Soluble ligands for NK cell receptors promote evasion of chronic lymphocytic leukemia cells from NK cell anti-tumor activity.

Natural killer (NK) cells are a major component of the anti-tumor immune response. NK cell dysfunctions have been reported in various hematologic malignancies, including chronic lymphocytic leukemia (CLL). Here we investigated the role of tumor cell-released soluble and exosomal ligands for NK cell receptors that modulate NK cell activity. Soluble CLL plasma factors suppressed NK cell cytotoxicity and down-regulated the surface receptors CD16 and CD56 on NK cells of healthy donors. The inhibition of NK cell cytotoxicity was attributed to the soluble ligand BAG6/BAT3 that engages the activating receptor NKp30 expressed on NK cells. Soluble BAG6 was detectable in the plasma of CLL patients, with the highest levels at the advanced disease stages. In contrast, NK cells were activated when BAG6 was presented on the surface of exosomes. The latter form was induced in non-CLL cells by cellular stress via an nSmase2-dependent pathway. Such cells were eliminated by lymphocytes in a xenograft tumor model in vivo. Here, exosomal BAG6 was essential for tumor cell killing because BAG6-deficient cells evaded immune detection. Taken together, the findings show that the dysregulated balance of exosomal vs soluble BAG6 expression may cause immune evasion of CLL cells.

Translational Development of a Zr-89-Labeled Inhibitor of Prostate-specific Membrane Antigen for PET Imaging in Prostate Cancer.

PURPOSE: We present here a Zr-89-labeled inhibitor of prostate-specific membrane antigen (PSMA) as a complement to the already established F-18- or Ga-68-ligands. PROCEDURES: The precursor PSMA-DFO (ABX) was used for Zr-89-labeling. This is not an antibody, but a peptide analogue of the precursor for the production of [177Lu]Lu-PSMA-617. The ligand [89Zr]Zr-PSMA-DFO was compared with [68Ga]Ga-PSMA-11 and [18F]F-JK-PSMA-7 in vitro by determination of the Kd value, cellular uptake, internalization in LNCaP cells, biodistribution studies with LNCaP prostate tumor xenografts in mice, and in vivo by small-animal PET imaging in LNCaP tumor mouse models. A first-in-human PET was performed with [89Zr]Zr-PSMA-DFO on a patient presenting with a biochemical recurrence after brachytherapy and an ambiguous intraprostatic finding with [18F]F-JK-PSMA-7 but histologically benign cells in a prostate biopsy 7 months previously. RESULTS: [89Zr]Zr-PSMA-DFO was prepared with a radiochemical purity ≥ 99.9% and a very high in vitro stability for up to 7 days at 37 °C. All radiotracers showed similar specific cellular binding and internalization, in vitro and comparable tumor uptake in biodistribution experiments during the first 5 h. The [89Zr]Zr-PSMA-DFO achieved significantly higher tumor/background ratios in LNCaP tumor xenografts (tumor/blood: 309 ± 89, tumor/muscle: 450 ± 38) after 24 h than [68Ga]Ga-PSMA-11 (tumor/blood: 112 ± 57, tumor/muscle: 58 ± 36) or [18F]F-JK-PSMA-7 (tumor/blood: 175 ± 30, tumor/muscle: 114 ± 14) after 4 h (p < 0.01). Small-animal PET imaging demonstrated in vivo that tumor visualization with [89Zr]Zr-PSMA-DFO is comparable to [68Ga]Ga-PSMA-11 or [18F]F-JK-PSMA-7 at early time points (1 h p.i.) and that PET scans up to 48 h p.i. clearly visualized the tumor at late time points. A late [89Zr]Zr-PSMA-DFO PET scan on a patient with biochemical recurrence (BCR) had demonstrated intensive tracer accumulation in the right (SUVmax 13.25, 48 h p.i.) and in the left prostate lobe (SUV max 9.47), a repeat biopsy revealed cancer cells on both sides. CONCLUSION: [89Zr]Zr-PSMA-DFO is a promising PSMA PET tracer for detection of tumor areas with lower PSMA expression and thus warrants further clinical evaluation.

