Toxocara canis Infection Alters lncRNA and mRNA Expression Profiles of Dog Bone Marrow.

Bone marrow is the main hematopoietic organ that produces red blood cells, granulocytes, monocyte/macrophages, megakaryocytes, lymphocytes, and myeloid dendritic cells. Many of these cells play roles in the pathogenesis of Toxocara canis infection, and understanding how infection alters the dynamics of transcription regulation in bone marrow is therefore critical for deciphering the global changes in the dog transcriptional signatures during T. canis infection. In this study, long non-coding RNA (lncRNA) and messenger RNA (mRNA) expression profiles in the bone marrow of Beagle dogs infected with T. canis were determined at 12 h post-infection (hpi), 24 hpi, 96 hpi, and 36 days post-infection (dpi). RNA-sequencing and bioinformatics analysis identified 1,098, 984, 1,120, and 1,305 differentially expressed lncRNAs (DElncRNAs), and 196, 253, 223, and 328 differentially expressed mRNAs (DEmRNAs) at 12 h, 24 h, 96 h, and 36 days after infection, respectively. We also identified 29, 36, 38, and 68 DEmRNAs potentially cis-regulated by 44, 44, 51, and 80 DElncRNAs at 12 hpi, 24 hpi, 96 hpi, and 36 dpi, respectively. To validate the sequencing findings, qRT-PCR was performed on 10 randomly selected transcripts. Many altered genes were involved in the differentiation of bone marrow cells. GO of DElncRNAs and GO and KEGG pathway analyses of DEmRNAs revealed alterations in several signaling pathways, including pathways involved in energy metabolism, amino acid biosynthesis and metabolism, Wnt signaling pathway, Huntington's disease, HIF-1 signaling pathway, cGMP-PKG signaling pathway, dilated cardiomyopathy, and adrenergic signaling in cardiomyocytes. These findings revealed that bone marrow of T. canis-infected dogs exhibits distinct lncRNA and mRNA expression patterns compared to healthy control dogs. Our data provide novel insights into T. canis interaction with the definitive host and shed light on the significance of the non-coding portion of the dog genome in the pathogenesis of toxocariasis.

Proteomic alterations in the plasma of Beagle dogs induced by Toxocara canis infection.

Toxocara canis causes ocular larva migrans and visceral larva migrans in humans. Knowledge about the molecular mechanism of T. canis-hosts interaction is limited. The proteomic alterations in the plasma of Beagle dogs induced by T. canis infection were studied by the quantitative mass spectrometry-based data-independent acquisition (DIA). 418, 414 and 411 plasma proteins were identified at 24 h post-infection (hpi), 96 hpi and 36 days post-infection (dpi), including 6, 5 and 23 proteins with differential abundance, respectively. At 24 hpi, the altered proteins, retinoic acid receptor responder protein 2 (RARRES2), WD repeat-containing protein 1 (WDR1), moesin and filamin-A, may participate in pro-inflammatory reaction or promote larvae migration. At 96 hpi, the altered protein C and fibroleukin may maintain the stability of the coagulation system to protect the lung. At 36 dpi, the alterations of C-reactive protein (CRP), ficolin (FCN), complement factor H-related protein 5 (CFHR5) and other complements can affect the three traditional complement system, including the classic pathway, lectin pathway and alternative pathway. These proteins may play important roles in the interaction between T. canis and its definitive hosts. Further study on these altered proteins triggered by T. canis infection may discovery novel therapeutic or diagnostic targets for toxocariasis. SIGNIFICANCE OF THE STUDY: Toxocara canis is one of the globally distributed soil-transmitted helminths, which causes ocular larva migrans and visceral larva migrans in humans and a wide range of warm-blooded animals. T. canis adapts to different microenvironments by resisting and adjusting various biological processes of the hosts. Knowledge about the molecular mechanism of T. canis-hosts interaction is limited. Plasma proteins are good marker for monitoring the occurrence and development of diseases. The proteomic alterations in the plasma of Beagle dogs induced by T. canis infection were studied by the quantitative mass spectrometry-based data-independent acquisition (DIA) in this study. A total of 418, 414 and 411 plasma proteins were identified at 24 h post-infection (hpi), 96 hpi and 36 days post-infection, respectively. Ten protein with differential abundances were validated by using parallel reaction monitoring (PRM). Collectively, our deep proteomic analysis of plasma revealed that proteins alterations were affected by disease development, and proteomic analysis is an ideal method for quantifying changes in circulating factors on a global scale in response to pathophysiological perturbations such as T. canis infection.

Global profiling of lncRNAs-miRNAs-mRNAs reveals differential expression of coding genes and non-coding RNAs in the lung of beagle dogs at different stages of Toxocara canis infection.

