The importance of MHC class II in allogeneic bone marrow transplantation and chimerism-based solid organ tolerance in a rat model.

Mixed hematopoietic chimerism enables donor-specific tolerance for solid organ grafts. This study evaluated the influence of different serological major histocompatibility complex disparities on chimerism development, graft-versus-host disease incidence and subsequently on solid organ tolerance in a rat model. For bone marrow transplantation conditioning total body irradiation was titrated using 10, 8 or 6 Gray. Bone marrow transplantation was performed across following major histocompatibility complex mismatched barriers: complete disparity, MHC class II, MHC class I or non-MHC mismatch. Recipients were clinically monitored for graft-versus-host disease and analyzed for chimerism using flow cytometry. After a reconstitution of 100 days, composition of peripheral leukocytes was determined. Mixed chimeras were challenged with heart grafts from allogeneic donor strains to define the impact of donor MHC class disparities on solid organ tolerance on the basis of stable chimerism. After myeloablation with 10 Gray of total body irradiation, chimerism after bone marrow transplantation was induced independent of MHC disparity. MHC class II disparity increased the incidence of graft-versus-host disease and reduced induction of stable chimerism upon myelosuppressive total body irradiation with 8 and 6 Gray, respectively. Stable mixed chimeras showed tolerance towards heart grafts from donors with MHC matched to either bone marrow donors or recipients. Isolated matching of MHC class II with bone marrow donors likewise led to stable tolerance as opposed to matching of MHC class I. In summary, MHC class II disparity was critically associated with the onset of graft-versus host disease and was identified as obstacle for successful development of chimerism after bone marrow transplantation and subsequent donor-specific solid organ tolerance

Courses of donor chimerism after MHC class II disparate BMT in recipients conditioned by 6 Gy of TBI and treated with immunosuppression.

<p>LEW.1AR2 male rats were irradiated with 6 Gy of TBI and received 1 x 10⁸ BMC from LEW.1AR1 donor rats one day after TBI. Recipients were additionally treated either with sirolimus (Srl) or cyclosporine A (CsA). Control animals did not receive any immunosuppression (no IS). Donor chimerism was detected within blood B cells by flow cytometry using donor-specific anti-RT1B/D^u mAb (1H1A) on days 14, 30, 60 and 100 post BMT. (<b>A</b>) Mean values of chimerism from groups receiving Srl for 14 days (Srl 14d; n = 13) or CsA for 14 days (CsA 14d; n = 11) as well as control animals (no IS; n = 11). (<b>B</b>) Mean values of chimerism from groups receiving Srl for 28 days (Srl 28d; n = 15) or CsA for 28 days (CsA 28d; n = 10). The statistical significance was appointed at * p < 0.05 for BMT groups receiving immunosuppression for 14 days and TBI alone (A) as well as for both groups receiving immunosuppression for 28 (B).</p

Application of Allogeneic Bone Marrow Cells in View of Residual Alloreactivity: Sirolimus but Not Cyclosporine Evolves Tolerogenic Properties

<div><p>Background</p><p>Application of bone marrow cells (BMC) is a promising strategy for tolerance induction, but usually requires strong depletion of the host immune system. This study evaluates the ability of immunosuppressants to evolve tolerogenic properties of BMC in view of residual alloreactivity.</p><p>Methods</p><p>The rat model used a major histocompatibility complex (MHC) class II disparate bone marrow transplantation (BMT) setting (LEW.1AR1 (RT1^auu) → LEW.1AR2 (RT1^aau)). Heart grafts (LEW.1WR1 (RT1^uua)) were disparate for the complete MHC to recipients and for MHC class I to BMC donors. Limited conditioning was performed by total body irradiation of 6 Gy. Cyclosporine (CsA) or Sirolimus (Srl) were administered for 14 or 28 days. Transplantation of heart grafts (HTx) was performed at day 16 or at day 100 after BMT. Chimerism and changes in the T cell pool were detected by flow cytometry.</p><p>Results</p><p>Mixed chimeras accepted HTx indefinitely, although the composition of the regenerated T cell pool was not changed to a basically donor MHC class II haplotype. Non-chimeric animals rejected HTx spontaneously. BMC recipients, who received HTx during T cell recovery at day 16, accepted HTx only after pre-treatment with Srl, although chimerism was lost. CsA pre-treatment led to accelerated HTx rejection as did isolated application of BMC.</p><p>Conclusion</p><p>Srl evolves tolerogenic properties of allogeneic BMC to achieve indefinite acceptance of partly MHC disparate HTx despite residual alloreactivity and in particular loss of chimerism.</p></div

Effects of 6 Gy of TBI on T cell pool.