Portal vein arterialization increases hepatocellular apoptosis and inhibits liver regeneration.

BACKGROUND: Portal vein arterialization is performed in particular situations to guarantee sufficient blood flow in the portal vein. In addition, some authors have postulated a proliferation-promoting influence of portal vein arterialization on the liver tissue. However, portal vein arterialization is an unphysiological procedure: It increases portal blood flow and blood pressure as well as oxygenation of the liver tissue. On the other hand, it reduces the influx of hepatotrophic factors from the portal venous blood. The aim of these experiments was to investigate apoptosis and proliferation of hepatocytes during various conditions of the portal perfusion. MATERIALS AND METHODS: After 70% liver resection in Lewis rats, the following four experimental groups were formed differing in portal perfusion: (I) hyperperfused, nonarterialized; (II) flow-regulated, nonarterialized; (III) hyperperfused, arterialized; (IV) flow-regulated, arterialized. A warm ischemia of 30 min was kept in all groups. RESULTS: Portal vein arterialization of 70% reduced rat livers significantly reduced liver regeneration as shown by a significant reduction in liver weight, body weight, and liver function after 6 wk, in contrast to the group with 70% liver mass reduction and portal venous inflow of the portal vein. Furthermore, we found a significantly elevated number of apoptotic hepatocytes after portal vein arterialization. These results were independent from blood flow regulation of the arterialized portal vein, which caused no improvement of the results. CONCLUSIONS: Portal vein arterialization should be performed only temporarily and is clinically not recommended as a permanent option, because of the increased hepatocellular apoptosis and the very distinctive, negative long-term effects on liver weight.

Simvastatin-dependent apoptosis in Hodgkin's lymphoma cells and growth impairment of human Hodgkin's tumors in vivo.

Statins are used to treat hypercholesterolemia and seem to have a preventive effect against cancer through pleiotropic effects including prenylation-inhibition. So far nothing is known about the activity of statins or more specific prenylation-inhibitors in Hodgkin's lymphoma (HL). We, therefore, evaluated the anti-HL activity of simvastatin and specific prenylation-inhibitors. 2 microM Simvastatin induced caspase-related apoptosis via depletion of prenylation-substrates in several HL-cell lines. Furthermore, it effectively impaired tumor growth in a mouse model for HL. Since the prenylation-inhibitors FTI-277 and GGTI-298 were also effective against HL-cells, we conclude that statins and specific prenylation-inhibitors should be evaluated in HL patients.

Terminal B cell differentiation is skewed by deregulated interleukin-6 secretion in beta2 integrin-deficient mice.

Absence of the common beta chain (CD18) of beta(2) integrins leads to leukocyte-adhesion deficiency type-1 (LAD1) in humans. Mice with a CD18 null mutation suffer from recurrent bacterial infections, impaired wound healing, and skin ulcers, closely resembling human LAD1. Previous findings in CD18(-/-) mice demonstrated a skewed terminal B cell differentiation with plasmacytosis and elevated serum immunoglobulin G (IgG). As interleukin-6 (IL-6) is a potent enhancer of plasma cell formation and Ig secretion, we assessed IL-6 serum levels of CD18(-/-) and wild-type (WT) mice kept under a conventional or barrier facility or specific pathogen-free (SPF) conditions. We detected an up to 20-fold increase in IL-6 in serum of CD18(-/-) mice compared with WT controls when kept under conventional or barrier facility conditions, respectively. Under SPF conditions, no significant differences in terms of IL-6 serum levels were found between CD18(-/-) and WT mice. However, histological alterations of secondary lymphoid tissues, plasmacytosis, abnormal plasmacytoid cells (Mott cells), and hypergammaglobulinemia persisted. To further analyze the role of IL-6 in these pathological alterations, we established a CD18(-/-) IL-6(-/-) double-deficient mouse mutant. In these mice, serum IgG levels were normal, and the altered plasma cell phenotype, including Mott cells, was no longer detectable. The CD18(-/-) IL-6(-/-) double-deficient mouse model thus demonstrated that IL-6 is responsible for parts of the phenotype seen in the CD18(-/-) mouse mutants. It may be of interest to examine human leukocyte-adhesion deficiency type-1 patients closer and search for pathological changes possibly induced via overproduction of IL-6.

