Sheraz Yaqub

Molecular mechanisms for protein kinase A-mediated modulation of immune function.

Protein kinase A (PKA) is a serine/threonine kinase that regulates a number of cellular processes important for immune activation and control. Modulation of signal transduction by PKA is a complex and diverse process, and differential isozyme expression, holoenzyme composition and subcellular localization contribute specificity to the PKA signalling pathway. In lymphocytes, phosphorylation by PKA has been demonstrated to regulate antigen receptor-induced signalling both by altering protein-protein interactions and by changing the enzymatic activity of target proteins. PKA substrates involved in immune activation include transcription factors, members of the MAP kinase pathway and phospholipases. The ability of PKA type I to regulate activation of signalling components important for formation of the immunological synapse, demonstrates that the cAMP signalling pathway can directly modulate proximal events in lymphocyte activation. Furthermore, the recent discovery that PKA regulates Src kinases through modulation of Csk, supports the notion that PKA is involved in the fine-tuning of immune receptor signalling in lipid rafts.

FOXP3+CD4+CD25+ Adaptive Regulatory T Cells Express Cyclooxygenase-2 and Suppress Effector T Cells by a Prostaglandin E2-Dependent Mechanism

CD4+CD25+ regulatory T (TR) cells suppress effector T cells by partly unknown mechanisms. In this study, we describe a population of human suppressive CD4+CD25+ adaptive TR (TRadapt) cells induced in vitro that express cyclooxygenase 2 (COX-2) and the transcription factor FOXP3. TRadapt cells produce PGE2 and suppress effector T cell responses in a manner that is reversed by COX inhibitors and PGE2 receptor-specific antagonists. In resting CD4+CD25− T cells, treatment with PGE2 induced FOXP3 expression. Thus, autocrine and paracrine effects of PGE2 produced by COX-2-positive TRadapt cells may be responsible for both the FOXP3+ phenotype and the mechanism used by these cells to suppress effector T cells.

Laparoscopic Lavage vs Primary Resection for Acute Perforated Diverticulitis: The SCANDIV Randomized Clinical Trial

IMPORTANCE Perforated colonic diverticulitis usually requires surgical resection, which is associated with significant morbidity. Cohort studies have suggested that laparoscopic lavage may treat perforated diverticulitis with less morbidity than resection procedures. OBJECTIVE To compare the outcomes from laparoscopic lavage with those for colon resection for perforated diverticulitis. DESIGN, SETTING, AND PARTICIPANTS Multicenter, randomized clinical superiority trial recruiting participants from 21 centers in Sweden and Norway from February 2010 to June 2014. The last patient follow-up was in December 2014 and final review and verification of the medical records was assessed in March 2015. Patients with suspected perforated diverticulitis, a clinical indication for emergency surgery, and free air on an abdominal computed tomography scan were eligible. Of 509 patients screened, 415 were eligible and 199 were enrolled. INTERVENTIONS Patients were assigned to undergo laparoscopic peritoneal lavage (n = 101) or colon resection (n = 98) based on a computer-generated, center-stratified block randomization. All patients with fecal peritonitis (15 patients in the laparoscopic peritoneal lavage group vs 13 in the colon resection group) underwent colon resection. Patients with a pathology requiring treatment beyond that necessary for perforated diverticulitis (12 in the laparoscopic lavage group vs 13 in the colon resection group) were also excluded from the protocol operations and treated as required for the pathology encountered. MAIN OUTCOMES AND MEASURES The primary outcome was severe postoperative complications (Clavien-Dindo score >IIIa) within 90 days. Secondary outcomes included other postoperative complications, reoperations, length of operating time, length of postoperative hospital stay, and quality of life. RESULTS The primary outcome was observed in 31 of 101 patients (30.7%) in the laparoscopic lavage group and 25 of 96 patients (26.0%) in the colon resection group (difference, 4.7% [95% CI, -7.9% to 17.0%]; P = .53). Mortality at 90 days did not significantly differ between the laparoscopic lavage group (14 patients [13.9%]) and the colon resection group (11 patients [11.5%]; difference, 2.4% [95% CI, -7.2% to 11.9%]; P = .67). The reoperation rate was significantly higher in the laparoscopic lavage group (15 of 74 patients [20.3%]) than in the colon resection group (4 of 70 patients [5.7%]; difference, 14.6% [95% CI, 3.5% to 25.6%]; P = .01) for patients who did not have fecal peritonitis. The length of operating time was significantly shorter in the laparoscopic lavage group; whereas, length of postoperative hospital stay and quality of life did not differ significantly between groups. Four sigmoid carcinomas were missed with laparoscopic lavage. CONCLUSIONS AND RELEVANCE Among patients with likely perforated diverticulitis and undergoing emergency surgery, the use of laparoscopic lavage vs primary resection did not reduce severe postoperative complications and led to worse outcomes in secondary end points. These findings do not support laparoscopic lavage for treatment of perforated diverticulitis. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01047462.

