Effect of donor-specific blood transfusions on allotransplanted teeth in a monkey model: Histoquantification of periodontal healing.

BACKGROUND/AIMS: Pre-transplant blood transfusions have previously shown a positive effect on organ allograft survival in humans and various animal species. The aims of this study were, first, to evaluate the effect of pre-transplant donor-specific blood transfusions on periodontal healing of fully developed allotransplanted teeth in monkeys; and second, to investigate the immune response against donor antigens and to determine a possible correlation between alloimmune reactions and histopathological signs of rejection. MATERIAL AND METHODS: Twenty monkeys (Cercopithecus aethiops) were divided into ten pairs with similar sizes of incisors. One monkey in each pair gave three transfusions to the other monkey with 1-week intervals. One week after the last transfusion, each pair exchanged simultaneously a central maxillary incisor and a lateral mandibular incisor. The corresponding central maxillary and mandibular lateral incisors were autotransplanted to the contralateral sockets. All teeth were treated endodontically per-operatively. Histocompatibility was evaluated by mixed lymphocyte culture before the first transfusion, while alloantibodies and cell-mediated alloresponses were measured before transfusions and at 2 and 8 weeks after transplantation. All animals were sacrificed 8 weeks after tooth transplantation. Serial sections of the transplanted teeth were quantified histologically. RESULTS: Mixed lymphocyte cultures showed positive reactions in 19 of 20 cases, indicating incompatibility. The majority of the monkeys developed antibodies towards the tooth donor. Cell-mediated cytotoxicity was negative in all monkeys. Histoquantification revealed a mean score of 70% normal periodontal ligament (PDL) in autotransplanted teeth, with 5% ankylosis. The allografts had a mean score of 17% normal PDL and 63% ankylosis, with no significant influence of transfusion. However, in the mandibular grafts, a tendency towards a positive transfusion effect was seen. CONCLUSIONS: Donor-specific blood transfusion did not reduce ankylosis in tooth allografts. The healing of mandibular incisor tooth allografts was improved, but not that of maxillary incisors. Donor-specific antibodies showed no effect on the survival of allograft PDL.

Mechanisms of human smooth muscle cell proliferation and transplant vasculopathy induced by HLA class I antibodies: in vitro and in vivo studies.

Vascular smooth muscle cells (SMC) play an important role in the pathophysiology of transplant vasculopathy (TV), a major cause of late death in patients receiving an organ transplant. In this review we describe the proliferative effect in vitro and in vivo of HLA class I antibodies on human SMC. We have developed an experimental model using segments of human mesenteric arteries transplanted in the position of the infrarenal aorta in immunodeficient mice (SCID/beige). Weekly injections of transplanted mice with a monoclonal antibody towards HLA class I provoked typical lesions of TV after 6 weeks in the human graft while transplanted mice receiving an irrelevant antibody did not develop any significant lesion. In vitro, the anti-HLA antibodies were mitogenic to SMC and we showed that they activate a stress-induced signaling pathway implicating matrix metalloproteinases (MMP) and neutral sphingomyelinase 2 (nSMase-2). The proliferative effect of anti-HLA antibodies could be blocked by pharmacological inhibitors or by siRNA. Administration of pharmacological inhibitors diminished the development of TV in grafted mice injected with anti-HLA antibodies demonstrating an important role of the MMP/nSMase-2 pathway in antibody-induced TV. This observation opens new perspectives for the management of TV in clinical settings.

A key role for matrix metalloproteinases and neutral sphingomyelinase-2 in transplant vasculopathy triggered by anti-HLA antibody.

