7 and 14 days post infection (d.p.i), from which whole lungs were harvested. A part of the lobes was fixed in 4% paraformaldehyde for histopathological assessment and the rest were split and stored at -80 centigrade for further analysis. Real-time quantitative PCR and cytometric bead array (CBA) were performed to detect viral loads in lungs and inflammatory cytokines in supernatants of lung homogenates. Results The mice in both groups showed severe symptoms after the infection of PR8. The maximum bodyweight loss of IL-1R1-/- mice [(24.22±0.80) % at 8 d. p.i] was lower than that of WT mice [(28.03±1.51)% at 9 d. p.i] (P<0.05). The IL-1R1-/- mice with PR8 infection showed a higher survival rate (90%) as compared with that of the control group (40%) (P<0.05). No statistical differences in virus loads were observed between the two groups at 3, 7 and 14 d. p.i. The lung weight to body weight ratio of IL-1R1-/- mice [(1.42±0.03) %] was lower than that of WT mice [(1.79±0.08) %] at 3 d. p.i (P<0.05). Pathological changes in IL-1R1-/- mice were less severe than those in WT mice. CBA detection assay revealed that the proinflammatory cytokines in lungs of IL-1R1-/- mice were less than those in WT mice. Conclusion IL-1R1 signaling plays a pathogenic role in mice infected with 2×104 TCID50 of influenza virus PR8 by promoting inflammatory responses. Key words: IL-1R1; Influenza virus; Inflammatory responses