W Timmermann

Validity of intra-operative neuromonitoring signals in thyroid surgery

BackgroundAlthough intra-operative neuromonitoring (IONM) is widely used in thyroid surgery, the validity of the received IONM signals are still unknown.MethodProspective collection of data forms in 29 hospitals from 8,534 patients with 15,403 nerves at risk, who underwent surgery for benign and malignant goitre disorders between August 1999 and January 2001. IONM was performed by indirect stimulation via the vagal nerve and by direct recurrent laryngeal nerve (RLN) stimulation in 12,486 cases. IONM signals were compared with early (<14 days) and late (6 months) postoperative vocal cord function findings.ResultsThe transient and permanent RLN palsy rate was 2.8% and 0.7%, respectively. Monitoring of the RLN function was significantly more reliable via the indirect IONM stimulation route than via the direct IONM stimulation route (specificity P<0.05). IONM by indirect stimulation via the vagal nerve reliably excluded postoperative, permanent, vocal cord palsy (specificity 97.6%, negative predictive value 99.6%). However, a changed IONM was insufficient to predict permanent RLN palsy (sensitivity 45.9%, positive predictive value 11.6%). IONM was not associated with increased general morbidity.ConclusionsFor intra-operative neuromonitoring, indirect stimulation of the RLN is superior to direct stimulation. An intact acoustic IONM signal is highly predictive of intact postoperative RLN function. When the IONM signal is abnormal or absent, a one-stage extensive thyroid resection should be performed only if the surgeon is absolutely convinced that the first RLN is not harmed or a total thyroidectomy is mandatory.

Apoptosis of T lymphocytes in liver and/or small bowel allografts during tolerance induction.

BACKGROUND Apoptosis of parenchymal cells has been described during allograft rejection. Immunologically privileged tissue in the mouse has been found to prevent rejection by initiating apoptosis of infiltrating lymphocytes. The aim of this study was to investigate whether apoptosis may play a role in T-cell regulation during rejection and subsequent tolerance induction after liver transplantation (LTx) and combined liver/small bowel transplantation (LSBTx). METHODS LTx and LSBTx (Brown Norway-->Lewis) were performed without immunosuppression. Cell migration, activation, and apoptosis were investigated by means of sequential histology, immunohistochemistry, and the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling assay. Donor (Brown Norway) and third-party (Dark Agouti) cardiac allografts were transplanted into LSBTx recipients to determine specific tolerance. RESULTS Transient acute cellular rejection occurred after LTx and LSBTx and was followed by specific tolerance. The kinetics of apoptosis were similar in liver allografts after LTx and LSBTx, but differed from the processes in small bowel allografts after LSBTx. Apoptosis of parenchymal cells in the grafted livers correlated directly with interleukin-2 receptor expression of the infiltrating T cells. During the late phase of rejection, a peak of apoptosis in the lymphocyte infiltrate was demonstrated, characterized as predominantly apoptotic CD8+ T lymphocytes. CONCLUSIONS These results demonstrate that specific tolerance is achieved in both LTx and LSBTx after a transient rejection crisis. Apoptosis is involved in graft rejection and tolerance induction. Activation of T lymphocytes correlates with parenchymal cell apoptosis in the allograft. T-cell inactivation seems to result in apoptosis of cytotoxic T cells and tolerance, which appears to be unique to the liver allograft.

Mechanisms of Tolerance Induction After Rat Liver Transplantation: Intrahepatic CD4+ T Cells Produce Different Cytokines During Rejection and Tolerance in Response to Stimulation☆☆☆

Increasing evidence supports the existence of regulatory T cells that may inhibit the allogeneic immune response after transplantation by secreting regulatory cytokines. To determine whether rat liver tolerance is associated with intrahepatic regulatory T cells secreting a characteristic cytokine profile, we analyzed the cytokine production of freshly isolated intragraft CD4+ T cells at different times postoperatively by semiquantitative reverse transcription-polymerase chain reaction and by enzyme-linked immunosorbent assay before and after in vitro stimulation. Orthotopic arterialized liver transplantation was performed in two allogeneic rat strain combinations, one with fatal acute rejection (DA-to-LEW) and one with long-lasting tolerance (LEW-to-DA) without immunosuppression despite a complete major histocompatibility complex mismatch (spontaneous liver tolerance). Liver allografts of both groups showed continuously increasing cellular infiltration between day 3 and day 7 after transplantation. In this inflammatory situation, very low levels of interleukin-13 were detectable directly after cell isolation, as well as after in vitro stimulation. However, after 30 days, intrahepatic CD4+T cells in the tolerance group were then able to express elevated messenger RNA levels of the anti-inflammatory cytokine interleukin-13 in response to stimulation. This result indicates the presence of an intragraft Th2-like CD4+T cell population, which may have a regulatory function in the induction of liver tolerance.

