The modified Glasgow prognostic score in Crohns diseasedoes it predict short-term outcome?

Background The modified Glasgow prognostic score (mGPS) has recently gained increased attention as a prognostic marker for malignant disease survival and postoperative short-term complications. Due to lacking data, the present study was conducted to correlate the mGPS with the postoperative course in patients following surgery for Crohns disease. Methods We enrolled 341 patients who underwent intestinal resection for symptomatic Crohns disease at a tertiary referral centre between 2000 and 2014. All relevant data were obtained from the institutional database and individual chart review. Thirty-day morbidity was defined according to the ClavienDindo classification. Results A total of 79 (23.17%) postoperative complications were identified (grade I and II: n = 54, 15.84%; grade III and IV: n = 23, 6.74%; grade V: n = 2, 0.59%). The mGPS did not show any correlation with an eventful postoperative course following surgery (no complication: median mGPS: 1, range 02; complications: median mGPS: 1, range 02; p = 0.8521). In addition, the occurrence of an anastomotic leakage was not associated with a higher mGPS (p = 0.8592). Patients with an acute indication for surgery (n = 29, 11.44%) had higher median mGPS (median: 2, range 02) in contrast to patients who were operated on electively (median: 1, range 02; p = 0.0003). No other correlation between surgical characteristics and mGPS was detected. Conclusions In the present study, we could clearly demonstrate that an acute indication for surgery in symptomatic Crohns disease is associated with higher mGPS scores. However, the mGPS did not correlate with postoperative complications. Further studies are required to define the prognostic value of mGPS in Crohns disease patients.(VLID)359159

Hybrid resistance to parental bone marrow grafts in nonlethally irradiated mice

Resistance to parental bone marrow (BM) grafts in F1 hybrid recipients is due to natural killer (NK) cellmediated rejection triggered through “missing self” recognition. “Hybrid resistance” has usually been investigated in lethally irradiated F1 recipients in conjunction with pharmacological activation of NK cells. Here, we investigated BMdirected NKcell alloreactivity in settings of reduced conditioning. Nonlethally irradiated (13 Gy) or nonirradiated F1 (C57BL6 BALB/c) recipient mice received titrated doses (520 x 106) of unseparated parental BALB/c BM without pharmacological NK cell activation. BM successfully engrafted in all mice and multilineage donor chimerism persisted longterm (24 weeks), even in the absence of irradiation. Chimerism was associated with the rearrangement of the NKcell receptor repertoire suggestive of reduced reactivity to BALB/c. Chimerism levels were lower after transplantation with parental BALB/c than with syngeneic F1 BM, indicating partial NKmediated rejection of parental BM. Activation of NK cells with polyinosinicpolycytidylic acid sodium salt poly(I:C), reduced parental chimerism in nonirradiated BM recipients but did not prevent hematopoietic stem cell engraftment. In contrast, equal numbers of parental lymph node cells were completely rejected. Hence, hybrid resistance leads to incomplete rejection of parental BM under reduced conditioning settings.(VLID)342372

IL-2/α-IL-2 Complex Treatment Cannot Be Substituted for the Adoptive Transfer of Regulatory T cells to Promote Bone Marrow Engraftment.

