Neoadjuvant therapy of gastric cancer with the human monoclonal IgM antibody SC-1: impact on the immune system.

Adjuvant therapies for minimal residual disease are a promising approach to improve the poor survival rates after surgery of gastric tumors. A pilot study of a neoadjuvant therapy was performed using a human monoclonal IgM antibody (SC-1) specifically inducing apoptosis in signet ring cell stomach carcinomas. However, scarce information exists on how such a treatment affects the immune system, in particular what are the effects of apoptosis induction and infusion of large amounts of IgM. Thus, the leukocyte composition (CD3, CD4, CD8, CD19, CD16+56, CD14) and several cytokines (TNF-alpha, IL6, IL12, IFN-gamma, GM-CSF, Neopterin) before and after SC-1 application were measured and compared to results of patients that underwent surgical removal of gastric carcinoma without antibody treatment. After SC-1 application, an increase in TNF-alpha and a decrease of lymphocytes and CD3+ T-cells but in the range obtained in healthy individuals was observed before surgery. After surgery, the IL6 levels increased and the TNF-alpha levels remained at the elevated level. Furthermore, there was a significant drop in lymphocytes and CD3+ T-cells. These effects were due to the surgical treatment. Other parameters did not show significant changes. It seems that the application of an apoptosis-inducing antibody prior to surgery of gastric tumors has mild if any effect on the immune system. Therefore, from an immunological point of view, the treatment with this monoclonal antibody is extremely safe.

Human antibody SC-1 reduces disseminated tumor cells in nude mice with human gastric cancer.

Advanced gastric cancer is a systemic disease that requires adjuvant therapy targeted at eliminating disseminated tumor cells (DTCs). We investigated whether the apoptosis-inducing human monoclonal IgM antibody SC-1 was able to reduce the number of disseminated gastric cancer cells in blood and bone marrow. Human gastric tumor specimens with positive expression of the SC-1 receptor were transplanted in nude mice with metastasizing gastric cancer. After tumor growth (4-6 weeks) animals were randomly allocated to intraperitoneal 100 microg SC-1 (n=23) or 100 microg human IgM (n=23). One week later, animals were sacrificed and blood and bone marrow specimens were obtained. A nested RT-PCR for cytokeratin 20 (CK-20) from blood and bone marrow of mice was performed for detection of disseminated tumor cells. Animals receiving SC-1 had significantly fewer DTCs than did control animals (p=0.0011). None of the SC-1 mice had DTCs simultaneously in both blood and bone marrow versus four of the control animals (p=0.0363). The reduction of DTCs in SC-1 animals was due to reduction in bone marrow (p=0.032 compared to controls), but not in blood (p=0.1158). Treatment with SC-1 significantly reduced the number of DTCs in bone marrow in this animal model.

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.

Allogeneic core amino acids of an immunodominant allopeptide are important for MHC binding and TCR recognition.

The indirect alloimmune response seems to be restricted to a few dominant major histocompatibility complex (MHC)-derived peptides responsible for T-cell activation in allograft rejection. The molecular mechanisms of indirect T-cell activation have been studied using peptide analogues derived from the dominant allopeptide in vitro, whereas the in vivo effects of peptide analogues have not been well characterized yet. In the present study, we generated allochimeric peptide analogues by replacing the three allogeneic amino acids 5L, 9L, and 10T in the sequence of the dominant MHC class I allopeptide P1. These allochimeric peptide analogues were used to define the allogeneic amino acids critical for the MHC binding and TCR recognition. We found that position 5 (5L) of the dominant allopeptide acts as an MHC-binding residue, while the other two allogeneic positions, 9 and 10, are important for the T-cell receptor (TCR) recognition. A peptide containing the MHC-binding residue 5L, as the only different amino acid between donor (RT1.A(u)) and recipient (RT1.A(l)) sequences, did not induce proliferation of lymph node cells primed with the dominant peptide and prevented dominant peptide-induced acceleration of allograft rejection. Identification of MHC and TCR contact residues should facilitate the development of antigen-specific therapies to inhibit or regulate the indirect alloimmune response.

Prolongation of small bowel allograft survival with a sequential therapy consisting of a synthetic MHC class II peptide and temporarily low-dose cyclosporine A.