Different kinetics of donor cell populations after isolated liver and combined liver/small bowel transplantation.

Abstract Spontaneous tolerance induction after liver transplantation also supports additional transplants, e. g. a small bowel graft, from the same donor (tolerogenic effect). Chimerism serves as a possible explanation of this phenomenon. Isolated liver (LTx) and combined liver/small bowel transplantation (LSBTx) are compared. LSBTx and LTx were performed in the BN → LEW rat strain combination without immunosuppression. Parenchymal damage during rejection was monitored by sequential standard histology. Donor/recipient populations were identified and further differentiated for immunhistochemical single and double staining. A small number of donor specific leukocytes can be detected on all days in host organs (microchimerism). A significantly larger donor leukocyte population survives long‐term in the sinusoids of liver (graft chimerism). Sinusoidal donor leukocytes survive rejection and recover in number after tolerance induction. Rejection of liver allografts and infiltration by host leukocytes are more pronounced after LSBTx than after LTx. Accordingly, during rejection a steeper decline of sinusoidal donor leukocytes is observed after LSBTx and recovery after tolerance induction is not as marked. Microchimerism apparently plays no significant role in either transplantation model. The number of sinusoidal donor leukocytes, however, mirrors closely host immune responses.

“Tolerogenic effect” of the liver for a small bowel allograft

Abstract A newly developed liver/small bowel transplantation model (LSBTx) was used to investigate the tolerogenic effect of a liver allograft toward a simultaneously transplanted small bowel. Small bowel transplantation (SBTx) under high‐dose immunosuppression was compared to LSBTx with a lower FK506 dosage. Syngeneic Lewis [(LEW) to LEW] and two fully allogeneic rat strain combinations (Brown Norway‐to‐LEW and Dark Agouti‐to‐LEW) were used. Clinical course and histological findings after SBTx demonstrated a chronic rejection of the small bowel allograft within 100 days. However, after LSBTx long‐term acceptance (> 150 days) was achieved after a transient rejection crisis, although initial immunosuppression was significantly lower. Furthermore, indicator heart transplantations demonstrated the induction of donor‐specific tolerance in both allogeneic strain combinations. In contrast to other LSBTx rat models, these results reflect observations after human LSBTx, in which the rate of acute and chronic rejection is also significantly lower than after human SBTx.

Detection of disseminated tumor cells in nude mice with human gastric cancer

Disseminated tumor cells (DTC) are a potential contributor to relapse of cancer. In the present study we developed a model for induction of disseminated tumor cells in nude mice, which can aid in the search for therapeutic approaches as well as improve our understanding of metastasizing gastric cancer. To detect DTC in blood and bone marrow we established a modified animal model of orthotopic transplantation. Two groups of nude mice were used for xenotransplantation of gastric cancer specimens. In group I tumor specimens originating from a gastric adenocarcinoma cell line were transplanted onto the stomach; in group II they were transplanted subcutaneously into both axillaries. Tumor growth, metastases and presence of DTC were compared in both groups. For detection of DTC a nested reverse transcriptase polymerase chain reaction (RT-PCR) for human cytokeratin (CK)-20 was performed on blood and bone marrow of all mice. Tumor growth occurred in both groups (9/10 animals in group I, 10/10 in group II) within 14 weeks. Only animals in group I developed local invasive tumor growth, stenosis of the stomach and distant metastases. Tumors in animals of group II grew with local displacement only and developed no metastases. There were no signals of CK-20 detected in the blood in both groups. In group I, 5 of 9 animals had positive signals of human CK-20 in their bone marrow as a sign of DTC. In group II no DTC were detected in bone marrow. We conclude that orthotopic transplantation is a prerequisite for the development of DTC and metastasizing tumor growth in this modified gastric cancer model.

Hierarchical immunogenicity of donor MHC class I peptides in allotransplantation.

The recognition of major histocompatibility complex (MHC) allopeptides by recipient MHC class II-restricted CD4(+) T cells via indirect pathway is a prerequisite for the generation of an immune response to the allograft. We tested 13-mer to 24-mer peptides from the MHC class I molecule for their possible immunogenicity in a fully MHC-mismatched rat strain combination. Our results confirm the hierarchical distribution of the immunogenicity of donor MHC class I peptides in the T cell alloactivation via indirect pathway. In addition, we show that allopeptide-induced immune response is critical for acute rejection of heart allografts. Among the seven allopeptides tested, peptide P1 was identified as immunodominant; it induced the greatest T cell proliferation and cytokine production in vitro as well as a significant reduction in allograft survival time. The TCR repertoire of T cells involved in the in vitro and in vivo responses induced by the dominant allopeptide P1 was found to be limited to the Vbeta10 and Vbeta 19 gene families. The identification of dominant allopeptides should greatly facilitate characterization of the specific T cell population responsible for allograft rejection and may be used to modulate the alloimmune response through antigen-specific therapy.