BACKGROUND: Outcomes for organ transplantation are constantly improving because of advances in organ preservation, surgical techniques, immune clinical monitoring, and immunosuppressive treatment preventing acute transplant rejection. However, chronic rejection including transplant vasculopathy still limits long-term patient survival. Transplant vasculopathy is characterized by progressive neointimal hyperplasia leading to arterial stenosis and ischemic failure of the allograft. This work sought to decipher the manner in which the humoral immune response, mimicked by W6/32 anti-HLA antibody, contributes to transplant vasculopathy. METHODS AND RESULTS: Studies were performed in vitro on cultured human smooth muscle cells, ex vivo on human arterial segments, and in vivo in a model consisting of human arterial segments grafted into severe combined immunodeficiency/beige mice injected weekly with anti-HLA antibodies. We report that anti-HLA antibodies are mitogenic for smooth muscle cells through a signaling mechanism implicating matrix metalloproteinases (MMPs) (membrane type 1 MMP and MMP2) and neutral sphingomyelinase-2. This mitogenic signaling and subsequent DNA synthesis are blocked in smooth muscle cells silenced for MMP2 or for neutral sphingomyelinase-2 by small interfering RNAs, in smooth muscle cells transfected with a vector coding for a dominant-negative form of membrane type 1 MMP, and after treatment by pharmacological inhibitors of MMPs (Ro28-2653) or neutral sphingomyelinase-2 (GW4869). In vivo, Ro28-2653 and GW4869 reduced the intimal thickening induced by anti-HLA antibodies in human mesenteric arteries grafted into severe combined immunodeficiency/beige mice. CONCLUSIONS: These data highlight a crucial role for MMP2 and neutral sphingomyelinase-2 in vasculopathy triggered by a humoral immune response and open new perspectives for preventing transplant vasculopathy with the use of MMP and neutral sphingomyelinase inhibitors, in addition to conventional immunosuppression.

Evaluation of 4-[18F]fluorobenzoyl-FALGEA-NH2 as a positron emission tomography tracer for epidermal growth factor receptor mutation variant III imaging in cancer.

INTRODUCTION: This study describes the radiosynthesis, in vitro and in vivo evaluation of the novel small peptide radioligand, 4-[(18)F]fluorobenzoyl-Phe-Ala-Leu-Gly-Glu-Ala-NH(2,) ([(18)F]FBA-FALGEA-NH(2)) as a positron emission tomography (PET) tracer for imaging of the cancer specific epidermal growth factor receptor (EGFR) variant III mutation, EGFRvIII. METHODS: For affinity, stability and PET measurements, H-FALGEA-NH(2) was radiolabelled using 4-[(18)F]fluorobenzoic acid ([(18)F]FBA). The binding affinity of ([(18)F]FBA)-FALGEA-NH(2) was measured on EGFRvIII expressing cells, NR6M. Stability studies in vitro and in vivo were carried out in blood plasma from nude mice. PET investigations of [(18)F]FBA-FALGEA-NH(2) were performed on a MicroPET scanner, using seven nude mice xenografted subcutaneously with human glioblastoma multiforme (GBM) tumours, expressing the EGFRvIII in its native form, and five nude mice xenografted subcutaneously with GBM tumours lacking EGFRvIII expression. Images of [(18)F]FDG were also obtained for comparison. The mice were injected with 5-10 MBq of the radiolabelled peptide or [(18)F]FDG. Furthermore, the gene expression of EGFRvIII in the tumours was determined using quantitative real-time PCR. RESULTS: Radiolabelling and purification was achieved within 180 min, with overall radiochemical yields of 2.6-9.8% (decay-corrected) and an average specific radioactivity of 6.4 GBq/µmol. The binding affinity (K(d)) of [(18)F]FBA-FALGEA-NH(2) to EGFRvIII expressing cells was determined to be 23 nM. The radiolabelled peptide was moderately stable in the plasma from nude mice where 53% of the peptide was intact after 60 min of incubation in plasma but rapidly degraded in vivo, where no intact peptide was observed in plasma 5 min post-injection. The PET imaging showed that [(18)F]FBA-FALGEA-NH(2) accumulated preferentially in the human GBM xenografts which expressed high amounts of the mutated receptor. The average tumour-to-muscle ratio (T/M) in the EGFRvIII tumours was 7.8 at 60 min post-injection, compared with 4.6 in the wild-type EGFR tumours. Furthermore, there was a strong correlation (R=0.86, P=.007) between the expression of EGFRvIII in the tumours and the tracer uptake expressed as T/M. CONCLUSION: Our results indicate that, despite its rapid metabolism, [(18)F]FBA-FALGEA-NH(2) binds preferentially to EGFRvIII in the tumours in vivo and is a promising lead for further development of EGFRvIII specific peptide radiopharmaceuticals.

