The relationship between 18 F-FDG-PETCT-derived markers of tumour metabolism and systemic inflammation in patients with recurrent disease following surgery for colorectal cancer.

AIM: 18 F-fluorodeoxyglucose positron emission tomography-computed tomography (18 F-FDG-PETCT)-derived markers of tumour metabolism have been reported to have prognostic significance in a variety of tumours. Host inflammation is also recognized to have prognostic significance. The aim of the present study was to investigate the relationship between these markers and host systemic inflammation in patients undergoing elective surgery for colorectal cancer. METHOD: Patients with histologically confirmed colorectal cancer who underwent elective surgery between 2008 and 2015 and also underwent 18 F-FDG-PETCT at a single centre were included (n = 103). The neutrophil-lymphocyte ratio (NLR) and modified Glasgow Prognostic Score (mGPS) were derived from routine blood tests. The maximum standardized uptake (SUVmax), peak standardized uptake (SUVpeak), metabolic tumour volume (MTV) and total lesion glycolysis (TLG) were measured. RESULTS: There was no association between 18 F-FDG-PETCT measures of tumour metabolism and systemic inflammation in the 33 patients who underwent preoperative imaging. Of the 70 patients with recurrent disease who underwent 18 F-FDG-PETCT during follow-up, patients with NLR ≥ 5 had a significantly higher SUVmax (20 vs 7, P = 0.002), SUVpeak (14 vs 5, P < 0.001), MTV (29 g vs 2 g, P = 0.001) and TLG (338 g vs 9 g, P < 0.001). Similarly, patients with a mGPS of 1-2 at the time of 18 F-FDG-PETCT had a significantly higher median SUVmax (11 vs 6, P = 0.048), SUVpeak (8 vs 4, P = 0.046), MTV (13 ml vs 2 ml, P = 0.005) and TLG (146 g vs 10 g, P = 0.004). CONCLUSION: The present study reports a direct association between 18 F-FDG-PETCT-derived measures of tumour metabolism and systemic inflammation in patients with recurrent colorectal cancer.

Unplanned reoperation within 30 days of surgery for colorectal cancer in NHS Lanarkshire.

AIM: A recent study of unplanned reoperation within 28 days after colorectal surgery in England found a mean rate of 6.5% and suggested that this be used as a performance indicator. We aimed to find the unplanned 30-day reoperation rate for patients having colorectal cancer surgery in NHS Lanarkshire. METHOD: This retrospective study identified all patients having surgery for colorectal cancer in NHS Lanarkshire between 2006 and 2008 from a prospective colorectal cancer database. Scottish Morbidity Record (SMR01) data were then examined for each patient to determine whether they returned to the operating theatre within 30 days of the index procedure. RESULTS: Five hundred and seventy-three patients had a primary operation for colorectal cancer during the period. The unplanned rate of reoperation within 30 days of surgery was 5.4%. There was no statistically significant difference between the hospital site, emergency or elective operation or laparoscopic resection or laparotomy. There was no statistically significant difference in reoperation rate between colorectal and general surgeons. CONCLUSION: The rate of unplanned reoperation in NHS Lanarkshire compares favourably with that of England; however, similar methodological problems exist. The accuracy of the data is dependent on coding and entry.

Effect of preoperative oral antibiotics in combination with mechanical bowel preparation on inflammatory response and short-term outcomes following left-sided colonic and rectal resections.

