OBJECTIVE: To develop monoclonal antibodies that can specifically recognize human von Willebrand factor (VWF) propeptide (VWFpp) in plasma, and establish a rapid and reliable method for the detection of VWFpp antigen in plasma by using the double-antibody sandwich ELISA with the obtained anti-VWFpp monoclonal antibody. METHODS: The recombinant human VWFpp (D1 and D2 regions) protein expressed in eukaryotic cells was used as immunogen to immunize BALB/c mice with routine method, so as to obtain clones of fusion cells. After screening and identification, hybridoma cell lines secreting monoclonal antibodies against VWFpp were selected, and then double-antibody sandwich ELISA assay was used to construct VWFpp antigen detection kit for the determination of VWFpp in human plasma. The levels of VWFpp antigen in plasma of 12 leukemia patients who underwent bone marrow transplantation were dynamically detected. RESULTS: Two hybridoma cell lines that can be subcultured continuously and secrete monoclonal antibodies against VWFpp were obtained and named SZ175 and SZ176 respectively. Identified by ELISA and Western blot, the antibodies could both specifically recognize VWFpp but couldn't recognize mature VWF (without propeptide). Based on the principle of double-antibody sandwich ELISA, monoclonal antibodies SZ175 and SZ176 were successfully made into a kit for detecting VWFpp antigen. The plasma VWFpp levels of leukemia patients before and after bone marrow transplantation were dynamically detected. The results showed that the plasma VWFpp levels of the patients after transplantation were significantly higher than those before transplantation. CONCLUSION Two monoclonal antibodies against VWFpp were successfully prepared, and a double-antibody sandwich ELISA detection kit for VWFpp antigen was constructed, which provides a powerful tool for further study on the biological function of VWFpp, the clinical diagnosis and classification of von Willebrand disease (VWD), and the prognostic monitoring of endothelial injury-related diseases.
Animals ; Mice ; Humans ; von Willebrand Factor ; Antibodies, Monoclonal ; Protein Precursors/metabolism* ; von Willebrand Diseases/diagnosis* ; Prognosis

The formation of most proteins consists of two steps: the synthesis of precursor proteins and the synthesis of functional proteins. In these processes, propeptides play important roles in assisting protein folding or inhibiting its activity. As an important polypeptide chain coded by a gene sequence in lipase gene, propeptide usually functions as an intramolecular chaperone, assisting enzyme molecule folding. Meanwhile, some specific sites on propeptide such as glycosylated sites, have important effect on the activity, stability in extreme environment, methanol resistance and the substrate specificity of the lipase. Studying the mechanism of propeptide-mediated protein folding, as well as the influence of propeptide on lipases, will allow to regulate lipase by alternating the propeptide folding behavior and in turn pave new ways for protein engineering research.
Lipase/metabolism* ; Molecular Chaperones/metabolism* ; Protein Folding ; Protein Precursors ; Substrate Specificity

OBJECTIVE: To discuss the clinical significance of antibacterial peptide LL-37 in the early diagnosis of patients with sepsis in emergency department. METHODS: Forty patients diagnosed with sepsis in the emergency department of the Affiliated Hospital of Zunyi Medical College from December 2017 to March 2018 were enrolled as sepsis group. Twenty healthy volunteers were enrolled contemporaneously in our hospital at medical center as healthy control group. Peripheral blood was collected immediately after diagnosis in sepsis group or during physical examination in healthy control group. The expression of antibacterial peptide LL-37 was determined by enzyme-linked immunosorbent assay (ELISA). Meanwhile, serum procalcitonin (PCT) and C-reactive protein (CRP) levels were determined. The differences in antibacterial peptide LL-37, PCT and CRP levels between the two groups were compared. Pearson correlation method was used to analyze the correlation between antibacterial peptide LL-37, PCT and CRP. Receiver operating characteristic (ROC) curve was drawn, and the early individually or jointly diagnostic value of each detected index for sepsis was analyzed. RESULTS: The levels of antimicrobial peptide LL-37, PCT and CRP in peripheral blood of sepsis group were significantly higher than those of healthy control group [LL-37 (μg/L): 1.34±0.69 vs. 0.10±0.06, PCT (μg/L): 46.67±39.51 vs. 0.03±0.02, CRP (mg/L): 129.68±49.83 vs. 3.16±2.85], with statistically significant differences (all P < 0.05). Pearson correlation analysis showed that the expression of antimicrobial peptide LL-37 was positively correlated with PCT and CRP levels (r₁ = 0.835, r₂ = 0.932, both P < 0.01). ROC curve analysis showed that the area under ROC curve (AUC) of LL-37, PCT and CRP for early diagnosis of sepsis was 0.885, 0.963 and 0.983, respectively, and the AUC of combined diagnosis of the three parameters was as high as 0.994, indicating that the value of combined diagnosis of sepsis was greater than that of single diagnosis; when the combined prediction probability of the three parameters was 0.92, the sensitivity was 97.5%, and the specificity was 95.0%. CONCLUSIONS Antibacterial peptide LL-37 has certain clinical value in early diagnosis of patients with sepsis, which can be used as early routine monitoring indicators for patients with early sepsis when combined with PCT and CRP.
Antimicrobial Cationic Peptides/metabolism* ; C-Reactive Protein ; Calcitonin ; Calcitonin Gene-Related Peptide ; Early Diagnosis ; Emergency Service, Hospital ; Humans ; Protein Precursors ; ROC Curve ; Sepsis/metabolism* ; Cathelicidins

