MicroRNA-192 suppresses liver metastasis of colon cancer.

Metastasis causes most deaths from colon cancer yet mechanistic understanding and therapeutic options remain limited. Here we show that expression of microRNA (miR)-192 is inversely correlated with metastatic potential of colon cancer cells. Ectopic expression of miR-192 sensitizes colon cancer cells to growth factor deprivation stress-induced apoptosis, whereas inhibition of miR-192 confers resistance. Overexpression of miR-192 inhibits metastatic colonization to the liver in an orthotopic mouse model of colon cancer. Alterations associated with the metastatic phenotype in the primary tumors include increased apoptosis, decreased proliferation and angiogenesis. Further studies indicate that miR-192 downregulates expression of Bcl-2, Zeb2 and VEGFA in vitro and in vivo, which is responsible for enhanced apoptosis, increased expression of E-cadherin and decreased angiogenesis in vivo, respectively. Finally, studies performed on human colonic adenocarcinoma show that expression of miR-192 is significantly reduced in neoplastic cells as compared with normal colonic epithelium. Importantly, there is a significant decrease in miR-192 expression in stage IV tumors when compared with stage I or II lesions. These findings indicate that miR-192 has an important role in colon cancer development and progression. Our studies underscore the clinical relevance and prognostic significance of miR-192 expression in colon cancer. Therefore, a major implication of our studies is that restoration of miR-192 expression or antagonism of its target genes (Bcl-2, Zeb2 or VEGFA) may have considerable therapeutic potential for anti-metastatic therapy in patients with colon cancer.

Lipoatrophic diabetes and end-stage liver disease secondary to nonalcoholic steatohepatitis with recurrence after liver transplantation.

BACKGROUND: Lipoatrophic diabetes is an insulin resistance syndrome characterized by the complete or partial lack of adipose tissue and disturbances in lipid and glucose metabolism. Nonalcoholic steatohepatitis (NASH) is a well-described change in liver pathology consisting of steatosis, hepatitis, and fibrosis that can be associated with lipoatrophic diabetes. RESULTS: This article describes the first reported case of lipoatrophic diabetes with NASH leading to liver failure and liver transplantation. Before transplantation, the patient required 600-700 U of insulin/day. After transplantation, a dramatic decline in her insulin requirements was observed, despite corticosteroids. Eighteen months after transplantation, her glycemic control worsened, and she developed recurrent NASH on serial liver biopsies. CONCLUSIONS: NASH associated with lipoatrophic diabetes can recur after liver transplantation, and in this case, was accompanied by increased insulin requirements. These results suggest that the development of NASH itself may contribute to the insulin resistance observed in lipoatrophic diabetes.

Natural DNA mixtures generated in fraternal twins in utero.

Analysis of multiple genetic loci using short tandem repeats (STR) is widely used in human identity testing because the extensive polymorphism at these loci allows for a high degree of discrimination among individuals. We recently received a forensic case that included several pieces of evidence and reference blood samples. Upon initial testing, one of the suspects had a DNA profile that included three alleles at four of the nine loci tested (vWA, FGA, TH01, and D5S818). At each locus, two of the alleles appeared to be "major" alleles with a third "minor" allele present. The profile appeared to be a mixture of two people. Contamination of this first reference sample was suspected and a second, unopened blood specimen was requested from this individual. The DNA profile from this second reference specimen was identical to that of the original specimen at each locus. One of the evidence samples also displayed an identical mixed DNA profile matching that of the reference specimens mentioned above. The relative peak heights of the two "major" and one "minor" allele remained constant in all three samples. Additional background information revealed that the suspect had not received a bone marrow transplant or blood transfusion. However, it was disclosed that this individual is a fraternal (dizygotic) twin. We hypothesize that an exchange of blood cells between the fetuses occurred in utero and that the additional alleles present in these reference samples are derived from cells contributed by his twin sibling. No additional specimens from the suspect or his twin could be obtained for confirmation, and our hypothesis remains untested. Forensic scientists should be aware of this possibility when faced with a DNA profile in which extra alleles at multiple loci are detected.

Loss of heterozygosity detected in a short tandem repeat (STR) locus commonly used for human DNA identification.

