Role of the pathologist in organ transplantation: the North Italy Transplant program experience.

The North Italy Transplant program (NITp) is one of the three organ exchange organizations in Italy together with AIRT and OCST, supervised by the Centro Nazionale Trapianti. It started its activity on June 18, 1972 and serves an area of about 18 million inhabitants in northern Italy. From June 18, 1972 to December 31, 2004, 5761 cadaveric donors have been used and 18,390 transplants performed in the NITp. At December 31, 2004, the NITp waiting list included 3407 patients (2261 kidney, 425 heart, 387 liver, 153 pancreas, 181 lung). From January 1 to August 31, 2005, 13 donors with cancer were used, namely, 4.2% of the overall number of procured donors. The yearly projection of this figure is more than twofold above that in the previous year. Pathologists play a crucial role in NITp activity, by assessing donor suitability and organ quality, by performing the autopsy control of donors, and by participating in transplant follow-up. In addition the pathologist responsible for the Veneto-centralized pathology unit plays the role of expert for second opinion for the NITp area. Pathologists are involved in expanding the pool of donors by analyzing organ biopsies in specific programs. Eight HBV(+) and/or HCV(+) liver biopsies have been evaluated during 2003 and 18 during 2004 and 12 livers, according to the protocol, were suitable for transplantation, and 14 double kidney transplantations were performed in 2003 and 35 in 2004.

Ferredoxin reductase affects p53-dependent, 5-fluorouracil-induced apoptosis in colorectal cancer cells.

Loss of p53 gene function, which occurs in most colon cancer cells, has been shown to abolish the apoptotic response to 5-fluorouracil (5-FU). To identify genes downstream of p53 that might mediate these effects, we assessed global patterns of gene expression following 5-FU treatment of isogenic cells differing only in their p53 status. The gene encoding mitochondrial ferredoxin reductase (protein, FR; gene, FDXR) was one of the few genes significantly induced by p53 after 5-FU treatment. The FR protein was localized to mitochondria and suppressed the growth of colon cancer cells when over-expressed. Targeted disruption of the FDXR gene in human colon cancer cells showed that it was essential for viability, and partial disruption of the gene resulted in decreased sensitivity to 5-FU-induced apoptosis. These data, coupled with the effects of pharmacologic inhibitors of reactive oxygen species, indicate that FR contributes to p53-mediated apoptosis through the generation of oxidative stress in mitochondria.

Secreted and cell surface genes expressed in benign and malignant colorectal tumors.

Serial analysis of gene expression was used to identify transcripts encoding secreted or cell surface proteins that were expressed in benign and malignant tumors of the colorectum. A total of 290,394 tags were analyzed from normal, adenomatous, and cancerous colonic epithelium. Of the 21,343 different transcripts observed, 957 were found to be differentially expressed between normal tissue and adenoma or between normal tissue and cancer. Forty-nine transcripts were elevated > or =20-fold in adenomas, 40 transcripts were elevated > or =20-fold in cancers, and 9 transcripts were elevated > or =20-fold in both. Products of six of these nine transcripts (TGFBI, LYS, RDP, MIC-1, REGA, and DEHL) were predicted to be secreted or to reside on the cell surface, and these were analyzed in more detail. The abnormal expression levels predicted by serial analysis of gene expression were confirmed by quantitative PCR analyses of each of these six genes. Moreover, the cell types responsible for the elevated expression were identified by in situ hybridization and by PCR analyses of epithelial cells immunoaffinity purified from primary tumors. This study extends knowledge of the differences in gene expression that underlie various stages of neoplasia and suggests specific diagnostic approaches that may be useful for the early detection of colorectal neoplasia.

A phosphatase associated with metastasis of colorectal cancer.

To gain insights into the molecular basis for metastasis, we compared the global gene expression profile of metastatic colorectal cancer with that of primary cancers, benign colorectal tumors, and normal colorectal epithelium. Among the genes identified, the PRL-3 protein tyrosine phosphatase gene was of particular interest. It was expressed at high levels in each of 18 cancer metastases studied but at lower levels in nonmetastatic tumors and normal colorectal epithelium. In 3 of 12 metastases examined, multiple copies of the PRL-3 gene were found within a small amplicon located at chromosome 8q24.3. These data suggest that the PRL-3 gene is important for colorectal cancer metastasis and provide a new therapeutic target for these intractable lesions.

