Characterising plasmacytoid and myeloid AXL+ SIGLEC-6+ dendritic cell functions and their interactions with HIV.

AXL+ Siglec-6+ dendritic cells (ASDC) are novel myeloid DCs which can be subdivided into CD11c+ and CD123+ expressing subsets. We showed for the first time that these two ASDC subsets are present in inflamed human anogenital tissues where HIV transmission occurs. Their presence in inflamed tissues was supported by single cell RNA analysis of public databases of such tissues including psoriasis diseased skin and colorectal cancer. Almost all previous studies have examined ASDCs as a combined population. Our data revealed that the two ASDC subsets differ markedly in their functions when compared with each other and to pDCs. Relative to their cell functions, both subsets of blood ASDCs but not pDCs expressed co-stimulatory and maturation markers which were more prevalent on CD11c+ ASDCs, thus inducing more T cell proliferation and activation than their CD123+ counterparts. There was also a significant polarisation of naïve T cells by both ASDC subsets toward Th2, Th9, Th22, Th17 and Treg but less toward a Th1 phenotype. Furthermore, we investigated the expression of chemokine receptors that facilitate ASDCs and pDCs migration from blood to inflamed tissues, their HIV binding receptors, and their interactions with HIV and CD4 T cells. For HIV infection, within 2 hours of HIV exposure, CD11c+ ASDCs showed a trend in more viral transfer to T cells than CD123+ ASDCs and pDCs for first phase transfer. However, for second phase transfer, CD123+ ASDCs showed a trend in transferring more HIV than CD11c+ ASDCs and there was no viral transfer from pDCs. As anogenital inflammation is a prerequisite for HIV transmission, strategies to inhibit ASDC recruitment into inflamed tissues and their ability to transmit HIV to CD4 T cells should be considered.

An in situ analysis pipeline for initial host-pathogen interactions reveals signatures of human colorectal HIV transmission.

The initial immune response to HIV determines transmission. However, due to technical limitations we still do not have a comparative map of early mucosal transmission events. By combining RNAscope, cyclic immunofluorescence, and image analysis tools, we quantify HIV transmission signatures in intact human colorectal explants within 2 h of topical exposure. We map HIV enrichment to mucosal dendritic cells (DCs) and submucosal macrophages, but not CD4+ T cells, the primary targets of downstream infection. HIV+ DCs accumulate near and within lymphoid aggregates, which act as early sanctuaries of high viral titers while facilitating HIV passage to the submucosa. Finally, HIV entry induces recruitment and clustering of target cells, facilitating DC- and macrophage-mediated HIV transfer and enhanced infection of CD4+ T cells. These data demonstrate a rapid response to HIV structured to maximize the likelihood of mucosal infection and provide a framework for in situ studies of host-pathogen interactions and immune-mediated pathologies.

Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue.

The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin+ and langerin-), newly described DC3, monocytes, Mf1, Mf2, Mf3 and Mf4. We also present a novel method to account for autofluorescent signal from tissue macrophages. Finally, we demonstrate that these methods can successfully be used to sort functional, immature intestinal DCs that can be used for functional assays such as cytokine production assays.

Human anogenital monocyte-derived dendritic cells and langerin+cDC2 are major HIV target cells.

Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2).

Identification of HIV transmitting CD11c+ human epidermal dendritic cells.

Langerhans cells (LC) are thought to be the only mononuclear phagocyte population in the epidermis where they detect pathogens. Here, we show that CD11c+ dendritic cells (DCs) are also present. These cells are transcriptionally similar to dermal cDC2 but are more efficient antigen-presenting cells. Compared to LCs, epidermal CD11c+ DCs are enriched in anogenital tissues where they preferentially interact with HIV, express the higher levels of HIV entry receptor CCR5, support the higher levels of HIV uptake and replication and are more efficient at transmitting the virus to CD4 T cells. Importantly, these findings are observed using both a lab-adapted and transmitted/founder strain of HIV. We also describe a CD33low cell population, which is transcriptionally similar to LCs but does not appear to function as antigen-presenting cells or acts as HIV target cells. Our findings reveal that epidermal DCs in anogenital tissues potentially play a key role in sexual transmission of HIV.

Transanal minimally invasive surgery: impact on quality of life and functional outcome.