Background: Preoperative oral antibiotics in addition to intravenous antibiotics and mechanical bowel preparation (MBP) may influence the gut microbiome and reduce both the postoperative systemic inflammatory response to surgery and postoperative infective complications following colorectal resection. This propensity score-matched study compared outcomes of patients undergoing left-sided colonic or rectal resection with or without a combination of oral antibiotics and MBP. Methods: The addition of oral antibiotics and MBP to prophylactic intravenous antibiotics in left-sided colonic and rectal resections was introduced in 2015-2016 at a single institution. Propensity score matching was undertaken to compare the effects of oral antibiotics plus MBP versus neither oral antibiotics nor MBP on the postoperative systemic inflammatory response and short-term outcomes in patients undergoing left-sided colonic or rectal resection between 2013 and 2018. Results: Of 396 patients who had propensity score matching for host, anaesthetic and operative factors, 204 matched patients were identified. The addition of oral antibiotics and MBP was associated with a significantly reduced postoperative inflammatory response (reduced postoperative Glasgow Prognostic Score) on day 3 (odds ratio (OR) 0·66, 95 per cent c.i. 0·44 to 0·99; P = 0·013) and day 4 (OR 0·46, 0·30 to 0·71; P = 0·001). Significantly reduced overall complications (OR 0·31, 0·17 to 0·56; P < 0·001), infective complications (OR 0·41, 0·22 to 0·77; P = 0·011), surgical-site infection (OR 0·37, 0·17 to 0·83; P = 0·024) and postoperative length of hospital stay (median 7 days versus 8 days in patients who had intravenous antibiotics alone; P = 0·050) were also observed. Conclusion: Preoperative oral antibiotics and MBP in addition to prophylactic intravenous antibiotics were associated with a reduction in the postoperative systemic inflammatory response and postoperative complications in patients undergoing resectional left-sided colonic or rectal surgery.

Antecedentes: La administración preoperatoria de antibióticos por vía oral (preoperative oral antibiotics, OAB), además de por vía intravenosa y de la preparación mecánica del colon (mechanical bowel preparation, MBP) puede afectar al microbioma intestinal y disminuir tanto la respuesta postoperatoria sistémica inflamatoria a la cirugía, como las complicaciones infecciosas postoperatorias tras una resección colorrectal. Este estudio emparejado por puntaje de propensión comparó los resultados de pacientes sometidos a resección del colon izquierdo o resección rectal con y sin una combinación de OAB y MBP. Métodos: La adición de OAB y MBP a la administración profiláctica de antibióticos por vía intravenosa fue introducida en 2015­2016 en un centro médico. Se llevó a cabo un estudio emparejado por puntaje de propensión para comparar los efectos de OAB con MBP versus la no administración de OAB ni el uso de MBP sobre la respuesta postoperatoria sistémica inflamatoria a la cirugía y los resultados a corto plazo en pacientes sometidos a resección del colon izquierdo o resección rectal desde el 2013 al 2018. Resultados: De los 396 pacientes incluidos en el emparejamiento por puntaje de propensión relativo a factores relacionados con el huésped, anestésicos y operatorios, se identificaron 204 pacientes emparejados. La adición de OAB y MBP se asoció con una disminución significativa de la respuesta inflamatoria postoperatoria (disminución postoperatoria de la puntuación pronóstica de Glasgow el día 3 (razón de oportunidades, odds ratio, OR 0,66, i.c. del 95% 0,44­0,99, P = 0,013) y el día 4 (OR 0,46, i.c. del 95% 0,30­0,71, P = 0,001). También se observaron reducciones significativas de las complicaciones globales (OR 0,31, i.c. del 95% 0,17­0,56, P < 0,001), complicaciones infecciosas (OR 0,41, i.c. del 95% 0,17­0,83, P = 0,011), infecciones del sitio quirúrgico (OR 0,37, i.c. del 95% 0,17­0,83, P = 0,024) y duración de la estancia hospitalaria postoperatoria (mediana 8 versus 7 días, P = 0,05). Conclusión: La adición preoperatoria de OAB y MBP a la administración profiláctica de antibióticos intravenosos se han asociado con una disminución de la respuesta inflamatoria sistémica postoperatoria y de las complicaciones postoperatorias en pacientes sometidos a resección del colon izquierdo o cirugía rectal.

Meta-analysis of oral antibiotics, in combination with preoperative intravenous antibiotics and mechanical bowel preparation the day before surgery, compared with intravenous antibiotics and mechanical bowel preparation alone to reduce surgical-site infections in elective colorectal surgery.

