Mechanistic Characterization of De Novo Generation of Variable Number Tandem Repeats in Circular Plasmids during Site-Directed Mutagenesis and Optimization for Coding Gene Application.

Site-directed mutagenesis for creating point mutations, sometimes, gives rise to plasmids carrying variable number tandem repeats (VNTRs) locally, which are arbitrarily regarded as polymerase chain reaction (PCR) related artifacts. Here, the alternative end-joining mechanism is reported rather than PCR artifacts accounts largely for that VNTRs formation and expansion. During generating a point mutation on GPLD1 gene, an unexpected formation of VNTRs employing the 31 bp mutagenesis primers is observed as the repeat unit in the pcDNA3.1-GPLD1 plasmid. The 31 bp VNTRs are formed in 24.75% of the resulting clones with copy number varied from 2 to 13. All repeat units are aligned with the same orientation as GPLD1 gene. 43.54% of the repeat junctions harbor nucleotide mutations while the rest don't. Their demonstrated short primers spanning the 3' part of the mutagenesis primers are essential for initial creation of the 2-copy tandem repeats (TRs) in circular plasmids. The dimerization of mutagenesis primers by the alternative end-joining in a correct orientation is required for further expansion of the 2-copy TRs. Lastly, a half-double priming strategy is established, verified the findings and offered a simple method for VNTRs creation on coding genes in circular plasmids without junction mutations.

Proteomic analysis of plasma proteins from patients with cardiac rupture after acute myocardial infarction using TMT-based quantitative proteomics approach.

BACKGROUND: Cardiac rupture (CR) is a rare but catastrophic mechanical complication of acute myocardial infarction (AMI) that seriously threatens human health. However, the reliable biomarkers for clinical diagnosis and the underlying signaling pathways insights of CR has yet to be elucidated. METHODS: In the present study, a quantitative approach with tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry was used to characterize the differential protein expression profiles of patients with CR. Plasma samples were collected from patients with CR (n = 37), patients with AMI (n = 47), and healthy controls (n = 47). Candidate proteins were selected for validation by multiple reaction monitoring (MRM) and enzyme-linked immunosorbent assay (ELISA). RESULTS: In total, 1208 proteins were quantified and 958 differentially expressed proteins (DEPs) were identified. The difference in the expression levels of the DEPs was more noticeable between the CR and Con groups than between the AMI and Con groups. Bioinformatics analysis showed most of the DEPs to be involved in numerous crucial biological processes and signaling pathways, such as RNA transport, ribosome, proteasome, and protein processing in the endoplasmic reticulum, as well as necroptosis and leukocyte transendothelial migration, which might play essential roles in the complex pathological processes associated with CR. MRM analysis confirmed the accuracy of the proteomic analysis results. Four proteins i.e., C-reactive protein (CRP), heat shock protein beta-1 (HSPB1), vinculin (VINC) and growth/differentiation factor 15 (GDF15), were further validated via ELISA. By receiver operating characteristic (ROC) analysis, combinations of these four proteins distinguished CR patients from AMI patients with a high area under the curve (AUC) value (0.895, 95% CI, 0.802-0.988, p < 0.001). CONCLUSIONS: Our study highlights the value of comprehensive proteomic characterization for identifying plasma proteome changes in patients with CR. This pilot study could serve as a valid foundation and initiation point for elucidation of the mechanisms of CR, which might aid in identifying effective diagnostic biomarkers in the future.

Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair.

The reliance on viral vectors for the production of genetically engineered immune cells for adoptive cellular therapies remains a translational bottleneck. Here we report a method leveraging the DNA repair pathway homology-mediated end joining, as well as optimized reagent composition and delivery, for the Cas9-induced targeted integration of large DNA payloads into primary human T cells with low toxicity and at efficiencies nearing those of viral vectors (targeted knock-in of 1-6.7 kb payloads at rates of up to 70% at multiple targeted genomic loci and with cell viabilities of over 80%). We used the method to produce T cells with an engineered T-cell receptor or a chimaeric antigen receptor and show that the cells maintained low levels of exhaustion markers and excellent capacities for proliferation and cytokine production and that they elicited potent antitumour cytotoxicity in vitro and in mice. The method is readily adaptable to current good manufacturing practices and scale-up processes, and hence may be used as an alternative to viral vectors for the production of genetically engineered T cells for cancer immunotherapies.

