Direct Current Electrical Stimulation Shifts THP-1-Derived Macrophage Polarization towards Pro-Regenerative M2 Phenotype.

A macrophage shift from the M1 to the M2 phenotype is relevant for promoting tissue repair and regeneration. In a previous in vivo study, we found that direct current (DC) electrical stimulation (EStim) increased the proportion of M2 macrophages in healing tissues and directed the balance of the injury response away from healing/scarring towards regeneration. These observations led us to hypothesize that DC EStim regulates macrophage polarization towards an M2 phenotype. THP-1-derived M0, M1 (IFN-γ and LPS), and M2 (IL-4 and IL-13) macrophages were exposed (or not: control group) to 100 mV/mm of DC EStim, 1 h/day for three days. Macrophage polarization was assessed through gene and surface marker expressions and cytokine secretion profiles. Following DC EStim treatment, M0 cells exhibited an upregulation of M2 marker genes IL10, CD163, and PPARG. In M1 cells, DC EStim upregulated the gene expressions of M2 markers IL10, TGM2, and CD206 and downregulated M1 marker gene CD86. EStim treatment also reduced the surface expression of CD86 and secretion of pro-inflammatory cytokines IL-1ß and IL-6. Our results suggest that DC EStim differentially exerts pro-M2 effects depending on the macrophage phenotype: it upregulates typical M2 genes in M0 and M1 cells while inhibiting M1 marker CD86 at the nuclear and protein levels and the secretion of pro-inflammatory interleukins in M1 cells. Conversely, M2 cells appear to be less responsive to the EStim treatment employed in this study.

Impaired Surface Expression of HLA-DR, TLR2, TLR4, and TLR9 in Ex Vivo-In Vitro Stimulated Monocytes from Severely Injured Trauma Patients.

Objective. Trauma patients (TP) frequently develop an imbalanced immune response that often causes infectious postinjury complications. Monocytes show a diminished capability of both producing proinflammatory cytokines and antigen presentation after trauma. TLR2, TLR4, and TLR9 recognize pathogens and subsequently activate monocytes. While there are conflictive data about TLR2 and TLR4 expression after trauma, no studies about the expression of TLR2, TLR4, TLR9, and HLA-DR on monocytes from TP after their secondary ex vivo-in vitro "hit" have been reported. Methods/Results. Ex vivo-in vitro lipopolysaccharide- (LPS-) stimulated blood from TP showed diminished interleukin- (IL-) 1ß-release in TP for five postinjury days compared to healthy volunteers (HV). The recovery was observed at day 5. In parallel, monocytes from TP showed an impaired capability of TLR2, TLR4, and TLR9 expression after secondary stimulation compared to HV, while the measurement of unstimulated samples showed significant reduction of TLR4 and TLR9 at ED. Furthermore, HLA-DR decreased after trauma and was even more profound by stimulation of monocytes. Ratio of monocytes to leukocytes was significantly increased at days 6 and 7 after trauma compared to HV. Conclusion. Impaired expression of TLRs and HLA-DR in acute inflammatory conditions may be responsible for the well-described monocyte paralysis after severe trauma.

Selective AKT Inhibition by MK-2206 Represses Colorectal Cancer-Initiating Stem Cells.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide. Growing evidence indicates that tumor-initiating cells (TICs) are responsible for tumor growth and progression. Conventional chemotherapeutics do not sufficiently eliminate TICs, leading to tumor relapse. We aimed to gain insight into TIC biology by comparing the transcriptome of primary TIC cultures and their normal stem cell counterparts to uncover expression differences. METHODS: We established colonosphere cultures derived from the resection of paired specimens of primary tumor and normal mucosa in patients with CRC. These colonospheres, enriched for TICs, were used for differential transcriptome analyses to detect new targets for a TIC-directed therapy. Effects of target inhibition on CRC cells were studied in vitro and in vivo. RESULTS: Pathway analysis of the regulated genes showed enrichment of genes central to PI3K/AKT and Wnt-signaling. We identified CD133 as a marker for a more aggressive CRC subpopulation enriched with TICs in SW480 CRC cells in an in vivo cancer model. Treatment of CRC cells with the selective AKT inhibitor MK-2206 caused a decrease in cell proliferation, particularly in the TIC fraction, resulting in a significant reduction of the stemness capacity to form colonospheres in vitro and to initiate tumor formation in vivo. Consequently, MK-2206 treatment of mice with established xenograft tumors exhibited a significant deceleration of tumor progression. Primary patient-derived tumorsphere growth was significantly inhibited by MK-2206. CONCLUSION: This study reveals that AKT signaling is critical for TIC proliferation and can be efficiently targeted by MK-2206 representing a preclinical therapeutic strategy to repress colorectal TICs.