Localization of 131I-labelled monoclonal antibody ERIC1 in a subcutaneous xenograft model of neuroblastoma in SCID mice.

PURPOSE: To evaluate a novel strategy of immunolocalization of human neuroblastoma by targeting the neural cell adhesion molecule (NCAM), which is over-expressed on neuroblastoma. METHODS: NCAM expression on the cell surface of established neuroblastoma cells was shown by flow cytometry. A SCID mouse model using IMR5-75 neuroblastoma cells to induce subcutaneous tumour growth was established. 131I was used to label monoclonal NCAM specific ERIC1 antibodies generating the 131I-ERIC1 antibody, which showed a high affinity to NCAM also after labelling (KD=9 x 10(-8) mol . l(-1)). RESULTS: Measurement of organ-specific radioactivity showed low organ-specific uptake (5.33%ID/g (percent of injected dose per gram of tissue) after 72 h), which continuously decreased over the 96 h investigation period, demonstrating clearance of radioactivity. In contrast, tumours accumulated radioactivity continuously up to a peak of 42.07%ID/g at the 96 h time point (31.07%ID/g at 72 h). This specific uptake could be blocked by application of unlabelled ERIC1 antibodies. Measurement of blood specific radioactivity revealed a characteristic clearance over the first 72 h. With 37 Gy, tumour-specific radioactivity reached therapeutic doses after 96 h. CONCLUSIONS: These results indicate that 131I-labelled ERIC1 has the ability to probe NCAM-expressing tumour cells in vivo with high efficiency and is a promising reagent for the diagnosis and treatment of NCAM-positive human tumours, especially for neuroblastoma.

Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activity in vitro and in vivo against chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia that affects B lymphocytes in adults. Natural killer (NK) cells in CLL patients are intrinsically potent but display poor in situ effector functions. NKG2D is an activating receptor found on NK and CD8+ T cells and plays a role in immunosurveillance of CLL. In this study, we developed mono- and dual-targeting triplebodies utilizing a natural ligand for human NKG2D receptor (ULBP2) to retarget NK cells against tumor cells. Triplebodies in both formats showed better ability to induce NK-cell-dependent killing of target cells compared to bispecific counterparts. A mono-targeting triplebody ULBP2-aCD19-aCD19 successfully triggered NK cell effector functions against CLL cell line MEC1 and primary tumor cells in allogenic and autologous settings. Additionally, a dual-targeting triplebody ULBP2-aCD19-aCD33 specific for two distinct tumor-associated antigens was developed to target antigen loss variants, such as mixed lineage leukemia (MLL). Of note, this triplebody exhibited cytotoxic activity against CD19/CD33 double positive cells and retained its binding features even in the absence of one of the tumor antigens. Further, ULBP2-aCD19-aCD19 showed significant in vivo activity in immune-deficient (NSG) mouse model transplanted with CLL cell line as target cells and human immune cells as an effector population providing a proof-of-principle for this therapeutic concept.

NK cell depletion diminish tumour-specific B cell responses.

Natural killer (NK) cells can exercise immediate cytotoxicity against malignant cells and thus far modulate the development of tumour directed T cell immunity. To investigate the impact of NK cells on the development of tumour directed B cell immunity mice were immunised with IMR5-75 human neuroblastoma cells with or without prior in vivo NK cell depletion. Flow cytometry analyses gave evidence for an impaired IgG response against the cells immunised with. Dissection of Th1 (IgG2a) and Th2 (IgG1) oriented B cell responses revealed Th1 responses as primarily affected, while Th2 oriented B cell responses as measured by flow cytometry and GD2 ganglioside-specific ELISA were enforced. The data reveal an unexpected impact of NK cells on the development of tumour directed B cell responses. Consequently, NK cell function has also to be taken into account when developing B cell-based cancer immunotherapy.