Cell therapy with recipient Tregs achieves engraftment of allogeneic bone marrow (BM) without the need for cytoreductive conditioning (i.e., without irradiation or cytotoxic drugs). Thereby mixed chimerism and transplantation tolerance are established in recipients conditioned solely with costimulation blockade and rapamycin. However, clinical translation would be substantially facilitated if Treg-stimulating pharmaceutical agents could be used instead of individualized cell therapy. Recently, it was shown that interleukin-2 (IL-2) complexed with a monoclonal antibody (mAb) (clone JES6-1A12) against IL-2 (IL-2 complexes) potently expands and activates Tregs in vivo. Therefore, we investigated whether IL-2 complexes can replace Treg therapy in a costimulation blockade-based and irradiation-free BM transplantation (BMT) model. Unexpectedly, the administration of IL-2 complexes at the time of BMT (instead of Tregs) failed to induce BM engraftment in non-irradiated recipients (0/6 with IL-2 complexes vs. 3/4 with Tregs, p0.05). CD8 T cells and NK cells of IL-2 complex-treated naïve mice showed an enhanced proliferative response towards donor antigens in vitro despite the marked expansion of Tregs. However, IL-2 complexes also expanded conventional CD4 T cells, CD8 T cells, NK cells, NKT cells and notably even B cells, albeit to a lesser extent. Notably, IL-2 complex expanded Tregs featured less potent suppressive functions than in vitro activated Tregs in terms of T cell suppression in vitro and BM engraftment in vivo. In conclusion, these data suggest that IL-2 complexes are less effective than recipient Tregs in promoting BM engraftment and in contrast actually trigger BM rejection, as their effect is not sufficiently restricted to Tregs but rather extends to several other lymphocyte populations

Scientific Reports / Phenotypes of Jackhammer esophagus in patients with typical symptoms of gastroesophageal reflux disease responsive to proton pump inhibitors

This trial was designed to assess the prevalence and characteristics of Jackhammer esophagus (JE), a novel hypercontractile disorder associated with progression to achalasia and limited outcomes following anti-reflux surgery in patients with typical symptoms of GERD and responsiveness to proton pump inhibitor (PPI) therapy. Consecutive patients, who were referred for surgical therapy because of PPI responsive typical symptoms of GERD, were prospectively assessed between January 2014 and May 2017. Patients diagnosed with JE subsequently underwent rigorous clinical screening including esophagogastroduodenoscopy (EGD), ambulatory pH impedance monitoring off PPI and a PPI trial. Out of 2443 evaluated patients, 37 (1.5%) subjects with a median age of 56.3 (51.6; 65) years were diagnosed with JE and left for final analysis. Extensive testing resulted in 16 (43.2%) GERD positive patients and 5 (13.9%) participants were observed to have an acid hypersensitive esophagus. There were no clinical parameters that differentiated phenotypes of JE. The prevalence of JE in patients with typical symptoms of GERD and response to PPI therapy is low. True GERD was diagnosed in less than half of this selected cohort, indicating the need for objective testing to stratify phenotypes of JE. (NCT03347903)(VLID)464183

Anti-Interleukin-6 Promotes Allogeneic Bone Marrow Engraftment and Prolonged Graft Survival in an Irradiation-Free Murine Transplant Model

Transfer of recipient regulatory T cells (Tregs) induces mixed chimerism and tolerance in an irradiation-free bone marrow (BM) transplantation (BMT) model involving short-course co-stimulation blockade and mTOR inhibition. Boosting endogenous Tregs pharmacologically in vivo would be an attractive alternative avoiding the current limitations of performing adoptive cell therapy in the routine clinical setting. Interleukin-6 (IL-6) potently inhibits Treg differentiation and its blockade was shown to increase Treg numbers in vivo. Therefore, we investigated whether IL-6 blockade can replace adoptive Treg transfer in irradiation-free allogeneic BMT. Treatment with anti-IL-6 instead of Treg transfer led to multi-lineage chimerism (persisting for ~12 weeks) in recipients of fully mismatched BM and significantly prolonged donor skin (MST 58 days) and heart (MST > 100 days) graft survival. Endogenous Foxp3+ Tregs expanded in anti-IL-6-treated BMT recipients, while dendritic cell (DC) activation and memory CD8+ T cell development were inhibited. Adding anti-IL-17 to anti-IL-6 treatment increased Treg frequencies, but did not further prolong donor skin graft survival significantly. These results demonstrate that IL-6 blockade promotes BM engraftment and donor graft survival in non-irradiated recipients and might provide an alternative to Treg cell therapy in the clinical setting

IL-2 complexes relocate B cells from the bone marrow to secondary lymphoid organs.