Fusarium Wilt Invasion Results in a Strong Impact on Strawberry Microbiomes.

Plant-endophytic microbes affect plant growth, development, nutrition, and resistance to pathogens. However, how endophytic microbial communities change in different strawberry plant compartments after Fusarium pathogen infection has remained elusive. In this study, 16S and internal transcribed spacer rRNA amplicon sequencing were used to systematically investigate changes in the bacterial and fungal diversity and composition in the endophytic compartments (roots, stems, and leaves) of healthy strawberries and strawberries with Fusarium wilt, respectively. The analysis of the diversity, structure, and composition of the bacterial and fungal communities revealed a strong effect of pathogen invasion on the endophytic communities. The bacterial and fungal community diversity was lower in the Fusarium-infected endophytic compartments than in the healthy samples. The relative abundance of certain bacterial and fungal genera also changed after Fusarium wilt infection. The relative abundance of the beneficial bacterial genera Bacillus, Bradyrhizobium, Methylophilus, Sphingobium, Lactobacillus, and Streptomyces, as well as fungal genera Acremonium, Penicillium, Talaromyces, and Trichoderma, were higher in the healthy samples than in the Fusarium wilt samples. The relative abundance of Fusarium in the infected samples was significantly higher than that in the healthy samples, consistent with the field observations and culture isolation results for strawberry wilt. Our findings provide a theoretical basis for the isolation, identification, and control of strawberry wilt disease.

Label-free detection of virus based on surface-enhanced Raman scattering.

Due to the background interference from biological samples, detecting viruses using surface-enhanced Raman scattering (SERS) in clinical samples is challenging. This study is based on SERS by reducing sodium borohydride and aggregating silver nanoparticles to develop suitable virus detection "hot spot." The monkeypox virus and human papillomavirus fingerprints were quickly obtained, tested, and identified in serum and artificial vaginal discharge, respectively, by combining the principal component analysis method. Therefore, these viruses were successfully identified in the biological background. In addition, the lowest detection limit was 100 copies/mL showing good reproducibility and signal-to-noise ratio. The concentration-dependent curve of the monkeypox virus had a good linear relationship. This method helps solve the SERS signal interference problem in complex biological samples, with low detection limits and high selectivity in virus characterization and quantitative analysis. Therefore, this method has a reasonable prospect of clinical application.

Chloroplast genome characteristics of Corylopsis microcarpa H.T. Chang (Hamamelidaceae).

Corylopsis microcarpa H.T. Chang 1960 is a relict species from China. The chloroplast genome of C. microcarpa is 159,438 bp in size and shows typical quadripartite structure, which includes a pair of inverted repeat regions (26,280 bp), a large single-copy region (88,185 bp), and a small single-copy region (18,693 bp). The whole chloroplast genome encodes 114 unique genes, including 80 protein-code genes, 30 transfer RNA (tRNA) genes, and four ribosomal RNA (rRNA) genes. Ninety-one SSRs were identified. The phylogenetic analysis revealed C. microcarpa diverged early in Corylopsis.

Imprime PGG Enhances Anti-Tumor Effects of Tumor-Targeting, Anti-Angiogenic, and Immune Checkpoint Inhibitor Antibodies.