Heterotopic auxiliary rat liver transplantation with flow-regulated portal vein arterialization in acute hepatic failure.

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.(1-3) The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.(4) In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.(5-6) We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2). A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.  The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor's portal vein was carried out via the recipient's right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient's aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. (7) In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3±0.8 g to 9.8±1 g. At this time, the graft's weight decreased from 3.3±0.8 g to 2.3±0.8 g. We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Re-vascularisation in human ovarian tissue after conventional freezing or vitrification and xenotransplantation.

OBJECTIVE: In ovarian tissue grafts there is a massive loss of follicles during the ischaemic period until re-vascularisation is established. The aim of our study was to investigate the influence of different cryopreservation techniques on the ability for the re-vascularisation of ovarian tissue transplanted to SCID mice. STUDY DESIGN: Ovarian fragments from five patients were cut into pieces (approximately 0.5 mm x 1.0 mm x 1.0 mm) and randomly distributed into three groups: fresh non-treated tissue (group A); tissue conventionally frozen in standard 0.5 ml insemination straws with 1.5 M ethylene glycol+0.1 M sucrose, with thawing in a 40 degrees C water bath and step-wise removal of cryoprotectants at room temperature in 0.5 M, 0.25 M and 0.15 M sucrose with gentle agitation (group B); tissue vitrified in 2.62 M dimethylsulphoxide+2.6 M acetamide+1.31 M propylene glycol+0.0075 M polyethylene glycol, with warming by direct plunging of solid specimens with ovarian pieces into 20 ml of 50% vitrification solution pre-warmed to 40 degrees C and dilution of cryoprotectants in a decreasing concentration of vitrification solution (25%, 12.5%) at room temperature (group C). We used a xenograft model in which ovarian tissue pieces of all three groups were subcutaneously transplanted in SCID mice. The animals were sacrificed on the third day after ovarian tissue transplantation and then weekly during 1 month to obtain the ovarian tissue grafts. These samples were examined by immunohistochemical staining with the endothelial cell-specific marker platelet endothelial cell adhesion molecule-1 (PECAM-1) to determine angiogenesis. Histological observation of tissue after explantation was performed and quality and quantity of follicles were assessed. RESULTS: No PECAM-1 staining was observed in all treatment groups prior to grafting. After warming and in vivo culture of ovarian tissue, the beginning of angiogenesis in pieces from all treatment groups on the third day was detected by PECAM-1 staining. After 4 weeks of in vivo culture the overall area of PECAM-1-positive blood vessels significantly increased (P<0.05), independent of the type of cryopreservation (groups B and C vs. group A). It was found that transplantation technique had negative influence on the integrity of follicles independent of the type of treatment during in vivo culture. The duration of in vivo culture has a negative, but not statistically significant, influence on follicle quality in long-cultured transplants inside each treatment group (P>0.5). CONCLUSION: The process of re-vascularisation of transplanted ovarian tissue is independent of the type of treatment and does not influence follicle quality.

The novel chimeric anti-NCAM (neural cell adhesion molecule) antibody ch.MK1 displays antitumor activity in SCID mice but does not activate complement-dependent cytolysis (CDC).

