GITR ligation enhances functionality of tumor-infiltrating T cells in hepatocellular carcinoma.

No curative treatment options are available for advanced hepatocellular carcinoma (HCC). Anti-PD1 antibody therapy can induce tumor regression in 20% of advanced HCC patients, demonstrating that co-inhibitory immune checkpoint blockade has therapeutic potential for this type of cancer. However, whether agonistic targeting of co-stimulatory receptors might be able to stimulate anti-tumor immunity in HCC is as yet unknown. We investigated whether agonistic targeting of the co-stimulatory receptor GITR could reinvigorate ex vivo functional responses of tumor-infiltrating lymphocytes (TIL) freshly isolated from resected tumors of HCC patients. In addition, we compared GITR expression between TIL and paired samples of leukocytes isolated from blood and tumor-free liver tissues, and studied the effects of combined GITR and PD1 targeting on ex vivo TIL responses. In all three tissue compartments, CD4+ FoxP3+ regulatory T cells (Treg) showed higher GITR- expression than effector T-cell subsets. The highest expression of GITR was found on CD4+ FoxP3hi CD45RA- activated Treg in tumors. Recombinant GITR-ligand as well as a humanized agonistic anti-GITR antibody enhanced ex vivo proliferative responses of CD4+ and CD8+ TIL to tumor antigens presented by mRNA-transfected autologous B-cell blasts, and also reinforced proliferation, IFN-γ secretion and granzyme B production in stimulations of TIL with CD3/CD28 antibodies. Combining GITR ligation with anti-PD1 antibody nivolumab further enhanced tumor antigen-specific responses of TIL in some, but not all, HCC patients, compared to either single treatment. In conclusion, agonistic targeting of GITR can enhance functionality of HCC TIL, and may therefore be a promising strategy for single or combinatorial immunotherapy in HCC.

Gene expression profiling of human tissue-resident immune cells: Comparing blood and liver.

In this study, we describe a method to reliably characterize intrahepatic leukocyte populations using flow cytometry and next-generation RNA sequencing on fresh human liver biopsies. Over the last decades, immune responses of viral hepatitis patients, and of other liver diseases, have been incompletely characterized. Most studies include peripheral blood samples only, foregoing the possibility to investigate the site of inflammation directly. Here, we show that with an optimized protocol that combines cell sorting and RNA sequencing, we can perform a side by side comparison of both intrahepatic and peripheral immune cells. Using core liver biopsies from chronic hepatitis B virus patients, we show that the expression levels of IFN-stimulated genes and leukocyte-specific genes are markedly different in the liver compartment as compared to the peripheral blood. These observations emphasize the need to sample the liver directly. The variation of gene expression profiles in these chronic hepatitis B patients was considerable, despite the uniform treatment with nucleotide analogs and absence of liver inflammation in these patients. Finally, we show that this method can provide a detailed characterization of previously undetected liver-specific effects of novel candidate therapeutic compounds.

Mitochondrial electron transport chain complex III sustains hepatitis E virus replication and represents an antiviral target.

Hepatitis E virus (HEV) infection has emerged as a global health problem. However, no approved medication is available, and the infection biology remains largely elusive. Electron transport chain (ETC), a key component of the mitochondria, is the main site that produces ATP and reactive oxygen species (ROS). By profiling the role of the different complexes of the mitochondrial ETC, we found that pharmacological inhibition of complex III, a well-defined drug target for the treatment of malaria and Pneumocystis pneumonia, potently restricts HEV replication. This effect demonstrated in our HEV models is equivalent to the anti-HEV potency of ribavirin, a widely used off-label treatment for patients with chronic HEV. Mechanistically, we found that this effect is independent of ATP production, ROS level, and pyridine depletion. By using pharmacological inhibitors and genetic approaches, we found that mitochondrial permeability transition pore (MPTP), a newly identified component of ETC, provides basal defense against HEV infection. HEV interferes with the opening of the MPTP. Furthermore, inhibition of the MPTP attenuated the anti-HEV effect of complex III inhibitors, suggesting that the MPTP mediates the antiviral effects of these inhibitors. These findings reveal new insights on HEV-host interactions and provide viable anti-HEV targets for therapeutic development.-Qu, C., Zhang, S., Wang, W., Li, M., Wang, Y., van der Heijde-Mulder, M., Shokrollahi, E., Hakim, M. S., Raat, N. J. H., Peppelenbosch, M. P., Pan, Q. Mitochondrial electron transport chain complex III sustains hepatitis E virus replication and represents an antiviral target.

Mucosal-Associated Invariant T Cells Are More Activated in Chronic Hepatitis B, but Not Depleted in Blood: Reversal by Antiviral Therapy.