Long-term small bowel allograft function induced by short-term FK 506 application is associated with split tolerance

Abstract Functional long‐term allograft survival after experimental small bowel transplantation (SBT) is limited by chronic rejection. Initial application of high‐dose FK 506 has been shown to induce stable long‐term graft function. In order to examine whether this long‐term function is associated with donor‐specific tolerance, we analyzed the functional status of recipient T cells in vivo and in vitro. One‐step orthotopic SBT was performed in the allogeneic Brown Norway (BN)‐to‐Lewis rat strain combination. FK 506 was given daily at a dose of 2 mg/kg from days 0‐5 in the rejection model and from days 0‐9 in the long‐term functional model. Mean survival time in the rejection model was 98 ± 2.8 days. Histological examination of these small bowel allografts disclosed signs of chronic rejection. In contrast, all animals of the long‐term functional model survived long term (> 250 days) without clinical signs of chronic rejection. The latter model, furthermore, produced evidence of donor‐specific tolerance. Whereas heterotopic Dark Agouti (DA) hearts were rejected regularly within 7 days, BN hearts survived indefinitely (> 70 days). In vitro, mixed leukocyte reactivity of CD4 + T cells was similarly strong against donor (BN) antigens as against third‐party (DA) antigens. The split tolerance revealed by our in vivo and in vitro results enabled acceptance of both the small bowel allograft without signs of chronic rejection and of donor‐specific heart allografts.

Videomicroscopic imaging of graft mucosa for monitoring immunosuppressive therapy after small intestinal transplantation in rats.

BACKGROUND Early diagnosis of rejection and effective immunosuppressive treatment are essential after small intestinal transplantation. To date little is known about microscopic alterations of the intestinal mucosa of the graft during rejection. We attempted to determine whether videomicroscopic imaging of the graft mucosa is a suitable method for monitoring immunosuppressive therapy. METHODS Real-time videomicroscopic imaging of an ileostoma was performed daily after allogeneic heterotopic small bowel transplantation in the rat (BN to LEW) with and without FK506 immunosuppression. Subsequently, the videomicroscopic findings were compared with the histologically determined grade of rejection. RESULTS A semiquantitative staging system was established for the intravital mucosal changes during graft rejection. The earliest changes related to rejection appeared on POD 6 in the untreated allogeneic group. The mucosa developed patchy paleness and the mucosal architecture was interrupted in places. The crypts were slightly widened and their color turned dark red (stage I). These alterations spread progressively over the mucosa on POD 7 (stage II). On POD 9 the mucosa appeared pale, the villi were shortened, and the crypts appeared wide and rounded (stage III). In the animals treated with FK506 similar changes were observed, but with a delayed onset. When FK506 was administered as antirejection therapy at the onset of rejection, a temporary improvement of mucosal alterations was observed (stage II --> stage I). The video-microscopic stages correlated with the histological grade of rejection. CONCLUSIONS The introduction of videomicroscopy has made computer-based high resolution imaging of mucosal microarchitecture possible. With videomi-croscopy beginning rejection can be detected, although it can still be reversed with antirejection therapy. This is a new noninvasive technique that might be of high clinical relevance.

Early T-cell inactivation and apoptosis—critical events for tolerance induction after allogeneic liver transplantation

DESPITE good results following human organ transplantation (eg, kidney, liver, heart, pancreas), transplantation of the small bowel is still not a routine procedure in humans. Because of the complex immunogeneic structure of this organ, the postoperative course after transplantation is complicated by high rejection and infection rates. Therefore, new immunosuppressive strategies inducing peripheral tolerance are of major interest for human small bowel transplantation. Rat liver transplantation (LTx) induces peripheral tolerance in rats. This phenomenon has been known for a long time, although its immunological mechanisms are still not completely understood. One thesis that may explain this phenomenon is “exhaustion” of the recipient’s immune response after LTx: the large number of liver alloantigens presented to recipient-type T cells may shift these cells to an anergic status. To verify this thesis and, on the other hand, to use tolerance induction after LTx for better small bowel allograft acceptance, we confronted the recipient’s immune system beside the liver with a simultaneous transplanted small bowel graft.