Genes expressed in human tumor endothelium.

To gain a molecular understanding of tumor angiogenesis, we compared gene expression patterns of endothelial cells derived from blood vessels of normal and malignant colorectal tissues. Of over 170 transcripts predominantly expressed in the endothelium, 79 were differentially expressed, including 46 that were specifically elevated in tumor-associated endothelium. Several of these genes encode extracellular matrix proteins, but most are of unknown function. Most of these tumor endothelial markers were expressed in a wide range of tumor types, as well as in normal vessels associated with wound healing and corpus luteum formation. These studies demonstrate that tumor and normal endothelium are distinct at the molecular level, a finding that may have significant implications for the development of anti-angiogenic therapies.

Chlorhexidine lavage in the treatment of experimental intra-abdominal infection.

HYPOTHESIS: Closed postoperative peritoneal lavage (CPPL) with chlorhexidine gluconate reduces the number of intraperitoneal bacteria and improves the outcome of intra-abdominal infection. DESIGN: Laboratory animal trial. INTERVENTIONS: Intra-abdominal infection was produced in mice by the cecal ligation and puncture technique. After 16 to 18 hours, the animals underwent relaparotomy and placement of an intra-abdominal catheter for CPPL. In the first experiment animals were randomly divided into 4 groups: no lavage (served as a control), CPPL with chlorhexidine. CPPL with cefoxitin, and CPPL with lactated Ringer solution (LR). Lavage was continued intermittently every 8 hours for 24 hours. All animals received systemic cefoxitin every 8 hours for 7 days. Mortality was recorded every 8 hours for 10 days. In the second experiment, animals were divided into 3 groups: no lavage (served as a control), CPPL with chlorhexidine, and CPPL with LR. Lavage was continued intermittently every 8 hours for 24 hours. The animals were killed 48 hours after reoperation. Bacterial counts from peritoneal fluid and biopsy specimens, as well as peritoneal white blood cell counts, were measured before and after lavage. RESULTS: Closed postoperative peritoncal lavage with chlorhexidine reduced mortality from 71% in a control group to 37% (P = .003). There was no survival benefit in either the CPPL with cefoxitin (91% mortality) (P = .14) or CPPL with LR groups (90% mortality) (P = .17). The statistically significant findings of analysis of variance evaluation demonstrated a decrease in bacterial counts after cecal excision in all 3 groups. There was a greater reduction in bacterial counts in the chlorhexidine group compared with the control group (P<.05). Bacterial counts decreased in peritoneal fluid, as well as in tissue biopsy specimens, after cecal excision. White blood cell counts significantly decreased after cecal excision in all 3 groups. There was no difference in white blood cell counts between the groups. Correlation analyses demonstrated weak interaction between bacterial and white blood cell counts before or after treatment in all the groups. Pearson r ranged from -0.37 to +0.35, none of which were statistically significant. CONCLUSIONS: In our experiments chlorhexidine lavage resulted in a 50% reduction in mortality and a significant reduction in bacterial counts compared with the control group. There was no survival benefit from lavage with either cefoxitin or LR. There was no reduction in bacterial counts in the LR group relative to the control group. Thus, the survival benefit and the reduction in bacterial numbers are attributed to the antibacterial properties of chlorhexidine rather than to the mechanical washing of the abdominal cavity. Closed postoperative peritoneal lavage with 0.05% chlorhexidine gluconate might be useful in the multimodal treatment of intra-abdominal infection.

Identification of CDK4 as a target of c-MYC.

The prototypic oncogene c-MYC encodes a transcription factor that can drive proliferation by promoting cell-cycle reentry. However, the mechanisms through which c-MYC achieves these effects have been unclear. Using serial analysis of gene expression, we have identified the cyclin-dependent kinase 4 (CDK4) gene as a transcriptional target of c-MYC. c-MYC induced a rapid increase in CDK4 mRNA levels through four highly conserved c-MYC binding sites within the CDK4 promoter. Cell-cycle progression is delayed in c-MYC-deficient RAT1 cells, and this delay was associated with a defect in CDK4 induction. Ectopic expression of CDK4 in these cells partially alleviated the growth defect. Thus, CDK4 provides a direct link between the oncogenic effects of c-MYC and cell-cycle regulation.

DBA/2J (Mls-1a) B-cell differentiation in BALB.xid recipients.