A monoclonal chimeric antibody ch.MK1 was generated by immunizing F004 mice expressing human instead of murine IgG1/kappa immunoglobulin constant regions. The novel antibody specifically binds cell surface-expressed human neural cell adhesion molecule (NCAM) as shown by immunoprecipitation, flow cytometry and cytospins. Functional analysis revealed nearly complete absence of complement-dependent cytolysis in ch.MK1 and in all other anti-NCAM antibodies tested for reference (UJ13a, ERIC1, 123C3, ch.5A2, B159), indicating an unexpected and group-specific property of anti-NCAM antibodies. As a most plausible mechanism, posttranslational modification of NCAM by complement-inhibiting polysialic acid is discussed. The antibody ch.MK1 demonstrated significant in vivo activity against NCAM-positive neuroblastoma in SCID mice in presence of human peripheral blood mononuclear cell. In absence of human peripheral blood mononuclear cell no distinct antitumor activity of the antibody alone was observed. In ch.MK1 the cellular component of the immune system seems to be the dominant effector mechanism, whereas complement-dependent cytolysis seems not to be necessarily required for antitumor activity. These observations help us to understand immunotherapeutic mechanisms of native anti-NCAM antibodies and may additionally contribute to the understanding of results of currently ongoing clinical studies with conjugated anti-NCAM antibodies.

Heat shock protein 90 inhibitor BIIB021 (CNF2024) depletes NF-kappaB and sensitizes Hodgkin's lymphoma cells for natural killer cell-mediated cytotoxicity.

PURPOSE: In Hodgkin's lymphoma, constitutive activation of NF-kappaB promotes tumor cell survival and proliferation. The molecular chaperone heat shock protein 90 (HSP90) has immune regulatory activity and supports the activation of NF-kappaB in Hodgkin's lymphoma cells. EXPERIMENTAL DESIGN: We analyzed the effect of HSP90 inhibition on viability and NF-kappaB activity in Hodgkin's lymphoma cells and the consequences for their recognition and killing through natural killer (NK) cells. RESULTS: The novel orally administrable HSP90 inhibitor BIIB021 (CNF2024) inhibited Hodgkin's lymphoma cell viability at low nanomolar concentrations in synergy with doxorubicin and gemcitabine. Annexin V/7-aminoactinomycin D binding assay revealed that BIIB021 selectively induced cell death in Hodgkin's lymphoma cells but not in lymphocytes from healthy individuals. We observed that BIIB021 inhibited the constitutive activity of NF-kappaB and this was independent of IkappaB mutations. Furthermore, we analyzed the effect of HSP90 inhibition on NK cell-mediated cytotoxicity. BIIB021 induced the expression of ligands for the activating NK cell receptor NKG2D on Hodgkin's lymphoma cells resulting in an increased susceptibility to NK cell-mediated killing. In a xenograft model of Hodgkin's lymphoma, HSP90 inhibition significantly delayed tumor growth. CONCLUSIONS: HSP90 inhibition has direct antitumor activity in Hodgkin's lymphoma in vitro and in vivo. Moreover, HSP90 inhibition may sensitize Hodgkin's lymphoma cells for NK cell-mediated killing via up-regulation of ligands engaging activating NK cell receptors.

Early detection of erlotinib treatment response in NSCLC by 3'-deoxy-3'-[F]-fluoro-L-thymidine ([F]FLT) positron emission tomography (PET).

BACKGROUND: Inhibition of the epidermal growth factor receptor (EGFR) has shown clinical success in patients with advanced non-small cell lung cancer (NSCLC). Somatic mutations of EGFR were found in lung adenocarcinoma that lead to exquisite dependency on EGFR signaling; thus patients with EGFR-mutant tumors are at high chance of response to EGFR inhibitors. However, imaging approaches affording early identification of tumor response in EGFR-dependent carcinomas have so far been lacking. METHODOLOGY/PRINCIPAL FINDINGS: We performed a systematic comparison of 3'-Deoxy-3'-[(18)F]-fluoro-L-thymidine ([(18)F]FLT) and 2-[(18)F]-fluoro-2-deoxy-D-glucose ([(18)F]FDG) positron emission tomography (PET) for their potential to identify response to EGFR inhibitors in a model of EGFR-dependent lung cancer early after treatment initiation. While erlotinib-sensitive tumors exhibited a striking and reproducible decrease in [(18)F]FLT uptake after only two days of treatment, [(18)F]FDG PET based imaging revealed no consistent reduction in tumor glucose uptake. In sensitive tumors, a decrease in [(18)F]FLT PET but not [(18)F]FDG PET uptake correlated with cell cycle arrest and induction of apoptosis. The reduction in [(18)F]FLT PET signal at day 2 translated into dramatic tumor shrinkage four days later. Furthermore, the specificity of our results is confirmed by the complete lack of [(18)F]FLT PET response of tumors expressing the T790M erlotinib resistance mutation of EGFR. CONCLUSIONS: [(18)F]FLT PET enables robust identification of erlotinib response in EGFR-dependent tumors at a very early stage. [(18)F]FLT PET imaging may represent an appropriate method for early prediction of response to EGFR TKI treatment in patients with NSCLC.