Imprime PGG (Imprime) is in late-stage clinical development as a combinatorial agent with several therapeutic modalities. Here we present pre-clinical mechanistic data supportive of Imprime, a soluble yeast ß-1,3/1,6-glucan pathogen-associated molecular pattern able to prime innate immune cells in a Dectin-1dependent manner. In tumor-free mice, Imprime evoked broad innate immune responses (type I interferon signature, mobilization of myeloid cells, dendritic cell and monocyte/macrophage expression of co-stimulatory ligands like CD86, and activation of natural killer cells). Imprime-mediated activation of myeloid cells also resulted in functional priming of antigen-specific CD8 T cell response. In tumor-bearing mice, Imprime monotherapy further resulted in activation of systemic and tumor infiltrating macrophages and enhanced cytotoxic CD8 T cell trafficking. Imprime enhanced the anti-tumor activity of several combinatorial agents in mouse cancer models; anti-tyrosinase-related protein 1 antibody in B16F10 melanoma experimental lung metastasis model, anti-vascular endothelial growth factor receptor 2 antibody in H1299 and H441 lung cancer, and anti-programmed cell death protein 1 antibody in MC38 colon cancer models. Mechanistically, combining Imprime with these combinatorial therapeutic agents elicited enhanced innate immune activation, supporting immunological synergy. Finally, Imprime treatment induced similar in vitro phenotypic and functional activation of human innate immune cells. Collectively, these data demonstrate Imprime's potential to orchestrate a broad, yet coordinated, anti-cancer immune response and complement existing cancer immunotherapies.

Plasma Proteome Profiling of Patients With In-stent Restenosis by Tandem Mass Tag-Based Quantitative Proteomics Approach.

Background: Despite the widespread application of new drug-eluting stents, a considerable portion of patients experience in-stent restenosis (ISR). To date, the pathophysiologic mechanisms of ISR remain poorly understood. Methods: In this study, we collected plasma samples from ISR patients (n = 29) and non-ISR patients (n = 36) after drug-eluting stent implantation, as well as from healthy controls (HCs) (n = 32). Our goal was to investigate differences in plasma protein profiles using tandem mass tag (TMT) labeling coupled with liquid chromatography and tandem mass spectrometry. The proteomic data were validated by enzyme-linked immunosorbent assay (ELISA). Bioinformatic analyses were conducted to analyze potential pathways and protein-protein interaction (PPI) involved in ISR. Results: A total of 1,696 proteins were identified, of which 278 differed in protein abundance between non-ISR and HCs, 497 between ISR and HCs, and 387 between ISR and non-ISR, respectively. Bioinformatic analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and PPI, further demonstrated that differentially abundant proteins between ISR and non-ISR are involved in several crucial biological processes and signaling pathways, such as focal adhesion, platelet activation, Rap1 signaling, regulation of actin cytoskeleton, and cholesterol metabolism. Among the identified differentially abundant proteins in ISR, 170 were increased in abundance relative to both non-ISR patients and HCs. Some of these proteins were identified to have critical functions for atherosclerosis development and might be involved in ISR pathology. Among these proteins, 3 proteins with increased abundance including fetuin-B, apolipoprotein C-III (APOC3), and cholesteryl ester transfer protein (CETP) were confirmed by ELISA. Conclusions: This is the first study provided a comprehensive proteomic profile to understand ISR pathology, which may help identify early diagnostic biomarkers and therapeutic targets.

CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells.

CRISPR-Cas9 cytidine and adenosine base editors (CBEs and ABEs) can disrupt genes without introducing double-stranded breaks by inactivating splice sites (BE-splice) or by introducing premature stop (pmSTOP) codons. However, no in-depth comparison of these methods or a modular tool for designing BE-splice sgRNAs exists. To address these needs, we develop SpliceR ( http://z.umn.edu/spliceR ) to design and rank BE-splice sgRNAs for any Ensembl annotated genome, and compared disruption approaches in T cells using a screen against the TCR-CD3 MHC Class I immune synapse. Among the targeted genes, we find that targeting splice-donors is the most reliable disruption method, followed by targeting splice-acceptors, and introducing pmSTOPs. Further, the CBE BE4 is more effective for disruption than the ABE ABE7.10, however this disparity is eliminated by employing ABE8e. Collectively, we demonstrate a robust method for gene disruption, accompanied by a modular design tool that is of use to basic and translational researchers alike.

Rapid and direct identification of the origin of white tea with proton transfer reaction time-of-flight mass spectrometry.