Generation of highly suppressive adaptive CD8+CD25+FOXP3+ regulatory T cells by continuous antigen stimulation

Continuous antigen stimulation of CD4+CD25– T cells leads to generation of adaptive CD4+CD25+FOXP3+ regulatory T (TR) cells. Here, we show that highly suppressive adaptive CD8+CD25+FOXP3+ T cells can be generated in the same manner by continuous antigen stimulation in the presence of CD14+ monocytes. During the course of stimulation, acquisition of immunosuppressive properties develops in parallel with up‐regulation and expression of cytotoxic molecules. The CD8+ TR cells inhibit CD4+ and CD8+ T cell proliferation and cytokine production, but do not alter the expression of granzyme A and granzyme B or perforin in CD8+ effector T cells. Although, the CD8+ TR cells express prostaglandin E2, IL‐10 and TGF‐β, the mechanism of suppression was independent of these soluble factors. In contrast to adaptive CD4+ TR cells, the CD8+ TR cells suppress mainly by a contact‐dependent mechanism as evident from transwell experiments. However, neither blocking antibodies to CTLA‐4, CD80 nor CD86 could reverse CD8+ TR‐mediated suppression, indicating that other mechanism(s) must be employed by these cells.

Regulatory T cells in colorectal cancer patients suppress anti-tumor immune activity in a COX-2 dependent manner

ObjectiveNaturally occurring regulatory T (TR) cells suppress autoreactive T cells whereas adaptive TR cells, induced in the periphery, play an important role in chronic viral diseases and cancer. Several studies indicate that cyclooxygenase (COX) inhibitors prevent cancer development of colon adenomas and delay disease progression in patients with colorectal cancer (CRC). We have shown that adaptive TR cells express COX-2 and produce PGE2 that suppress effector T cells in a manner that is reversed by COX-inhibitors.Methods and resultsHere we demonstrate that CRC patients have elevated levels of PGE2 in peripheral blood, and CRC tissue samples and draining lymph nodes display increased numbers of FOXP3+ TR cells. Depletion of TR cells from PBMC enhanced anti-tumor T-cell responses to peptides from carcinoembryonic antigen. Furthermore, the COX inhibitor indomethacin and the PKA type I antagonist Rp-8-Br-cAMPS significantly improved the anti-tumor immune activity.ConclusionWe suggest that adaptive TR cells contribute to an immunosuppressive microenvironment in CRC and inhibit effector T cells by a COX-2–PGE2-dependent mechanism and thereby facilitate tumor growth. Therapeutic strategies targeting TR cells and the PGE2–cAMP pathway may be interesting to pursue to enhance anti-tumor immune activity in CRC patients.

Differentiation of naive CD4+ T cells into CD4+CD25+FOXP3+ regulatory T cells by continuous antigen stimulation

Human CD4+CD25+ regulatory T (TR) cells express the transcription factor forkhead box p3 (FOXP3) and have potent immunosuppressive properties. While naturally occurring TR cells develop in the thymus, adaptive TR cells develop in the periphery from naive CD4+ T cells. Adaptive TR cells may express cyclooxygenase type 2 (COX‐2) and suppress effector T cells by a PGE2‐dependent mechanism, which is reversible with COX inhibitors. In this study we have characterized the differentiation of naive CD4+ T cells into adaptive TR cells in detail during 7 days of continuous antigen stimulation. After 2 days of stimulation of CD4+CD25– T cells, the cells expressed FOXP3 and COX‐2 and displayed potent immunosuppressive properties. The suppressive phenotype was present at all observed time‐points from Day 2, although suppression was merely present at Day 7. The adaptive TR cells expressed cell surface markers consistent with an activated phenotype and secreted high levels of TGF‐β, IL‐10, and PGE2. However, the suppressive phenotype was found exclusively in cells that proliferated upon activation. These data support the notion that activation of naive CD4+ T cells leads to concomitant acquisition of effector and suppressive properties.

Activation of C-terminal Src kinase (Csk) by phosphorylation at serine-364 depends on the Csk-Src homology 3 domain

In the present study, we investigate the mechanism for the protein kinase A (PKA)-mediated activation of C-terminal Src kinase (Csk). Although isolated Csk kinase domain was phosphorylated at Ser(364) by PKA to the same stoichiometry as wild-type Csk, significant activation of the isolated Csk kinase domain by PKA was observed only in the presence of the purified Src homology 3 domain (SH3 domain). Furthermore, the interaction between the SH3 and kinase domains was facilitated by PKA-mediated phosphorylation of the kinase domain, as evaluated by surface plasmon resonance. This suggests that an overall structural domain organization and interaction between the kinase and SH3 domains are important for the activity of Csk and its regulation by PKA.