BACKGROUND: Transanal minimally invasive surgery (TAMIS) is emerging as an alternative to transanal endoscopic microsurgery. Quality of life (QOL) and functional outcome are important aspects when valuing a new technique. The aim of this prospective study was to assess both functional outcome and QOL after TAMIS. METHODS: From 2011 to 2013, patients were prospectively studied prior to and at least 6 months after TAMIS for rectal adenomas and low-risk T1 carcinomas using a single-site laparoscopy port. Functional outcome was determined using the Faecal Incontinence Severity Index (FISI). Quality of life was measured using functional [Faecal Incontinence Quality of Life (FIQL)] and generic (EuroQol EQ-5D) questionnaires. RESULTS: The study population consisted of 24 patients 13 men, median age 59 (range 42-83) with 24 tumours [median distance from the dentate line 8 cm (range 2-17 cm); median tumour size 6 cm(2) (range 0.25-51 cm(2)); 20 adenomas; 4 low-risk T1 carcinomas]. Post-operative complications occurred in one patient (4 %; grade IIIb according to Clavien Dindo classification). Compared to baseline, FISI remained unaffected (9.8 vs 7.3; P = 0.26), FIQL remained unaffected, and EuroQol EQ-5D improved (EQ-VAS: 77 vs 83; P = 0.04). CONCLUSION: There was no detrimental effect of TAMIS on anorectal function. Overall QOL was improved after TAMIS, probably due to removal of the tumour, and at 6 months was equal to the general population.

Dynamic Article: Permanent Sacral Nerve Stimulation Under Local Anesthesia: Feasibility, Best Practice, and Patient Satisfaction.

BACKGROUND: The increasing incidence of fecal incontinence and the use of sacral neuromodulation have an increasing impact on health care providers and health care costs. OBJECTIVE: The purpose of this study was to investigate the technical and clinical success rates, complications, and patient satisfaction of the implantation of permanent sacral nerve stimulation under local anesthesia. DESIGN: A cohort analysis of consecutive patients with sacral nerve stimulation for fecal incontinence over a period of 1 year was performed. SETTINGS: This study was conducted at a specialized pelvic floor unit in a tertiary care center. PATIENTS: Sixty-one patients were available for the assessment after 1-year follow-up. MAIN OUTCOME MEASURES: Technical success, procedural time, and complications were noted. Clinical outcome (including Fecal Incontinence Severity Index, Fecal Incontinence Quality of Life scale, and Gastrointestinal Quality of Life Index were collected prospectively before and after treatment. RESULTS: All procedures were successfully completed under local anesthesia, with a median total procedural time of 50 minutes (range, 26-72 minutes). All patients were discharged on the day of their procedure. Postoperative complications occurred in 3 patients (4.9%). At 3 months follow-up, the median Fecal Incontinence Severity Index score was reduced from 37 to 27 (p = 0.001). Both the Fecal Incontinence Quality of Life scale and the Gastrointestinal Quality of Life Index had improved from 63 to 82 (p < 0.001) and 72 to 90 (p = 0.012). At a mean follow-up of 13 months, both the Fecal Incontinence Quality of Life scale and the Gastrointestinal Quality of Life Index improved further to 90 (p < 0.001) and 94 (p < 0.001). All patients would recommend the procedure under local anesthesia to other patients. No patients experienced leg pain during follow-up. LIMITATIONS: This study involved a relatively small group of patients, and patient satisfaction was only recorded for the last 22 patients. No exact cost calculations were made. CONCLUSIONS: Permanent sacral nerve stimulation implantation under local anesthesia has high technical and clinical success rates. It is safe, well tolerated by patients, and has obvious logistical and financial benefits.

Sacral neuromodulation for faecal incontinence: is the outcome compromised in patients with high-grade internal rectal prolapse?