<p>Three male LEW.1AR2 rats were irradiated with 6 Gy of TBI. Analysis was performed on day 3 after TBI. Control animals of identical strain, gender and age did not receive any treatment (ctrl). After counting vital mononuclear cells per μl blood as well as per spleen, analyses were performed by flow cytometry. T cells with α/β TCR expression (R73⁺) were divided into CD8⁺ and CD8^-. Propidium-iodid counter-staining was used to exclude avital cells. (<b>A</b>) Mean values of absolute numbers of α/β TCR+ cells in peripheral blood. (<b>B</b>) Absolute numbers of vital α/β TCR+ cells in spleen subdivided according to CD8 expression as mean values.</p

Detection of donor-derived MHC class II^high cells.

<p>Three colour flow cytometry was used to detect highly expressing MHC class II donor cells of BMT recipients. Mononuclear cells were pre-gated concerning scatter properties. (<b>A</b>) Leukocytes expressing high density of MHC class II antigens were gated using anti-CD45 mAb (OX-1) and anti-MHC class II mAb (OX-6). (<b>B</b>) Anti-RT1B/D^u mAb (1H1A) was used to detect cells of donor origin (1H1A⁺). Data from a representative chimeric recipient treated with 6 Gy and Srl for 28 days are shown.</p

SOCIUS Mentoring&mdash;A Novel Course to Encourage Students for a Career as Surgical Oncologists

Surgical disciplines are affected by an increasing shortage of young doctors. Studies show that formerly interested students decide against a career in surgical disciplines at the end of their studies or during practical year. Measures to counteract this development are urgently needed. As a joint project between gynecology, urology, and general surgery, SOCIUS mentoring was designed to prepare and encourage students for a career in surgical oncology. The structured curriculum of SOCIUS mentoring contains six modules, including surgical skills, soft skills, mentoring, theory, clinical visitation, and congress participation and runs over one year. Effects on confidence towards physician skills and plans for a future career were evaluated with questionnaires. After participation, students reported increased confidence in surgical and soft skills. In addition, participants noted that they have specified their career goals and gained more confidence in surgery, as well as seeing more development potential for a career in surgery. We describe the implementation of a novel extracurricular program for motivated students that combines individual mentoring with surgical and soft skills training. Due to its modular structure, this concept can easily be transferred to other disciplines. SOCIUS mentoring, with its combination of mentoring and skills training, is a promising measure to prepare and motivate students for their surgical career and thus counteract the shortage of young talent

CD4⁺/CD4^- T cell ratio in peripheral blood of T cell regenerated BMT recipients at day 100.

<p>Peripheral blood of T cell regenerated BMT recipients at day 100 was analyzed by flow cytometry (n = 5 per group). Naïve rats of LEW.1AR1 donor and LEW.1AR2 recipient strain with same gender and similar age were analyzed as controls (n = 5 per group). Anti-α/β TCR mAb and anti-CD4 mAb were used. Mean values of CD4⁺/CD4^- ratios in α/β TCR⁺ cells are given. The type of chimerism is indicated (X = stable chimerism, no X = no persistent chimerism). Irradiation dosages and immunosuppressants (sirolimus: Srl; cyclosporine: CsA) including duration of application in days are given. The statistical significance was appointed at * p < 0.05 for naïve LEW.1AR1 as well as X 10 Gy group versus naïve LEW.1AR2 and all other groups.</p

A Depleting Anti-CD45 Monoclonal Antibody as Isolated Conditioning for Bone Marrow Transplantation in the Rat

