Multiparameter flow cytometry analysis of leukocyte markers for diagnosis in preterm neonatal sepsis.

PURPOSE: Neonatal sepsis is an important public health concern worldwide due to its immediate lethality and long-term morbidity rates, Clinical evaluation and laboratory analyses are indispensable for diagnosis of neonatal sepsis. However, assessing multiple biomarkers in neonates is difficult due to limited blood availability. The aim is to investigate if the neonatal sepsis in preterm could be identified by multiparameter analysis with flow cytometry. MATERIALS AND METHODS: The expression of activation-related molecules was evaluated by flow cytometry in newborn with or without risk factors for sepsis. RESULTS: Our analysis revealed that several markers could be useful for sepsis diagnosis, such as CD45RA, CD45RO, or CD71 on T cells; HLA-DR on NKT or classic monocytes, and TREM-1 on non-classic monocytes or neutrophils. However, ROC analysis shows that the expression of CD45RO on T lymphocytes is the only useful biomarker for diagnosis of neonatal late-onset sepsis. Also, decision tree analyses showed that CD45RO plus CD27 could help differentiate the preterm septic neonates from those with risk factors. CONCLUSIONS: Our study shows a complementary and practical strategy for biomarker assessment in neonatal sepsis.

Effect of fresh frozen plasma on the in vitro activation of U937 monocytes: a potential role for the age of blood donors and their underlying cytokine profile.

BACKGROUND: Fresh frozen plasma (FFP) administration may increase the risk of nosocomial infections in parallel with the development of immune modulation. This could be driven by soluble mediators, possibly influencing the in vitro activation of human U937 monocyte cells, in a manner dependent on the age of the donors. METHODS: FFP donors were stratified into groups of 19-30 years, 31-40 years or 41-50 years, and U937 cells were cultured with FFP (alone or plus lipopolysaccharide-LPS) for 24 h. Both in FFP and supernatants, TNF, IL-1ß, IL-6, and IL-10 levels were measured by ELISA. Additionally, CD11B, TLR2, and CASP3 gene expression were measured by qtPCR in U937 cells. Total phagocytic activity was also assayed. RESULTS: Elevated IL-10, but low TNF and IL-1ß levels were measured in FFP from individuals aged 19-40 years, whereas in individuals aged 41-50 years FFP were characterized by equalized TNF and IL-10 levels. Elevated IL-6 levels were found in all FFP samples, especially in those from the oldest individuals. FFP stimulation was associated with striking modifications in cytokine production in an age-dependent way. Exposure to FFP attenuates the response to LPS. TLR2 and CD11B expression were enhanced regardless of the age of plasma donors, although CASP3 expression was increased only when FFP from individuals aged 19-40 years were tested. Phagocytosis decreased after exposure to FFP regardless of donor age. CONCLUSION: Our results suggest that soluble mediators in FFP may modulate the functioning of monocytes. Interestingly, this effect appears to be partially influenced by the age of donors.

Effect of fresh frozen plasma on the in vitro activation of U937 monocytes: a potential role for the age of blood donors and their underlying cytokine profile

BACKGROUND: Fresh frozen plasma (FFP) administration may increase the risk of nosocomial infections in parallel with the development of immune modulation. This could be driven by soluble mediators, possibly influencing the in vitro activation of human U937 monocyte cells, in a manner dependent on the age of the donors. METHODS: FFP donors were stratified into groups of 19-30 years, 31-40 years or 41-50 years, and U937 cells were cultured with FFP (alone or plus lipopolysaccharide-LPS) for 24 h. Both in FFP and supernatants, TNF, IL-1ß, IL-6, and IL-10 levels were measured by ELISA. Additionally, CD11B, TLR2, and CASP3 gene expression were measured by qtPCR in U937 cells. Total phagocytic activity was also assayed. RESULTS: Elevated IL-10, but low TNF and IL-1ß levels were measured in FFP from individuals aged 19-40 years, whereas in individuals aged 41-50 years FFP were characterized by equalized TNF and IL-10 levels. Elevated IL-6 levels were found in all FFP samples, especially in those from the oldest individuals. FFP stimulation was associated with striking modifications in cytokine production in an age-dependent way. Exposure to FFP attenuates the response to LPS. TLR2 and CD11B expression were enhanced regardless of the age of plasma donors, although CASP3 expression was increased only when FFP from individuals aged 19-40 years were tested. Phagocytosis decreased after exposure to FFP regardless of donor age. CONCLUSION: Our results suggest that soluble mediators in FFP may modulate the functioning of monocytes. Interestingly, this effect appears to be partially influenced by the age of donors.