Transarterial chemoembolization of hepatocellular carcinoma in a rat model: the effect of additional injection of survivin siRNA to the treatment protocol.

BACKGROUND: Transarterial chemoembolization is one of the most widely accepted interventional treatment options for treatment of hepatocellular carcinoma. Still there is a lack of a standard protocol regarding the injected chemotherapeutics. Survivin is an inhibitor of Apoptosis protein that functions to inhibit apoptosis, promote proliferation, and enhance invasion. Survivin is selectively up-regulated in many human tumors. Small interfering RNA (siRNA) can trigger an RNA interference response in mammalian cells and induce strong inhibition of specific gene expression including Survivin. The aim of the study is to assess the effectiveness of the additional injection of Survivin siRNA to the routine protocol of Transarterial Chemoembolization (TACE) for the treatment of hepatocellular carcinoma in a rat model. METHODS: The study was performed on 20 male ACI rats. On day 0 a solid Morris Hepatoma 3924A was subcapsullary implanted in the liver. On day 12 MRI measurement of the initial tumor volume (V1) was performed. TACE was performed on day 13. The rats were divided into 2 groups; Group (A, n = 10) in which 0.1 mg mitomycin, 0.1 ml lipiodol and 5.0 mg degradable starch microspheres were injected in addition 2.5 nmol survivin siRNA were injected. The same agents were injected in Group (B,=10) without Survivin siRNA. MRI was repeated on day 25 to assess the tumor volume (V2). The tumor growth ratio (V2/V1) was calculated. Western blot and immunohistochemical analysis were performed. RESULTS: For group A the mean tumor growth ratio (V2/V1) was 1.1313 +/- 0.1381, and was 3.1911 +/- 0.1393 in group B. A statistically significant difference between both groups was observed regarding the inhibition of tumor growth (P < 0.0001) where Group A showed more inhibition compared to Group B. Similarly immunohistochemical analysis showed significantly lower (p < 0.002) VEGF staining in group A compared to group B. Western Blot analysis showed a similar difference in VEGF expression (P < 0.0001). CONCLUSION: The additional injection of Survivin siRNA to the routine TACE protocol increased the inhibition of the hepatocellular carcinoma growth in a rat animal model compared to regular TACE protocol.

Chronic ethanol feeding modulates inflammatory mediators, activation of nuclear factor-κB, and responsiveness to endotoxin in murine Kupffer cells and circulating leukocytes.

Chronic ethanol abuse is known to increase susceptibility to infections after injury, in part, by modification of macrophage function. Several intracellular signalling mechanisms are involved in the initiation of inflammatory responses, including the nuclear factor-κB (NF-κB) pathway. In this study, we investigated the systemic and hepatic effect of chronic ethanol feeding on in vivo activation of NF-κB in NF-κB(EGFP) reporter gene mice. Specifically, the study focused on Kupffer cell proinflammatory cytokines IL-6 and TNF-α and activation of NF-κB after chronic ethanol feeding followed by in vitro stimulation with lipopolysaccharide (LPS). We found that chronic ethanol upregulated NF-κB activation and increased hepatic and systemic proinflammatory cytokine levels. Similarly, LPS-stimulated IL-1 ß release from whole blood was significantly enhanced in ethanol-fed mice. However, LPS significantly increased IL-6 and TNF-α levels. These results demonstrate that chronic ethanol feeding can improve the responsiveness of macrophage LPS-stimulated IL-6 and TNF-α production and indicate that this effect may result from ethanol-induced alterations in intracellular signalling through NF-κB. Furthermore, LPS and TNF-α stimulated the gene expression of different inflammatory mediators, in part, in a NF-κB-dependent manner.

