Sara Cajander

Preliminary results in quantitation of HLA-DRA by real-time PCR: a promising approach to identify immunosuppression in sepsis

IntroductionReduced monocyte human leukocyte antigen (mHLA)-DR surface expression in the late phase of sepsis is postulated as a general biomarker of sepsis-induced immunosuppression and an independent predictor of nosocomial infections.However, traditional monitoring of mHLA-DR by flow cytometry has disadvantages due to specific laboratory requirements. An mRNA-based HLA-DR monitoring by polymerase chain reaction (PCR) would improve the clinical usage and facilitate conduction of large multicenter studies. In this study, we evaluated an mRNA-based HLA-DR monitoring by quantitative real-time PCR (qRT-PCR) as an alternative method to traditional flow cytometry.MethodsFifty-nine patients with sepsis and blood culture growing pathogenic bacteria were studied. Blood samples were collected at day 1 or 2 after admission, for measurement of mHLA-DR by flow cytometry and mRNA expression of HLA-DRA and class II transactivator (CIITA) by qRT-PCR. Blood samples from blood donors were used as controls (n = 30).ResultsA significant reduced expression of mHLA-DR, HLA-DRA, and CIITA was seen in septic patients compared with controls. HLA-DRA mRNA level in whole blood was highly correlated with surface expression of mHLA-DR.ConclusionsPatients with sepsis display a diminished expression of HLA-DR at the monocyte surface as well as in the gene expression at the mRNA level. The mRNA expression level of HLA-DRA monitored by qRT-PCR correlates highly with surface expression of HLA-DR and appears to be a possible future biomarker for evaluation of immunosuppression in sepsis.

Quantitative Real-Time Polymerase Chain Reaction Measurement of HLA-DRA Gene Expression in Whole Blood Is Highly Reproducible and Shows Changes That Reflect Dynamic Shifts in Monocyte Surface HLA-DR Expression during the Course of Sepsis

Introduction A decrease in the expression of monocyte surface protein HLA-DR (mHLA-DR), measured by flow cytometry (FCM), has been suggested as a marker of immunosuppression and negative outcome in severe sepsis. However, FCM is not always available due to sample preparation that limits its use to laboratory operational hours. In this prospective study we evaluated dynamic changes in mHLA-DR expression during sepsis in relation to changes in HLA-DRA gene expression and Class II transactivator (CIITA), measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Aims The aims of this study were: 1. to validate the robustness of qRT-PCR measurement of HLA-DRA- and CIITA–mRNA expression, in terms of reproducibility; and 2. to see if changes in expression of these genes reflect changes in mHLA-DR expression during the course of severe and non-severe bacteraemic sepsis. Methods and Findings Blood samples were collected from 60 patients with bacteraemic sepsis on up to five occasions during Days 1–28 after hospital admission. We found the reproducibility of the qRT-PCR method to be high by demonstrating low threshold variations (<0.11 standard deviation (SD)) of the qRT-PCR system, low intra-assay variation of Ct-values within triplicates (≤0.15 SD) and low inter-assay variations (12%) of the calculated target gene ratios. Our results also revealed dynamic HLA-DRA expression patterns during the course of sepsis that reflected those of mHLA-DR measured by FCM. Furthermore, HLA-DRA and mHLA-DR recovery slopes in patients with non-severe sepsis differed from those in patients with severe sepsis, shown by mixed model for repeated measurements (p<0.05). However, during the first seven days of sepsis, PCR-measurements showed a higher magnitude of difference between the two sepsis groups. Mean differences (95% CI) between severe sepsis (n = 20) and non-severe sepsis (n = 40) were; on day 1–2, HLA-DRA 0.40 (0.28–0.59) p<0.001, CIITA 0.48 (0.32–0.72) p = 0.005, mHLA-DR 0.63 (0.45–1.00) p = 0.04, day 7 HLA-DRA 0.59 (0.46–0.77) p<0.001, CIITA 0.56 (0.41–0.76) p<0.001, mHLA-DR 0.81 (0.66–1.00) p = 0.28. Conclusion We conclude that qRT-PCR measurement of HLA-DRA expression is robust, and that this method appears to be preferable to FCM in identifying patients with severe sepsis that may benefit from immunostimulation.

Dynamics of monocytic HLA-DR expression differs between bacterial etiologies during the course of bloodstream infection

Objective In the pathogenesis of sepsis, activation of both pro- and anti-inflammatory responses are key components, but knowledge is lacking on the association between bacterial etiology and development of dysregulated responses with sustained immunosuppression. The aim of this study was to evaluate how the immunosupression marker HLA-DR on monocytes (mHLA-DR) is associated with bacterial etiology and markers of inflammation during the clinical trajectory of bloodstream infection (BSI). Methods Ninety-one adults, predominantly non-ICU patients, with BSI caused by Streptococcus pneumoniae (n = 27), Staphylococcus aureus (n = 22), Escherichia coli/Klebsiella pneumoniae (n = 23), and other species (n = 19) were prospectively included, and sampled on admission (day 0) and on days 1–2, 3, 7±1, 14±2, and 28±4. Results The dynamics of mHLA-DR, measured by flow cytometry, differed significantly between etiology groups (p<0.001). Patients with S. pneumoniae and S. aureus BSI demonstrated low initial mHLA-DR, with the S. aureus group showing delayed recovery over time. Eleven patients (55% S. aureus) had negative outcome (secondary bacteremia or death) and they demonstrated sustained C-reactive protein elevation, neutrophilia, lymphocytopenia, and loss of mHLA-DR. Conclusions Dynamics of mHLA-DR varied according to the bacterial etiology of infection, with delayed recovery in patients with S. aureus BSI. Patients with negative outcome showed sustained CRP elevation, neutrophilia, lymphocytopenia, and low levels of mHLA-DR, supporting the theory of a dysregulated host response with persistent inflammation and immunosuppression in late stages of deleterious sepsis.

Expression of HLA-DRA and CD74 mRNA in whole blood during the course of complicated and uncomplicated Staphylococcus aureus bacteremia

To improve management of Staphylococcus aureus bacteremia (SAB), better understanding of host–pathogen interactions is needed. In vitro studies have shown that S. aureus bacteria induce dose‐dependent immunosuppression that is evidenced by reduced expression of major histocompatibility complex (MHC) class II on antigen presenting cells. Thus, the aim of this study was to determine whether expression of the MHC class II‐related genes HLA‐DRA and CD74 is more greatly reduced in complicated SAB, with its probable higher loads of S. aureus, than in uncomplicated SAB. Adult patients with SAB were prospectively included and blood samples taken on the day of confirmation of SAB (Day 1) and on Days 2, 3, 5 and 7. HLA‐DRA and CD74 mRNA expression was determined by quantitative reverse transcription PCR. Sepsis was defined according to the Sepsis‐3 classification and SAB was categorized as complicated in patients with deep‐seated infection and/or hematogenous seeding. Twenty patients with SAB were enrolled and samples obtained on all assessment days. HLA‐DRA and CD74 expression did not differ significantly between patients with SAB and sepsis (n = 13) and those without sepsis (n = 7) on any assessment day. However, patients with complicated SAB (n = 14) had significantly weaker HLA‐DRA expression on all five assessment days than patients with uncomplicated SAB (n = 6). Additionally, they tended to have weaker CD74 expressions. Neutrophil, monocyte and leukocyte counts did not differ significantly between complicated and uncomplicated SAB. In conclusion, patients with complicated SAB show weaker HLA‐DRA expression than those with uncomplicated SAB during the first week of bacteremia.