RATIONALE: White tea has become very popular in recent years, but there has been no scientific identification of white tea from different origins. For product authentication and valorization, every kind of white tea must be marked with an indication of its origin. METHODS: Volatile profiles of white tea leaf samples from their main origins in China (Fuding City, Zhenghe City and Jianyang City) were analyzed using proton transfer reaction time-of-flight mass spectrometry (PTR-TOFMS). Tentative identifications of the volatile organic compounds (VOCs) were obtained by PTR-TOFMS of the headspace. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed to evaluate the differences among the various origins. RESULTS: Teas from different origins were shown to have characteristic VOCs and profiles. Thus, white teas from different origins could be separated by characterizing the volatile emissions from the dry tea leaves. The ability of the two classification models to use the volatile fingerprints in origin discrimination was investigated. CONCLUSIONS: Two classification models (PCA and OPLS-DA) were applied to the PTR-TOFMS data obtained from the VOCs of various white teas. The classification models were shown to be useful in identifying the origin of white tea samples, providing a reference for white tea identification.

Loss of tumor suppressor inositol polyphosphate 4-phosphatase type B impairs DNA double-strand break repair by destabilization of DNA tethering protein Rad50.

Genome instability is the fundamental hallmark of malignant tumors. Tumor suppressors often play a role in maintaining genome stability. Our previous genetic screen identified inositol polyphosphate 4-phosphatase type B (INPP4B), primarily hydrolyzing phosphatidylinositol 3, 4-disphosphate, is a potential tumor suppressor in lung cancer cells. How INPP4B regulates the genome stability of lung cancer cells is unclear. Here we report knockout of INPP4B in lung adenocarcinoma A549 cells by Crispr-Cas9 gene editing leads to sensitization to ionizing radiation (IR), PARP inhibitor olaparib and impaired DNA homologous recombination repair. Re-introduction of a Crispr-Cas9 resistant INPP4B gene in the INPP4B knockout cells partially restored their resistance to IR, indicating loss of INPP4B protein is relevant to the increased IR sensitivity. Furthermore, we showed ectopic expressed INPP4B in A549 cells responds to IR irradiation by redistribution from cytoplasm to nucleus and endogenous INPP4B protein interacts with Rad50, a crucial MRN complex component for tethering DNA double-strand breaks. Loss of INPP4B protein results in decreased stability of Rad50 in vivo, suggesting an unanticipated role of tumor suppressor INPP4B in maintaining genome integrity via facilitating Rad50 mediated DNA double-strand break repair. Taken together, our findings support a dual role of INPP4B in suppression of tumorigenesis by safeguarding genome stability, as well as inhibiting of PI3K-Akt-mTOR signaling, and offer a new therapeutic strategy for personalized cancer treatment to patients with INPP4B defects or deficiency in the clinic.

Immunoglobulin Restores Immune Responses to BTH1677 in Patients With Low Levels of Antibodies to Beta-glucan.

BACKGROUND: BTH1677 is a beta-glucan pathogen-associated molecular pattern (PAMP) being evaluated as a novel immunotherapy of cancer. We previously described that the presence of antibodies against beta-glucan (ABA) in serum is necessary for BTH1677 antitumoral activity. We hypothesized that infusion of immunoglobulin can reinstate responses to BTH1677 in individuals with low ABA levels. PATIENTS AND METHODS: We report two single-patient studies: one in a patient with metastatic colorectal cancer who received BTH1677, combined with tumor targeting antibody cetuximab; and a second in a patient with metastatic neuroendocrine tumor who received BTH1677 combined with immune checkpoint inhibitor pembrolizumab. RESULTS: The patients had low serum titers of ABA and low innate immune effector functionality induced by BTH1677. Addition of intravenous immunoglobulins restored innate immune activity of BTH1677 and induced clinically meaningful anti-tumoral activity, with long-term disease control. CONCLUSION: Infusion of immunoglobulin can restore activity of BTH1677 in individuals with low serum ABA level.

A germline mutation in Rab43 gene identified from a cancer family predisposes to a hereditary liver-colon cancer syndrome.