Extracellular vesicles as potential biomarkers for diagnosis and recurrence detection of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor and a leading cause of cancer-related deaths worldwide. However, current diagnostic tools are often invasive and technically limited. In the last decade, non-invasive liquid biopsies have transformed the field of clinical oncology, showcasing the potential of various liquid-biopsy derived analytes, including extracellular vesicles (EVs), to diagnose and monitor HCC progression and metastatic spreading, serving as promising novel biomarkers. A prospective single-center cohort study including 37 HCC patients and 20 patients with non-malignant liver disease (NMLD), as a control group, was conducted. Serum EVs of both groups were analyzed before and after liver surgery. The study utilized microbead-based magnetic particle sorting and flow cytometry to detect 37 characteristic surface proteins of EVs. Furthermore, HCC patients who experienced tumor recurrence (R-HCC) within 12 months after surgery were compared to HCC patients without recurrence (NR-HCC). EVs of R-HCC patients (n = 12/20) showed significantly lower levels of CD31 compared to EVs of NR-HCC patients (p = 0.0033). EVs of NMLD-group showed significantly higher expressions of CD41b than EVs of HCC group (p = 0.0286). The study determined significant short-term changes in CD19 dynamics in EVs of the NMLD-group, with preoperative values being significantly higher than postoperative values (p = 0.0065). This finding of our pilot study suggests EVs could play a role as potential targets for the development of diagnostic and therapeutic approaches for the early and non-invasive detection of HCC recurrence. Further, more in-depth analysis of the specific EV markers are needed to corroborate their potential role as diagnostic and therapeutic targets for HCC.

Transglutaminase 2 is associated with adverse colorectal cancer survival and represents a therapeutic target.

Molecular markers for predicting prognosis of colorectal cancer (CRC) patients are urgently needed for effective disease management. We reported previously that the multifunctional enzyme Transglutaminase 2 (TGM2) is essential for CRC cell survival by inactivation of the tumor suppressor p53. Based on these data, we determined the clinical relevance of TGM2 expression and explored its potential as prognostic marker and therapeutic target in CRC. We profiled TGM2 protein expression in tumor samples of 279 clinically characterized CRC patients using immunohistochemical staining. TGM2 expression was upregulated in matched tumor samples in comparison to normal tissue. A strong TGM2 expression was associated with advanced tumor stages and predicted worse prognosis regarding progression-free and overall-survival, even at early stages. Inhibition of TGM2 in CRC cell lines by the inhibitors LDN27219 and Tyrphostin resulted in a strong reduction of cancer cell proliferation and tumorsphere formation in vitro by induction of p53-mediated apoptosis. Primary patient-derived tumorsphere formation was significantly reduced by inhibition of TGM2. Treatment of mice with TGM2 inhibitors exhibited a significant deceleration of tumor progression. Our data indicate that high TGM2 expression in CRC is associated with worse prognosis and may serve as a therapeutic target in CRC patients with strong TGM2 expression.

Integrin α2 and ß1 Cross-Communication with mTOR/AKT and the CDK-Cyclin Axis in Hepatocellular Carcinoma Cells.

