Elisabet Tina

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.

Thoracic epidural analgesia inhibits the neuro-hormonal but not the acute inflammatory stress response after radical retropubic prostatectomy

BACKGROUND Epidural anaesthesia and analgesia has been shown to suppress the neuro-hormonal stress response, but its role in the inflammatory response is unclear. The primary aim was to assess whether the choice of analgesic technique influences these processes in patients undergoing radical retropubic prostatectomy. METHODS Twenty-six patients were randomized to Group P (systemic opioid-based analgesia) or Group E (thoracic epidural-based analgesia) perioperatively. Induction and maintenance of anaesthesia followed a standardized protocol. The following measurements were made perioperatively: plasma cortisol, glucose, insulin, C-reactive proteins, leucocyte count, plasma cytokines [interleukin (IL)-6, tumour necrosis factor (TNF)-α], and pokeweed mitogen-stimulated cytokines [interferon (IFN)-γ, IL-2, IL-12p70, IL-10, IL-4, and IL-17]. Other parameters recorded were pain, morphine consumption, and perioperative complications. RESULTS Plasma concentration of cortisol and glucose were significantly higher in Group P compared with Group E at the end of surgery, the mean difference was 232 nmol litre(-1) [95% confidence interval (CI) 84-381] (P=0.004) and 1.6 mmol litre(-1) (95% CI 0.6-2.5) (P=0.003), respectively. No significant differences were seen in IL-6 and TNF-α at 24 h (P=0.953 and 0.368, respectively) and at 72 h (P=0.931 and 0.691, respectively). IL-17 was higher in Group P compared with Group E, both at 24 h (P=0.001) and 72 h (P=0.018) after operation. Pain intensity was significantly greater in Group P compared with Group E (P<0.05) up to 24 h. CONCLUSIONS In this small prospective randomized study, thoracic epidural analgesia reduced the early postoperative stress response but not the acute inflammatory response after radical retrobupic prostatectomy, suggesting that other pathways are involved during the acute phase reaction.

The mitochondrial transporter SLC25A43 is frequently deleted and may influence cell proliferation in HER2-positive breast tumors

BackgroundOverexpression of the human epidermal growth factor receptor (HER) 2 is associated with poor prognosis and shortened survival in breast cancer patients. HER2 is a potent activator of several signaling pathways that support cell survival, proliferation and metabolism. In HER2-positive breast cancer there are most likely unexplored proteins that act directly or indirectly downstream of well established pathways and take part in tumor development and treatment response.MethodsIn order to identify novel copy number variations (CNVs) in HER2-positive breast cancer whole-genome single nucleotide polymorphism (SNP) arrays were used. A PCR-based loss of heterozygosis (LOH) assay was conducted to verify presence of deletion in HER2-positive breast cancer cases but also in HER2 negative breast cancers, cervical cancers and lung cancers. Screening for mutations was performed using single-strand conformation polymorphism (SSCP) followed by PCR sequencing. Protein expression was evaluated with immunohistochemistry (IHC).ResultsA common deletion at chromosome Xq24 was found in 80% of the cases. This locus harbors the gene solute carrier (SLC) family 25A member 43 (SLC25A43) encoding for a mitochondrial transport protein. The LOH assay revealed presence of SLC25A43 deletion in HER2-positive (48%), HER2-negative (9%), cervical (42%) and lung (67%) cancers. HER2-positive tumors with negative or low SLC25A43 protein expression had significantly lower S-phase fraction compared to tumors with medium or high expression (P = 0.024).ConclusionsWe have found deletion in the SLC25A43 gene to be a common event in HER2-positive breast cancer as well as in other cancers. In addition, the SLC25A43 protein expression was shown to be related to S-phase fraction in HER2-positive breast cancer. Our results indicate a possible role of SLC25A43 in HER2-positive breast cancer and support the hypothesis of altered mitochondrial function in cancer.

The mitochondrial transport protein SLC25A43 affects drug efficacy and drug-induced cell cycle arrest in breast cancer cell lines