OBJECTIVETo study the expression of serum cytokines, interleukin-38 (IL-38) and interleukin-1β (IL-1β) in the acute phase of Kawasaki disease (KD) in children and the association of IL-38 and IL-1β with inflammatory response in the acute phase and the development of coronary artery lesion (CAL).

METHODSA total of 40 children with KD who were hospitalized in the hospital between July 2015 and June 2016 were enrolled, with 21 children in the CAL group and 19 in the non-CAL (NCAL) group. Thirty healthy children and 19 children with infection and pyrexia, who were matched for sex and age, were enrolled as healthy control group and pyrexia control group respectively. ELISA was used to measure the serum levels of IL-38 and IL-1β in the 40 children in the acute phase of KD. Spearman's rank correlation analysis was used to investigate the correlations of IL-1β and IL-38 with interleukin-6 (IL-6), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), procalcitonin (PCT), N-terminal pro-brain natriuretic peptide (NT-proBNP), triglyceride (TG), and total cholesterol (TC).

RESULTSThe serum level of IL-38 in the children in the acute phase of KD was significantly lower than that in the healthy control group (P<0.05), but significantly higher than that in the pyrexia control group (P<0.05). There was no significant difference in the level of IL-38 between the CAL and NCAL groups (P>0.05). The children in the acute phase of KD had a significantly higher level of IL-1β than the healthy control group (P<0.05), while there was no significant difference between this group and the pyrexia control group (P>0.05). There was also no significant difference in the level of IL-1β between the CAL and NCAL groups (P>0.05). Serum IL-1β and IL-38 levels were not correlated with serum levels of CRP, ESR, PCT, IL-6, and NT-ProBNP or blood lipids (TG and TC) (P>0.05).

CONCLUSIONSIL-38 is involved in an inflammatory response in the acute phase of KD and may exert an anti-inflammatory effect, which is opposite to the effect of IL-1β to promote inflammatory response. However, there is no significant correlation between these two cytokines and the development of CAL in KD.

Acute Disease ; Atrial Natriuretic Factor ; blood ; Blood Sedimentation ; C-Reactive Protein ; metabolism ; Case-Control Studies ; Child ; Child, Preschool ; Cholesterol ; blood ; Coronary Artery Disease ; blood ; etiology ; pathology ; Coronary Vessels ; pathology ; Female ; Humans ; Infant ; Interleukin-1beta ; blood ; Interleukins ; blood ; Male ; Mucocutaneous Lymph Node Syndrome ; blood ; complications ; Procalcitonin ; blood ; Protein Precursors ; blood ; Triglycerides ; blood

BACKGROUNDThe clinical significance of acute vasoreactivity testing (AVT) in patients with chronic thromboembolic pulmonary hypertension (CTEPH) remains unclear. We analyzed changes in hemodynamics and oxygenation dynamics indices after AVT in patients with CTEPH using patients with pulmonary arterial hypertension (PAH) as controls.

METHODSWe analyzed retrospectively the results of AVT in 80 patients with PAH and 175 patients with CTEPH registered in the research database of Beijing Chao-Yang Hospital between October 2005 and August 2014. Demographic variables, cardiopulmonary indicators, and laboratory findings were compared in these two subgroups. A long-term follow-up was conducted in patients with CTEPH. Between-group comparisons were performed using the independent-sample t-test or the rank sum test, within-group comparisons were conducted using the paired t-test or the Wilcoxon signed-rank test, and count data were analyzed using the Chi-squared test. Survival was estimated using the Kaplan-Meier method and log-rank test.