Integrin receptors contribute to hepatocellular carcinoma (HCC) invasion, while AKT-mTOR signaling controls mitosis. The present study was designed to explore the links between integrins and the AKT-mTOR pathway and the CDK-Cyclin axis. HCC cell lines (HepG2, Huh7, Hep3B) were stimulated with soluble collagen or Matrigel to activate integrins, or with insulin-like growth factor 1 (IGF1) to activate AKT-mTOR. HCC growth, proliferation, adhesion, and chemotaxis were evaluated. AKT/mTOR-related proteins, proteins of the CDK-Cyclin axis, focal adhesion kinase (FAK), and integrin-linked kinase (ILK) were determined following IGF1-stimulation or integrin knockdown. Stimulation with collagen or Matrigel increased tumor cell growth and proliferation. This was associated with significant alteration of the integrins α2, αV, and ß1. Blockade of these integrins led to cell cycle arrest in G2/M and diminished the number of tumor cell clones. Knocking down the integrins α2 or ß1 suppressed ILK, reduced FAK-phosphorylation and diminished AKT/mTOR, as well as the proteins of the CDK-Cyclin axis. Activating the cells with IGF1 enhanced the expression of the integrins α2, αV, ß1, activated FAK, and increased tumor cell adhesion and chemotaxis. Blocking the AKT pathway canceled the enhancing effect of IGF on the integrins α2 and ß1. These findings reveal that HCC growth, proliferation, and invasion are controlled by a fine-tuned network between α2/ß1-FAK signaling, the AKT-mTOR pathway, and the CDK-Cyclin axis. Concerted blockade of the integrin α2/ß1 complex along with AKT-mTOR signaling could, therefore, provide an option to prevent progressive dissemination of HCC.

Critical analysis of simultaneous blockage of histone deacetylase and multiple receptor tyrosine kinase in the treatment of prostate cancer.

BACKGROUND: The concept of molecular tumor targeting might be an innovative option to treat advanced prostate cancer. We analyzed the effect of combining the multiple receptor tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) on adhesion and growth properties of prostate cancer cell lines. METHODS: PC-3, DU-145, and LNCaP cells were treated with AEE788, VPA or with an AEE788-VPA combination, and cell cycle progression investigated. Furthermore, tumor cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins was evaluated, and integrin α and ß subtypes were analyzed. Finally, effects of drug treatment on cell signaling pathways were determined. RESULTS: AEE788 moderately and VPA strongly reduced tumor cell adhesion and growth. VPA impaired cell cycle progression and altered the expression level of the cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin B, D1, cyclin E, p21, and p27. VPA also acted on the membranous, cytoplasmic, and gene expression pattern of various integrin α and ß subtypes. AEE788 acted likewise, but more moderately. Combining AEE788 and VPA did not result in an additive anti-tumor effect. Signaling analysis revealed that the EGFr downstream target Akt was similarly modified in the presence of VPA or the VPA-AEE788 combination, but not influenced by AEE788 alone. CONCLUSIONS: The AEE788-VPA combination has no advantage over VPA monotreatment in vitro. The non-responsiveness of Akt

Inactivating SOCS1 mutations are caused by aberrant somatic hypermutation and restricted to a subset of B-cell lymphoma entities.

STATs are constitutively activated in several malignancies. In primary mediastinal large B-cell lymphoma and Hodgkin lymphoma (HL), inactivating mutations in SOCS1, an inhibitor of JAK/STAT signaling, contribute to deregulated STAT activity. Based on indications that the SOCS1 mutations are caused by the B cell-specific somatic hypermutation (SHM) process, we analyzed B-cell non-HL and normal B cells for mutations in SOCS1. One-fourth of diffuse large B-cell lymphoma and follicular lymphomas carried SOCS1 mutations, which were preferentially targeted to SHM hotspot motifs and frequently obviously inactivating. Rare mutations were observed in Burkitt lymphoma, plasmacytoma, and mantle cell lymphoma but not in tumors of a non-B-cell origin. Mutations in single-sorted germinal center B cells were infrequent relative to other genes mutated as byproducts of normal SHM, indicating that SOCS1 inactivation in primary mediastinal large B-cell lymphoma, HL, diffuse large B-cell lymphoma, and follicular lymphoma is frequently the result of aberrant SHM.

Significantly high expression of platelet-derived growth factor (PDGF) in benign nodules of the thyroid: relevance in the development of goitre recurrence?