BACKGROUND: Hereditary cancer syndromes have inherited germline mutations which predispose to benign and malignant tumors. Understanding of the molecular causes in hereditary cancer syndromes has advanced cancer treatment and prevention. However, the causal genes of many hereditary cancer syndromes remain unknown due to their rare frequency of mutation. METHODS: A large Chinese family with a history of hereditary liver-colon cancer syndrome was studied. The genomic DNA was extracted from the blood samples of involved family members, whole-exome sequencing was performed to identify genetic variants. Functional validation of a candidate variant was carried out using gene expression, gene knockout and immunohistochemistry. RESULTS: The whole-exome of the proband diagnosed with colon cancer was sequenced in comparison with his mother. A total of 13 SNVs and 16 InDels were identified. Among these variants, we focused on a mutation of Rab43 gene, a GTPase family member involving in protein trafficking, for further validation. Sanger DNA sequencing confirmed a mutation (c: 128810106C > T, p: A158T) occurred in one allele of Rab43 gene from the proband, that heterozygous mutation also was verified in the genome of the proband's deceased father with liver cancer, but not in his healthy mother and sister. Ectopic expression of the Rab43 A158T mutant in Huh7 cells led to more enhanced cell growth, proliferation and migration compared to the expression of wild type Rab43. Conversely, knockout of Rab43 in HepG2 cells resulted in slow cell growth and multiple nuclei formation and impaired activation of Akt. Finally, a positive correlation between the expression levels of Rab43 protein and cancer development in that family was confirmed. CONCLUSIONS: A germline mutation of Rab43 gene is identified to be associated with the onset of a familial liver-colon cancer syndrome. Our finding points to a potential role of protein trafficking in the tumorigenesis of the familial cancer syndrome, and helps the genetic counseling to the affected family members.

Identification and comparison of oligopeptides during withering process of White tea by ultra-high pressure liquid chromatography coupled with quadrupole-orbitrap ultra-high resolution mass spectrometry.

Peptides could have specific tastes or bioactivities depending on the length and sequence of amino acids. Till date it remains unknown what peptides are formed during the white tea manufacturing process and whether they contribute to the flavor or bio-activities of white tea. As a first step to address these questions, we applied ultra-high pressure liquid chromatography coupled with quadrupole-orbitrap ultra-high resolution mass spectrometry (UPLC-Quadrupole-Orbitrap-UHRMS) to monitor peptides dynamic changes during the withering process. A total of 196 abundant peptides were identified. Most of them were oligopeptides within a molecular weight of 1000 Da. Four of them were randomly selected, synthesized peptides were applied for further confirmation and quantification. Sequence analysis suggested that some of them were potential taste contributors. Proteinase cleave site analysis identified two separate periods of active proteins degradation at 0-12 h and 30-42 h of the withering processes. Further analysis of cleavage sites also suggested that protein degradation during withering steps were random rather than a stepwise reaction.

Immune Pharmacodynamic Responses of the Novel Cancer Immunotherapeutic Imprime PGG in Healthy Volunteers.

Imprime PGG (Imprime) is an i.v. administered, yeast ß-1,3/1,6 glucan in clinical development with checkpoint inhibitors. Imprime-mediated innate immune activation requires immune complex formation with naturally occurring IgG anti-ß glucan Abs (ABA). We administered Imprime to healthy human volunteers to assess the necessity of ABA for Imprime-mediated immunopharmacodynamic (IPD) changes. Imprime (4 mg/kg) was administered i.v. in single and multiple infusions. Subsets of subjects were premedicated with antihistamine and corticosteroid. Peripheral blood was measured before, during and after Imprime administration for IPD changes (e.g., ABA, circulating immune complexes, complement activation, complete blood counts, cytokine/chemokine, and gene expression changes). IPD changes were analyzed based on pretreatment serum ABA levels: low-ABA (<20 µg/ml), mid-ABA (≥20-50 µg/ml), and high-ABA (≥50 µg/ml). At the end of infusion, free serum ABA levels decreased, circulating immune complex levels increased, and complement activation was observed. At ∼1-4 h after end of infusion, increased expression of cytokines/chemokines, a 1.5-4-fold increase in neutrophil and monocyte counts and a broad activation of innate immune genes were observed. Low-ABA subjects typically showed minimal IPD changes except when ABA levels rose above 20 µg/ml after repeated Imprime dosing. Mild-to-moderate infusion-related reactions occurred in subjects with ABA ≥20 µg/ml. Premedications alleviated some of the infusion-related reactions, but also inhibited cytokine responses. In conclusion, ABA levels, being critical for Imprime-mediated immune activation may provide a plausible, mechanism-based biomarker to identify patients most likely to respond to Imprime-based anticancer immunotherapy.