RESULTSThe rates of positive response to AVT were similar in the CTEPH (25/175, 14.3%) and PAH (9/80, 11.3%) groups (P > 0.05). Factors significantly associated a positive response to AVT in the CTEPH group were level of N-terminal pro-brain natriuretic peptide (≤1131.000 ng/L), mean pulmonary arterial pressure (mPAP, ≤44.500 mmHg), pulmonary vascular resistance (PVR, ≤846.500 dyn·s-1·m-5), cardiac output (CO, ≥3.475 L/min), and mixed venous oxygen partial pressure (PvO2, ≥35.150 mmHg). Inhalation of iloprost resulted in similar changes in mean blood pressure, mPAP, PVR, systemic vascular resistance, CO, arterial oxygen saturation (SaO2), mixed venous oxygen saturation, partial pressure of oxygen in arterial blood (PaO2), PvO2, and intrapulmonary shunt (Qs/Qt) in the PAH and CTEPH groups (all P > 0.05). The survival time in patients with CTEPH with a negative response to AVT was somewhat shorter than that in AVT-responders although the difference was not statistically significant (χ2 =3.613, P = 0.057). The survival time of patients with CTEPH who received calcium channel blockers (CCBs) was longer than that in the group with only basic treatment and not shorter than that of patients who receiving targeted drugs or underwent pulmonary endarterectomy (PEA) although there was no significant difference between the four different treatment regimens (χ2 =3.069, P = 0.381).

CONCLUSIONSThe rates of positive response to AVT were similar in the CTEPH and PAH groups, and iloprost inhalation induced similar changes in hemodynamics and oxygenation dynamics indices. A positive response to AVT in the CTEPH group was significantly correlated with milder disease and better survival. Patients with CTEPH who cannot undergo PEA or receive targeted therapy but have a positive response to AVT might benefit from CCB treatment.

Administration, Inhalation ; Adult ; Aged ; Arterial Pressure ; drug effects ; Atrial Natriuretic Factor ; metabolism ; Calcium Channel Blockers ; administration & dosage ; therapeutic use ; Endarterectomy ; Familial Primary Pulmonary Hypertension ; drug therapy ; physiopathology ; Female ; Hemodynamics ; drug effects ; Humans ; Hypertension, Pulmonary ; drug therapy ; physiopathology ; Iloprost ; administration & dosage ; therapeutic use ; Male ; Middle Aged ; Protein Precursors ; metabolism ; Retrospective Studies ; Software ; Vasodilator Agents ; administration & dosage ; therapeutic use

PURPOSE: Patients with gout are similar to those with bacterial infection in terms of the nature of inflammation. Herein we compared the differences in procalcitonin (PCT) levels between these two inflammatory conditions and evaluated the ability of serum PCT to function as a clinical marker for differential diagnosis between acute gouty attack and bacterial infection. MATERIALS AND METHODS: Serum samples were obtained from 67 patients with acute gouty arthritis and 90 age-matched patients with bacterial infection. Serum PCT levels were measured with an enzyme-linked fluorescent assay. RESULTS: Serum PCT levels in patients with acute gouty arthritis were significantly lower than those in patients with bacterial infection (0.096±0.105 ng/mL vs. 4.94±13.763 ng/mL, p=0.001). However, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels showed no significant differences between the two groups. To assess the ability of PCT to discriminate between acute gouty arthritis and bacterial infection, the areas under the curves (AUCs) of serum PCT, uric acid, and CRP were 0.857 [95% confidence interval (CI), 0.798-0.917, p<0.001], 0.808 (95% CI, 0.738-0.878, p<0.001), and 0.638 (95% CI, 0.544-0.731, p=0.005), respectively. There were no significant differences in ESR and white blood cell counts between these two conditions. With a cut-off value of 0.095 ng/mL, the sums of sensitivity and specificity of PCT were the highest (81.0% and 80.6%, respectively). CONCLUSION: Serum PCT levels were significantly lower in patients with acute gouty attack than in patients with bacterial infection. Thus, serum PCT can be used as a useful serologic marker to differentiate between acute gouty arthritis and bacterial infections.
Area Under Curve ; Arthritis, Gouty/*diagnosis ; Bacterial Infections/*diagnosis ; Biomarkers/blood ; Blood Sedimentation ; C-Reactive Protein/metabolism ; Calcitonin/*blood ; Case-Control Studies ; Cross-Sectional Studies ; Diagnosis, Differential ; Female ; Humans ; Inflammation ; Leukocyte Count ; Male ; Middle Aged ; Protein Precursors/*blood ; Sensitivity and Specificity ; Uric Acid/blood

Folate, vitamin B12, and homocysteine (HCY) are involved in the metabolism of nucleic acid precursors and it has been hypothesized that they also influence telomere length, a biomarker of aging. However, previous studies have reported inconsistent findings, and data for older adults are limited. Our study aimed to evaluate associations between leukocyte telomere length (LTL) and serum folate, vitamin B12, and HCY levels among adults aged 55 years and over. In a cross-sectional study in 798 men and women aged 55-79 years, serum folate, vitamin B12, and HCY levels were measured using chemiluminescent immunometric assays, and relative LTL was assessed using quantitative real-time polymerase chain reaction. To evaluate associations between LTL and serum folate, vitamin B12, and HCY levels, multiple linear regression models were used. In multiple models adjusted for age, sex, serum high sensitive C-reactive protein (hs-CRP) levels, and other potential confounding factors, we found no association between LTL and serum folate, vitamin B12, and HCY levels. However, we did find a significant inverse association between HCY levels and LTL in participants with serum hs-CRP levels of > or = 2 mg/L (p < 0.05). Moreover, there was a trend toward an association between HCY and vitamin B12 levels in these individuals (p = 0.08). In those with serum hs-CRP levels of < 2 mg/L, HCY was inversely associated with vitamin B12 levels (p < 0.001) and had no association with LTL. Our findings suggest that increased serum HCY levels, when combined with the presence of systemic inflammation, may play a role in accelerating biological aging.
Adult* ; Aging ; C-Reactive Protein ; Cross-Sectional Studies ; Female ; Folic Acid ; Homocysteine* ; Humans ; Inflammation* ; Leukocytes* ; Linear Models ; Male ; Metabolism ; Nucleic Acid Precursors ; Real-Time Polymerase Chain Reaction ; Telomere* ; Vitamin B 12* ; Vitamin B Complex ; Vitamins*

Animals ; Cell Differentiation ; Hepatocyte Growth Factor ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; metabolism ; Protein Precursors ; genetics ; metabolism ; Serine Endopeptidases ; genetics ; metabolism

Epidermal growth factor (EGF) is an epithelial cell growth factor that can stimulate intestinal development, repair the damage of epidermal cells as well as reduce the incidence of pathogen infection and diarrhea. In order to produce a recombinant Lactobacillus plantarum (L. plantarum) expressing porcine epidermal growth factor (pEGF), we constructed a recombinant vector stably expressing pEGF in L. plantarum strains. First, L. plantarum strain Lp-1 was isolated from intestinal contents of piglets. Then the functional domain of pEGF, M6 precursor protein signal peptide (SP) and super strong constitutive promoter (SCP) were connected with the backbone plasmid pIAβ8 to construct the recombinant vector that was transformed into Lp-1 by electroporation. Afterwards, pEGF was expressed in Lp-1 and detected by Tricine-SDS-PAGE and ELISA. After orally irrigated early-weaned BALB/c mice with the recombinant L. plantarum every morning and late afternoon for 10 consecutive days, body weight, villous height and crypt depth in the intestine were measured to examine the influence of the recombinant bacteria on the intestinal development of early-weaned mice in vivo. Finally, the results of our experiments demonstrated that pEGF was successfully expressed in Lp-1 and the molecular weight of pEGF was 6 kDa. In addition, the recombinant pEGF can enhanced the daily gain and exerted significance influence (P < 0.05) to the small intestinal morphology of early-weaned BALB/c mice. In conclusion, pEGF could be expressed in L. plantarum and the recombinant pEGF possesses good biological activity.
Animals ; Electrophoresis, Polyacrylamide Gel ; Epidermal Growth Factor ; biosynthesis ; Genetic Vectors ; Intestines ; microbiology ; Lactobacillus plantarum ; metabolism ; Mice ; Mice, Inbred BALB C ; Plasmids ; Promoter Regions, Genetic ; Protein Precursors ; Protein Sorting Signals ; Recombinant Proteins ; biosynthesis ; Swine

OBJECTIVETo explore the effect of Compound Tongtu Granule (CTG) on intestinal permeability in elderly sepsis patients.

METHODSEighty elderly sepsis patients were randomly assigned to the experimental group and the control group by randomized double blinded method, 40 in each group. On the basis of conventional antiseptic treatment program, patients in the experimental group took CTG, while those in the control group took placebos. The dosage for CTG or placebos was 14.3 g each package, one package each time, twice daily for 14 successive days. Patients' abdominal symptoms and signs, levels of serum inflammatory factors (high-sensitivity C-reactive protein and procalcitonin), levels of plasma endotoxin, and the intestinal permeability (IP, represented by urinary lactulose/mannitol excretion rate) were compared between the two groups before and after treatment.

RESULTSAfter 14-day treatment, patients in the experimental group had improved abdominal symptoms, increased frequency of defecation, significantly decreased levels of plasma endotoxin and IP, when compared with the control group (P < 0.05).

CONCLUSIONCTG could improve the intestinal barrier function in elderly sepsis patients.

Aged ; C-Reactive Protein ; metabolism ; Calcitonin ; metabolism ; Calcitonin Gene-Related Peptide ; Defecation ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Endotoxins ; metabolism ; Humans ; Intestines ; metabolism ; Permeability ; Protein Precursors ; metabolism ; Sepsis ; drug therapy ; physiopathology