The roundworm Toxocara canis causes toxocariasis in dogs and larval migrans in humans. Better understanding of the lung response to T. canis infection could explain why T. canis must migrate to and undergoes part of its development inside the lung of the definitive host. In this study, we profiled the expression patterns of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs in the lungs of Beagle dogs infected by T. canis, using high throughput RNA sequencing. At 24 h p.i., 1,012 lncRNAs, 393 mRNAs and 10 miRNAs were differentially expressed (DE). We also identified 883 DElncRNAs, 264 DEmRNAs and 20 DEmiRNAs at 96 h p.i., and 996 DElncRNAs, 342 DEmRNAs and eight DEmiRNAs at 36 days p.i., between infected and control dogs. Significant changes in the levels of expression of transcripts related to immune response and inflammation were associated with the antiparasitic response of the lung to T. canis. The remarkable increase in the expression of scgb1a1 at all time points after infection suggests the need for consistent moderation of the excessive inflammatory response. Also, upregulation of foxj1 at 24 h p.i., and downregulation of IL-1ß and IL-21 at 96 h p.i., suggest an attenuation of the humoral immunity of infected dogs. These results indicate that T. canis pathogenesis in the lung is mediated through contributions from both pro-inflammatory and anti-inflammatory mechanisms. Competing endogenous RNA (ceRNA) network analysis revealed significant interactions between DElncRNAs, DEmiRNAs and DEmRNAs, and improved our understanding of the ceRNA regulatory mechanisms in the context of T. canis infection. These data provide comprehensive understanding of the regulatory networks that govern the lung response to T. canis infection and reveal new mechanistic insights into the interaction between the host and parasite during the course of T. canis infection in the canine.

Serum metabolomic alterations in Beagle dogs experimentally infected with Toxocara canis.

BACKGROUND: Toxocara canis, a globally distributed roundworm, can cause debilitating disease in dogs and humans; however, little is known about the metabolomic response of the hosts to T. canis infection. There is an increasing need to understand the metabolic mechanisms underlying the pathogenesis of T. canis infection in dogs. Here, we examined the metabolomic changes in Beagle dogs' serum following T. canis infection using LC-MS/MS. RESULTS: The metabolic profiles of Beagle dogs' serum were determined at 12 h, 24 h, 10 d and 36 d after oral infection with 300 infectious T. canis eggs by LC-MS/MS. We tested whether the T. canis-associated differentially abundant metabolites could distinguish the serum of infected dogs from controls, as measured by the area under the receiver operating characteristic (ROC) curve (AUC). The differentially expressed metabolites were further evaluated by principal components analysis and pathway enrichment analysis. A total of 5756 and 5299 ions were detected in ESI+ and ESI- mode, respectively. ROC curve analysis revealed nine and five metabolite markers, at 12 hpi and 24 hpi to 36 dpi, respectively, with potential diagnostic value for toxocariasis. The levels of taurocholate, estradiol, prostaglandins and leukotriene were significantly changed. Primary bile acid biosynthesis pathway, steroid hormone biosynthesis pathway and biosynthesis of unsaturated fatty acids pathway were significantly altered by T. canis infection. CONCLUSIONS: These findings show that T. canis infection can induce several changes in the dog serum metabolome and that the metabolic signature associated with T. canis infection in dogs has potential for toxocariasis diagnosis.

[Clinical and prognostic significance of preoperative serum CA153, CEA and TPS levels in patients with primary breast cancer].

OBJECTIVE: To investigate the clinical and prognostic values of preoperative serum CA153, CEA and TPS levels in patients with primary breast cancer. METHODS: A total of 386 hospitalized patients with stage I ∼ IV breast cancer from Nov 1998 to Feb 2009 were followed up, and their clinicopathological data were analyzed retrospectively to determine the factors affecting their prognosis. RESULTS: First, preoperative serum CA153 expression level was significantly associated with the age of onset and tumor size (P < 0.05), the expression of serum CEA was correlated with tumor size (P < 0.05), and the expression of serum tissue polypeptide specific antigen (TPS) was correlated with tumor size and lymph node metastases (P < 0.05). Second, the overall survival was significantly shorter among patients with elevated serum CA153, CEA or TPS, respectively (P < 0.05 for overall). Finally, multivariate Cox regression analysis indicated that estrogen receptor status (ER) and elevated preoperative values of CA 153 are independent prognostic factors for overall survival (P < 0.05), and CA 153 is a risk factor but estrogen receptor status is a protective factor for overall survival. CONCLUSIONS: Higher preoperative expression of serum CA153, CEA or TPS is closely correlated with clinicopathological characteristics and overall survival. The prognosis is poorer in primary breast cancer patients with higher CA15-3 expression level, and pre-treatment CA153 expression level can be used as an independent prognostic parameter in patients with primarily breast cancer.

[Clinical research of AFP variant with microcentrifugalcolumn method and crossed affinity immunoelectrophoresis autoradiography method].