<p>The mechanisms of B cell expansion in secondary lymphoid organs were investigated. Naive C57BL/6 mice received IL-2 complexes (5μg IL-2 / 25μg α-IL-2) for three days (d0, d1, d2) and distinct cell populations were analyzed in indicated tissues 2 days (d4) after the last dose. <b>(A)</b> CD3⁺ CD4⁺ Foxp3⁻ T cells, CD3⁺ CD8⁺ T cells, CD3⁻ Nk1.1⁺ NK cells and CD3⁺ NK1.1⁺ cells but not CD19⁺ B cells proliferated upon IL-2 complex treatment in the spleen as assessed by their expression of Ki67. Histograms are shown from representative mice (n = 4). <b>(B)</b> IL-2 complexes (n = 4) reduced the proportion and absolute number of CD19⁺ B cells within CD45⁺ leukocytes in the BM as compared with untreated mice (n = 4). Two color dot plots illustrate representative mice (left). Data are pooled from 2 independent experiments (right) [p = 0.014].</p

Rapamycin reduces the expansion and activation of CD8 effector cells.

<p>IL-2 complexes were combined with selected agents to alleviate the unintended expansion and activation of CD8 T cells. IL-2 complexes (5μg IL-2 / 25μg α-IL-2) were administered to female C57BL/6 mice three times in succession (d0, d1,0 d2) together with specified agents. The effect of combined therapy was determined on splenic CD8⁺ T cells and CD4⁺ Foxp3⁺ Tregs 2 (d4) and 4 (d6) days after the last injection. <b>(A)</b> Co-administration of rapamycin (n = 4) reduced the absolute cell number of splenic CD8⁺ T cells at both measured time points compared to mice treated only with IL-2 complexes (n = 4) [p = 0.0142 for d4; p = 0.0244 for d6]. Data are pooled from two independent experiments. Groups receiving α -IL-6 and IL-15-Fc consisted of 2 mice from one experiment at each time point. <b>(B)</b> Rapamycin (n = 4) but not α-IL-6 (n = 2) or IL-15-Fc (n = 2) considerably decreased the amount of effector memory CD8⁺ T cells (d6) defined as CD44^hi CD62^lo compared to IL-2 complex treatment alone (n = 4). Two color dot plots depict representative mice. <b>(C)</b> Addition of α-IL-6 (n = 2) decreased the fraction of Foxp3⁺ cells within splenic CD4 T cell population while rapamycin (n = 4) and IL-15-Fc (n = 2) had no profound effect (d6). Histograms illustrate representative mice of each group.</p

IL-2 complexes preferentially induce the proliferation of Helios⁺ Nrp1⁺ Tregs.

<p>To determine the effect of IL-2 complexes (5μg IL-2 / 25μg α-IL-2) on thymus derived Tregs, naïve C57BL/6 mice received three successive injections (d0, d1, d2) and Treg specific markers were measured in the spleen two days (d4) after the last injection. <b>(A)</b> IL-2 complexes (n = 6) slightly raised the amount of thymus derived Helios⁺ Nrp1⁺ Tregs compared to untreated mice (n = 6). Representative mice are displayed in two color dot plots (left). Data are pooled from three independent experiments (right) [p = 0.0287]. <b>(B)</b> Helios⁺ Nrp1⁺ Tregs exhibited a higher degree of proliferation than Helios⁻ Nrp1⁻ Tregs as measured by their expression of the proliferation marker Ki67 both before and after treatment with IL-2 complexes. Histograms show representative mice (left). The bars compare the expression of Ki67 between Helios⁺ Nrp1⁺ (n = 6) and Helios⁻ Nrp1⁻ (n = 6) Tregs upon IL-2 complex treatment (right) [p<0.0001]. Data are pooled from three independent experiments. <b>(C)</b> Helios⁺ Nrp1⁺ Tregs exhibit a higher surface expression of CD25. Representative mice were selected for histograms (left). The bars show the MFI of CD25-PE-Cy7 on Helios⁺ Nrp1⁺ and Helios- Nrp1- Tregs before (n = 3) and after (n = 3) IL-2 complex treatment (right) [untreated: p = 0.0003; IL-2 complexes: p = 0.0003]. The bars represent the mean of three mice from one experiment. <b>(D)</b> Blockade of MHC class II molecules decreased the amount and absolute cell number of CD4⁺ Tregs in the spleen compared to single giving of IL-2 complexes. Two color dot plots illustrate representative mice (left). Each bar shows the mean of 3 mice from one experiment (right) [p = 0.0031]. <b>(E)</b> Co-administration of α-MHC-II did not change the proportion of Helios⁺ Nrp1⁺ Tregs after IL-2 complex treatment. Representative mice are shown in the two color dot plots (n = 3).</p

IL-2 complexes promote BM rejection.