Maintenance of EGFR and EGFRvIII expressions in an in vivo and in vitro model of human glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common, and most aggressive primary brain tumor among adults. A vast majority of the tumors express high levels of the epidermal growth factor receptor (EGFR) as a consequence of gene amplification. Furthermore, gene amplification is often associated with mutation of EGFR, and the constitutive activated deletion variant EGFRvIII is the most common EGFR mutation found in GBM. Activated EGFR signaling, through overexpression and/or mutation, is involved in increased tumorigenic potential. As such, EGFR is an attractive target for GBM therapy. However, clinical studies with EGFR inhibitors have shown inconsistent results, and as such, further knowledge regarding the role of EGFR and EGFRvIII in GBM is needed. For this, an appropriate in vivo/in vitro tumor model is required. Here, we report the establishment of an experimental GBM model in which the expressions of EGFR and EGFRvIII are maintained both in xenograft tumors growing subcutaneously on mice and in cell cultures established in stem cell conditions. With this model it will be possible to further study the role of EGFR and EGFRvIII, and response to targeted therapy, in GBM.

Apolipoprotein E-deficient mice develop an anti-Chlamydophila pneumoniae T helper 2 response and resist vascular infection.

BACKGROUND: Hypercholesterolemia could inhibit the immune response against various pathogens. No information is available about its impact on the immune response toward Chlamydophila pneumoniae. METHODS: Apolipoprotein E (apoE)-deficient and wild-type mice fed a normal diet were infected with a single intranasal inoculation of viable C. pneumoniae. RESULTS: Whereas interferon gamma concentrations (T helper 1 response) were similar in the lungs and spleen of apoE-deficient and wild-type mice, increased concentrations of interleukin 10, interleukin 6, and interleukin 4 (T helper 2 response) were found in the lungs of apoE-deficient mice. The spleen B lymphocyte percentage and interleukin 4 levels and serum specific antibody titers were higher in apoE-deficient mice. C. pneumoniae infection was facilitated neither in the lungs nor in the aorta of these mice. On the contrary, the number of apoE-deficient mice with detectable levels of bacterial DNA in the aorta was clearly decreased. When low-density lipoprotein receptor-deficient mice fed a normal diet were similarly infected, no difference in the interleukin 4 concentration and infection level was observed in the lungs and no protection was found in the aorta. CONCLUSIONS: Mild hypercholesterolemia in mice does not facilitate C. pneumoniae persistence in the vascular wall. ApoE deficiency, rather than mild hypercholesterolemia, probably favors the development of an unusual anti-C. pneumoniae T helper 2 response and protects against vascular infection.

Polyoma BK virus-associated nephropathy in kidney-transplant patients: Effects of leflunomide on T-cell functions and disease outcome.

BACKGROUND: In kidney-transplant recipients, leflunomide has been shown to be efficient for treating polyomavirus BK virus-associated-nephropathy (PVAN). However, it is unknown whether the beneficial effect of leflunomide is related to it having a lower immunosuppressive effect than mycophenolate mofetil (MMF), or to its anti-viral activity. The aim of this study was to assess i) T-cell functions before and after conversion from MMF to leflunomide in kidney-transplant patients with PVAN, and ii) effects of leflunomide on PVAN outcome. PATIENTS AND METHODS: Twelve patients were enrolled in this study. At PVAN diagnosis, MMF was replaced by leflunomide. Other immunosuppressive drug doses and levels were maintained unchanged. T-cell functions, i.e., intralymphocyte cytokine expression (IL-2 and TNF-alpha), T-cell activation [i.e., transferrin receptor (CD71) and interleukin (IL)-2 alpha-chain (CD25) expression], and T-cell proliferation were measured using a flow-cytometry whole-blood assay before and at one month after conversion. RESULTS: Despite a slight decrease in tacrolimus trough levels, no significant change in T-cell-function biomarkers was observed after conversion. After a follow-up of 6 (4-30) months, five patients were cleared of the virus, and decreased viral load was observed in four patients. Only one patient suffered a graft loss. No difference in immunological parameters was observed between patients who were cleared or not of BKV. CONCLUSION: Results of this pilot study suggest that the potential benefits of leflunomide to treat PVAN in kidney-transplant patients is not related to reduced immunosuppression induced by replacing MMF by leflunomide. Virological studies are required to determine the anti-BKV effect of leflunomide.

Cytokines correlate with age in healthy volunteers, dialysis patients and kidney-transplant patients.