Short tandem repeat (STR) markers are commonly used in basic genetic research and in human identification testing. Clinically, STRs can be used to study genetic alterations in tumors. A genetic deletion common to many types of cancer is referred to as the loss of heterozygosity (LOH). Numerous examples of LOH in cancer have been described and some have been mapped to areas located in close proximity to markers employed in human identity testing. Despite this fact, LOH has rarely been observed for STR loci commonly employed in forensic testing. Recently, for medico-legal purposes, we were asked to determine whether a tissue biopsy originated from a particular individual. For a reference source we assessed two specimens, one from normal tissue and one from cancerous tissue. When both reference specimens were used to generate DNA profiles, we observed LOH at one STR locus, D13S317. As demonstrated in other cancers only the cancerous biopsy demonstrated LOH. The forensic community should be cognizant of these unusual circumstances because, as identification of human DNA continues to be used more extensively, certain instances will arise in which reference material will not be readily available. In these situations, archived specimens may be employed as a reference source. Clinical specimens such as tissue biopsies should be used with caution if they have not been confirmed to contain normal tissue.

Acute liver failure associated with prolonged use of bromfenac leading to liver transplantation. The Acute Liver Failure Study Group.

Bromfenac, a nonnarcotic analgesic nonsteroidal anti-inflammatory drug, was associated with reversible, minor elevations in serum aminotransferase levels during clinical trials. The aim of this study is to describe the clinical, laboratory, and histological features of 4 patients with severe bromfenac hepatotoxicity identified at 3 tertiary care centers participating in the US Acute Liver Failure Study Group. Bromfenac was administered for chronic musculoskeletal disorders to 4 women in therapeutic doses of 25 to 100 mg/d for a minimum of 90 days. All patients reported a prodrome of malaise and fatigue and presented with severe, symptomatic hepatocellular injury with associated hypoprothrombinemia. None of the subjects had underlying liver or kidney disease, and there was no evidence of a hypersensitivity reaction. Other identifiable causes of acute liver failure were uniformly excluded. Despite supportive measures, all the subjects developed progressive liver failure over 5 to 37 days, leading to emergency liver transplantation in 3 patients and death in 1 patient while awaiting transplantation. Extensive confluent parenchymal necrosis that appeared to begin in the central zones and was accompanied by a predominantly lymphocytic infiltrate was noted in all the livers examined. Nodular regeneration was seen in the 2 patients with a more protracted clinical course. Administration of therapeutic doses of bromfenac for greater than 90 days was associated with the development of acute liver failure leading to liver transplantation or death in 4 adult women. The poor outcomes observed in this series, coupled with the inability to identify individuals at risk for severe, idiosyncratic bromfenac hepatotoxicity, preclude further use of bromfenac in the medical community.

Correlation of mucosal disaccharidase activities with histology in evaluation of rejection following intestinal transplantation.

Following intestinal transplantation, we have found that recovery from severe rejection may be difficult to identify. In this study we sought to ascertain whether concurrent determination of mucosal disaccharidase activities and histologic assessment improves the accuracy of diagnosis of rejection. Histologic changes were graded blindly using a standard set of diagnostic criteria, and these changes were compared over time to maltase, sucrase, lactase, and palatinase activities in four pediatric patients under treatment for severe rejection. The histologic criteria, which included magnitude of enterocyte loss, degree of granulation tissue, severity of villus atrophy, and frequency of apoptosis and cryptitis, were found to correlate with one another over time irrespective of outcome (r = 0.72 to r = 0.85). Enzyme activities were also correlated with each other over time (r = 0.64 to r = 0.80). However, the correlation between histologic diagnosis and enzyme activity was weaker (r = -0.48 to r = -0.57). Furthermore, neither histologic nor enzyme evaluation early in the course of rejection predicted ultimate clinical outcome. The results of this investigation show that determination of mucosal disaccharidase activity provides no additional useful information concerning efficacy of anti-rejection therapy as compared to histologic analysis alone.

Human leukocyte antigen class II associations in patients with idiopathic dilated cardiomyopathy. Myocarditis Treatment Trial Investigators.