PURPOSE: Platelet-derived growth factor (PDGF) is a critical regulator of cell proliferation and influences the development of tumors. The role of PDGF in benign thyroid diseases is presently not well-determined. The purpose is to evaluate PDGF isoforms and receptors in primary culture of thyrocytes isolated from human thyroid tissue. METHODS: Forty patients with uninodular (n = 11), multinodular (n = 15) and recurrent goitre (n = 14) were investigated. Nodular and corresponding paranodular thyroid tissues were characterized. RNA and protein were extracted from primary thyrocyte monoculture. RT-PCR, western blot and ELISA were performed to evaluate PDGF isoforms AA, BB, CC, DD and PDGF receptors α and ß. RESULTS: Significantly higher mRNA expression of PDGF-AA, -BB, -CC and PDGFR molecules α and ß was measured by RT-PCR in thyrocytes from uninodular and recurrent nodular tissue compared with corresponding paranodular tissue. Elevated PDGF protein and PDGFR-α and -ß were confirmed by western blot. Likewise, higher secretion of PDGF-AA and -BB was detected in the supernatant of thyrocyte culture from all nodular tissue compared with paranodular tissue. Interestingly, comparison of nodular and corresponding paranodular tissues in multinodular goitre did not show significant difference of expression levels of PDGF isoforms or receptors. CONCLUSION: These findings suggest that the overexpression of PDGF isoforms and receptors may play a crucial role in the development of thyroid nodules and recurrent goitre.

Carbonic-anhydrase IX expression is increased in thyroid cancer tissue and represents a potential therapeutic target to eradicate thyroid tumor-initiating cells.

The expression of Carbonic-anhydrase IX (CAIX) in thyroid cancer (TC) subtypes was determined and its role in cancer cell growth and tumor-initiating cells (TICs) investigated. Immunohistochemistry in 114 TC patients revealed that CAIX expression was increased in tumor specimens of papillary, follicular and anaplastic TCs compared to normal thyroid tissue. Clinicopathological data indicated that lymph node metastases were more frequent in patients with high CAIX expression. The Cancer Genome Atlas database analysis demonstrated that a strong CAIX-mRNA expression was associated with advanced tumor stages and poor survival in TCs. In TC cell lines, CAIX expression was elevated in tumorspheres compared to monolayer cultures and was associated with an increased expression of stemness markers. Genetic knockdown or pharmacological inhibition of CAIX suppressed cell proliferation and the TIC ability to form tumorspheres by an induction of apoptosis and cell-cycle arrest. These findings suggest CAIX as a potential prognostic marker and a therapeutic target for thyroid cancer.

Early dynamic changes in circulating tumor cells and prognostic relevance following interventional radiological treatments in patients with hepatocellular carcinoma.

The aim of this study was to investigate the dynamic changes of circulating tumor cells (CTCs) in patients with hepatocellular carcinoma (HCC) before and immediately after conducting a microwave ablation (MWA) and conventional transarterial chemoembolization (C-TACE). Additionally, the CTCs short-term dynamics were compared with the clinical course of the HCC-patients. Blood samples from 17 patients with HCC who underwent MWA (n = 10) or C-TACE (n = 7) were analyzed. Venous blood was taken before and immediately after the radiological interventions to isolate and quantify CTCs using flow cytometry. CTCs were identified as CD45- and positive for the markers ASGPR, CD146 and CD274 (PD-L1). Patients were followed of up to 2.2 years after the radiological intervention. CTCs were detected in 13 HCC patients (76%) prior to the radiological interventions. The rate of CTCs was significantly decreased after the intervention in patients treated with MWA (0.4 CTCs/mL of blood, p = 0.031). However, no significant differences were observed in patients who received C-TACE (0.3 CTCs/mL of blood, p = 0.300). Overall, no correlation was found between the CTCs rate before and after the radiological intervention and recurrence rate of HCC. This preliminary data could confirm the tumoricidal effects of MWA in patients with HCC by significantly decreasing CTCs rate. In our study, we were able to detect CTCs in HCC patients using 3 different tumor markers. This preliminary data shows significant lower CTCs detected in response to MWA. However, large-scale randomized clinical trials are needed to determine the future role and the prognostic relevance of CTCs following this treatment.

Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53.

Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein-protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.

Neutrophil phenotype and function in partial hepatectomy in man.

PURPOSE: Hepatic resections are still associated with considerable morbidity mainly because of postoperative infection. Adequate function of neutrophils is a crucial element in host defense. The aim of the study was to characterize neutrophils during partial hepatectomy. METHODS: Fourteen patients undergoing partial liver resection were enrolled. Twenty-four hours pre-, intra- (after induction of anesthesia, after preparation of the liver, and 15 min after release of the Pringle maneuver), as well as postoperatively (3 h after Pringle; 24, 48, and 120 h after surgery), blood samples were obtained. In addition, healthy volunteers (n = 5) were investigated. Adhesion molecules (CD 62, CD 18), Fcy receptors (CD 16, CD 32), and phagocytosis by neutrophils were characterized by fluorescence-activated cell sorter analysis. Spontaneous and stimulated (formyl-methionyl-leucyl-phenylalanine) oxygen radical generation was measured by lucigenin-enhanced chemiluminescence. RESULTS: Numeric upregulation of CD 62 and CD 18 on neutrophils was seen before the use of Pringle maneuver and persisted thereafter (p < 0.05). Spontaneous numeric expression of Fcy receptors (CD16 and CD 32) was unchanged during liver dissection but downregulated after Pringle maneuver was opened (p < 0.05). Although numeric Fcy receptors were downregulated, phagocytosis of heterologous opsonized Escherichia coli bacteria by neutrophils was unaffected. Spontaneous oxygen radical production peaked sharply 15 min after release of the Pringle maneuver (p < 0.05), contrary to stimulated oxygen radical production, which was depressed 3 h after the release of the Pringle maneuver (ns). CONCLUSIONS: Uneventful partial hepatectomy in man resulted already in a significant change in the phenotype but in less significant changes in the functions of neutrophils.

Regulatory T Cells Modulate CD4 Proliferation after Severe Trauma via IL-10.

OBJECTIVE: Severely injured patients frequently develop an immunological imbalance following the traumatic insult, which might result in infectious complications evoked by a persisting immunosuppression. Regulatory T cells (Tregs) maintain the immune homeostasis by suppressing proinflammatory responses, however, their functionality after trauma is unclear. Here, we characterized the role of Tregs in regulating the proliferation of CD4+ lymphocytes in traumatized patients (TP). METHODS: Peripheral blood was obtained daily from 29 severely injured TP (Injury Severity Score, ISS ≥16) for ten days following admission to the emergency department (ED). Ten healthy volunteers (HV) served as controls. The frequency and activity of Tregs were assessed by flow cytometry. Proliferation of CD4+ cells was analyzed either in presence or absence of Tregs, or after blocking of either IL-10 or IL-10R1. RESULTS: The frequencies of CD4+CD25high and CD4+CD25+CD127- Tregs were significantly decreased immediately upon admission of TP to the ED and during the following 10 post-injury days. Compared with HV CD4+ T cell proliferation in TP increased significantly upon their admission and on the following days. As expected, CD4+CD25+CD127- Tregs reduced the proliferation of CD4+ cells in HV, nevertheless, CD4+ proliferation in TP was increased by Tregs. Neutralization of IL-10 as well as blocking the IL-10R1 increased further CD4+ T cell proliferation in Tregs-depleted cultures, thereby confirming an IL-10-mediated mechanism of IL-10-regulated CD4+ T cell proliferation. Neutralization of IL-10 in TP decreased CD4+ T cell proliferation in Tregs-depleted cultures, whereas blocking of the IL-10R1 receptor had no significant effects. CONCLUSIONS: The frequency of Tregs in the CD4+ T lymphocyte population is reduced after trauma; however, their inductiveness is increased. The mechanisms of deregulated influence of Tregs on CD4+ T cell proliferation are mediated via IL-10 but not via the IL-10R1.