One step generation of fully chimeric antibodies using Cgamma1- and Ckappa mutant mice.

Humanized antibodies (Abs) are effective drugs against a variety of diseases such as cancer, autoimmune diseases, transplant rejection and others. The most powerful technology to develop humanized Abs is the use of mice that produce humanized Abs. By modifying the genetic background of F004 mice a new mouse substrain was developed for optimized "one step" generation of chimeric humanized monoclonal Abs. The new mice (F004-Jen) demonstrated improved fertility still expressing the human locus at the same level as the parental F004 mouse. The value of these mice for the generation of chimeric Abs was exemplified for a panel of chimeric Abs against the human neural cell adhesion molecule (NCAM): The fully chimeric human IgG1/kappa Ab Ch.MK1 bound to NCAM expressing cells with a K(D)=4.3-8.7 x 10(-8) M and was functionally active as demonstrated by depleting NCAM expressing cells. We also demonstrated that chimeric IgG1/kappa Abs can be induced by hybridoma class switching of IgM producing hybridoma cells, providing an alternative way to chimeric Abs. The present data highlight F004-Jen mice as an efficient tool for "one step" generation of chimeric Abs.

Improved technique of heterotopic auxiliary rat liver transplantation with portal vein arterialization.

BACKGROUND AND AIMS: In acute, potentially reversible hepatic failure, auxiliary liver transplantation is a promising alternative approach. Using the auxiliary partial orthotopic liver transplantation (APOLT) method--the orthotopic implantation of auxiliary segments--most of the technical problems (lack of space for the additional liver mass, the portal vein reconstruction, and the venous outflow) are avoided, but extensive resections of the native liver and the graft are necessary. Erhard described the heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA). Initial clinical results demonstrated that an adequate liver function can be achieved using this technique. We developed and improved a technique of HALT with flow-regulated PVA in the rat to perform further investigations. The aim of this paper is to explain in detail this improved experimental surgical technique. MATERIALS AND METHODS: Liver transplantations were performed in 122 male Lewis rats: After a right nephrectomy, the liver graft, which was reduced to about 30% of the original size, was implanted into the right upper quadrant of the recipient's abdomen. The infrahepatic caval vein was anastomosed end-to-side. The donor's portal vein was completely arterialized to the recipient's right renal artery in stent technique. Using a stent with an internal diameter of 0.3 mm, the flow in the arterialized portal vein was regulated to achieve physiologic parameters. The celiac trunk of the graft was anastomosed to the recipient's aorta, end-to-side. The bile duct was implanted into the duodenum. RESULTS: After improvements of the surgical technique, we achieved a perioperative survival of 90% and a 6-week survival of 80% in the last 112 transplantations. CONCLUSION: We developed a standardized and improved technique, which can be used for experiments of regeneration and inter-liver competition in auxiliary liver transplantation. Furthermore, this technique is suitable for the investigation of the influence of portal vein arterialization and portal hyperperfusion on liver microcirculation, function, and morphology.

Auxiliary liver transplantation with flow-regulated portal vein arterialization offers a successful therapeutic option in acute hepatic failure--investigations in heterotopic auxiliary rat liver transplantation.

Heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) was proposed in acute hepatic failure (AHF). However, clinical results of PVA are controversial because of lacking standardized flow-regulation. In rats, we examined HALT with flow-regulated PVA in AHF. Group A: HALT with flow-regulated PVA and 85% resection of the native liver to induce AHF [acute experiments (n = 8), killing after 7 days (n = 8) and after 6 weeks (n = 11)]. Group B: 85% liver-resection (n = 10). The average blood-flow in the arterialized portal vein in HALT achieved normal values (1.7 +/- 0.4 ml/min/g liver-weight). After reperfusion, the diameters of the sinusoids (6.4 +/- 0.6 microm), the postsinusoidal venules (31.1 +/- 3.3 microm) and the intersinusoidal distance (17.9+/-0.7 microm) also achieved normal values. The functional sinusoidal density amounted to 335 +/- 48/cm. The 6-week survival was nine of 11 with excellent liver function (Quick's value: 110% +/- 7.8%). The hepatobiliary radioisotope scanning with (99mTc) ethyl hepatic iminodiacetic acid (EHIDA) showed no significant differences between the native livers and grafts. The hepatocellular morphology was regular, apart from low-grade necroses in two grafts. The grafts' sinusoidal endothelial cells did not show any morphological changes. In group B, however, all rats died from AHF within 6 days. HALT with flow-regulated PVA achieved good results regarding microcirculation, morphology and function and can reliably bridge AHF.

Physiologic microcirculation of the heterotopically transplanted rat liver with portal vein arterialization depending on optimal stent diameter.

BACKGROUND: Clinical experience with portal vein arterialization (PVA) in liver transplantation is controversial. One reason for this is the lack of standardized flow regulation. The present experiments aimed to establish flow regulation in the arterialized portal vein for heterotopic auxiliary liver transplantation (HALT), to obtain physiological portal blood flow, and to compare this technique with orthotopic liver transplantation. MATERIAL/METHODS: Lewis rats were divided into 7 groups (n = 8 transplantations/group). Group: A I-IV: In HALT, the portal vein was anastomosed to the right renal artery using stents with different diameters (0.2, 0.3, 0.4, 0.5 mm). Afterwards, HALT with PVA using the stent diameter that had achieved the most physiological portal blood flow (group B II) was compared with orthotopic liver transplantation with porto-portal anastomosis (group B III) and to the sham group (B I). RESULTS: After reperfusion, only the 0.3 mm stent resulted in an average blood flow in the arterialized portal vein in HALT which was within the normal range (1.7+/-0.4 ml/min/g liver weight). The parameters of microcirculation and early graft function were significantly better in group B II than in group B III (functional sinusoidal density: 335+/-48 vs. 224+/-31/cm, diameter of sinusoids: 6.4+/-0.6 vs. 5.2+/-0.6 microm, diameter of postsinusoidal venules: 31.1+/-3.3 vs. 25.5+/-2.0 microm, bile-production: 27+/-8 vs. 19+/-5 microl/h/g liver weight). CONCLUSIONS: Using an optimal stent diameter in HALT with portal vein arterialization, an adequate flow-regulation can be achieved. Avoiding portal hyper- and hypoperfusion, good results for microcirculation and early graft function can be obtained.

Magnetic resonance imaging in an orthotopic rat model: blockade of epidermal growth factor receptor with EMD72000 inhibits human pancreatic carcinoma growth.