BACKGROUND: Surgical-site infection (SSI) is a potentially serious complication following colorectal surgery. The present systematic review and meta-analysis aimed to investigate the effect of preoperative oral antibiotics and mechanical bowel preparation (MBP) on SSI rates. METHODS: A systematic review of PubMed, Cochrane Database of Systematic Reviews and Cochrane Central Register of Controlled Trials was performed using appropriate keywords. Included were RCTs and observational studies reporting rates of SSI following elective colorectal surgery, in patients given preoperative oral antibiotic prophylaxis, in combination with intravenous (i.v.) antibiotic prophylaxis and MBP, compared with patients given only i.v. antibiotic prophylaxis with MBP. A meta-analysis was undertaken. RESULTS: Twenty-two studies (57 207 patients) were included, of which 14 were RCTs and eight observational studies. Preoperative oral antibiotics, in combination with i.v. antibiotics and MBP, were associated with significantly lower rates of SSI than combined i.v. antibiotics and MBP in RCTs (odds ratio (OR) 0·45, 95 per cent c.i. 0·34 to 0·59; P < 0·001) and cohort studies (OR 0·47, 0·44 to 0·50; P < 0·001). There was a similarly significant effect on SSI with use of a combination of preoperative oral aminoglycoside and erythromycin (OR 0·40, 0·25 to 0·64; P < 0·001), or preoperative oral aminoglycoside and metronidazole (OR 0·51, 0·39 to 0·68; P < 0·001). Preoperative oral antibiotics were significantly associated with reduced postoperative rates of anastomotic leak, ileus, reoperation, readmission and mortality in the cohort studies. CONCLUSION: Oral antibiotic prophylaxis, in combination with MBP and i.v. antibiotics, is superior to MBP and i.v. antibiotic prophylaxis alone in reducing SSI in elective colorectal surgery.

Divergent behavior of mucosal memory T cells.

Memory CD4 T cells are strategically positioned at mucosal surfaces to initiate a robust adaptive immune response. The detection of specific antigen via the T-cell receptor causes these memory T cells to unleash a potent antimicrobial response that includes rousing local innate immune populations for tissue-specific defense. Paradoxically, these same memory T cells can also be stimulated by nonantigen-specific signals that are generated by the activity of local innate immune cells. This versatility of mucosal memory T cells in both the initiation and the sensing of local innate immunity could be a vitally important asset during pathogen defense but alternatively could be responsible for initiating and maintaining chronic inflammation in sensitive mucosal tissues.

CD103-CD11b+ dendritic cells regulate the sensitivity of CD4 T-cell responses to bacterial flagellin.

Toll-like receptor 5 (TLR5) has been widely studied in an inflammatory context, but the effect of TLR5 on the adaptive response to bacterial flagellin has received considerably less attention. Here, we demonstrate that TLR5 expression by dendritic cells (DCs) allows a 1,000-fold enhancement of T-cell sensitivity to flagellin, and this enhancement did not require the expression of NLRC4 or Myd88. The effect of TLR5 on CD4 T-cell sensitivity was independent of the adjuvant effect of flagellin and TLR5 ligation did not alter the sensitivity of ovalbumin (OVA)-specific T cells to OVA. In the spleen, the exquisite T-cell sensitivity to flagellin was regulated by CD4-CD8α- DCs and was blocked by a monoclonal antibody to TLR5. In the mesenteric lymph nodes, flagellin-specific T-cell activation was regulated by a population of CD103-CD11b+ DCs. Thus, TLR5 expression by mucosal and systemic DC subsets controls the sensitivity of the adaptive immune response to flagellated pathogens.

Development of protective immunity to Salmonella, a mucosal pathogen with a systemic agenda.

Salmonella infections can cause a range of intestinal and systemic diseases in human and animal hosts. Although some Salmonella serovars initiate a localized intestinal inflammatory response, others use the intestine as a portal of entry to initiate a systemic infection. Considerable progress has been made in understanding bacterial invasion and dissemination strategies, as well as the nature of the Salmonella-specific immune response to oral infection. Innate and adaptive immunity are rapidly initiated after oral infection, but these effector responses can also be hindered by bacterial evasion strategies. Furthermore, although Salmonella resides within intramacrophage phagosomes, recent studies have highlighted a surprising collaboration of CD4 Th1, Th17, and B-cell responses in mediating resistance to Salmonella infection.

Feeding by the tick, Ixodes scapularis, causes CD4(+) T cells responding to cognate antigen to develop the capacity to express IL-4.

