CyProQuant-PCR: a real time RT-PCR technique for profiling human cytokines, based on external RNA standards, readily automatable for clinical use

BACKGROUND: Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency. RESULTS: To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-γ, MIF, TGF-β1 and TNF-α mRNA. We showed that the beta-2 microglobulin (β2-MG) gene was suitable for data normalisation since the level of β2-MG transcripts in naïve PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-β1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-β1 mRNA in response to LPS. CONCLUSION: The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications

Father’s perceptions and care involvement for their very preterm infants at French neonatal intensive care units

ObjectivesWe aimed to evaluate (1) fathers’ perceptions and care involvement for their very premature infants and their views of the hospitalization period based on parental reports and (2) their evolution over time.MethodsWe used an online parental survey to assess answers from parents of very preterm infants who were successfully discharged from French neonatal units. We analysed answers from February 2014 to January 2019 to an anonymous internet-based survey from the GREEN committee of the French Neonatal Society. Responses were compared for period 1 (P1, 1998 to 2013) and period 2 (P2, 2014 to 2019).ResultsWe analyzed 2,483 surveys, 124 (5%) from fathers and 2,359 (95%) from mothers. At birth, 1,845 (80%) fathers were present in the hospital, but only 879 (38%) were near the mother. The presence of fathers in the NICU increased from P1 to P2 (34.5% vs. 43.1%, p = 0.03). Nearly two thirds of fathers accompanied their infants during transfer to the NICU (1,204 fathers, 60.6%). Fathers and mothers had similar perceptions regarding relationships with caregivers and skin-to-skin contact with their infants. However, more fathers than mothers felt welcome in the NICU and in care involvement regarding requests for their wishes when they met their infant (79% vs. 60%, p = 0.02) and in the presentation of the NICU (91% vs. 76%; p = 0.03). Mothers and fathers significantly differed in the caring procedures they performed (p = 0.01), procedures they did not perform but wanted to perform (p < 0.001), and procedures they did not perform and did not want to perform (p < 0.01).ConclusionMost fathers were present at the births of their very preterm infants, but fewer fathers were near the mother at this time. Less than two thirds of fathers accompanied their infants to the NICU. There should be further changes to better meet the specific needs of the fathers of infants requiring care in the NICU. Continuing assessment with an online questionnaire may be useful to monitor changes over time in father’s involvement in NICUs

Evolution of Gut Microbiome and Metabolome in Suspected Necrotizing Enterocolitis: A Case-Control Study

Background: Necrotizing enterocolitis (NEC) is a devastating condition in preterm infantsdue to multiple factors, including gut microbiota dysbiosis. NEC development is poorly understood,due to the focus on severe NEC (NEC-2/3). Methods: We studied the gut microbiota, microbiomeand metabolome of children with suspected NEC (NEC-1). Results: NEC-1 gut microbiota had ahigher abundance of the Streptococcus (second 10-days of life) and Staphylococcus (third 10-days oflife) species. NEC-1 children showed a microbiome evolution in the third 10-days of life being themost divergent, and were associated with a dierent metabolomic signature than in healthy children.The NEC-1 microbiome had increased glycosaminoglycan degradation and lysosome activity bythe first 10-days of life, and was more sensitive to childbirth, low birth weight and gestational age,than healthy microbiome. NEC-1 fecal metabolome was more divergent by the second month of life.Conclusions: NEC-1 gut microbiota and microbiome modifications appear more distinguishable bythe third 10-days of life, compared to healthy children. These data identify a precise window of time(i.e., the third 10-days of life) and provide microbial targets to fight/blunt NEC-1 progression

Father’s perceptions and care involvement for their very preterm infants at French neonatal intensive care units

International audienceObjectives We aimed to evaluate (1) fathers’ perceptions and care involvement for their very premature infants and their views of the hospitalization period based on parental reports and (2) their evolution over time. Methods We used an online parental survey to assess answers from parents of very preterm infants who were successfully discharged from French neonatal units. We analysed answers from February 2014 to January 2019 to an anonymous internet-based survey from the GREEN committee of the French Neonatal Society. Responses were compared for period 1 (P1, 1998 to 2013) and period 2 (P2, 2014 to 2019). Results We analyzed 2,483 surveys, 124 (5%) from fathers and 2,359 (95%) from mothers. At birth, 1,845 (80%) fathers were present in the hospital, but only 879 (38%) were near the mother. The presence of fathers in the NICU increased from P1 to P2 (34.5% vs. 43.1%, p = 0.03). Nearly two thirds of fathers accompanied their infants during transfer to the NICU (1,204 fathers, 60.6%). Fathers and mothers had similar perceptions regarding relationships with caregivers and skin-to-skin contact with their infants. However, more fathers than mothers felt welcome in the NICU and in care involvement regarding requests for their wishes when they met their infant (79% vs. 60%, p = 0.02) and in the presentation of the NICU (91% vs. 76%; p = 0.03). Mothers and fathers significantly differed in the caring procedures they performed ( p = 0.01), procedures they did not perform but wanted to perform ( p < 0.001), and procedures they did not perform and did not want to perform ( p < 0.01). Conclusion Most fathers were present at the births of their very preterm infants, but fewer fathers were near the mother at this time. Less than two thirds of fathers accompanied their infants to the NICU. There should be further changes to better meet the specific needs of the fathers of infants requiring care in the NICU. Continuing assessment with an online questionnaire may be useful to monitor changes over time in father’s involvement in NICUs