It has been extensively documented the role of the indirect pathway of allorecognition in allograft rejection. However, recent data demonstrate that the manipulation of this pathway could be also sufficient to promote prolongation of allograft survival. In the present study we evaluated the effect of preoperative immunization with the WF-specific MHC class II peptides RT1.D2 and RT1.B2 in combination with low-dose CsA from days 0 to 7 (5 mg/kg/day) and from days 8 to 30 (1 mg/kg/day) after WF small bowel transplantation. Seven days before and on the day of transplantation, LEW recipients were immunized with the two WF MHC class II peptides RT1.B2 and RT1.D2. The CsA monotherapy induced an allograft survival of 49.3 +/- 6.1 days. MHC class II peptide immunization had a limited effect on allograft survival for RT1.D2 (47.1 +/- 3.8 days) and induced prolongation of allograft survival for RT1.B2 (73.6 +/- 34.6 days). This effect seems to be based on the absence or silence of RT1.B2-reactive T cells and rejection seems to be correlated with the presence of RT1.B2-specific T cells in the late phase. Therefore, the combination of RT1.B2 with low-dose CsA shifts the immunological response and protects small bowel allograft rejection.

Well-being, mood and calcium homeostasis in patients with hypoparathyroidism receiving standard treatment with calcium and vitamin D.

OBJECTIVE: Standard treatment in hypoparathyroidism consists of calcium and vitamin D (or vitamin D analogs) but does not employ replacement of the actual missing hormone. Only few studies have evaluated the efficacy of calcium/vitamin D treatment in hypoparathyroidism; the impact of chronic hypoparathyroid disease on well-being has not been investigated previously. DESIGN: Cross-sectional, controlled study in 25 unselected women with postsurgical hypoparathyroidism since 6.4plus minus8.0 years (s.d.) on stable treatment with calcium and vitamin D (or analogs) and in 25 controls with a history of thyroid surgery but intact parathyroid function, who were matched for sex, age and time since surgery. METHODS: Assessment of well-being and mood using validated questionnaires (the revised version Symptom Checklist 90 (SCL-90-R); the Giessen Complaint List (GBB-24); and the von Zerssen Symptom List (B-L Zerssen)), serum and urinary calcium/phosphorus homeostasis, and in the hypoparathyroid patients also screening for secondary disease by kidney ultrasound, ophthalmological split lamp examination, and measurement of bone mineral density. RESULTS: Serum calcium was in the accepted therapeutic range in the majority of hypoparathyroid patients. However, calcium/phosphorus homeostasis as a whole was clearly non-physiological. Nephrolithiasis was detected in 2 and cataracts in 11 of 25 hypoparathyroid patients. As compared with controls, hypoparathyroid patients had significantly higher global complaint scores in GBB-24 (P=0.036), B-L Zerssen (P=0.002) and SCL-90-R (P=0.020) with predominant increases in the subscale scores for anxiety, phobic anxiety and their physical equivalents. CONCLUSIONS: Current standard treatment in hypoparathyroidism is not only associated with an altered calcium/phosphorus homeostasis but also fails to restore well-being in these patients. Future studies need to address the impact of more physiological treatment options like parathyroid hormone(1-34) or parathyroid transplantation on well-being and mood in these patients.

Risk factors of paralysis and functional outcome after recurrent laryngeal nerve monitoring in thyroid surgery.

BACKGROUND: Recurrent laryngeal nerve monitoring (RLNM) has been suspected to reduce postoperative RLN paralysis (RLNP). However, functional outcome of RLNM in comparison with no nerve identification and visual nerve identification only has not been analyzed. METHODS: Analysis of 16,448 consecutive multi-institutional operations resulted in 29,998 nerves at risk. Three groups of different RLN treatment were compared: group 1, no RLN identification; group 2, visual RLN identification; and group 3, visual RLN identification and electromyographic monitoring. RLNM was performed with a bipolar needle electrode that was placed through the cricothyroid ligament into the vocal muscle. RESULTS: Risk factors for permanent RLNP were recurrent benign and malignant goiter (odds ratios, [ORs]), 4.7, and 6.7, respectively), primary surgery in thyroid malignancy (OR, 2.0), lobectomy (OR, 1.8), no nerve identification (OR, 1.4), low or medium volume hospital (OR, 1.3), and low volume surgeons (OR, 1.2). CONCLUSIONS: Based on these data, visual nerve identification was identified to be the gold standard of RLN treatment in thyroid surgery. RLNM is a promising tool for nerve identification and protection in extended thyroid resection procedures. However, because of the overall low frequency of RLNP, no statistical difference compared with visual nerve identification only was reached in the setting of this study.

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.

Ultrastructural alterations of primary human liver sinusoidal cells in patients treated for peritonitis.