T-cell functions are currently used as biomarkers for the pharmacodynamic monitoring of immunosuppressive drugs or as disease biomarkers of inflammation/sepsis and organ rejection. In order to evaluate co-factors potentially influencing the expression of the immunological biomarkers, we explored T-cell proliferation, T-cell activation (CD25 and CD71 expressions) and intra-lymphocyte cytokine production (interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha) in healthy volunteers, dialysis patients and stable kidney-transplant patients treated with standard immunosuppressive therapy, i.e. tacrolimus, mycophenolic acid with or without steroids. Age was positively correlated with TNF-alpha expression in all three patient populations, and with IL-2 expression in healthy volunteers and kidney-transplant patients. Further age was correlated with inhibition of lymphocyte proliferation in healthy volunteers and with the T-cell activation marker CD25 in kidney-transplant patients. In healthy volunteers lymphocyte proliferation was higher in woman as compared to men. Other biomarkers of T-cell function were independent of the gender. In the kidney-transplant patient group a significantly lower expression of all biomarkers of T-cell functions compared to healthy volunteers and dialysis patients. In dialysis patients we found significant increased IL-2 expression compared to healthy volunteers, while the other T-cell functions were not significantly different. Further time on dialysis had no effect on the level of biomarker expression. In conclusion we found decreased T-cell functions in kidney-transplant patients compared to healthy volunteers and dialysis patients, increased IL-2 expression in dialysis patients compared to healthy volunteers and in all three populations we found a correlation of age and intra-T-lymphocyte TNF-alpha expression.

Interleukin-6 deficiency fails to prevent chronic rejection after aortic allografts in apolipoprotein E-deficient mice.

BACKGROUND: Chronic vascular rejection (CVR) is characterized by an intimal thickening in the arteries of allografts due to immunoinflammatory reactions and smooth muscle cell proliferation. Interleukin 6 (IL-6) levels are increased in patients with graft rejection, however the role of IL-6 in CVR is not known. We investigated if IL-6 deficiency in the recipient could prevent CVR after an aortic allograft. METHODS: Donor aortas from wild-type DBA/2 mice were transplanted into C57BL/6 recipients, either wild-type mice or mice deficient for IL-6 (IL-6(-/-)), apolipoprotein E (ApoE(-/-)), or both (IL-6(-/-)ApoE(-/-)). Alloantibody titers were determined at Day 30, 60, or 90 after grafting. The grafts were examined for CVR lesions by morphometry and immunohistology. RESULTS: All recipient allografts displayed lesions typical for CVR. The lesions were larger in IL-6-deficient strains, and significantly so in IL-6(-/-)ApoE(-/-) recipients. Early immunoglobulin (Ig) G1 alloantibody deposits were observed in the grafts of ApoE-deficient strains and late IgG2a deposits in the grafts of IL-6-deficient strains. A rapid and sustained type 1 helper T cell (Th1; IgG2a) alloresponse in IL-6(-/-) mice, and a strong type 2 helper T cell (Th2; IgG1) response in ApoE(-/-) mice were observed. IL-6(-/-)ApoE(-/-) mice displayed the highest alloantibody titer, with a Th1 dominance. CONCLUSIONS: Unexpectedly, IL-6 deficiency in the recipient mice did not prevent CVR lesions but even aggravated them in IL-6(-/-)ApoE(-/-) recipients. This was associated with increased local and systemic alloresponses.

Thymidine selectively enhances growth suppressive effects of camptothecin/irinotecan in MSI+ cells and tumors containing a mutation of MRE11.

PURPOSE: DNA synthesis inhibitors and damaging agents are widely used in cancer therapy; however, sensitivity of tumors to such agents is highly variable. The response of tumor cells in culture to these agents is strongly influenced by the status of DNA damage response pathways. Here, we attempt to exploit the altered response of mismatch repair (MMR)-deficient colon cancer cells and tumors to camptothecin or irinotecan and thymidine by combining them to improve therapeutic response. EXPERIMENTAL DESIGN: A panel of colon cancer cell lines was assayed for response to camptothecin-thymidine combinations by measuring colony formation, cell cycle distribution, and senescence. Cell strains defective in p53, p21, or Mre11 were used in these assays to investigate the role of these cell cycle regulators. The in vivo antitumor response of xenografts to irinotecan and thymidine combinations was assessed in nude mice. RESULTS: Camptothecin-thymidine combinations suppress colony formation of MMR-deficient tumor cells 10- to 3,000-fold relative to that obtained with camptothecin alone and significantly reduce the concentrations of the agents required to induce late S/G(2) arrest and senescence. Sensitivity is not a direct result of MMR, p53, or p21 status. However MMR-deficient cell lines containing an intronic frameshift mutation of MRE11 show greatest sensitivity to these agents. Increased sensitivity to this combination is also evident in vivo as thymidine enhances irinotecan-induced growth suppression of MMR-deficient tumors carrying the MRE11 mutation in mouse xenografts. CONCLUSION: Irinotecan-thymidine combinations may be particularly effective when targeted to MSI+ tumors containing this readily detectable MRE11 mutation.