Hydrophilic interaction liquid chromatography coupled with quadrupole-orbitrap ultra high resolution mass spectrometry to quantitate nucleobases, nucleosides, and nucleotides during white tea withering process.

Nucleotides, nucleosides, and nucleobases are important bioactive compounds. Recent studies suggested that they possess taste activity. However, it remains unknown about their presence in white tea and how they change during white tea manufacture. Here, we first established method based on hydrophilic interaction liquid chromatography coupled with quadrupole-orbitrap ultra high resolution mass spectrometry (HILIC-Quadrupole-Orbitrap-UHRMS) platform, then applied it to study the dynamic changes of nucleotides, nucleosides, and nucleobases during white tea withering process. Five compounds, including adenosine 5'-monophosphate monohydrate (AMP), guanosine 5'-monophosphate disodium salt hydrate (GMP), adenosine, cytidine, thymine and uracil, were detected from withering samples. They showed a general decline trend during white tea withering process, however, considerable amount of them was retained after withering for 48 h except adenosine which was below detection limit after withering for 21 h. This study provided a complete picture about nucleotides, nucleosides and nucleobases changes during white tea withering process.

Imprime PGG-Mediated Anti-Cancer Immune Activation Requires Immune Complex Formation.

Imprime PGG (Imprime), an intravenously-administered, soluble ß-glucan, has shown compelling efficacy in multiple phase 2 clinical trials with tumor targeting or anti-angiogenic antibodies. Mechanistically, Imprime acts as pathogen-associated molecular pattern (PAMP) directly activating innate immune effector cells, triggering a coordinated anti-cancer immune response. Herein, using whole blood from healthy human subjects, we show that Imprime-induced anti-cancer functionality is dependent on immune complex formation with naturally-occurring, anti-ß glucan antibodies (ABA). The formation of Imprime-ABA complexes activates complement, primarily via the classical complement pathway, and is opsonized by iC3b. Immune complex binding depends upon Complement Receptor 3 and Fcg Receptor IIa, eliciting phenotypic activation of, and enhanced chemokine production by, neutrophils and monocytes, enabling these effector cells to kill antibody-opsonized tumor cells via the generation of reactive oxygen species and antibody-dependent cellular phagocytosis. Importantly, these innate immune cell changes were not evident in subjects with low ABA levels but could be rescued with exogenous ABA supplementation. Together, these data indicate that pre-existing ABA are essential for Imprime-mediated anti-cancer immune activation and suggest that pre-treatment ABA levels may provide a plausible patient selection biomarker to delineate patients most likely to benefit from Imprime-based therapy.

Multilocus Sequence Types and Virulence Determinants of Hypermucoviscosity-Positive Klebsiella pneumoniae Isolated from Community-Acquired Infection Cases in Harbin, North China.

We investigated the molecular epidemiologic characteristics and virulence of hypermucoviscosity-positive Klebsiella pneumoniae in mainland China. We detected 16 hypermucoviscosity-positive strains in 65 total clinical isolates (24.62%). We found that 68.75% (11/16) of the positive strains had K2 genotype and carried the rmpA and iucA genes. Multilocus sequence typing revealed 5 sequence types (STs): ST65 [7], ST23 [4], ST86 [3], ST412 [1], ST375 [1], whereas the remaining 4 isolates were defined as other STs. The order of the median lethal dose values for the ST types was ST23 (2.19 × 10(3) CFU/mouse) < ST86 (1.70 × 10(4) CFU/mouse) < ST65 (5.05 × 10(7) CFU/mouse) < the other STs (1.90 × 10(8) CFU/mouse). In conclusion, the K2 with ST65 carrying rmpA and iucA was the most predominant among the hypermucoviscosity-positive K. pneumoniae strains obtained from community-acquired infection cases in Harbin, North China. Sequence types are a valuable tool to predict the risk of K. pneumoniae infection.