BACKGROUND: Idiopathic dilated cardiomyopathy (IDC) is a disease of unknown etiology for which immune abnormalities, possibly related to viral infections, are suspected but unproven. Previous serologic studies have reported associations between human leukocyte antigen DR4 and IDC. A molecular study of human leukocyte antigen associations was undertaken in patients with IDC to further explore the possibility of susceptibility markers of genetically determined disease. METHODS AND RESULTS: In this study, 36 patients from the Myocarditis Treatment Trial (32 IDC and 4 myocarditis patients) were examined using restriction fragment length polymorphism analysis and polymerase chain reaction amplification with sequence-specific primers to perform class II typing. All 4 myocarditis patients were DQ5 positive and 3 possessed the allele DQB1*0501. In the IDC group, the frequency of human leukocyte antigen DR4 was similar to that reported in the normal population. In addition, there was no excess prevalence of any molecularly defined DR4 alleles (0401-0419). There was an increase in the frequency of DR12 in IDC patients. The frequencies of the alleles DQB1 *0503 and DQB1*0301 and/or *0304 were also increased in IDC patients versus the normal population. CONCLUSION: The molecular studies point to a relationship between the DQ locus and IDC.

Biochemical and functional characterization of soluble multivalent MHC L(d)/Fc gamma 1 and L(d)/Fc mu chimeric proteins loaded with specific peptides.

Central to the specificity of the immune system is the interaction between the T cell receptor and the major histocompatibility complex (MHC)-peptide ligand complex. To better understand the nature of this interaction, and to investigate possible avenues for specific therapeutic intervention, we have produced soluble recombinant molecules that can modulate antigen-specific T cells. Our approach involved the construction of recombinant murine genes composed of the MHC class I gene H-2L(d) and the Fc portion of immunoglobulin (Ig) heavy chain genes mu or gamma1. Stable transfectants of these L(d)/Fc gamma1 and L(d)/Fc mu genes generated correctly spliced transcripts and were capable of secreting chimeric protein. Immunoprecipitation analyses demonstrated the presence of chimeric L(d)/ Fc gamma1 and L(d)/Fc mu monomers of approximately 69 kDa and 90 kDa, respectively, as well as chimeric dimers under nonreducing conditions. The capacity of L(d)/Ig molecules to bind specific peptide ligands was demonstrated using radiolabeled peptides or with monoclonal reagents that specifically identify peptide-induced conformational changes in the L(d) ligand binding site. Soluble divalent L(d)/Fc gamma1 molecules were loaded with the murine cytomegalovirus-derived peptide and other L(d)-specific peptide ligands and subsequently isolated and purified. Peptide-loaded L(d)/Fc gamma1 molecules were capable of inhibiting the response of class I-restricted T cells in vitro in a peptide-specific fashion. The development of soluble multivalent chimeric proteins that possess unique properties of both the MHC class I and Ig molecules provides a valuable reagent for the study of potential mechanisms of in vitro and in vivo immune modulation.

Experience with protocol biopsies after solitary pancreas transplantation.

The early detection of allograft rejection remains elusive after solitary pancreas transplantation (PTX). We have previously described a modified technique of cystoscopic transduodenal PTX biopsy using the Biopty gun under ultrasound guidance. During the last 2 years, we performed 24 solitary PTXs with prospective protocol biopsy monitoring as well as biopsies performed whenever clinically indicated. The study group included 17 pancreas transplants alone, 6 sequential pancreas after kidney transplants, and 1 sequential pancreas after liver transplant. Five patients received pancreas retransplants. A total of 92 cystoscopically directed core PTX biopsies were performed, including 50 protocol biopsies (mean 2.1 per patient). Protocol biopsies were performed at 1 month (19), 2 months (3), 3 months (20), 6 months (7), and 12 months (1) after PTX. Adequate PTX tissue for histopathologic examination was obtained in 49 cases (98%). Biopsy findings included no rejection (34), mild rejection (13), pancreatitis (1), and cytomegalovirus infection (1). Overall, 15 of the 49 evaluable biopsies (31%) had significant histopathologic findings. All but 1 of the cases of mild rejection were treated with bolus steroids. Eight of these patients subsequently developed recurrent biopsy-proven rejection within 2 months; 5 grafts were subsequently lost to rejection between 3 and 13 months after PTX. Three biopsy complications occurred: 1 hematoma, 1 pancreatitis, and 1 ileus. Patient survival is 96% and PTX graft survival (complete insulin independence) is 75% after a mean follow-up of 15 months. In the remaining 42 clinically indicated biopsies, 3 were insufficient, 8 showed no rejection, and 31 (79%) had rejection. In half of these cases, the rejection was graded as moderate to severe. In conclusion, prospective monitoring with protocol PTX biopsies may result in the earlier detection of allograft rejection and have a direct effect on improving results after solitary PTX.