In vitro mitogen-stimulated T-cell from hepatitis C virus-positive liver transplantation candidates, increases T-cell activation markers and T-cell proliferation.

The incidence of acute rejection is significantly higher in hepatitis C virus (HCV) liver-transplant patients than in patients who have received a graft for other liver diseases, i.e., mainly alcoholic cirrhosis. The aim of this study was to assess T-cell function, i.e., intralymphocyte cytokine expression (IL-2 and TNF-alpha), T-cell activation [i.e., transferrin receptor (CD71) and interleukin (IL)-2 alpha-chain (CD25) expression], and T-cell proliferation using a flow-cytometry whole-blood assay in patients waiting for a liver transplantation (n=49). Our data suggest that, in mitogen-stimulated T-cells, (i) intra-lymphocyte cytokine expression is significantly higher in patients with liver disease than in healthy volunteers (n=25); (ii) the expression of T-cell activation markers is decreased in patients with liver cirrhosis compared to healthy volunteers, and (iii) the expression of T-cell activation markers and T-cell proliferation are increased in patients with HCV infection (n=15) compared to those without HCV infection (n=34), particularly compared to patients with alcoholic liver disease (n=19). Circulating CD19-positive cells count was also significantly higher in HCV-positive patients. In conclusion, in vitro, mitogen-stimulated T-cell seem to induce a higher immune response in the blood from patients waiting for a liver transplant for HCV-related liver disease than those without HCV infection, and particularly those with alcoholic liver disease.

Pharmacodynamic monitoring of the conversion from mycophenolate mofetil to enteric-coated mycophenolate sodium in stable kidney-allograft recipients.

BACKGROUND: The formulations of mycophenolic acid, i.e., mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS), seem to have different pharmacokinetic profiles. The aim of this study was to compare the effects MMF and EC-MPS on T-cell proliferation, T-cell activation, T-cell function, and lymphocyte subsets. CLINICAL STUDY AND METHODS: Ten stable kidney-transplant patients on standard maintenance therapy of tacrolimus and MMF (1 g/d), with or without steroids, were converted from MMF to EC-MPS at equivalent dose (720 mg/d). Tacrolimus and steroid doses remained unchanged before, and at 1, 2, 3, and 6 months (M) after conversion. Intra T-lymphocyte cytokines IL-2 and TNF-alpha, lymphocyte-activation surface markers (CD25 and CD71), T-cell proliferation (PCNA+ PI(high)), total lymphocyte count, as well as lymphocytes subsets (CD2, CD3, CD4, CD8, CD19, NK cells) were measured by flow cytometry before conversion and at M1, M2, M3, and M6. RESULTS: We found no significant differences of MMF versus EC-MPS on lymphocyte function. T-cell proliferation and T-cell activation (CD25 and CD71 expression), but not cytokine expression (TNF-alpha and IL-2), showed a trend to increase after conversion from MMF to EC-MPS. Total lymphocyte, CD2, CD3, CD4, CD8, and NK cells counts were not significantly modified. CONCLUSION: This study revealed a trend to a lower immunosuppression with EC-MPS as compared to MMF in stable renal transplant patients.

Reconstitution of immunodeficient SCID/beige mice with human cells: applications in preclinical studies.