Complement and neutrophil function changes after liver resection in humans.

BACKGROUND: Complement activation contributes to the regulation of liver regeneration after liver resection (LR) in mice. METHODS: We hypothesized that complement activation and changes in C5a-receptors (C5aR, C5L2) on polymorphonuclear cells (PMN) and monocytes are important in clinical LR. Anaphylatoxin and C5b9 plasma levels were measured (bead-array, ELISA) (25 patients) and receptor expression was assessed after LR (19 patients) (FACS). In vitro PMN C5a-dependent chemotactic response (7 patients) as well as L-selectin shedding and Mac-1 expression (3 patients) was determined. RESULTS: C3a increased after LR (31.1 +/- 4 before LR vs. 41.6 +/- 5 ng/ml, 30 min after LR, P < 0.01), as did C5b9 (12.7 +/- 1 before LR vs. 26.9 +/- 3 ng/ml, 60 min after LR, P < 0.001). C4a and C5a decreased after LR, by 25% 24 h after LR and 30% 2 h after LR, respectively (P < 0.01). C5L2 expression decreased at 4 h, rising at 24 h after LR (PMN: 6.3 +/- 1 before LR, 3.1 +/- 1, 4 h, 8.3 +/- 2, 24 h; P < 0.01). The receptor-related changes accompanied a diminished C5a-dependent chemotactic response by PMN (42.1 +/- 17 before LR vs. 2.1 +/- 3 4 h after LR; P < 0.01) and a reduction of activation upon C5a-R stimulation as measured by L-selectin shedding and Mac-1 expression on PMN. Changes in C5L2 expression on monocytes paralleled postoperative impairment of liver function. CONCLUSIONS: These results indicate that complement components are released after clinical LR and subsequently PMN display altered C5a-dependent functional responses.

Comparative Analysis of the Regulatory T Cells Dynamics in Peripheral Blood in Human and Porcine Polytrauma.

Background: Severely injured patients experience substantial immunological stress in the aftermath of traumatic insult, which often results in systemic immune dysregulation. Regulatory T cells (Treg) play a key role in the suppression of the immune response and in the maintenance of immunological homeostasis. Little is known about their presence and dynamics in blood after trauma, and nothing is known about Treg in the porcine polytrauma model. Here, we assessed different subsets of Treg in trauma patients (TP) and compared those to either healthy volunteers (HV) or data from porcine polytrauma. Methods: Peripheral blood was withdrawn from 20 TP with injury severity score (ISS) ≥16 at the admittance to the emergency department (ED), and subsequently on day 1 and at day 3. Ten HV were included as controls (ctrl). The porcine polytrauma model consisted of a femur fracture, liver laceration, lung contusion, and hemorrhagic shock resulting in an ISS of 27. After polytrauma, the animals underwent resuscitation and surgical fracture fixation. Blood samples were withdrawn before and immediately after trauma, 24 and 72 h later. Different subsets of Treg, CD4+CD25+, CD4+CD25+FoxP3+, CD4+CD25+CD127-, and CD4+CD25+CD127-FoxP3+ were characterized by flow cytometry. Results: Absolute cell counts of leukocytes were significantly increasing after trauma, and again decreasing in the follow-up in human and porcine samples. The proportion of human Treg in the peripheral blood of TP admitted to the ED was lower when compared to HV. Their numbers did not recover until 72 h after trauma. Comparable data were found for all subsets. The situation in the porcine trauma model was comparable with the clinical data. In porcine peripheral blood before trauma, we could identify Treg with the typical immunophenotype (CD4+CD25+CD127-), which were virtually absent immediately after trauma. Similar to the human situation, most of these cells expressed FoxP3, as assessed by intracellular FACS stain. Conclusion: Despite minor percental differences in the recovery of Treg populations after trauma, our findings show a comparable decrease of Treg early after polytrauma, and strengthen the immunological significance of the porcine polytrauma model. Furthermore, the Treg subpopulation CD4+CD25+CD127- was characterized in porcine samples.

