Adam G Testro

Toll-like receptors and their role in gastrointestinal disease.

The innate immune response to invading pathogens is centred upon a family of non‐clonal, germline‐encoded pattern recognition receptors (PRRs), the Toll‐like receptors (TLRs). These provide specificity for a vast range of microbial pathogens, and offer an immediate anti‐microbial response system. Thirteen mammalian TLRs have been described; 10 are expressed in humans, each responsible for the recognition of distinct, invariant microbial structures originating from bacteria, viruses, fungi and protozoa. The two most thoroughly studied are TLR4 and TLR2, the PRRs for Gram‐negative and Gram‐positive bacterial products, respectively. TLR4 is also the major receptor recognising endogenous ligands released from damaged or dying cells. Activation of a TLR by its relevant ligand rapidly ignites a complex intracellular signaling cascade that ultimately results in upregulation of inflammatory genes and production of proinflammatory cytokines, interferons and recruitment of myeloid cells. It also stimulates expression, upon antigen presenting cells, of co‐stimulatory molecules required to induce an adaptive immune response. Whilst a robust TLR response is critical for survival and defence against invading pathogens, inappropriate signaling in response to alterations in the local microflora environment can be detrimental. Such ‘unhelpful TLR responses’ could form the basis for a large number of gastrointestinal and liver disorders, including inflammatory bowel disease, viral hepatitis, autoimmune liver diseases and hepatic fibrosis. As our understanding of TLRs expands, the pathogenesis of a number of gastrointestinal disorders will be further elucidated, and this offers potential for specific therapies aimed directly at TLR signaling.

Hepatitis B virus reactivation following immunosuppressive therapy: guidelines for prevention and management

It is well known that immunosuppressive drugs or cancer chemotherapy can stimulate replication of hepatitis B virus (HBV) and precipitate severe flares of HBV infection. The risk of this syndrome of ‘reactivation hepatitis B’ is highest in haematopoietic stem cell or solid organ transplant recipients and in those undergoing chemotherapy for haematological malignancies; however, it has been described following almost any form of immunosuppressive treatment. Fortunately, it can be largely prevented by prophylactic therapy with oral anti‐HBV nucleoside/nucleotide analogues. Importantly, chronic HBV infection is usually asymptomatic, and most patients at risk are likely to be unaware that they carry the infection. Thus, the key to avoiding this potentially fatal complication of immunosuppressive treatment is to ensure that all patients at risk of chronic HBV infection are screened for the disease before commencing immunosuppressive treatment or chemotherapy.

Faecal microbiota transplantation for severe Clostridium difficile infection in the intensive care unit.

We describe a case of faecal microbiota transplantation (FMT) used for severe binary toxin-positive Clostridium difficile infection in an intensive care setting. The patient was admitted to the ICU of a tertiary hospital and failed traditional maximal pharmacological management. Adjunctive therapy with FMT given through gastroscopy resulted in resolution of the C. difficile-related symptoms. Although there is a growing experience with FMT for recurrent C. difficile infection, published evidence in severe disease is very limited. In a landscape of increasingly severe C. difficile infection, adjunctive FMT may be considered a useful early treatment option.

Acute allograft rejection in human liver transplant recipients is associated with signaling through toll-like receptor 4.

Background and Aims:  Toll‐like receptor (TLR) signaling is a crucial step in initiating adaptive immune responses. In addition to recognizing endotoxin, TLR4 also recognizes endogenous ligands (‘damage‐associated structures’), which are released into the circulation in the peri‐transplantation period. TLR2 to a lesser extent also recognizes these endogenous ligands. Multiple studies involving solid organ transplants demonstrate a clear association between TLR4 and allograft rejection. In the present study we assessed whether an association exists between TLR4 and TLR2‐dependent responses and acute liver allograft rejection.