Participation of CD161(+) and invariant natural killer T cells in pediatric asthma exacerbations.

Asthma has been defined as a disease of chronic airway inflammation in which many cells and cellular products participate with variable degrees of airflow obstruction and hyperresponsiveness that lead to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing. Prominent among these cellular elements are two cell types referred to as the invariant natural killer T (iNKT) cells and a subpopulation of T cells expressing the molecule CD161, which are both thought to play a role in the pathogenesis of asthma. Although the presence of iNKT and other CD161(+) cells in murine models has been associated with asthma, relatively few studies have been performed in the adult patient with asthma that have been often conflicting and even fewer studies are available in children. The present study was performed to investigate the peripheral blood frequencies of iNKT and CD161(+) T cells in children with asthma. A total of 35 children, 19 stable asthmatic patients, 6 who had experienced an asthmatic attack within 24 hours and had not received any treatment, and 10 healthy controls, aged 6-12 years, were enrolled in the study. iNKT and CD161(+) T-cell frequencies in blood were measured together with quantitative levels of IL-4 and interferon (IFN) γ using a cytofluorimetric approach. The results show that iNKT cells are increased in pediatric asthmatic patients undergoing exacerbations of asthma. These cells also produced less IFN-γ and more IL-4 than children with stable asthma and in healthy control children. These results suggest that iNKT cells might participate in the development of the asthmatic exacerbations. The increased production of IL-4 in conjunction with the decrease of IFN-γ may be mechanistically responsible, at least partially, for the heightening of the immunologic response leading to the asthmatic attack in children. Knowledge of these interactive mechanisms involving the iNKT cell and our understanding of its role in the exacerbation of asthma hold great promise in the development of better diagnostic predictive markers of disease progression as well as new forms of therapeutic interventions.

[RH hybrid box identification in subjects with Rh negative phenotype from Mexico's Valley]. / Identificación de la caja RH híbrida en el fenotipo Rh negativo de sujetos del valle de México.

BACKGROUND: The prevalence of the RhD and RhCE gene alleles is related to the ethnic mixture. The aim of this report is to describe the predominant molecular mechanisms in RhD negative subjects residents from Mexico's valley according to the phenotype of RhCE. METHODS: Blood samples from RhD negative women and men were studied. The RhD/RhCE phenotype was identified by hemagglutination and Rh hybrid box by PCR-FRLP with PstI. RESULTS: 216 subjects were included. The RhD phenotypes were ccdee in 179 cases (82.8%), Ccdee in 15 cases (11.6%), ccdEe in seven (3.2%), CcdEe in four (1.9%), and CcdEE in a single subject (0.5%). In five cases, RhD hybrid box was not amplified (2.3%), 21 cases were hemizygotes (9.7%), and 188 cases homozygotes (87%), for RhD hybrid box. The homozygote condition was more frequent in those individuals with phenotype ccdee (87%). The allelic frequency of RhD hybrid box was 0.928. The frequency of Rhcc haplotype was higher in those subjects homozygotes for RhD hybrid box (chi2 = 4.658, p < 0.05). CONCLUSIONS: In this population, RhD gene deletion is the main molecular mechanism to generate to RhD negative condition and this depends on the European mixture.

Phase I enzyme induction in Girardinichthys viviparus, an endangered goodeid fish, exposed to water from native localities enriched with polychlorinated biphenyls.

The present study examines the induction of mixed-function oxidase (MFO) enzymes, including CYP content CYP1A (EROD) activity and alcohol dehydrogenase activity (ADH), in an endemic Mexican fish species, the black-fin goodeid Girardinichthys viviparus, exposed to the water of two localities, Lake Texcoco (LTX) and Lake Zumpango, and to the same matrices enriched in polychlorinated biphenyls (PCBs) to simulate the potential toxic effects of sublethal increases in these xenobiotics. Fishes of both sexes born in the laboratory were exposed for 1, 2, 4, 8, and 16 days. Water from the two types of localities of the black-fin goodeid contains MFO inducers. Of the two, the most contaminated is LTX water, which also contains PCBs. EROD activity was higher in all treatments with female compared with male fish. This suggests greater metabolic compromise in female fish as a response to damage caused by these xenobiotics. In this species, CYP induction displayed two patterns that were not always concurrent with higher CYP1A activity. In the enriched matrix system, biotransformation processes were notably altered. Increased ADH may indicate that this enzyme is involved in the biotransformation of PCBs and their metabolites, particularly in male fish, and provides at least a part of reductive power required by the MFO enzymes; however, specific studies are needed to clarify this point.