OBJECTIVE: To compare the clinical value of microcentrifugalcolumn method with crossed affinity immunoelectrophoresis autoradiography method for the measurement of alpha-fetoprotein variant (AFP-L3) in differentiation of benign and malignant liver. METHODS: Serum AFP-L3 variants in 102 primary hepatocellular carcinoma patients and 41 chronic and cirrhosis patients were separated by microcentrifugalcolumn method and crossed affinity immunoelectrophoresis autoradiography method and the clinical value of both method were compared. RESULTS: In 102 primary hepatocellular carcinoma patients, the sensitivity of AFP-L3 was 79.4% and 91.2% respectively, in 41 chronic and cirrhosis patients. The specificity of AFP-L3 was 70.7% and 29.3% respectively. The diagnostic accuracy was 76.9% and 73.4% respectively. The area in the ROC curve was 0.791 and 0.758 respectively. In the 4 primary hepatocellular carcinoma patients with lower AFP-L3, the AFP-L3 was positive by useing microcentrifugalcolumn method, but none of AFP-L3 were found by useing crossed affinity immunoelectrophoresis autoradiography method. CONCLUSION: The micro centrifugalcolumn method is more simple and rapid, it may be more useful in differentiation of benign and malignant liver than traditional crossed affinity immunoelectrophoresis autoradiography method.

[Multicenter study on screen method for gestational diabetes].

OBJECTIVE: To investigate the feasibility of using random blood glucose to screen gestational diabetes mellitus (GDM). METHOD: The random blood glucose was determined in 1 038 pregnant women between 24 and 32 gestational weeks. Then 50 gram glucose challenge test (50 g GCT) was performed followed immediately. Finally, 75 gram oral glucose tolerance test (75 g OGTT) was done without dietary control for 3 days. If two values of four were abnormal, GDM was diagnosed. Impaired glucose tolerance (IGT) was diagnosed if only one value was abnormal or the 2nd hour value ranged from 120 to 164 mg/dl. RESULTS: (1) The determination of the three steps was completed in 948 cases. Among them, 42 cases (4.4%) were GDM, 372 cases (39.2%) were IGT and other 534 cases were normal. (2) In the normal group, the random blood glucose were different in fasting and postprandial times. No difference was found among blood glucose values determined of 50 g GCT at different times except that the value of 50 g GCT 1 hour postprandial was higher than the value at other times. There was no significant association between random blood glucose and 50 g GCT. (3) The sensitivity and specificity were 50.0% and 67.7%, when IGT was diagnosed using the cut point of 6.4 mmol/L (115 mg/dl) of random blood glucose, which was similar with 51.1% of sensitivity and 71.2% of specificity when using >or= 7.8 mmoL/L (140 mg/dl) as the cut point of 50 g GCT. If 6.4 mmol/L (115 mg/dl) was used as the cut point in GDM group the sensitivity would be 80.0%, which was much higher than that of IGT group and the specificity was 61.2%. In this study, if the value of >or= 8.3 mmoL/L (150 mg/dl) was used as the cut-point of 50 g GCT to screen the GDM, the sensitivity decreased only 2.0% while the specificity increased more than 10.0%. CONCLUSIONS: (1) The determination of random blood glucose to screen GDM couldn't replace the 50 g GCT, but it can be used as a complemental method and can be used repeatedly at any gestational age and convenience the pregnant women and the doctors. (2) The value of 8.3 mmol/L (150 mg/dl) was used as the cut-point of 50 g GCT, the specificity would be increased and the requirement for OGTT would be lowered markedly, which would reduce economic and psychological stress.

[Postpartum reclassification in women with gestational diabetes and analyzing the high risk factors associated with them].

OBJECTIVE: To follow up the 75 g oral glucose tolerance test (OGTT) in gestational diabetes mellitus (GDM) at 2 month after birth for reclassifying and analyzing the risk factors associated with the abnormal blood glucose level. METHODS: 294 cases of GDM underwent a 2 hours 75 g OGTT two months postpartum. According to the WHO diagnostic criteria, they were divided into three groups: (1) type 2 diabetic mellitus (DM), impaired glucose tolerance (IGT) and normal. The related factors during pregnancy were analysed. RESULTS: 160 cases (54.4%) were normal, 75 cases (25.5%) and 59 cases (20.1%) were IGT and DM respectively. (2) In the DM group, the gestational age at diagnosis was much earlier than those of the other two groups (P < 0.01), the glucose level of 50 gram glucose challenge test (GCT) and fasting blood glucose values in OGTT and the value of HbAlc at diagnosis were evidently higher than the other two groups (P < 0.01, P < 0.01, P < 0.01), the gestational age of use of insulin for treatment was earlier than normal group, the dosage used markedly greater than the other two groups, the fasting blood glucose and 2 hours postprandial glucose were significantly higher than other two groups (P < 0.01) within 7 days after birth. Maternal age, body weight and family history did not show difference in the three groups. CONCLUSION: Among the GDM, about 1/4 were IGT and 1/5 were type 2 DM. The DM group showed early diagnosis, high fasting blood glucose, high frequency to use insulin during pregnancy.