Effects of tick feeding on an early antigen-specific T cell response were studied by monitoring a clonotypic population of adoptively transferred T cell receptor (TCR) transgenic CD4 cells responding to a tick-associated antigen. When recipient mice were infested with pathogen-free Ixodes scapularis nymphs several days prior to T cell transfer and intradermal injection of soluble cognate antigen at the feeding site, the clonotypic CD4 cells gained the ability to express the Th2 effector cytokine IL-4. Notably, this effect was not only observed in BALB/c mice predisposed towards developing Th2 responses but also in B10.D2 mice predisposed towards Th1 responsiveness. Furthermore, tick feeding was able to superimpose IL-4 expression potential onto a strong Th1 response (indicated by robust IFN-gamma expression potential) elicited by immunization with a vaccinia virus expressing the cognate antigen. The magnitude to which tick feeding was able to programme IL-4 expression potential in CD4 cells was partially reduced in mice that had been previously exposed to pathogen-free tick nymphs 6 weeks earlier, as well as when the nymphs were infected with Borrelia burgdorferi. Intradermal injection of salivary gland extract programmed IL-4 expression potential similar to that of tick infestation, suggesting that IL-4 programming activity is contained within tick saliva.

Antibody is required for protection against virulent but not attenuated Salmonella enterica serovar typhimurium.

Resolution of infection with attenuated Salmonella is an active process that requires CD4(+) T cells. Here, we demonstrate that costimulation via the surface molecule CD28, but not antibody production by B cells, is required for clearance of attenuated aroA Salmonella enterica serovar typhimurium. In contrast, specific antibody is critical for vaccine-induced protection against virulent bacteria. Therefore, CD28(+) CD4(+) T cells are sufficient for clearance of avirulent Salmonella in naive hosts, whereas CD4(+) T cells and specific antibodies are required for protection from virulent Salmonella in immune hosts.

Selective down-regulation of Th2 immune responses following treatment with antigen-coupled splenocytes.

Intravenous injection of antigen-coupled splenocytes has been widely used to induce specific tolerance to a variety of antigens. In this study, we investigated the effects of such a treatment on Th1 and Th2 antigen-specific immune responses. Using both well-characterized model antigens and crude homogenates from Leishmania major promastigotes, we found that intravenous injection of antigen-coupled splenocytes strongly down-regulated antigen-specific Th2 responses but had no or only moderate effects on Th1 responses. Because the susceptibility of inbred strains of mice to murine leishmaniasis has been found to be correlated with a strong Th2 response against parasite antigens, we investigated whether administration of splenocytes chemically coupled to parasite antigens could protect susceptible mice from murine leishmaniasis. We found that this was indeed the case and further demonstrated that protection was associated with a strong decrease in the number of parasite-specific Th2-like cells. Because administration of antigen-coupled splenocytes is believed to induce ligation of the T cell receptor complex without inducing a co-stimulatory signal, our results further suggest that priming of Th1 cells is less dependent on co-stimulatory signals than the priming of Th2 cells.

Vaccine efficacy of Salmonella strains expressing glycoprotein 63 with different promoters.

The development of Salmonella vaccine vectors has been hindered by both the requirement for multiple doses to induce immune responses and a lack of plasmid stability. Direct comparisons of different promoter systems with the same antigen are necessary to address these important issues. We have previously described an AroA- AroD- deletion mutant of Salmonella typhimurium (GID101) which expresses the gene encoding the Leishmania major promastigote surface glycoprotein gp63 (GID101). While this construct provided significant protection against L. major challenge to highly susceptible BALB/c mice, this required at least two oral doses. We report here the use of two different inducible promoters, the nirB and osmC promoters, to improve vaccine efficacy. These constructs (termed GID105 and GID106, respectively) expressed gp63 in vitro under inducible conditions and colonized BALB/c mice after oral administration. GID105 demonstrated greater plasmid stability in vitro and in vivo than did either GID106 or GID101, which expresses gp63 constitutively. Spleen and lymph node cells from mice immunized with a single oral dose of GID105 proliferated in vitro in response to L. major and secreted gamma interferon, whereas cells from mice given the other constructs did not. Mice immunized with a single oral dose of GID1O5 or GID106 developed significantly smaller lesions upon challenge with L. major, whereas mice administered GID101 did not. Mice administered GID105 also showed considerable resistance to Leishmania donovani infection. These data provide a direct comparison of promoter systems and demonstrate that the use of inducible promoters such as the nirB promoter allows a considerable improvement over the previous vaccine construct in terms of protection against infection.