Binding of Soluble Yeast ß-Glucan to Human Neutrophils and Monocytes is Complement-Dependent.

The immunomodulatory properties of yeast ß-1,3/1,6 glucans are mediated through their ability to be recognized by human innate immune cells. While several studies have investigated binding of opsonized and unopsonized particulate ß-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble ß-glucans. Using a well-characterized, pharmaceutical-grade, soluble yeast ß-glucan, this study evaluated and characterized the binding of soluble ß-glucan to human neutrophils and monocytes. The results demonstrated that soluble ß-glucan bound to both human neutrophils and monocytes in a concentration-dependent and receptor-specific manner. Antibodies blocking the CD11b and CD18 chains of CR3 significantly inhibited binding to both cell types, establishing CR3 as the key receptor recognizing the soluble ß-glucan in these cells. Binding of soluble ß-glucan to human neutrophils and monocytes required serum and was also dependent on incubation time and temperature, strongly suggesting that binding was complement-mediated. Indeed, binding was reduced in heat-inactivated serum, or in serum treated with methylamine or in serum reacted with the C3-specific inhibitor compstatin. Opsonization of soluble ß-glucan was demonstrated by detection of iC3b, the complement opsonin on ß-glucan-bound cells, as well as by the direct binding of iC3b to ß-glucan in the absence of cells. Binding of ß-glucan to cells was partially inhibited by blockade of the alternative pathway of complement, suggesting that the C3 activation amplification step mediated by this pathway also contributed to binding.

[Follow-up of pediatric inflammatory bowel disease].

OBJECTIVE: To analyze the clinical characteristics and prognosis of pediatric inflammatory bowel disease (IBD) through a long-term follow-up so as to improve the diagnosis and management of IBD in children. METHODS: Seventy-three IBD patients admitted into our hospital from May 2000 to September 2010 were re-evaluated with the uniform diagnostic criteria proposed by the 2010 consensus diagnostic criteria for pediatric IBD. All patients were followed up by questionnaire, telephone and face-to-face interview. RESULTS: Among them, 56 cases (76.7%) (ulcerative colitis (UC): n = 34, Crohn's disease (CD): n = 22) were available for follow-up study. Among 34 UC cases, 13 cases had their diagnosis confirmed and 21 cases were diagnosed as probable UC. Meanwhile, among 22 CD cases, 14 and 8 had definite and probable diagnoses respectively. At diagnosis, 46.9% (15/32) of UC patients had extensive colitis, 40.6% (13/32) left-sided colitis while 72.7% (16/22) of CD patients with had ileocolonic. And 28 cases (82.4%) of UC patients and 20 cases (90.9%) of CD patients fulfilled the criteria for moderate to severe grade. Among 56 IBD cases, there was no death for CD, but 5 died for UC (14.7%). In the remaining 29 UC and 22 CD patients, 16 cases (55.2%) and 15 cases (68.2%) stayed symptom-free (P > 0.05). Moreover, 8 cases (27.6%) of UC and 3 cases (13.6%) of CD patients belonged to chronic relapsing type while 16 cases (55.2%) of UC and 15 cases (68.2%) of CD patients were of chronic persistent type. The physical activities of most IBD patients (n = 49) were unrestricted. The surgical rate for IBD was 19.6% (n = 11), 8.8% for UC (n = 3) and 36.4% for CD (n = 8) (P < 0.05). The incidences of surgical complications such as intestinal obstruction, intestinal perforation and hemorrhage of gastrointestinal tract were 7.1% (n = 4), 7.1% (n = 4) and 1.8% (n = 1). And it was more common in the CD group. CONCLUSIONS: Most IBD patients belong to chronic persistent type and then chronic relapsing type. Their physical activities are unrestricted. The surgical rate for CD is significantly higher than UC. And surgical complications such as intestinal obstruction, intestinal perforation and hemorrhage of gastrointestinal tract occur more frequently in the CD group.