Antibody-secreting plasma cells persist for decades in human intestine

Plasma cells (PCs) produce antibodies that mediate immunity after infection or vaccination. In contrast to PCs in the bone marrow, PCs in the gut have been considered short lived. In this study, we studied PC dynamics in the human small intestine by cell-turnover analysis in organ transplants and by retrospective cell birth dating measuring carbon-14 in genomic DNA. We identified three distinct PC subsets: a CD19+ PC subset was dynamically exchanged, whereas of two CD19− PC subsets, CD45+ PCs exhibited little and CD45− PCs no replacement and had a median age of 11 and 22 yr, respectively. Accumulation of CD45− PCs during ageing and the presence of rotavirus-specific clones entirely within the CD19− PC subsets support selection and maintenance of protective PCs for life in human intestine.

Aspirin As Secondary Prevention in Patients With Colorectal Cancer: An Unselected Population-Based Study

PURPOSE Regular use of aspirin (acetylsalicylic acid) is associated with reduced incidence and mortality of colorectal cancer (CRC). However, aspirin as primary prevention is debated because of the risk of hemorrhagic adverse effects. Aspirin as secondary prevention may be more justified from a risk-benefit perspective. We have examined the association between aspirin use after the diagnosis of CRC with CRC-specific survival (CSS) and overall survival (OS). MATERIALS AND METHODS An observational, population-based, retrospective cohort study was conducted by linking patients diagnosed with CRC from 2004 through 2011 (Cancer Registry of Norway) with data on their aspirin use (The Norwegian Prescription Database). These registries cover more than 99% of the Norwegian population and include all patients in an unselected and consecutive manner. Exposure to aspirin was defined as receipt of aspirin prescriptions for more than 6 months after the diagnosis of CRC. Multivariable Cox-proportional hazard analyses were used to model survival. The main outcome measures of the study were CSS and OS. RESULTS A total of 23,162 patients diagnosed with CRC were included, 6,102 of whom were exposed to aspirin after the diagnosis of CRC (26.3%). The median follow-up time was 3.0 years. A total of 2,071 deaths (32.9%, all causes) occurred among aspirin-exposed patients, of which 1,158 (19.0%) were CRC specific. Among unexposed patients (n = 17,060), there were 7,218 deaths (42.3%), of which 5,375 (31.5%) were CRC specific. In multivariable analysis, aspirin exposure after the diagnosis of CRC was independently associated with improved CSS (hazard ratio [HR], 0.85; 95% CI, 0.79 to 0.92) and OS (HR, 0.95; 95% CI, 0.90 to 1.01). CONCLUSION Aspirin use after the diagnosis of CRC is independently associated with improved CSS and OS.

Laparoscopic Versus Open Resection for Colorectal Liver Metastases: The OSLO-COMET Randomized Controlled Trial.

Objective: To perform the first randomized controlled trial to compare laparoscopic and open liver resection. Summary Background Data: Laparoscopic liver resection is increasingly used for the surgical treatment of liver tumors. However, high-level evidence to conclude that laparoscopic liver resection is superior to open liver resection is lacking. Methods: Explanatory, assessor-blinded, single center, randomized superiority trial recruiting patients from Oslo University Hospital, Oslo, Norway from February 2012 to January 2016. A total of 280 patients with resectable liver metastases from colorectal cancer were randomly assigned to undergo laparoscopic (n = 133) or open (n = 147) parenchyma-sparing liver resection. The primary outcome was postoperative complications within 30 days (Accordion grade 2 or higher). Secondary outcomes included cost-effectiveness, postoperative hospital stay, blood loss, operation time, and resection margins. Results: The postoperative complication rate was 19% in the laparoscopic-surgery group and 31% in the open-surgery group (12 percentage points difference [95% confidence interval 1.67–21.8; P = 0.021]). The postoperative hospital stay was shorter for laparoscopic surgery (53 vs 96 hours, P < 0.001), whereas there were no differences in blood loss, operation time, and resection margins. Mortality at 90 days did not differ significantly from the laparoscopic group (0 patients) to the open group (1 patient). In a 4-month perspective, the costs were equal, whereas patients in the laparoscopic-surgery group gained 0.011 quality-adjusted life years compared to patients in the open-surgery group (P = 0.001). Conclusions: In patients undergoing parenchyma-sparing liver resection for colorectal metastases, laparoscopic surgery was associated with significantly less postoperative complications compared to open surgery. Laparoscopic resection was cost-effective compared to open resection with a 67% probability. The rate of free resection margins was the same in both groups. Our results support the continued implementation of laparoscopic liver resection.