Objective A monoclonal antibody (mAb) against the leukocyte common antigen CD45 (RT7 in rats) could facilitate bone marrow transplantation (BMT). This study in rats evaluates a depletive rat anti-RT7(a) mAb as isolated tool for BMT conditioning without using irradiation or any chemotherapeutic /immunosuppressive agent. Methods The model used a CD45 di-allelic polymorphism (RT7(a)/RT7(b)). The anti-RT7(a) mAb was intravenously administered to LEW. 1W rats (RT1(u)RT7(a)) at 5, 10 and 15 mg/kg. 1x10(8) BM cells of MHC syngeneic (RT1(u)), MHC disparate (RT1(l)) or MHC haploidentical (RT1(u/l)) donors were transplanted. All BM donor strains carried the RT7(b) allele so that their CD45(+) cells were not affected by the anti-RT7(a) mAb. Recipients were monitored for reconstitution and donor-chimerism in blood leukocytes. Results mAb dosages of 5 or 10mg/kg were myelosuppressive, whereas 15mg/kg was myeloablative. Multi-lineage donor-chimerism at day 100 indicated engraftment ofMHC syngeneic BM after any used mAb dosage (5 mg/kg: 46+/-7%; 10mg/kg: 62+/-5%; 15mg/kg: 80+/-4%). MHC disparate BM resulted in autologous reconstitution after conditioning by 10mg/kg of the mAb and caused transient chimerism ending up in death associated with aplasia after conditioning by 15mg/kg of the mAb. MHC haploidentical BM (F1 to parental) engrafted only after conditioning by 15 mg/kg (chimerism at day 100: 78+/-7%). Abandonment of alpha/beta TCR+ cell depletion fromBMgrafts impaired the engraftment process after conditioning using 15 mg/kg of the mAb in theMHC syngeneic setting (2 of 6 recipients failed to engraft) and the MHC haploidentical setting (3 of 6 recipients failed). Conclusion This depletive anti-RT7(a) mAb ismyelosuppressive and conditions for engraftment of MHC syngeneic BM. The mAb also facilitates engraftment ofMHC haploidentical BM, if amyeloablative dose is used. RT7(b) expressing, BM- seeded alpha/beta TCR+ cells seem to impair the engraftment process after myeloablative mAb conditioning

Preoperative leukocytosis and the resection severity index are independent risk factors for survival in patients with intrahepatic cholangiocarcinoma

Purpose!#!The incidence of intrahepatic cholangiocarcinoma is increasing worldwide. Despite advances in surgical and non-surgical treatment, reported outcomes are still poor and surgical resection remains to be the only chance for long-term survival of affected patients. The identification and validation of prognostic factors and scores, such as the recently introduced resection severity index, for postoperative morbidity and mortality are essential to facilitate optimal therapeutic regimens.!##!Methods!#!This is a retrospective analysis of 269 patients undergoing resection of histologically confirmed intrahepatic cholangiocarcinoma between February 1996 and September 2018 at a tertiary referral center for hepatobiliary surgery. Regression analyses were performed to evaluate potential prognostic factors, including the resection severity index.!##!Results!#!Median postoperative follow-up time was 22.93 (0.10-234.39) months. Severe postoperative complications (≥ Clavien-Dindo grade III) were observed in 94 (34.9%) patients. The body mass index (p = 0.035), the resection severity index (ASAT in U/l divided by Quick in % multiplied by the extent of liver resection graded in points; p = 0.006), additional hilar bile duct resection (p = 0.005), and number of packed red blood cells transfused during operation (p = 0.036) were independent risk factors for the onset of severe postoperative complications. Median Kaplan-Meier survival after resection was 27.63 months. Preoperative leukocytosis (p = 0.003), the resection severity index (p = 0.005), multivisceral resection (p = 0.001), and T stage ≥ 3 (p = 0.013) were identified as independent risk factors for survival.!##!Conclusion!#!Preoperative leukocytosis and the resection severity index are useful variables for preoperative risk stratification since they were identified as significant predictors for postoperative morbidity and mortality, respectively