Study on the effect of chemoembolization combined with microwave ablation for the treatment of hepatocellular carcinoma in rats.

PURPOSE: We aimed to evaluate the combining effects of transarterial chemoembolization (TACE) and open local thermal microwave ablation in a hepatocellular carcinoma animal model. METHODS: Tumor cubes were implanted into the liver of 30 male inbred ACI rats. Groups of 10 animals were treated at 13 days (TACE or microwave ablation) and 16 days (microwave ablation) postimplantation with combined therapy of TACE (0.1 mg mitomycin C; 0.1 mg iodized oil; 5.0 mg degradable starch microspheres) and microwave ablation (2450 Mhz; 45 s; 35 W) (study group A), TACE alone (control group B), or microwave ablation alone (control group C). At day 12 and day 25 tumor size was measured via magnetic resonance imaging and the relative growth ratio was calculated. Hepatic specimens were immunohistochemically examined for the expression of vascular endothelial growth factor (VEGF). RESULTS: Mean growth rates were 1.34±0.19 in group A, 3.19±0.13 in group B, and 4.18±0.19 in group C. Compared with control groups B and C, tumor growth rate in group A was significantly inhibited (P < 0.01). The VEGF-antibody reaction in peritumoral tissue (staining intensity at portal triad, percent antibody reaction and staining intensity at central vein) was significantly lower in group A compared with group B (P < 0.01). No significant difference between group A and group C could be observed. CONCLUSION: This investigation shows improved results of TACE followed by microwave ablation as treatment of hepatocellular carcinoma in a rat model, compared with single therapy regimen regarding the inhibition of growth rate and reduction of VEGF-level in peritumoral tissue.

c-Met responsiveness of isolated hepatocytes evaluated in an in vitro de-differentiation model.

Hepatocyte growth factor (HGF) accelerates tissue regeneration and ameliorates tissue fibrosis through its ligand c-Met receptor tyrosine kinase. Hence, HGF is currently discussed as an attractive therapeutic candidate for fatal liver diseases. However, it remains unclear whether c-Met of de-differentiated hepatocytes adequately responds to HGF in an impaired liver. Therefore, we investigated c-Met expression and c-Met responsiveness to HGF in an experimental de-differentiation cell culture system. Primary rat hepatocytes were seeded on a two-dimensional collagen matrix or embedded within a three dimensional collagen gel to guarantee intact cell geometry. Cells were cultivated in a growth factor enriched extracellular milieu (de-differentiation medium), or in a chemically defined differentiation medium, representing physiologically intact hepatocytes. c-Met surface expression was determined by flow cytometry. Receptor localisation was examined by confocal microscopy, c-Met and phosphorylated c-Met protein were determined by western blotting. Hepatocyte-specific asialoglycoprotein receptor (ASGPr) was examined to control the differentiation status of the cells. Growth factor enriched milieu induced a rapid loss of ASGPr with a significant increase of c-Met surface level and a decrease in c-Met protein level. Surprisingly, the increased amount of c-Met surface expression was associated with its loss of responsiveness to HGF. The addition of bile acids into the culture medium had significantly delayed the process of de-differentiation and restrained the drastic elevation of c-Met (tauroursodeoxycholic acid > ursodeoxycholic acid). Application of the three-dimensional hepatocellular architecture stabilized the c-Met surface receptor level and rendered c-Met activation. We have demonstrated that growth factor enriched extracellular milieu and loss of intact liver architecture seems to be accompanied by an up-regulation of c-Met surface level. Our findings suggest that irresponsiveness of c-Met to soluble HGF was possibly caused by an excessive HGF production and receptor over-stimulation. Both events should be considered when establishing an HGF-based therapy for fibrosis/cirrhosis.