Background: Mucosal-associated invariant T (MAIT) cells might play a role in control of viral replication during chronic hepatitis B (cHBV) infection, but little is known of their number, phenotype, or function in cHBV patients. Methods: We performed flow cytometry on CD3+Vɑ7.2+CD161+ MAIT cells in blood of 55 cHBV patients. Nine patients were sampled before and on entecavir treatment. Six patients on therapy underwent a liver biopsy for flow cytometric analysis. Measurements included MAIT cell frequency, phenotype, and cytokine-producing capacity. Results: The MAIT cells were not deleted in blood or liver of cHBV patients compared with healthy controls, but they had higher percentages of CD38+ MAIT cells in blood, which declined on entecavir treatment. Peripheral MAIT cells of patients in the HBeAg-negative phase were least activated. Cytokine-producing MAIT cells were as frequent, but granzyme B-producing MAIT cells were more frequent upon stimulation with Escherichia coli compared with healthy controls. Conclusions: We demonstrate that, in sharp contrast to hepatitis C virus and human immunodeficiency virus patients, MAIT cells isolated from HBV patients are not deleted but are more activated, which can be normalized by nucleoside analog therapy. These observations may aid in deciphering the role of MAIT cells in immune responses to HBV.

Prebiotic galactooligosaccharides activate mucin and pectic galactan utilization pathways in the human gut symbiont Bacteroides thetaiotaomicron.

Galactooligosaccharides (GOS) are prebiotic carbohydrates that impart changes in the gut bacterial composition of formula-fed infants to more closely resemble that of breast-fed infants. Consuming human milk oligosaccharides (HMOs) provides specific bacterial strains with an advantage for colonizing the infant intestine. These same effects are seen in infants after GOS consumption, however GOS are very complex mixtures and the underlying molecular mechanisms of how GOS mimic HMOs are relatively unknown. Here we studied the effects of GOS utilization on a prominent gut symbiont, Bacteroides thetaiotaomicron, which has been previously shown to consume HMOs via mucin O-glycan degradation pathways. We show that several pathways for targeting O-mucin glycans are activated in B. thetaiotaomicron by GOS, as well as the galactan utilization sytem. Characterization of the endo-galactanase from this system identified activity on various longer GOS substrates while a subset of GOS compounds were identified as potential activators of mucin glycan metabolism in B. thetaiotaomicron. Our results show that GOS functions as an inducer of mucin-glycan pathways while providing a nutrient source in the form of ß-(1 → 4)-galactan. These metabolic features of GOS mixtures may serve to explain the beneficial effects that are seen for GOS supplemented infant formula.

A simple colorimetric assay for measuring fructosamine 3 kinase activity.

BACKGROUND: Fructosamine 3 kinase (FN3K) is a deglycating enzyme, which may play a key role in reducing diabetes-induced organ damage by removing bound glucose from glycated proteins. We wanted to develop a simple colorimetric method for assaying FN3K activity in human body fluids. METHODS: Glycated bovine serum albumin (BSA) was obtained by glycation with a 10% glucose solution at 37 °C. After 72 h, glycated BSA was dialyzed against phosphate buffered saline (0.1 mol/L, pH 7.4). The dialyzed solution (containing ±1000 µmol/L fructosamine) was used as an FN3K substrate. In the assay, 300 µL of substrate was incubated with 50 µL of serum and 100 µL of MgCl2 (0.7 mmol/L)/ATP (3.2 mmol/L). The fructosamine concentration was determined at the start and after incubation (120 min, 25 °C). The decrease in fructosamine concentration over time is a measure for the FN3K activity (1 U corresponding to 1 µmol/min). Concomitantly, the FN3K SNP rs1056534 and the ferroportin SNP rs1156350 were genotyped. RESULTS: Within-assay CV was 6.0%. Reference values for FN3K activity in serum were 14.2±1.6 U/L (n=143). Reference values for FN3K were neither age- nor sex-dependent. The various FN3K SNP rs1056534 genotypes showed no significant differences in serum FN3K activity. In diabetics (n=191), values (14.0±2.2 U/L) were comparable to those of the controls. FN3K activity in erythrocytes was significantly higher (170.3±7.6 U/L). The intra-erythrocytic FN3K activity makes the results prone to hemolysis. FN3K activity depended on the ferroportin Q248H genotypes, with the highest value for the wild type genotype. Neither transferrin saturation nor ferritin were confounders for the FN3K activity. FN3K activity was significantly (p<0.0001) correlated with HbA1c values, although the correlation between FN3K and HbA1c was weak. CONCLUSIONS: The simple colorimetric method allows determining FN3K activity in human serum. The assay may be useful for studying the impact of deglycation processes in diabetes mellitus.