Transfusion-induced graft-versus-host disease after liver transplantation. Documentation using polymerase chain reaction with HLA-DR sequence-specific primers.

Graft-versus-host disease (GVHD) occurring after liver transplantation can pose a difficult diagnostic dilemma. Similar clinical and pathologic skin and gastrointestinal manifestations can result from other causes (i.e., drugs, infections). Treatment for each of these entities differs, and the high mortality associated with GVHD makes this distinction critical. GVHD has been assumed to result from the cotransplantation of donor lymphoid tissue along with the allograft. In most instances, the patient also receives blood products during the operation, and occasionally during the postoperative period, and the lymphoid cells in these products are also a potential source of concern. In this report, we describe a patient who developed GVHD after liver transplantation. Using molecular diagnostic techniques, we determined that the source for this GVHD was not the organ donor, but was most likely nonirradiated blood products received during the hospital course. Our results suggest that transplant recipients with concomitant hematopoietic dysfunction would benefit from irradiated blood products to reduce the likelihood of this complication.

Frozen section evaluation of donor livers before transplantation.

Frozen section examination was performed on 385 donor livers before transplantation. Exclusion criteria were applied to the donor livers examined to exclude potentially dysfunctional livers. The exclusion criteria included the following: severe macrovesicular steatosis, ischemic necrosis, prominent chronic portal inflammation, prominent periductular fibrosis, granulomatous inflammation, bridging fibrosis, and malignancy. Twenty-seven of the 385 donor livers examined were excluded before transplantation. The following histologic features were present in the excluded livers: severe steatosis (22), ischemic necrosis (2), portal inflammation (1), and periductular fibrosis (2). Steatosis was present in 51 of the 385 (13.25%) organs examined, including 22 of the donor organs excluded before transplantation. Twenty-nine livers with mild to moderate steatosis were implanted into size and blood type-matched recipients. Indicators of allograft function (prothrombin time and bilirubin) and damage (aspartate aminotransferase and alanine aminotransferase) were measured daily for the first 10 days after transplant. There was no statistically significant difference between the group of nonfat livers and donor livers containing mild steatosis. Statistically significant higher posttransplant serum alanine aminotransferase and prothrombin time levels were present in the patients with livers implanted with mild versus moderate steatosis. The 1-year survival rate for patients receiving fatty versus nonfatty donor livers was not statistically different (Kaplan-Meier, P = 0.592). No significant differences were found in the clinical and laboratory characteristics of donors whose organs were implanted compared with the clinical and laboratory characteristics of donors whose organs were excluded. The primary nonfunction rate after applying the exclusion criteria was 1.4%, which is a significant decrease compared with our primary nonfunction rate of 8.5% before using frozen section examination. Frozen section examination is useful in excluding donor organs which may become dysfunctional after transplantation.

Marking characteristics of anti-nuclear matrix protein NM200.4 in human breast carcinomas and normal human tissues.

Nuclear matrix proteins are a group of recently described proteins that are thought to be cell-type specific. Using a monoclonal antibody (NM 200.4; Matritech, Cambridge, MA) generated against nuclear matrix proteins isolated from a human breast carcinoma cell line, we examined frozen tissue sections from 30 breast carcinomas, and a variety of normal tissues to determine the antibody specificity, and to assess the relationship with the staining pattern and tumor type and hormone receptor status. Most breast carcinomas marked with the antibody, but stromal and vascular endothelial cells in the tissues surrounding these lesions also marked focally. Marking of vascular endothelium in a variety of benign tissues, renal tubular epithelium, and occasionally uterine smooth muscle cells was also observed. Normal breast tissue from 4 patients without breast cancer did not react. Studies on breast tumors revealed that 15/20 invasive ductal, 3/4 in situ ductal, 3/3 medullary, 2/2 invasive lobular, and 1/1 colloid carcinomas marked with this antibody. Image analysis revealed that the staining intensity of medullary carcinoma was twice that found in invasive ductal carcinoma (avg pixel density 76.6 vs. 30.1; P < 0.05). Invasive lobular and in situ ductal carcinoma also expressed higher staining intensities than invasive ductal carcinoma, but these differences were not significant. Invasive ductal carcinomas had heterogeneity in staining intensity (avg. pixel intensity range: 0-94 units). Tumors with multiple aneuploid populations had significantly higher stain intensity values than either diploid lesions or lesions containing a single aneuploid population (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)