Intestinal transplantation: Current status and future directions

Three decades after the first intestinal transplant was performed in humans, this life‐saving procedure has come of age and now offers hope of long‐term survival in a small group of patients with life‐threatening complications of intestinal failure and parenteral nutrition. Success rates have greatly improved, largely through advances in immunosuppression protocols, improved surgical technique and postoperative care, and accumulated experience. Management of the intestinal transplant recipient entails careful surveillance, prevention, and treatment of rejection and infection, as well as optimization of feeding and nutrition. With this approach, survival and quality of life are demonstrably improved, such that intestinal transplantation is now an established and accepted procedure for this very select group of highly‐complex patients.

Long-term outcome of patients treated with terlipressin for types 1 and 2 hepatorenal syndrome

Background:  Studies suggest that terlipressin is effective in the treatment of hepatorenal syndrome (HRS). However, factors predicting response to therapy and the long‐term outcome of patients have not been defined.

Cyclosporine and tacrolimus have inhibitory effects on toll-like receptor signaling after liver transplantation.

Toll‐like receptors (TLRs) play a key role in transplantation biology. The effect of immunosuppression on TLR function after liver transplantation is unknown. Peripheral blood mononuclear cells (PBMCs) from 113 post–liver transplant patients and 13 healthy controls were stimulated with TLR‐specific ligands [lipopolysaccharide (TLR4), pan‐3‐cys (P3C) (TLR2), Poly (I:C) (PIC) (TLR3), R848 (TLR7/8), and CpG (TLR9)] for 24 hours. PBMCs from 5 healthy controls were also cultured with therapeutic concentrations of cyclosporine A (CYA) and tacrolimus (TAC). Cytokine production was measured with enzyme‐linked immunosorbent assays and flow cytometry. PBMCs from patients on calcineurin inhibitors after liver transplantation produced less interleukin‐6 (IL‐6) and tumor necrosis factor α (TNFα) in response to TLR2 stimulation (IL‐6: P=0.02; TNFα: P=0.01), TLR4 stimulation (IL‐6: P=0.02; TNFα: P=0.01), and TLR7/8 stimulation (IL‐6: P=0.02; TNFα: P=0.02), compared with healthy controls. Both CD56bright and CD56dim natural killer (NK) cells from patients on calcineurin inhibitors also produced less interferon‐γ (IFNγ) with TLR7/8 stimulation compared with healthy controls (CD56bright: P=0.002; CD56dim: P=0.004). Similar findings were demonstrated in healthy PBMCs cultured with CYA (PBMCs: TLR2, IL‐6: P=0.005; TLR4, IL‐6: P=0.03, TNFα: P=0.03; TLR7/8, IL‐6: P=0.02, TNFα: P=0.01; CD56dim NK cells: TLR7/8, IFNγ: P=0.03). TAC impaired TLR4‐mediated IL‐6 and TNFα production by PBMCs (IL‐6; P = 0.02; TNFα P = 0.009). In conclusion, patients on calcineurin inhibitors had impaired inflammatory cytokine production in response to TLR2, TLR4, and TLR7/8 stimulation compared comparison with healthy controls. Importantly, TAC and CYA appear to have different effects on TLR signaling. Impaired TLR function has important repercussions for risk of infection, graft rejection, and disease recurrence after transplantation, and the different immunosuppressive profiles of CYA and TAC may guide the choice of therapy to improve disease outcomes. Liver Transpl 19:1099‐1107, 2013.

Immune monitoring post liver transplant

Many of the causes of short and late morbidity following liver transplantation are associated with immunosuppression or immunosuppressive medications. Current care often involves close monitoring of liver biochemistry as well as therapeutic drug levels. However, the postoperative course following liver transplantation can often be associated with significant complications including infection and rejection, suggesting an inadequacy in current immune function monitoring. Many assays have been tested in the research setting to identify possible biomarkers that may be used to predict clinical events such as acute cellular rejection, and therefore allow modification of a patients immunosuppressive regimen prior to a clinical event. However, these generally require significant laboratory processing and have had difficulty becoming established in common clinical use outside the research setting. One assay, Cylex ImmuKnow has been food and drug administration approved but has had variable results. In this review we discuss the assays that have been used to assess monitoring of immune function after liver transplantation and consider possible future directions.