Acute peritonitis is still associated with a high mortality rate. Bacterial toxins are rapidly cleared from the peritoneal cavity and may induce general sepsis. The hepatic sinusoidal cells are part of the primary defence against these toxins. The object of this study was to examine ultrastructural alterations of human sinusoidal liver cells from patients undergoing surgical treatment for peritonitis. Liver specimens, obtained from five patients treated with programmed interval peritoneal lavages for peritonitis, were analyzed by electron microscopy. Despite interindividual differences in etiology of peritonitis, the detected ultrastructural alterations displayed a high degree of similarity. Kupffer cells displayed enhanced phagocytotic activity. Numerous Kupffer cell-lymphocyte contacts were observed. Notably, the morphological appearance of the endothelial cells resembled that of an activated phagocytotic cell. The ultrastructural alterations peaked on day 7, and regressed during the course of treatment. Our findings demonstrate that major changes occur in the ultrastructural appearance of both Kupffer cells and sinusoidal endothelial cells in patients with acute peritonitis treated successfully with programmed interval peritoneal lavages. Our data suggest that in peritonitis, a septic spread of toxins and antigens may be modulated by sinusoidal liver cells.

Ten-year follow-up of a prospective trial for the targeted therapy of gastric cancer with the human monoclonal antibody PAT-SC1.

The fully human monoclonal antibody PAT-SC1 is specific for an isoform of CD55 (decay-accelerating factor) designated CD55PAT-SC1. This antigen is expressed in the majority (80%) of gastric cancers (GCs), and the antibody induces tumour cell-specific apoptosis in vitro as well as in vivo. PAT-SC1, therefore, has been deemed promising as a therapeutic agent. Here, we describe the results of an academic clinical study performed in a neoadjuvant setting with resectable GC patients. Patients undergoing treatment for GC between 1997 and 2001 were tested for CD55PAT-SC1 expression. Fifty-one resectable patients that tested positively received a single administration of 20 mg PAT-SC1 48 h prior to surgery. They underwent standard surgery with either subtotal or total gastrectomy with bursectomy, omentectomy and a modified D2-lymphadenectomy, aimed at R0 resection. Primary endpoints of the present study were to evaluate side-effects of the PAT-SC1 antibody treatment and to evaluate histopathological effects such as tumour regression and induction of apoptosis. Long-term survival was a secondary endpoint. Administration of PAT-SC1 appeared safe with only reversible side-effects according to WHO grade I and II. Despite the low­dose of the antibody, 81.6% of the patients showed signs of increased apoptosis within the primary tumour and 60% showed signs of tumour cell regression. Comparison of the 10-year survival rates of the R0-resected CD55PAT-SC1-positive patients treated with the PAT-SC1 antibody with a historical collective of R0-resected CD55PAT-SC1-positive patients not treated with PAT-SC1 indicated a survival benefit in the treated patients. Furthermore, comparison of the patient survival of CD55PAT­SC1-positive vs. CD55PAT-SC1-negative groups suggested that CD55PAT-SC1 antigen expression is an independent predictor of poor survival in a Cox regression analysis. Antibody PAT-SC1 may be a useful additive therapeutic agent in the treatment of patients with CD55PAT-SC1-expressing GCs. In combination with radical standard surgery, PAT-SC1 given as an adjuvant or neoadjuvant immunotherapeutic agent induces apoptosis in tumour cells which may improve survival of these patients. Because of the human origin and its specific binding to the CD55PAT-SC1 antigen, PAT-SC1 was well tolerated in this trial.

Disseminated tumor cells in the blood of patients with gastric cancer are an independent predictive marker of poor prognosis.

OBJECTIVE: Gastric cancer carries a poor prognosis even after curative resection (R0). Tumor progression in gastric cancer patients has been attributed to the persistence of disseminated tumor cells (DTC) in various body compartments as a sign of minimal residual disease, although the prognostic relevance of DTC is still unclear. In this study the prognostic relevance of DTC in the blood of gastric cancer patients was investigated. MATERIALS AND METHODS: Venous blood samples of 70 cancer patients were taken intraoperatively before surgical manipulation and examined by reverse transcription-polymerase chain reaction (RT-PCR) for expression of cytokeratin 20 (CK20) as a marker for DTC. Tumor-related survival was analyzed using univariate and multivariate models assessing occurrence of DTC, residual tumor classification, and tumor stage. Median follow-up was 20 months (range 1-57 months). RESULTS: Twenty-eight of the 70 patients (40%) were CK20 positive. The prevalence of DTC in patients following R0 resection (15/41, 37%) was similar to that in patients with residual tumor (13/29, 45%, NS). Furthermore, expression of CK20 was independent of TNM stage. Univariate analysis of R0-resected patients revealed CK20 to be a marker for significantly shorter tumor-related survival (p = 0.0363). In a multivariate analysis, CK20 was an independent prognostic marker. Detection of CK20 had greatest impact for early tumor stages (T1 and T2, N0; p < 0.0032). CONCLUSIONS: Detection of DTC in venous blood of gastric cancer patients is an independent predictive marker of poor prognosis and thus could help to define patients for adjuvant therapy with this tumor entity.