Studies of superantigens (SAg) have focused primarily on their impact on CD4+ T cells, largely bypassing the impact of the sequelae of this interaction upon the antigen-presenting cell (APC). Sequelae of SAg-induced CD4+ T-cell activation include the 'bathing' of the SAg-presenting cell with cytokines that promote the differentiation of the APC. In this report, the SAg-induced differentiation of Mls+ DBA/2J B cells was studied in vivo by their transplantation into B-cell-defective BALB.xid recipients. Rapid, high-level serum immunoglobulin M (IgM) production was noted shortly after transfer, disappearing by 3 weeks. Donor B cells, as evidenced after their chemical and genetic impairment and by the use of an IgM allotype-disparate donor-recipient combination, contributed to this transient IgM production. These results clarify a discrepancy in the literature regarding donor B-cell contribution to IgM production and illustrate a model system to utilize SAg to study B-lymphocyte diversity.

Superantigen disruption of CD8+ T and B lymphocyte homeostasis.

Superantigens (SAgs) activate TH cells and induce their differentiation into cytokine-producing effector cells. Supranormal cytokine production is characteristic of SAg-induced polyclonal TH activation. Study of this interaction has focused upon TH cell function to the relative exclusion of other lymphocyte populations. SAgs also impact cells dependent upon TH cells for their differentiation and disrupt the normal homeostasis of the immune system. In this report, several changes in lymphocyte biology that result from SAg activation of TH cells are described. SCID mice, reconstituted with the SAg-expressing cells of DBA/2J mice, were employed as secondary recipients of SAg-reactive TH cells. Significant increases in serum IgM and IgG2a production were noted after the transfer of SAg-reactive It cells. Both B and CD8 T lymphocyte numbers increased with those of CD8 T cells surpassing levels found in normal mice. These results illustrate the ability of the TH-SAg interaction to disrupt B and CD8+ T lymphocyte homeostasis.

Identification and classification of p53-regulated genes.

Sequence-specific transactivation by p53 is essential to its role as a tumor suppressor. A modified tetracycline-inducible system was established to search for transcripts that were activated soon after p53 induction. Among 9,954 unique transcripts identified by serial analysis of gene expression, 34 were increased more than 10-fold; 31 of these had not previously been known to be regulated by p53. The transcription patterns of these genes, as well as previously described p53-regulated genes, were evaluated and classified in a panel of widely studied colorectal cancer cell lines. "Class I" genes were uniformly induced by p53 in all cell lines; "class II" genes were induced in a subset of the lines; and "class III" genes were not induced in any of the lines. These genes were also distinguished by the timing of their induction, their induction by clinically relevant chemotherapeutic agents, the absolute requirement for p53 in this induction, and their inducibility by p73, a p53 homolog. The results revealed substantial heterogeneity in the transcriptional responses to p53, even in cells derived from a single epithelial cell type, and pave the way to a deeper understanding of p53 tumor suppressor action.

Increased serum IgG1 levels and reduced numbers of B-1 B cells in DBA/2J mice.

B-cell heterogeneity studies have historically focused upon BALB/c mice and their derivatives. In contrast, the B cells of DBA/2J mice, a prototype strain for the study of the endogenous minor lymphocyte stimulatory (Mls) viral superantigen Mls-1a, have not been extensively investigated. DBA/2J B cells, by functioning as Mls-1a antigen-presenting cells, influence their own differentiation and diversity by inducing the proliferation and differentiation of specific CD4 T-cell subsets. In this report, the B cells of DBA/2J and BALB/c mice were compared for their ability to restore B-cell function in severe combined immunodeficient (SCID) recipients. Although spleen and bone marrow cells from these strains exhibited similar restoration of serum IgM production, the transfer of DBA/2J B cells into SCID mice led to greater IgG1 production. The peritoneal cells of DBA/2J mice consisted of a lower percentage of B-1 B cells and were less capable of restoring B-cell function after transfer into SCID recipients. These differences are discussed with respect to the possible role of viral superantigens in influencing B-lymphocyte diversity.

Identification of c-MYC as a target of the APC pathway.

The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. Here, the c-MYC oncogene is identified as a target gene in this signaling pathway. Expression of c-MYC was shown to be repressed by wild-type APC and activated by beta-catenin, and these effects were mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers.