Association between Adverse Maternal Clinical Outcomes and Imbalance of Cytokines and Angiogenic Factors in Preterm Preeclampsia / Associação entre desfechos maternos adversos e desbalanço de citocinas e fatores angiogênicos em pré-eclâmpsia pré-termo

Abstract Objective Preeclampsia (PE) is a pregnancy-specific syndrome characterized by abnormal levels of cytokines and angiogenic factors, playing a role in the disease development. The present study evaluated whether immunological markers are associated with the gestational age and with the disease severity in preeclamptic women. Methods Ninety-five women who developed PE were stratified for gestational age as preterm PE (< 37 weeks) and term PE (≥ 37 weeks of gestation) and compared for disease severity as well as plasma concentration of angiogenic factors and cytokines. The concentrations of placental growth factor (PlGF), vascular endothelial growth factor (VEGF), Fms-like soluble tyrosine kinase (sFlt-1) and soluble endoglin (sEng), as well as the cytokines, tumor necrosis factor-α (TNF-α) and interleukin 10 (IL-10), were determined by enzyme-linked immunosorbent assay (ELISA). Results The comparison between preeclamptic groups showed a higher percentage of severe cases in preterm PE (82.1%) than in term PE (35.9%). Similarly, the concentrations of TNF-α, sFlt-1, and sEng, as well as TNF-α/IL-10 and sFlt-1/PlGF ratios were significantly higher in the preterm PE group. In contrast, concentrations of PlGF, VEGF, and IL-10 were significantly lower in women with preterm PE. Negative correlations between TNF-α and IL-10 (r = 0.5232) and between PlGF and sFlt1 (r = 0.4158) were detected in the preterm PE. Conclusion In pregnant women with preterm PE, there is an imbalance between immunological markers, with the predominance of anti-angiogenic factors and TNF-α, associated with adverse maternal clinical outcomes.

Resumo Objetivo A pré-eclâmpsia (PE) é uma síndrome específica da gravidez caracterizada por níveis anormais de citocinas e fatores angiogênicos, que desempenham um papel no desenvolvimento da doença. Este estudo avaliou se os marcadores imunológicos estão associados à idade gestacional e à gravidade da doença em mulheres com pré-eclâmpsia. Métodos Noventa e cinco mulheres que desenvolveram PE foram estratificadas pela idade gestacional em PE pré-termo (< 37 semanas) e PE a termo (≥ 37 semanas de gestação) e comparadas quanto à gravidade da doença, bem como à concentração plasmática de fatores angiogênicos e citocinas. As concentrações de fator de crescimento placentário (PlGF), fator de crescimento endotelial vascular (VEGF), tirosina quinase solúvel semelhante a Fms (sFlt-1) e endoglina solúvel (sEng), bem como as citocinas, fator de necrose tumoral alfa (TNF- α) e interleucina 10 (IL-10), foram determinados porensaio de imunoabsorção enzimática (ELISA, na sigla em inglês). Resultados A comparação entre os grupos com pré-eclâmpsia mostrou maior porcentagem de casos graves em PE pré-termo (82,1%) do que em PE a termo (35,9%). Da mesma forma, as concentrações de TNF-α, sFlt-1 e sEng, bem como as razões TNF-α/IL-10 e sFlt-1/PlGF foram significativamente maiores no grupo de PE pré-termo. Em contraste, as concentrações de PlGF, VEGF e IL-10 foram significativamente menores em mulheres com PE pré-termo. Correlações negativas entre TNF-α e IL-10 (r = 0.5232) e entre PlGF e sFlt1 (r = 0.4158) foram detectadas no grupo de PE pré-termo. Conclusão Em gestantes com PE pré-termo, ocorre um desequilíbrio entre os marcadores imunológicos, com predomínio de fatores antiangiogênicos e TNF-α, associados a desfechos clínicos maternos adversos.

Evaluation of angiogenic factors (PlGF and sFlt-1) in pre-eclampsia diagnosis / Avaliação dos fatores angiogênicos (PlGF e sFlt-1) no diagnóstico de pré-eclâmpsia

Abstract Objective: Recent observations support the hypothesis that an imbalance between angiogenic factors has a fundamental role in the pathogenesis of pre-eclampsia and is responsible for the clinical manifestations of the disease. The goal of the present study was to evaluate the sensitivity, specificity, and the best accuracy level of Soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), and sFlt-1/PlGF ratio in maternal serum and protein/creatinine ratio in urine sample to define the best cutoff point of these tests to discriminate between the patients with gestational hypertension and the patients with pre-eclampsia, to evaluate the possibility of using them as diagnostic methods. Methods: A prospective longitudinal study was performed, and blood samples were collected from 95 pregnant patients with hypertension to measure serum concentrations of biomarkers sFlt-1 and PlGF. Urine samples were collected for protein screening. Significance was set as p < 0.05. Results: The sFlt-1/PlGF ratio demonstrated a sensitivity of 57.5% and a specificity of 60% using 50.4 as a cutoff point. The test that showed the best accuracy in the diagnosis of pre-eclampsia was protein/creatinine ratio, with a sensitivity of 78.9% and a specificity of 70% using 0.4 as a cutoff point and showing an area under the receiver operating characteristic curve of 0.80 (p < 0.001). Conclusion: No studied laboratory test proved to be fairly accurate for the diagnosis of pre-eclampsia, except for the protein/creatinine ratio. The evidence is insufficient to recommend biomarkers sFlt-1 and PlGF to be used for the diagnosis of pre-eclampsia.

Resumo Objetivo: Pesquisas recentes sustentam a hipótese de que um desequilíbrio entre fatores angiogênicos desempenhe um papel fundamental na patogênese da pré-eclâmpsia e seja responsável pelas manifestações clínicas da doença. O objetivo do presente estudo foi avaliar a sensibilidade, a especificidade e o nível de melhor acurácia do Fator semelhante a tirosina quinase 1 (sFlt-1), Fator de crescimento placentário (PlGF), e relação sFlt-1/PlGF no soro materno e relação proteína/creatinina em amostra de urina e definir o melhor ponto de corte desses testes para distinguir pacientes com hipertensão gestacional daquelas com pré-eclâmpsia, a fim de avaliar a possibilidade de utilizá-los como métodos diagnósticos. Métodos: Foi realizado um estudo prospectivo longitudinal e foram coletadas amostras de sangue de 95 gestantes com hipertensão arterial para dosar as concentrações séricas dos biomarcadores sFlt-1 e PlGF. Amostras de urina foram coletadas para pesquisa de proteinúria. Foram consideradas significativas as diferenças com p < 0,05. Resultados: A relação sFlt-1/PlGF demonstrou sensibilidade de 57,5% e especificidade de 60% utilizando 50,4 como ponto de corte. O teste que apresentou a melhor acurácia no diagnóstico de pré-eclâmpsia foi a relação proteína/creatinina, com sensibilidade de 78,9% e especificidade de 70%, utilizando 0,4 como ponto de corte e demostrando uma área sob a curva receiver operating characteristic (ROC, na sigla em inglês) de 0,80 (p < 0,001). Conclusão: Nenhum método de rastreamento isolado se mostrou com boa acurácia para o diagnóstico de pré-eclâmpsia, exceto a relação proteína/creatinina. As evidências são insuficientes para recomendar os biomarcadores sFlt-1 e PlGF como diagnóstico de pré-eclâmpsia.

Coexistence of proangiogenic potential and increased MMP-9, TIMP-1, and TIMP-2 levels in the plasma of patients with critical limb ischemia / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The objective of this study was to assess the angiogenic potential expressed as a quotient of vascular endothelial growth factor A (VEGF-A), as an indicator of proangiogenic activity, and the circulating receptors (soluble VEGF receptor protein R1 (sVEGFR-1) and sVEGFR-2), as indicators of the effect of angiogenic inhibition, depending on the concentrations of matrix metalloproteinase 2 (MMP-2) and MMP-9 and their tissue inhibitor 1 (TIMP-1) and TIMP-2 in the plasma of patients with lower extremity artery disease (LEAD). These blood parameters in patients with intermittent claudication (IC) and critical limb ischemia (CLI) were compared for select clinical and biochemical features. Stimulation of angiogenesis in the plasma of individuals with LEAD was evident as indicated by the significant increase in VEGF-A concentration along with reduced inhibition depending on circulating receptors sVEGFR-1 and sVEGFR-2. Critical ischemia was associated with higher VEGF-A, MMP-9, TIMP-1, and TIMP-2 concentrations than in the case of IC.

Aryl Hydrocarbon Receptor Ligands Indoxyl 3-sulfate and Indole-3-carbinol Inhibit FMS-like Tyrosine Kinase 3 Ligand-induced Bone Marrow-derived plasmacytoid Dendritic Cell Differentiation

Aryl hydrocarbon receptor (AhR) regulates both innate and adaptive immune responses by sensing a variety of small synthetic and natural chemicals, which act as its ligands. AhR, which is expressed in dendritic cells (DCs), regulates the differentiation of DCs. However, effects of AhR on the differentiation of DCs are variable due to the heterogeneity of DCs in cell surface marker expression, anatomical location, and functional responses. The plasmacytoid DCs (pDCs), one of DC subsets, not only induce innate as well as adaptive immune responses by secreting type I interferons and pro-inflammatory cytokines, but also induce IL-10 producing regulatory T cell or anergy or deletion of antigen-specific T cells. We showed here that AhR ligands indoxyl 3-sulfate (I3S) and indole-3-carbinol (I3C) inhibited the development of pDCs derived from bone marrow (BM) precursors induced by FMS-like tyrosine kinase 3 ligand (Flt3L). I3S and I3C downregulated the expression of signal transducer and activator of transcription 3 (STAT3) and E2-2 (Tcf4). In mice orally treated with I3S and I3C, oral tolerance to dinitrofluorobenzene was impaired and the proportion of CD11c⁺B220⁺ cells in mesenteric lymph nodes was reduced. These data demonstrate that AhR negatively regulates the development of pDCs from BM precursors induced by Flt3L, probably via repressing the expression of STAT3.

Evaluation of sFlt-1/PlGF Ratio for Predicting and Improving Clinical Management of Pre-eclampsia: Experience in a Specialized Perinatal Care Center

BACKGROUND: Management of pregnant women at high risk of pre-eclampsia (PE) requires frequent monitoring, with referral to specialized perinatal care centers. Reliable tests are necessary to improve prediction of PE and related complications and to assess disease severity and progression. An imbalance in two biomarkers, soluble fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF), is involved in PE pathogenesis. The sFlt-1 to PlGF ratio is increased in pregnant women before the onset of PE. An elevated ratio is highly predictive of PE, whereas the diagnosis of PE can be ruled out within one week for low ratios. The main objective of this study was to assess whether a low sFlt-1/PlGF ratio, below a cutoff of 38, can predict the absence of PE within one week. METHODS: We performed a prospective, monocentric, observational study to evaluate serum sFlt-1/PlGF ratio (Roche Diagnostics Cobas e411 system) for predicting -PE in a group of 67 high-risk pregnant women (20–37 gestation weeks). RESULTS: Among the 67 patients included, 53 had a sFlt-1/PlGF ratio lower than 38; none developed subsequent PE leading to a negative predictive value of 100%. Eight patients developed clinical PE. The positive predictive value was 21% at one week and 18% at four weeks, in accordance with previous studies. CONCLUSIONS: The serum sFlt-1/PlGF ratio showed highly predictive performances for ruling out PE. Using these biomarkers in routine management of PE may improve clinical care and avoid inappropriate hospitalization, which has a significant economic impact.

Report on the External Quality Assessment Scheme for Molecular Diagnostics in Korea (2017)

Quality control for genetic analysis has become more important with a drastic increase in testing volume and clinical demands. The molecular diagnostics division of the Korean Association of Quality Assurance for Clinical Laboratory conducted two trials in 2017 on the basis of molecular diagnostics surveys, involving 53 laboratories. The molecular diagnostics surveys included 37 tests: gene rearrangement tests for leukemia (BCR-ABL1, PML-RARA, AML1-ETO, and TEL-AML1), genetic tests for Janus kinase 2, FMS-like tyrosine kinase 3-internal tandem duplication, FMS-like tyrosine kinase 3-tyrosine kinase domain, nucleophosmin, cancer-associated genes (KRAS, EGFR, KIT, and BRAF), hereditary breast and ovarian cancer genes (BRCA1 and BRCA2), Li-Fraumeni syndrome (TP53), Wilson disease (ATP7B), achondroplasia (FGFR3), hearing loss and deafness (GJB2), Avellino (TGFBI), multiple endocrine neoplasia 2 (RET), Huntington disease, spinocerebellar ataxia, spinal and bulbar muscular atrophy, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes, myoclonic epilepsy ragged red fibre, Leber hereditary optic neuropathy, Prader-raderd Angelman syndrome, Duchenne muscular dystrophy, spinal muscular atrophy, fragile X syndrome, apolipoprotein E genotyping, methylenetetrahydrofolate reductase genotyping, and ABO genotyping. Molecular genetic surveys revealed excellent results for most participants. The external quality assessment program for genetic analysis in 2017 proved useful for continuous education and the evaluation of quality improvement.

Ventilation-induced changes correlate to pulmonary vascular response and VEGF, VEGFR-1/2, and eNOS expression in the rat model of postnatal hypoxia

Neonatal asphyxia occurs due to reduction in oxygen supply to vital organs in the newborn. Rapid restoration of oxygen to the lungs after a long period of asphyxia can cause lung injury and decline of respiratory function, which result from the activity of molecules that induce vascular changes in the lung such as nitric oxide (NO) and vascular endothelial growth factors (VEGF). In this study, we evaluated the pulmonary and vascular morphometry of rats submitted to the model of neonatal asphyxia and mechanical ventilation, their expression of pulmonary VEGF, VEGF receptors (VEGFR-1/VEGFR-2), and endothelial NO synthase (eNOS). Neonate Sprague-Dawley rats (CEUA #043/2011) were divided into four groups (n=8 each): control (C), control submitted to ventilation (CV), hypoxia (H), and hypoxia submitted to ventilation (HV). The fetuses were harvested at 21.5 days of gestation. The morphometric variables measured were body weight (BW), total lung weight (TLW), left lung weight (LLW), and TLW/BW ratio. Pulmonary vascular measurements, VEGFR-1, VEGFR-2, VEGF, and eNOS immunohistochemistry were performed. The morphometric analysis showed decreased TLW and TLW/BW ratio in HV compared to C and H (P<0.005). Immunohistochemistry showed increased VEGFR-2/VEGF and decreased VEGFR-1 expression in H (P<0.05) and lower eNOS expression in H and HV. Median wall thickness was increased in H, and the expression of VEGFR-1, VEGFR-2, VEGF, and eNOS was altered, especially in neonates undergoing H and HV. These data suggested the occurrence of arteriolar wall changes mediated by NO and VEGF signaling in neonatal hypoxia.

Effect of Anti-vascular Endothelial Growth Factor Antibody on the Survival of Cultured Retinal Ganglion Cells

PURPOSE: To investigate the effects of anti-vascular endothelial growth factor (VEGF) antibody on the survival of retinal ganglion cell (RGC)-5 cells differentiated with staurosporine under oxidative stress. METHODS: We used real-time polymerase chain reaction and Western blot to confirm the expression of VEGF, VEGF receptor (VEGFR)-1 and VEGFR-2 in RGC-5 cells differentiated with staurosporine for 6 hours. The differentiated RGC-5 cells were treated with 800 µM hydrogen peroxide (H₂O₂) for 24 hours to induce oxidative stress. Then, the survival rate of RGC-5 was confirmed by lactate dehydrogenase assay at each concentration (0, 0.01, 0.1, and 1 mg) using bevacizumab as the anti-VEGF antibody. The expression of VEGF, VEGFR-1, and VEGFR-2 was confirmed using real-time polymerase chain reaction. RESULTS: VEGF, VEGFR-1, and VEGFR-2 were all expressed in differentiated RGC-5 cells. When RGC-5 cells were simultaneously treated with bevacizumab and 800 µM H₂O₂, survival of RGC-5 decreased with bevacizumab concentration. VEGF expression in RGC-5 cells increased with increasing concentration of bevacizumab. Similar patterns were observed for VEGFR-1 and VEGFR-2, but the degree of increase was smaller than that for VEGF. CONCLUSIONS: When bevacizumab was administered to differentiated RGC-5 cells, the cell damage caused by oxidative stress increased. Therefore, given these in vitro study results, caution should be exercised with bevacizumab treatment.

Fragment Analysis for Detection of the FLT3-Internal Tandem Duplication: Comparison with Conventional PCR and Sanger Sequencing

BACKGROUND: We evaluated a sensitive and quantitative method utilizing fragment analysis of the fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD), simultaneously measuring mutant allele burden and length, and verified the analytical performance. METHODS: The number and allelic burden of FLT3-ITD mutations was determined by fragment analysis. Serial mixtures of mutant and wild-type plasmid DNA were used to calculate the limit of detection of fragment analysis, conventional PCR, and Sanger sequencing. Specificity was evaluated using DNA samples derived from 50 normal donors. Results of fragment analysis were compared to those of conventional PCR, using 481 AML specimens. RESULTS: Defined mixtures were consistently and accurately identified by fragment analysis at a 5% relative concentration of mutant to wild-type, and at 10% and 20% ratios by conventional PCR and direct sequencing, respectively. No false positivity was identified. Among 481 AML specimens, 40.1% (193/481) had FLT3-ITD mutations. The mutant allele burden (1.7-94.1%; median, 28.2%) and repeated length of the mutation (14-153 bp; median, 49 bp) were variable. The concordance rate between fragment analysis and conventional PCR was 97.7% (470/481). Fragment analysis was more sensitive than conventional PCR and detected 11 additional cases: seven had mutations below 10%, three cases represented conventional PCR failure, and one case showed false negativity because of short ITD length (14 bp). CONCLUSIONS: The new fragment analysis method proved to be sensitive and reliable for the detection and monitoring of FLT3-ITD in patients with AML. This could be used to simultaneously assess ITD mutant allele burden and length.

Survival and treatment response in adults with acute promyelocytic leukemia treated with a modified International Consortium on Acute Promyelocytic Leukemia protocol

ABSTRACT Acute promyelocytic leukemia has good prognosis in view of the high complete remission and survival rates achieved with therapies containing all-trans retinoic acid or arsenic trioxide. However, there is a significant risk of death during induction due to hemorrhage secondary to disseminated intravascular coagulation. This has contributed to a gap in the prognosis of patients between developed and developing countries. The International Consortium on Acute Promyelocytic Leukemia was created in 2005 and proposed a treatment protocol based on daunorubicin and all-trans retinoic acid stratified by risk geared toward developing countries. Herein are presented the results from the first patient cohort treated in a single developing country hospital employing a slightly modified version of the International Consortium protocol in a real life setting. Twenty patients with acute promyelocytic leukemia were enrolled: 27.8% had low-risk, 55.6% intermediate risk and 16.7% high-risk. The complete remission rate was 94.4% after a median of 42 days. Both relapse rates and death rates were one patient (5.5%) each. No deaths were observed during consolidation. After a median follow-up of 29 months, the overall survival rate was 89.1%. Efficacy and safety of the International Consortium on Acute Promyelocytic Leukemia protocol has been reproduced in acute promyelocytic leukemia patients from a developing country.