Experimental studies of the in vivo behaviour of human cells and tissues have become possible with the development of immunodeficient mice strains. Such mice accept readily allogeneic or xenogeneic grafts, including grafts of human cells or tissues, without rejection. In this review we describe different immunodeficient mouse strains that have been used for reconstitution by human immune cells. We subsequently go through the experience that we and others have had with reconstitution, and mention the adverse effects, in particular xenogeneic graft versus host reactions. The use of haematopoietic stem cells avoids such reactions but the immunological reconstitution may take several months. We then report the use of immunodeficient mice for the study of chronic vascular rejection of human mesenteric arteries due to cellular or humoral alloreaction. We have shown that SCID/beige mice grafted with a human artery at the place of the aorta developed a thickening of the intima of the human artery after 5-6 weeks, when they were reconstituted with spleen cells from another human donor. The thickening is mainly due to a proliferation of smooth muscle cells. The same type of lesion developed if they received injection of antibodies towards HLA class I antigens. The arteries of the mouse did not develop any lesion. The arterial lesions closely resembled those seen after clinical organ transplantation. Mice that received spleen cells from the same human donor developed little or no lesions. An important aspect of this experimental transplantation model is the possibility to test drugs that may be used in clinical transplantation. In recent experiments we have shown that novel immunosuppressive drugs can inhibit the hyperproliferation of smooth muscle cells in vitro. Preclinical testing in reconstituted SCID/beige mice grafted with human arteries will permit the evaluation of the potential use of these drugs to prevent chronic vascular rejection. The model also allows pharmacodynamic studies that give information on the biological impact of different drugs that may be used in experimental or clinical transplantation.

Multiple human mesenteric arterial grafts from the same donor to study human chronic vascular rejection in humanized SCID/beige mice.

BACKGROUND: Chronic vascular rejection (CVR) is a major problem in clinical transplantation. Studies in experimental animals have been important to understand some of its mechanisms, but they are hampered by the difficulty of extrapolating the results into clinical practice. METHODS: We created a new experimental model for the study of human CVR by grafting multiple human mesenteric arteries from the same human donor into different SCID/beige mice in the infrarenal aortic position. Twenty-seven different mice were successfully grafted with a human artery from 6 donors. One week later, 23 of the mice received an intraperitoneal injection of 40 million human spleen cells, either from the same donor (autologous) or from another donor (allogeneic). RESULTS: In 81% of the mice an immune reconstitution was obtained, shown by the presence of human T, B and NK cells and IgG in circulating blood. At the time of sacrifice, 5 weeks after the arterial transplantation, a typical CVR with infiltration of human immune cells and deposit of human immunoglobulin was observed in the reconstituted mice that received allogeneic cells, whereas only minor lesions were noted in autologous combinations. No CVR was observed without injection of human splenocytes. We did not observe lymphoma or graft-vs-host reactions during the experiment. CONCLUSIONS: We show that it is feasible to graft multiple human arteries from the same donor into SCID/beige mice, and that a specific and typical CVR is observed after reconstitution with allogeneic spleen cells. Our method allows for pre-clinical testing of new therapeutics in controlled series.

Chlamydia pneumoniae alters mildly oxidized low-density lipoprotein-induced cell death in human endothelial cells, leading to necrosis rather than apoptosis.

BACKGROUND: Atherosclerosis is characterized by oxidative stress that induces lipid and protein oxidation in the vascular wall. Oxidized low-density lipoproteins (oxLDLs) are present in lesions, and one of their actions is to induce apoptosis or necrosis in vascular cells. A role for Chlamydia pneumoniae in atherosclerosis has been proposed, but the mechanisms involved remain largely unknown. METHODS: The in vitro effect of C. pneumoniae infection on apoptosis induced by mildly oxidized LDLs (moxLDLs) in human endothelial cells was studied. RESULTS: Infection inhibited apoptosis, as was demonstrated by a decrease in such apoptotic features as cytochrome c release, caspase activity, 89-kilodalton poly(ADP-ribose) polymerase (PARP) fragment formation, nuclear condensation and fragmentation, and DNA fragmentation. However, the inhibition of apoptosis did not favor cell survival, because infection promoted cell death with necrotic features, as was illustrated by an increase in lactate dehydrogenase release, an enhancement of necrotic cellular morphological characteristics, and generation of low-molecular-mass PARP fragments. The increase in occurrence of necrosis-like cell death was correlated with a strong increase in intracellular reactive oxygen species (ROS) concentration. Vitamin E inhibited ROS production and promoted cell survival, underscoring the involvement of ROS in cell death induced by the combination of C. pneumoniae and moxLDLs. CONCLUSION: C. pneumoniae infection enhances the inflammatory action of oxLDLs in the vascular wall, leading to cell necrosis rather than apoptosis.

Analysis of the epidermal growth factor receptor specific transcriptome: effect of receptor expression level and an activating mutation.