The mitochondria have been identified as key players of apoptosis, cell proliferation and cell cycle regulation. However, the role of mitochondria in breast cancer and treatment failure remains unclear. We have previously shown a common deletion of the gene SLC25A43 in human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This gene is coding for a mitochondrial inner membrane transporter and, to date, little is known about the function of this protein. We have also found that low protein expression of SLC25A43 significantly correlates with a lower S phase fraction in HER2-positive breast cancer. The aim of this study was to investigate whether knockdown (KD) of SLC25A43 could have an effect on the cytotoxicity of different cytostatic drugs using MCF10A, MCF7 and BT-474 cells. Following siRNA-mediated KD of SLC25A43, one non-malignant and two breast cancer cell lines were exposed to the anthracycline epirubicin or the taxane paclitaxel. The HER2-positive breast cancer cells were also exposed to the targeted therapy trastuzumab and dual exposure to trastuzumab and paclitaxel. We found that KD of SLC25A43 resulted in a decreased cytotoxic effect of paclitaxel in the two cancer cell lines (P<0.05). Further analysis of cell cycle phase distribution showed that KD increased the paclitaxel-induced G2/M block in these two cell lines (P<0.05). KD of SLC25A43 also reduced the inhibitory effect of trastuzumab on cell proliferation in the HER2-positive cancer cell line BT-474 (P<0.05), and the drug-induced G0/G1 block (P<0.05). Moreover, SLC25A43 influenced the percentage of Ki-67-positive cells. Our findings demonstrate that the mitochondrial protein SLC25A43 affects drug efficacy and cell cycle regulation following drug exposure in breast cancer cell lines.

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.

HER4 tumor expression in breast cancer patients randomized to treatment with or without tamoxifen

The human epidermal growth factor receptor (HER) 4 is a relative of HER2 and has been associated to endocrine breast cancer and prediction of tamoxifen response. In addition to PI3K/Akt and MAPK pathway activation, ligand binding to HER4 triggers proteolytic cleavage and release of an intracellular receptor domain (4ICD) with signaling properties. The aim of the present study was to analyze HER4 protein expression and intracellular localization in breast cancer tissue from patients randomized to treatment with or without adjuvant tamoxifen. To investigate HER4 expression and localization in response to estradiol (E2) and 4-hydroxytamoxifen (4-OHT) exposure, we also performed in vitro studies. Cytoplasmic, nuclear and membrane expression of HER4 protein was evaluated by immunohistochemical staining in tumor tissue from 912 breast cancer patients. Three different breast epithelia cancer cell lines were exposed to E2 and 4-OHT and mRNA expression was analyzed using qPCR. Further, nuclear and cytoplasmic proteins were separated and analyzed with western blotting. We found an association between nuclear HER4 protein expression and ER-positivity (P=0.004). Furthermore, significant association was found between cytoplasmic HER4 and ER-negativity (P<0.0005), PgR-negativity (P<0.0005), tumor size >20 mm (P=0.001) and HER2-negativity (P=0.008). However, no overall significance of HER4 on recurrence-free survival was found. After E2 exposure, HER4 mRNA and protein expression had decreased in two cell lines in vitro yet no changes in nuclear or cytoplasmic protein fractions were seen. In conclusion, nuclear HER4 seem to be co-located with ER, however, we did not find support for overall HER4 expression in independently predicting response of tamoxifen treatment. The possible influence of separate isoforms was not tested and future studies may further evaluate HER4 significance.

Expression profile of the amino acid transporters SLC7A5, SLC7A7, SLC7A8 and the enzyme TDO2 in basal cell carcinoma

The incidence of basal cell carcinoma (BCC) is increasing and the costs for care rising. Therefore, the need for simplified and cost‐effective treatment choices is substantial. Aberrant signalling in several pathways, induced by ultraviolet radiation, is of importance in the development of BCC. Alterations in tumour metabolic activity are part of general carcinogenesis; however, these alterations are only partially recognized in skin cancer.

Decreased expression of the mitochondrial solute carrier SLC25A43 in basal cell carcinoma compared with healthy skin

Basal cell carcinoma is the most common type of cancer in fair-skinned individuals, and its incidence is rapidly increasing. The aim of the present study was to investigate the gene and protein expression of the mitochondrial solute carrier family 25 member 43 (SLC25A43) in basal cell carcinoma. SLC25A43 has previously been identified to be genetically altered and associated with cell proliferation in human epidermal growth factor receptor 2-positive breast cancer. However, the knowledge about SLC25A43 is limited, and its role in other cancers is unknown. The SLC25A43 gene and protein expression was analysed in 14 basal cell carcinomas and healthy skin samples from the same individuals by quantitative polymerase chain reaction and immunohistochemistry, respectively. The results demonstrated a significantly lower (≥50%) SLC25A43 gene expression in all carcinomas compared with that in healthy skin. In addition, SLC25A43 protein expression was absent in >90% of all visual fields in the basal cell carcinomas, and the H-score was significantly lower in tumours compared with the adjacent epidermis. These results demonstrate that SLC25A43 expression is altered at the gene and protein levels in basal cell carcinoma. The underlying mechanisms and the clinical relevance of these data must be elucidated in additional experimental and clinical studies.

Early perioperative immunological effects of anaesthesia and analgesia in patients undergoing prostate cancer surgery: A randomised pilot study.

Early perioperative immunological effects of anaesthesia and analgesia in patients undergoing prostate cancer surgery : A randomised pilot study.