Application of Molecular Adsorbent Recirculating System in patients with severe liver failure after hepatic resection or transplantation: initial single-centre experiences.

Acute liver failure after hepatic surgery is still plaqued with high mortality rate. Recently, a liver dialysis system (MARS) that allows detoxification of albumin-bound substances and may hereby support liver regeneration and patient's recovery has been developed. In the present study, we report our experiences with MARS dialysis in patients with liver failure after hepatic resection or transplantation. Between September 1999 and January 2001, five patients were treated with MARS (2-5 courses). Though beneficial effects such as improvement of encephalopathy and renal function as well as reduced bilirubin levels were recorded during MARS therapy, only one patient survived. Neither significant technical problems nor adverse effects occurred by using MARS dialysis. We conclude that in surgical patients, acute liver failure is usually part of a complicated clinical course affecting multipleorgan systems. Thus, it is difficult to determine the specific influence of MARS on patient's outcome. However, beneficial effects observed in our patients justify its continuous use and may stimulate further evaluation in controlled studies with surgical patients.

Validity of intra-operative neuromonitoring signals in thyroid surgery.

BACKGROUND: Although intra-operative neuromonitoring (IONM) is widely used in thyroid surgery, the validity of the received IONM signals are still unknown. METHOD: Prospective 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. RESULTS: The 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. CONCLUSIONS: For 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.

Microsurgical training course for clinicians and scientists at a German University hospital: a 10-year experience.

Microsurgical techniques are being increasingly applied in almost all surgical disciplines. However, the opportunities to learn these skills in a structured course are rare. We have conducted a 5-day microsurgical training course on a yearly basis since 1991. The course follows step-by-step training, starting with nonvital models for vascular and nerval microanastomoses. As the participants improve, exercises on laboratory animals are offered to close the gap between nonliving models and the clinical situation. Lectures provide theoretical and clinical background information. Clinical and experimental applications can be witnessed and practiced in a second part of the course. With this step-by-step curriculum, we conduct a successful training program, e.g., each participant is able to perform microvascular and nerval anastomoses on a reproducible basis. The organization, program, and execution of the training course are presented, together with an evaluation of the course concept by the participants concerning expectations, learning success, and level of satisfaction.

Optimization of a metastasizing human gastric cancer model in nude mice.

Our purpose was to optimize the surgical orthotopic implantation (SOI) technique to create a reproducible gastric cancer model in nude mice with stable tumor growth and metastasizing course. We performed xenotransplantation of primary human tumor specimens from patients with gastric cancer (series 1) and orthotopic transplantation of tumor specimens originating from the gastric cancer cell line 23132/87 (series 2). All specimens were transplanted using microsurgical techniques. The two series were compared with regard to tumor growth rates and kinetics, development of metastases, and induction of minimal residual disease (MRD), as determined by histology and PCR techniques. In series 1 mice, the tumor growth rate was slow; in series 2 mice, it was both fast and reproducible. Unlike animals in series 1, animals in series 2 developed metastases and MRD. In conclusion, the optimized SOI technique presented here represents a reproducible and reliably metastasizing gastric cancer model.

Short-term immunosuppression after rat parathyroid allotransplantation.

The aim of this study was to evaluate whether short-term postoperative immunosuppression is able to sufficiently prolong graft survival after experimental allogeneic parathyroid transplantation. Heterotopic parathyroid transplantation was performed in 6 groups: 1) syngeneic control Lewis (LEW) to LEW; 2) allogeneic control Wistar-Furth (WF) to LEW; 3-5) WF to LEW plus short-term immunosuppression, postoperative days 1-13 (cyclosporine 5/10/20 mg/kg); and 6) WF to LEW plus 10 mg/kg CyA from preoperative day 7 to postoperative day 7. Graft function was examined up to 60 days; histological and immunohistological examination was performed on all grafts with impaired function. Graft function after syngeneic transplantation was indefinite, while recipients of allogeneic grafts turned hypocalcemic after 13 +/- 2 days. With immunosuppression, graft function was 21 +/- 2 days (groups 5 and 6) and 28 +/- 3 days (groups 3 and 4). Histologically, a cellular infiltrate responsible for graft destruction was found. The results show that indefinite parathyroid allograft survival cannot be achieved by short-term immunosuppression alone. Whether the combination of an additional graft pretreatment and immunosuppression has an impact on graft function will be further examined.