The purpose of our research was to investigate the antiangiogenic effect of the epidermal growth factor receptor monoclonal antibody (anti-EGF-R MAB) EMD72000, in an orthotopic human pancreatic carcinoma model in rats, assessed by magnetic resonance (MR) imaging using angiogenic surrogate markers in comparison with histopathologic findings. Human pancreatic adenocarcinoma cells L3.6pl were injected orthotopically in the pancreas of 12 athymic nude rats. Through a 21-day course, groups of 6 rats were treated intraperitoneally with either EMD72000 or with saline solution for control animals. Dynamic contrast-enhanced MR imaging was performed before and after the treatment to assess microvascular permeability, estimated by the endothelial transfer coefficient (KPS) and fractional plasma volumes (fPV) of the pancreatic tumors. EMD72000-treated animals showed significantly less tumor volume progression (1,080 mm3 +/- 1,244; p = 0.012) and significantly lower values for microvascular permeability (KPS = 4.2 ml min(-1) 100 ml(-1) of tissue +/- 2.8; p = 0.015), fractional plasma volume (fPV = 0.018 ml ml(-1) of tissue +/- .015; p = 0.003) and microvessel density (MVD = 13 +/- 4 (0.159 mm2); p = 0.001) than saline-treated animals (6,544 mm3 +/- 5,202; 9.5 ml min(-1) 100 ml(-1) of tissue +/- 4.3, 0.056 ml ml(-1) of tissue +/- 0.019 and 25 +/- 5 (0.159 mm2), respectively). KPS and fPV values showed moderate positive correlation with MVD (r = 0.5, p = 0.103; r = 0.6, p = 0.065, respectively). Intraperitoneal injection of EMD72000 inhibits orthotopic human pancreatic carcinoma growth in rats. Antiangiogenic effects of anti-EGF-R MAB EMD72000 can be quantified and monitored noninvasively by dynamic MR imaging.

Mutational analysis of the IkappaBalpha gene in activated B cell-like diffuse large B-cell lymphoma.

The lymphoma cells of the activated B cell-like (ABC-) subtype of diffuse large B-cell lymphoma (DLBCL) show constitutive activity of the transcription factor, nuclear factor kappaB (NFkappaB). We sought to determine whether mutations in the IkappaBalpha gene - the predominant inhibitor of NFkappaB - might play a role in the pathogenesis of ABC-DLBCL. All exons of the IkappaBalpha gene were directly sequenced from 10 cases of immunohistochemically classified ABC-DLBCL and from six non-ABC-DLBCL cases. Two novel polymorphisms were identified, based on their presence in tumour as well as non-tumour DNA of the respective patients: a duplication near the transcriptional start and a single nucleotide exchange in exon 1. A somatic missense mutation was identified in exon 3, in addition to a wild-type sequence in only one ABC-DLBCL case. Thus, also in this case no clonal biallelic inactivating mutation was present in the IkappaBalpha gene. We conclude that mutations in the IkappaBalpha gene do not play a dominant role in the pathogenesis of ABC-DLBCL.

Improved microcirculation of a liver graft by controlled portal vein arterialization.

BACKGROUND: The clinical results of portal vein arterialization (PVA) in liver transplantation are controversial without a standardized portal flow regulation. The aim of these experiments was to perform a flow-regulated PVA in liver transplantation, to examine the microcirculation and early graft function after heterotopic auxiliary liver transplantation (HALT) with flow-regulated PVA, and to compare this technique with HALT with porto-portal anastomosis. Using the recently developed orthogonal polarization spectral (OPS) imaging, for the first time the microcirculation of liver grafts with PVA was visualized. MATERIALS AND METHODS: HALT was performed in Lewis rats. The portal vein was either completely arterialized via the right renal artery in a standardized splint-technique (Group I, n = 8) or anastomosed end-to-end to the recipient's portal vein (Group II, n = 8). RESULTS: After reperfusion, the average blood flow in the portal vein was within the normal range in Group I (1.7 +/- 0.4 ml/min/g liver weight) and significantly higher than in Group II (1.2 +/- 0.2 ml/min/g liver weight). The functional sinusoidal density in Group I (335 +/- 48/microm) was significantly higher than in Group II (232 +/- 58/microm), whereas the diameter of the sinusoids and the postsinusoidal venules yielded no significant differences between both groups. The bile production was comparable (27 +/- 8 versus 29 +/- 11 microl/h/g liver weight). CONCLUSIONS: In our experiments it was possible to achieve an adequate flow regulation in the arterialized portal vein with good results concerning microcirculation and early graft function. We recommend that further investigations on liver transplantation with PVA should be performed with portal flow regulation, before PVA is employed in clinical transplantation.