Effects of antibiotics on expression and function of Toll-like receptors 2 and 4 on mononuclear cells in patients with advanced cirrhosis

BACKGROUND & AIMS Toll-like receptors (TLRs) are critical to innate immune responses. TLR4 recognises Gram-negative bacteria, whilst TLR2 recognises Gram-positive. We examined TLR expression and function in cirrhosis, and whether this is affected by antibiotic therapy. METHODS Sixty-four subjects were included (23 controls and 41 Child-Pugh C cirrhotic patients). Thirty patients were taking norfloxacin or trimethoprim-sulfamethoxazole as prophylaxis against bacterial peritonitis and 11 were not. In a second study, 8 patients were examined before and after commencement of antibiotics. Monocyte expression of TLR2 and 4 was determined by flow cytometry. Monocytes from the patients with paired samples were stimulated using TLR ligands and TNF-alpha production measured. RESULTS Patients not taking antibiotics had significantly decreased TLR4 expression compared with controls (0.74 vs. 1.0, p=0.009) and patients receiving antibiotics (0.74 vs. 0.98, p=0.02). There were no differences with regard to TLR2. In the patients with paired samples, TLR4 expression increased (0.74-1.49, p=0.002) following antibiotic use, whilst again, there was no change in TLR2 expression (0.99 vs. 0.92, p=0.20). TLR4-dependent TNF-alpha production increased following antibiotic use (1077 vs. 3620pg/mL, p<0.05), whilst TLR2-dependent production was unchanged. CONCLUSIONS TLR4 expression is decreased in patients with Child-Pugh C cirrhosis, but is restored by antibiotics targeting enteric Gram-negative bacteria. TLR4-dependent cytokine production also increases significantly following antibiotic therapy. This suggests that the high incidence of Gram-negative infection in cirrhotic patients is in part due to down-regulation of the TLR4-dependant immune response and that the efficacy of antibiotic prophylaxis is contributed to by modulation of innate immunity.

Targeted individual prophylaxis offers superior risk stratification for cytomegalovirus reactivation after liver transplantation

Cytomegalovirus (CMV) can reactivate following liver transplantation. Management of patients currently considered low risk based on pretransplant serology remains contentious, with universal prophylaxis and preemptive strategies suffering from significant deficiencies. We hypothesized that a CMV‐specific T cell assay performed early after transplant as part of a preemptive strategy could better stratify “low‐risk” (recipient seropositive) patients. We conducted a prospective, blinded, observational study in 75 adult recipients. QuantiFERON‐cytomegalovirus was performed both before and at multiple times after transplant. Low‐risk patients (n = 58) were monitored as per unit protocol and treatment was commenced if CMV > 1000 copies/mL (DNAemia). Twenty patients needed antiviral treatment for other reasons and were censored (mainly for rejection or herpes simplex virus infection); 19/38 (50%) of the remaining low‐risk patients developed DNAemia at mean 34.6 days after transplant. A week 2 result of <0.1 IU/mL was significantly associated with risk of subsequent DNAemia (hazard ratio [HR], 6.9; P = 0.002). The positive predictive value of 80% suggests these patients are inappropriately labeled low risk and are actually at high likelihood of CMV reactivation. A secondary cutoff of <0.2 IU/mL was associated with moderate risk (HR, 2.8; P = 0.01). In conclusion, a protocol based on a single early CMV‐specific T cell based assay would offer improved risk stratification and individualization of patient management after transplant. This could offer improved drug and service utilization and potentially result in significant improvements over both currently used protocols to manage supposedly low‐risk patients. Liver Transpl 21:1478‐1485, 2015.