<p>The ability of IL-2 complexes to replace Treg therapy was tested in different BMT models. Donor chimerism was analyzed in blood 14 days after transplantation by staining the BALB/c specific marker H2-D^d on myeloid (Mac-1) cells. <b>(A)</b> IL-2 complexes were less effective than Treg therapy in promoting BM engraftment. Naïve C57BL/6 mice were grafted with 20×10⁶ unseparated BALB/c BM cells (d0) under the cover of costimulation blockade (α-CD154, CTLA4Ig) and a short course of rapamycin. The recipients were additionally treated with either <i>in vitro</i> activated Tregs (3×10⁶) (n = 4), IL-2 complex expanded Tregs (3×10⁶) (n = 4) or IL-2 complexes (5μg IL-2 / 25μg α-IL-2; d-4, d-3, d-2) (n = 6). Two color flow cytometry plots are shown from representative BMT recipients (left). Each dot of the scatter diagram represents one mouse from one experiment (right) [<i>in vitro</i> Tregs vs. <i>in vivo</i> Tregs: p = 0.0341; <i>in vitro</i> Tregs vs. IL-2 complexes: p = 0.0187]. <b>(B)</b> IL-2 complexes enhanced BM rejection. Naïve C57BL/6 mice received a total body irradiation of 1Gy, costimulation blockade (α-CD154, CTLA4-Ig), as well as 20×10⁶ unseparated BALB/c BM cells (d0) with varying doses (1μg IL-2 / 5μg α-IL-2, n = 8; 0.5 μg IL-2 / 2.5μg α-IL-2, n = 5, 0.25 μg IL-2 / 1.25μg α-IL-2, n = 5) of IL-2 complexes; (d3, d5) or without IL-2 complexes (n = 11). Two-color flow cytometry plots are shown from representative BMT recipients (left). Each dot in the scatter diagram depicts one mouse from two individual experiments (right) [no IL-2 complexes vs. 1μg IL-2 / 5μg α-IL-2 p = 0.0004; no IL-2 complexes vs. 0.5μg IL-2 / 2.5μg α-IL-2: p = 0.7769, no IL-2 complexes vs. 0.25μg IL-2 / 1.25μg α-IL-2: p = 0.8966]. <b>(C)</b> Omission of CTLA4-Ig did not reverse the detrimental effect of IL-2 complexes. Naïve C57BL/6 mice were irradiated with 1Gy TBI before receiving costimulation blockade (α-CD154) and 20×10⁶ unseparated BALB/c BM cells (d0) with (1μg IL-2 / 5μg α-IL-2; d3, d5) (n = 6) or without IL-2 complexes (n = 6). Two-color flow cytometry plots are shown from representative BMT recipients (left). Each dot in the scatter diagram shows one mouse from one experiment (right) [p = 0.0627]. <b>(D)</b> IL-2 complexes increased the reactivity of CD8 T cells and NK cells toward donor antigens. Splenocytes from untreated mice or mice treated with IL-2 complexes were stimulated <i>in vitro</i> with irradiated BALB/c (allogeneic) or C57BL/6 (syngeneic) BM cells. The proliferation of CD8 T and NK cells was assessed by measuring the proliferation marker Ki67. Each symbol represents 2 mice from one experiment. <b>(F)</b> 4×10⁵ responder splenocytes from congenic CD45.1 mice were stimulated <i>in vitro</i> with α-CD3 and co-cultured with equal number of either <i>in vitro</i> activated or IL-2 complex expanded Tregs.</p