BACKGROUND: High-grade internal rectal prolapse appears to be one of the contributing factors in the multifactorial origin of faecal incontinence. Whether it affects the outcome of sacral neuromodulation is unknown. We compared the functional results of sacral neuromodulation for faecal incontinence in patients with and without a high-grade internal rectal prolapse. METHOD: One hundred six consecutive patients suffering from faecal incontinence, who were eligible for sacral neuromodulation between 2009 and 2012, were identified from a prospective database. All patients underwent preoperative defaecating proctography, anorectal manometry and ultrasound. Symptoms were assessed preoperatively and at 12 months after operation using a standardized questionnaire incorporating the Faecal Incontinence Severity Index (FISI range = 0-61) and the Gastrointestinal Quality of Life Index (GIQLI). Success was defined as a decrease in the FISI score of 50 % or more. RESULTS: High-grade internal rectal prolapse (HIRP) was found in 36 patients (34%). The patient characteristics were similar in both groups. Temporary test stimulation was successful in 60 patients without HIRP (86%) and in 25 patients with HIRP (69 %) (p = 0.03). A permanent pulse generator was then implanted on these patients. After 1-year follow-up, the median FISI was reduced in patients without HIRP from 37 to 23 (p < 0.01). No significant change in FISI score was observed in patients with a HIRP (FISI, 38 to 34; p = 0.16). Quality of life (GIQLI) was only improved in patients without HIRP. A successful outcome per protocol was achieved in 31 patients without HIRP (52%) versus 4 patients with HIRP (16%) (p < 0.01). CONCLUSION: The presence of a high-grade internal rectal prolapse has a detrimental effect on sacral neuromodulation for faecal incontinence.

Predictors of outcome after transanal advancement flap repair for high transsphincteric fistulas.

BACKGROUND: Transanal advancement flap repair fails in 1 of every 3 patients with a high transsphincteric fistula. It has been reported that smoking, obesity, and previous attempts at repair adversely affect the outcome of transanal advancement flap repair. Because these findings could not be confirmed by other studies, it is still unclear whether these and other factors have an impact on the outcome. OBJECTIVE: The aim of this study was to identify predictors of outcome in a large cohort of patients who underwent transanal advancement flap repair for a high transsphincteric fistula. DESIGN: This study was performed as a retrospective review. SETTINGS: The study was conducted at the Division of Colon and Rectal Surgery, Erasmus MC, between 2000 and 2012. PATIENTS: A consecutive series of 252 patients with a high transsphincteric fistula of cryptoglandular origin were included. Patients with a rectovaginal or Crohn fistula were excluded. INTERVENTIONS: All patients underwent transanal advancement flap repair. Preoperatively, patients underwent endoanal MRI. MAIN OUTCOME MEASURES: Healing was defined as complete wound healing with absence of symptoms. Patients were followed up to assess failure. Seventeen patient- and fistula-related variables were assessed. RESULTS: Median duration of follow-up was 21 months (range, 6-136 months). The failure rate at 3 years was 41% (95% CI, 34-48). None of the studied variables predicted the outcome of flap repair except horseshoe extension. In univariate and multivariate analyses, significantly less failures were observed in patients with a horseshoe extension (p < 0.05). LIMITATIONS: Retrospective design, a single surgeon series, and potential selection bias caused by the tertiary referral center status are the limitations of this study. CONCLUSIONS: Of all studied variables, horseshoe extension was found to be the only positive predictor of outcome after flap repair for high transsphincteric fistulas.

Is microvessel density correlated with anastomotic leakage after low anterior resection?

BACKGROUND/AIMS: Anastomotic leakage after low anterior resection may be the result of poor vascular supply from the proximal anastomotic loop. The purpose of this study was to investigate the correlation between colonic microvessel density and anastomotic breakdown. METHODOLOGY: Between 2006 and 2009, a consecutive series of 81 patients underwent double-stapled low anterior resection followed by a colorectal anastomosis. Symptomatic anastomotic leakage occurred in 14 patients (17%). In these patients, microvascular density was determined by image analysis of CD-31-immunostained sections from the proximal resection site. The results were compared with a sample of the remaining 67 patients without anastomotic leakage closely matched for age, gender, ASA-classification, pathological stage and neoadjuvant treatment. RESULTS: The mean percentage of anti-CD31 stained area, obtained from the proximal resection site was similar between patients with or without anastomotic leakage (4.0% +/- 1.8% versus 4.4% +/- 1.6% respectively, P = 0.53). With respect to neo-adjuvant therapy, no differences in the density of CD31 positive were observed (pre-operative radiotherapy = 4.3% +/- 1.8% versus pre-operative chemoradiotherapy 4.1% +/- 1.6%, P = 0.77). The mean vessel density reached borderline statistical significance in women (5.0% +/- 1.8%) compared to men (3.8% +/- 1.8%) (P = 0.06). CONCLUSIONS: Microvessel density quantification with immunohistochemical analysis of CD31 expression of the proximal anastomotic region did not show any correlation with anastomotic leakage in the clinical setting.