Overexpression or expression of activating mutations of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. The present study employed Affymetrix oligonucleotide arrays to profile genes induced by ligand-activated EGFR with the receptor either moderately expressed or overexpressed at an in-itself transforming level. These changes were compared to those induced by the naturally occurring constitutively active variant EGFRvIII. This study provides novel insight on the activities and mechanisms of EGFRvIII and EGFR mediated transformation, as genes encoding proteins with functions in promoting cell proliferation, invasion, antiapoptosis, and angiogenesis featured prominently in the EGFRvIII- and EGFR-expressing cells. Surprisingly, it was found that ligand-activated EGFR induced the expression of a large group of genes known to be inducible by interferons. Expression of this module was absent in the EGFRvIII-expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor-mediated events. Furthermore, activation of this module correlated with activation of STAT1 and STAT3. The results thus demonstrate that ligand-activated EGFR at different expression levels results in different kinetics of signaling and induction of gene expression. In addition, the constitutively active variant EGFRvIII seems to activate only a subset of signal pathways and induce a subset of genes as compared to the ligand-activated EGFR.

Caspase-10 triggers Bid cleavage and caspase cascade activation in FasL-induced apoptosis.

In contrast to caspase-8, controversy exists as to the ability of caspase-10 to mediate apoptosis in response to FasL. Herein, we have shown activation of caspase-10, -3, and -7 as well as B cell lymphoma-2-interacting domain (Bid) cleavage and cytochrome c release in caspase-8-deficient Jurkat (I9-2) cells treated with FasL. Apoptosis was clearly induced as illustrated by nuclear and DNA fragmentation. These events were inhibited by benzyloxycarbonyl-VAD-fluoromethyl ketone, a broad spectrum caspase inhibitor, indicating that caspases were functionally and actively involved. Benzyloxycarbonyl-AEVD-fluoromethyl ketone, a caspase-10 inhibitor, had a comparable effect. FasL-induced cell death was not completely abolished by caspase inhibitors in agreement with the existence of a cytotoxic caspase-independent pathway. In subpopulations of I9-2 cells displaying distinct caspase-10 expression levels, cell sensitivity to FasL correlated with caspase-10 expression. A robust caspase activation, Bid cleavage, and DNA fragmentation were observed in cells with high caspase-10 levels but not in those with low levels. In vitro, caspase-10, as well as caspase-8, could cleave Bid to generate active truncated Bid (p15). Altogether, our data strongly suggest that caspase-10 can serve as an initiator caspase in Fas signaling leading to Bid processing, caspase cascade activation, and apoptosis.

Engraftment of human T, B and NK cells in CB.17 SCID/beige mice by transfer of human spleen cells.

Models of severe combined immuno-deficient (SCID) mice reconstituted with a competent human immune system represent a valuable tool for the study of human immune responses in vivo. Reconstitution with human cells can be achieved using large numbers of peripheral blood lymphocytes, but levels of engraftment are poor and graft versus host disease (GVHD) frequently occurs. SCID/beige mice are at the same time deficient for adaptive and innate immunity and the objective of this study was to develop a safe and efficient way to achieve human lymphocyte engraftment in these mice using human spleen cells. After institutional authorisations and informed consent of relatives, a piece of spleen was obtained from cadaveric organ donors and the splenocytes were isolated and cryopreserved for later use. Single intraperitoneal injections of 5-100 x10(6) splenocytes were performed into SCID/beige mice. Reconstitution of a human immune system was monitored weekly by the presence of human cells and IgG in peripheral blood. The mice were sacrificed 4 weeks after the injection and the engraftment in lymphoid organs was studied. A reproducible reconstitution was obtained with intraperitoneal injection of 30-40 x10(6) spleen cells. Human T, B and NK cells as well as human IgG were present in peripheral blood. In lymphoid tissues, the same lymphocytic subpopulations were detected and in addition some antigen presenting cells. The reconstitution was functional because graft rejection was observed after transplantation of human allogeneic tissues. When less than 30 x10(6) cells were injected, the reconstitution was variable. When more than 40 x10(6) cells were injected, GVHD occurred with increasing frequency. In conclusion, we show that intraperitoneal injection of 30-40 x10(6) human splenocytes into SCID/beige mice induces a quick and functional engraftment of human T, B and NK cells with no risk of GVHD. This model may be used to study human transplantation immunobiology in vivo.