Characterization of CD4+ T cell responses during natural infection with Salmonella typhimurium.

CD4+ T cells are important for resistance to infection with Salmonella typhimurium. However, the Ag specificity of this T cell response is unknown. Here, we demonstrate that a significant fraction of Salmonella-specific CD4+ T cells respond to the flagellar filament protein, FliC, and that this Ag has the capacity to protect naive mice from lethal Salmonella infection. To characterize this Ag-specific response further, we generated FliC-specific CD4+ T cell clones from mice that had resolved infection with an attenuated strain of Salmonella. These clones were found to respond to an epitope from a constant region of FliC, enabling them to cross-react with flagellar proteins expressed by a number of distinct Salmonella serovars.

Protection against Leishmania major infection in genetically susceptible BALB/c mice by gp63 delivered orally in attenuated Salmonella typhimurium (AroA- AroD-).

The gene encoding the Leishmania major (L. major) promastigote surface glycoprotein, gp63, was introduced into the Salmonella typhimurium (S. typhimurium) aroA- aroD- live oral vaccine strain BRD509 and expressed under the control of a constitutive tac promoter in plasmid pKK233-2. This construct (GID101) expressed gp63 in vitro and was used to immunize highly susceptible BALB/c mice by the oral route. The plasmid was relatively stably inherited by bacteria growing or persisting in the mesenteric lymph nodes of immunized mice. Mice immunized with GID101 developed significant resistance against a challenge infection with L. major compared to controls immunized with BRD509 alone. Spleen and lymph node cells from immunized mice developed a strong in vitro proliferative T-cell response to killed or live L. major. The activated T cells secreted interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) which was abrogated by treatment with anti-CD4 but not with anti-CD8 antibody. The cells did not produce detectable levels of interleukin-4 (IL-4). The immunized mice also produced significant amounts of leishmanial specific IgG2a antibody but did not develop delayed-type hypersensitivity (DTH) to live parasites. No IgG1 antibody was detected. These data therefore demonstrate that gp63 gene delivered orally by a vaccine strain of S. typhimurium can preferentially induce the development of Th-1 subset of CD4+ T cells and protective immunity in the highly susceptible BALB/c mice.

Selective tolerization of Th1-like cells after nasal administration of a cholera toxoid-LACK conjugate.

Recent reports have suggested that after infection of BALB/c mice with Leishmania major, CD4+ T cells responding to a single antigen, LACK (Leishmania homologue of receptors for activated C kinase), drive the differentiation of other responding T cells to the Th2 phenotype and so allow lesion development to occur. Transgenic mice expressing LACK in the thymus are tolerant to LACK and thus resolve infection with L. major. The oral administration of soluble protein to mice has been shown to result in the peripheral tolerance of antigen-specific CD4+ T cells. We therefore sought to tolerize LACK reactive T cells in non-transgenic BALB/c mice in order to determine the effectiveness of this tolerization approach as an alternative to standard vaccination protocols against L. major infection. Surprisingly, oral or nasal administration of up to 8 mg of recombinant LACK did not affect the outcome of infection. We therefore conjugated LACK to cholera toxin beta subunit (CTB-LACK) which has previously been shown to improve the effectiveness of oral tolerance to conjugated antigens. Nasal administration of as little as 12 microg of CTB-LACK effectively diminished the capacity of mice to mount a subsequent proliferative response to LACK and further delayed the onset of lesion development in infected mice. However, pretreatment with CTB-LACK did not prevent the eventual onset and progression of disease in these mice. An examination of cytokine responsiveness to LACK after tolerization with CTB-LACK revealed that while the Th1 response to LACK was suppressed, Th2 cytokine production was unaffected. Similar experiments using an ovalbumin-CTB conjugate suggested that this selective tolerance of Th1 cells was not specific to the LACK protein but may be an effect common to CTB-conjugated proteins.

Immunological tolerance to a pancreatic antigen as a result of local expression of TNFalpha by islet beta cells.