Laparoscopic ventral rectopexy for fecal incontinence associated with high-grade internal rectal prolapse.

BACKGROUND: The role of internal rectal prolapse in the origin of fecal incontinence remains to be defined. In our institution, laparoscopic ventral rectopexy is offered to patients with high-grade internal prolapse and fecal incontinence. OBJECTIVE: The present study was designed to evaluate the functional outcome after laparoscopic ventral rectopexy in patients with fecal incontinence associated with high-grade internal rectal prolapse. DESIGN: This study was designed as a prospective observational study. SETTINGS: The study took place in a university hospital. PATIENTS: Between 2009 and 2011, 72 patients with fecal incontinence not responding to maximum medical treatment (including biofeedback) were included. All patients had a grade III or grade IV rectal prolapse. INTERVENTION: Laparoscopic ventral rectopexy was performed. MAIN OUTCOME MEASURES: Preoperative endoanal ultrasonography and anorectal manometry were performed. Fecal continence was evaluated by using the Rockwood Fecal Incontinence Severity Index score before and 1 year after surgery. RESULTS: The median fecal incontinence severity index score 1 year after surgery was lower than the median score before surgery (15 versus 31; p < 0.01), representing an improvement in fecal continence. LIMITATIONS: This was a preliminary observational study with no control group, no postoperative proctography, and no postoperative anal physiology. CONCLUSION: Laparoscopic ventral rectopexy can improve symptoms of fecal incontinence in patients with a high-grade internal rectal prolapse. Internal rectal prolapse contributes to the multifactorial origin of fecal incontinence.

[Intersphincteric ligation of perianal fistula]. / Intersfincterische ligatie van perianale fistels.

Fistulotomy is inappropriate for patients with a high transsphincteric fistula, passing through the upper or middle third of the external anal sphincter, because this procedure requires division of a large part of the anal sphincter, with subsequent risk of fecal incontinence. Therefore, sphincter preserving procedures have been developed for the treatment of high transsphincteric fistulas, such as flap repair. In most hospitals, fistulotomy is still the treatment of choice for low transsphincteric fistula, passing through the lower third of the external anal sphincter. Although this procedure is considered simple and effective with a minimal risk of incontinence, data suggest that the risk of diminished fecal continence is not insignificant. Ligation of the intersphincteric fistula tract (LIFT) is a new sphincter preserving technique. This technique may be a sphincter preserving alternative for fistulotomy in low transsphincteric fistulas and for flap repair in high transsphincteric fistulas.

Treatment of anal fistulas with high intersphincteric extension.

BACKGROUND: Intersphincteric fistulas with a high upward extension, up to or above the level of the puborectal muscle, in the intersphincteric plane are rare. Most of these fistulas have no external opening and they are frequently associated with a high intersphincteric and/or supralevator abscess. Division of a large amount of internal anal sphincter by extended fistulotomy has a potential risk of diminished fecal continence. OBJECTIVE: The aim of this study was to evaluate flap repair combined with drainage of associated abscesses in high intersphincteric fistulas. DESIGN: This study was performed as a retrospective review. SETTINGS: The study was conducted at the Division of Colon and Rectal Surgery, Erasmus MC, between March 1995 and February 2011. PATIENTS: Fourteen patients with a cryptoglandular fistula with high intersphincteric extension were included. INTERVENTIONS: Transanal advancement flap repair combined with intersphincteric and/or extrasphincteric drainage of associated abscesses was performed. Preoperatively, patients underwent endoanal MRI. MAIN OUTCOME MEASURES: Healing was defined as complete wound healing with absence of symptoms. Patients were followed up to assess the recurrence rate and Rockwood fecal continence score. RESULTS: In 1 patient the fistula was not associated with an abscess. In 10 patients the fistula tract ended in a high intersphincteric abscess. Three patients presented with a high intersphincteric abscess and a supralevator abscess. Primary healing was observed in 79% of the patients. The 3 patients without primary healing had a supralevator abscess. In these patients, healing was obtained after a second, third, and fourth procedure. The overall healing rate was 100%. Median postoperative Rockwood score was 0 (range, 0-15). LIMITATIONS: Retrospective design and lack of baseline continence data were the limitations of this study. CONCLUSIONS: Since most high intersphincteric fistulas have no external opening and are frequently associated with abscesses, preoperative imaging is useful. Flap repair with adequate drainage of the abscesses is successful, except in fistulas with supralevator extension. However, healing may be achieved by additional procedures.