Cytomegalovirus-Induced Expression of CD244 after Liver Transplantation Is Associated with CD8+ T Cell Hyporesponsiveness to Alloantigen.

The chronic presence of viral Ags can induce T cell exhaustion, which is characterized by upregulation of coinhibitory receptors and loss of T cell function. We studied whether a similar phenomenon occurs after liver transplantation (LTx), when there is continuous exposure to alloantigen. Expression of coinhibitory receptors on circulating CD4(+) and CD8(+) T cells was analyzed longitudinally in 19 patients until 6 mo after LTx and cross-sectionally in 38 patients late (1-12 y) after LTx. Expression of the coinhibitory receptors CD160 and CD244 on circulating CD8(+) T cells was already higher 6 mo after LTx compared with pre-LTx, and the elevated expression was sustained late after LTx, with CD244 showing the more prominent increase. The strongest upregulation of CD244 on circulating CD8(+) T cells was observed in patients who experienced CMV infection after LTx. CMV infection also was associated with reduced CD8(+) T cell proliferation and cytotoxic degranulation in response to alloantigen late after LTx. Purified CD244(+)CD8(+) T cells from LTx patients showed lower proliferative responses to alloantigen, as well as to polyclonal stimulation, than did their CD244(-) counterparts. In addition, the CD244(+)CD8(+) T cell population contained the majority of CMV peptide-loaded MHC class I tetramer-binding cells. In conclusion, CMV infection after LTx, rather than persistence of alloantigen, induces the accumulation of dysfunctional CD244(+)CD8(+) T cells in the circulation that persist long-term, resulting in reduced frequencies of circulating alloreactive CD8(+) T cells. These results suggest that CMV infection restrains CD8(+) T cell alloresponses after LTx.

Deep vein thrombosis after arthroscopic anterior cruciate ligament reconstruction: a prospective cohort study of 100 patients.

PURPOSE: To establish the incidence of venous thromboembolic complications as detected by bilateral complete compression ultrasonography (CCUS) after arthroscopic anterior cruciate ligament (ACL) reconstruction without thromboprophylaxis. METHODS: We performed a prospective cohort study to establish the incidence of venous thromboembolic complications after arthroscopic ACL reconstruction, as detected by bilateral CCUS at 14 days (range, 11 to 17 days) postoperatively. One hundred consecutive patients underwent bilateral extended ultrasonography. RESULTS: One hundred predominantly European patients with a mean age of 30 ± 10 years and mean body mass index of 25 ± 4 underwent ACL reconstruction with a mean operative duration of 68 ± 23 minutes and a tourniquet time of 76 ± 23 minutes. In 84% of patients an autologous hamstring graft was used, in 14% a bone-patellar tendon-bone graft was used, and 2 patients received an allograft. Of 100 patients, 9 (incidence, 9%; 95% confidence interval, 4.2 to 16.4) showed asymptomatic proximal or distal deep vein thrombosis on CCUS, of whom 4 (incidence, 4%; 95% confidence interval, 1.1 to 9.9) were symptomatic. A nonfatal pulmonary embolus developed in 1 patient during the 8-week follow-up period. CONCLUSIONS: This study shows that the incidence of venous thromboembolism after arthroscopic ACL reconstruction is relatively high; a 9% incidence of asymptomatic proximal or distal deep vein thrombosis was found, whereas 4% of patients were symptomatic. Further research is recommended to assess the need for thromboprophylaxis in patients undergoing ACL reconstruction, especially when risk factors are present. LEVEL OF EVIDENCE: Level IV, case series.

Awake craniotomy induces fewer changes in the plasma amino acid profile than craniotomy under general anesthesia.

In this prospective, observational, 2-armed study, we compared the plasma amino acid profiles of patients undergoing awake craniotomy to those undergoing craniotomy under general anesthesia. Both experimental groups were also compared with a healthy, age-matched and sex-matched reference group not undergoing surgery. It is our intention to investigate whether plasma amino acid levels provide information about physical and emotional stress, as well as pain during awake craniotomy versus craniotomy under general anesthesia. Both experimental groups received preoperative, perioperative, and postoperative dexamethasone. The plasma levels of 20 amino acids were determined preoperative, perioperative, and postoperatively in all groups and were correlated with subjective markers for pain, stress, and anxiety. In both craniotomy groups, preoperative levels of tryptophan and valine were significantly decreased whereas glutamate, alanine, and arginine were significantly increased relative to the reference group. Throughout time, tryptophan levels were significantly lower in the general anesthesia group versus the awake craniotomy group. The general anesthesia group had a significantly higher phenylalanine/tyrosine ratio, which may suggest higher oxidative stress, than the awake group throughout time. Between experimental groups, a significant increase in large neutral amino acids was found postoperatively in awake craniotomy patients, pain was also less and recovery was faster. A significant difference in mean hospitalization time was also found, with awake craniotomy patients leaving after 4.53+/-2.12 days and general anesthesia patients after 6.17+/-1.62 days; P=0.012. This study demonstrates that awake craniotomy is likely to be physically and emotionally less stressful than general anesthesia and that amino acid profiling holds promise for monitoring postoperative pain and recovery.