Recent experiments have suggested that tumor necrosis factor alpha (TNFalpha) can down-regulate islet-specific T cells and prevent the development of autoimmune diabetes. Here we demonstrate that transgenic mice expressing both TNFalpha and the Leishmania major LACK antigen in the pancreas (RIP-TNFalpha/RIP-LACK) exhibit an impaired ability to mount a CD4+ T cell response against LACK. In addition, peripheral CD4+ T cells from TCR transgenic mice (TCR-LACK/RIP-TNFalpha/RIP-LACK) produced reduced interleukin-2 but elevated levels of T helper 2 cytokines in response to LACK peptide in vitro. Taken together, our data suggest that TNFalpha may act in vivo to modulate a potentially damaging self-reactive T cell response by inducing tolerance to pancreatic antigens.

Priming by microbial antigens from the intestinal flora determines the ability of CD4+ T cells to rapidly secrete IL-4 in BALB/c mice infected with Leishmania major.

Infection of BALB/c mice with Leishmania major results in the rapid accumulation of IL-4 transcripts within CD4(+) T cells that react to the parasite Leishmania homologue of mammalian RACK1 (LACK) Ag. Because memory/effector cells secrete IL-4 more rapidly than naive cells, we sought to analyze the phenotype of these lymphocytes before infection. Indeed, a fraction of LACK-specific CD4(+) T cells expressed a typical CD62 ligand(low)CD44(high)CD45RB(low) phenotype in uninfected mice. LACK-specific T cells were primed in gut-associated lymphoid tissues by cross-reactive microbial Ags as demonstrated by their reactivity with bacterial extracts and by the ability of APCs from the mesenteric LN of BALB/c mice to induce their proliferation. Also, mice in which the digestive tract has been decontaminated exhibited a reduced proportion of LACK-specific T cells expressing a memory/effector phenotype and did not exhibit the early accumulation of IL-4 transcripts induced by L. major. Thus, LACK-specific T cells represent a subset of CD4(+) T cells which have acquired the ability to rapidly secrete IL-4 as the result of their priming by cross-reactive microbial Ags. Tracking the fate of these cells may provide information about the regulation of cell-mediated immune responses to gut Ags in physiological and pathological situations.

Regulation of the immune response by nitric oxide differentially produced by T helper type 1 and T helper type 2 cells.

The balance between T helper type 1 (Th 1) and T helper type 2 (Th2) cells determines the outcome of many important diseases. Using cloned murine T cell lines, evidence is provided that Th1, but not Th2, cells can be activated by specific antigens or a T cell mitogen, concanavalin A, to produce large amounts of nitric oxide (NO). Furthermore, NO can inhibit the secretion of interleukin (IL)-2 and interferon-gamma by Th1 cells but has no effect on IL-4 production by Th2 cells. Th1 and Th2 cells can, thus, be distinguished by their differential production of and susceptibility to NO. NO exerts a self-regulatory effect on Th1 cells which are implicated in immunopathology.

In vivo activation of antigen-specific CD4 T cells.

Physical detection of antigen-specific CD4 T cells has revealed features of the in vivo immune response that were not appreciated from in vitro studies. In vivo, antigen is initially presented to naïve CD4 T cells exclusively by dendritic cells within the T cell areas of secondary lymphoid tissues. Anatomic constraints make it likely that these dendritic cells acquire the antigen at the site where it enters the body. Inflammation enhances in vivo T cell activation by stimulating dendritic cells to migrate to the T cell areas and display stable peptide-MHC complexes and costimulatory ligands. Once stimulated by a dendritic cell, antigen-specific CD4 T cells produce IL-2 but proliferate in an IL-2--independent fashion. Inflammatory signals induce chemokine receptors on activated T cells that direct their migration into the B cell areas to interact with antigen-specific B cells. Most of the activated T cells then die within the lymphoid tissues. However, in the presence of inflammation, a population of memory T cells survives. This population is composed of two functional classes. One recirculates through nonlymphoid tissues and is capable of immediate effector lymphokine production. The other recirculates through lymph nodes and quickly acquires the capacity to produce effector lymphokines if stimulated. Therefore, antigenic stimulation in the presence of inflammation produces an increased number of specific T cells capable of producing effector lymphokines throughout the body.