Blood transfusions and prognosis in colorectal cancer: long-term results of a randomized controlled trial.

OBJECTIVE: Perioperative blood transfusions may adversely affect survival in patients with colorectal malignancy, although definite proof of a causal relationship has never been reported. BACKGROUND: We report the long-term outcomes of a randomized controlled trial performed between 1986 and 1991 to compare the effects of allogeneic blood transfusions and an autologous blood transfusion program in colorectal cancer patients. METHODS: All 475 randomized patients operated upon for colorectal cancer were tracked via a national computerized record-linkage system to investigate survival and cause of death. Kaplan-Meier survival curves were constructed and multivariate Cox regression analysis was performed to study 20 years' overall survival. Colorectal cancer-specific survival was analyzed over the 10-year time period after surgery. RESULTS: The overall survival percentage at 20 years after surgery was worse in the autologous group (21%) compared to the allogeneic group (28%) (P = 0.041; log-rank test). Cox regression, allowing for tumor stage, age, and sex, resulted in a hazard ratio (autologous vs allogeneic group) for overall mortality of 1.24 (95% confidence interval 1.00-1.54; P = 0.051). Colorectal cancer-specific survival at 10 years for the whole study group was 48% and 60% for the autologous and allogeneic group, respectively (P = 0.020; log-rank test). The adjusted hazard ratio was 1.39 (95 confidence interval 1.05-1.83; P = 0.045). CONCLUSIONS: At long-term follow-up colorectal cancer patients did not benefit from autologous transfusion compared with standard allogeneic transfusion. On the contrary, the overall and colorectal cancer-specific survival rates were worse in the patients in the autologous transfusion group.

Local application of gentamicin-containing collagen implant in the prophylaxis of surgical site infection following gastrointestinal surgery.

BACKGROUND: Gastrointestinal surgery is associated with one of the highest rates of SSI due to the nature of the surgery and to the added complication of operating on patients with significant co-morbidities. This high rate of SSI may negatively impact wound healing, patient recovery time, length of hospital stay and associated healthcare costs. This article provides an overview of the efficacy and safety of prophylactic application of resorbable gentamicin-containing collagen implants (GCCI) in the prevention of SSI following GI surgical procedures. METHOD: Thirteen publications were identified using the PubMed online database and search terms 'gentamicin collagen implant' plus 'surgical site infection', 'wound infection' and 'gastrointestinal surgery'. RESULTS: Eleven out of 13 studies have demonstrated that prophylactic use of GCCI can reduce the wound infection rate in high-risk GI surgical procedures (e.g. abdominoperineal resection [APR]) and improve wound healing after pilonidal sinus excision. GCCI may also have a role to play in preventing anastomotic leakage following mesorectal excision for rectal carcinoma. It is recommended that GCCI are used dry in line with the manufacturer's recommendations. CONCLUSION: This review demonstrates that GCCI can significantly reduce surgical site infection following GI surgery including pilonidal sinus excision and high-risk procedures such as APR.

High tie versus low tie in rectal surgery: comparison of anastomotic perfusion.

PURPOSE: Both "high tie" (HT) and "low tie" (LT) are well-known strategies in rectal surgery. The aim of this study was to compare colonic perfusion after HT to colonic perfusion after LT. METHODS: Patients undergoing rectal resection for malignancy were included. Colonic perfusion was measured with laser Doppler flowmetry, immediately after laparotomy on the antimesenterial side of the colon segment that was to become the afferent loop (measurement A). This measurement was repeated after rectal resection (measurement B). The blood flow ratios (B/A) were compared between the HT group and the LT group. RESULTS: Blood flow was measured in 33 patients, 16 undergoing HT and 17 undergoing LT. Colonic blood flow slightly decreased in the HT group whereas the flow increased in the LT group. The blood flow ratio was significantly higher in the LT group (1.48 vs. 0.91; p = 0.04), independent of the blood pressure. CONCLUSION: This study shows the blood flow ratio to be higher in the LT group. This suggests that anastomoses may benefit from better perfusion when LT is performed.