Dutch orthopedic thromboprophylaxis: a 5-year follow-up survey.

BACKGROUND AND PURPOSE: Previous surveys in the Netherlands have revealed that guidelines regarding orthopedic thromboprophylaxis were not followed and that a wide variation in protocols exists. This survey was performed to assess the current use of thromboprophylactic modalities and to compare it with the results of a previous survey. METHODS: All departments of orthopedic surgery in the Netherlands were sent a follow-up survey on venous thromboprophylaxis, and the data obtained were compared to the results of a survey performed 5 years earlier. RESULTS: All departments used pharmacological thromboprophylaxis following arthroplasties of the hip and knee. Low-molecular-weight heparin (LMWH) was used most frequently (79%) of the departments, followed by fondaparinux (13%). 5 years earlier, coumarin treatment was the predominant prophylaxis (79%). All departments prescribed pharmacological prophylaxis after femoral and tibial fractures; 78% used LMWH. Prophylaxis was continued for 6 weeks in 85% of cases. LMWH treatment was initiated on the day before surgery in 31% of cases (65% in the previous survey), perioperatively in 55%, and in the evening following surgery in 24%. In general, for daycare surgery and arthroscopies either no prophylaxis was given or a LMWH was given for 1 day. After anterior cruciate ligament reconstruction, 94% of departments prescribed some form of pharmacological prophylaxis. INTERPRETATION: The use of pharmacological prophylaxis after arthroplasty of the hip and knee and also after fracture surgery around the hip and knee is common practice in the Netherlands. In 5 years, the widely used coumarin derivates have been largely replaced with LMWH.

High revision rate after treatment of femoral neck fractures with an optionally (un)cemented stem.

BACKGROUND: The advantages of uncemented and cemented components in hip arthroplasty have been subject of debate. We have studied on a hemiprosthesis, which can be optionally implanted with or without cement. Since the stem geometry and surface in cemented arthroplasty differs from the uncemented one and cannot be fused into one general design, we hypothesised that this hemiprosthesis used without cement has a considerable high revision rate, based on aseptic loosening. METHODS: A hemiprosthesis, which is designed for both cemented and uncemented fixation, was used (Conquest, Smith&Nephew). Preoperatively, the choice of whether to use cement or not was based on the shape and bone quality of the femoral canal. Revision rate and indication, mortality, perioperative complications and radiographic features of 151 consecutive hips in 146 patients were evaluated. RESULTS: Twenty-three stems (15%) were implanted with cement and 128 (85%) without. After a mean follow-up of 2 years, a revision rate of 8.6% and a survival percentage of 90% (CI 85-95) were observed. Twelve uncemented stems warranted revision, compared with one cemented stem. Revision because of aseptic loosening was necessary in 7 (6%) stems, all uncemented. No differences in operation-related mortality and morbidity were observed. CONCLUSION: Because of the rather high revision rate, the authors advice not to use this hemiprosthesis without cement.

Frontal lobe structure and executive function in migraine patients.

Neuroimaging studies have identified frontal lobe brain abnormalities in migraineurs. Neuropsychological investigations highlighted frontal lobe related cognitive impairments in migraineurs, including working memory and executive function deficits. The relationship between brain anatomy and cognitive function in migraine, however, is unclear. The aim of this study was to simultaneously investigated cortex structure and executive function (EF) in patients with migraine and control subjects. Thus, we assessed grey matter (GM) density in 25 adult patients with migraine, compared to age and sex-matched control subjects, using magnetic resonance imaging (MRI) and voxel-based-morphometry (VBM), and we measured EF in the same population, employing three EF tasks of the Maudsley attention and response suppression (MARS) battery. Migraineurs, compared to control subjects, showed decreased frontal and parietal lobe GM density and slower response time to task set-shifting and, the delayed response time correlated significantly with reduced GM density of the frontal lobes in migraineurs. Frontal and parietal lobe abnormalities in migraineurs could be an underlying cause of significantly slower response time during cognitive set-shifting.