N. Cary

Evidence that the death of macrophage foam cells contributes to the lipid core of atheroma

Sections of human atherosclerotic lesions of different stages show that, in early lesions, the acellular lipid core is usually immediately adjacent to the deepest edge of a collection of macrophage foam cells. Advanced lesions with a large lipid core have variable numbers of macrophage foam cells, close to the lateral edges, or shoulders, of the core. In both early and advanced lesions, some of the macrophages nearest the core appear to be dying. Lipid cores contain two materials which in earlier lesions are found only in macrophages, namely ceroid and CD68 antigen, but do not contain recognisable smooth muscle cell actin. It is concluded that death of macrophage foam cells contributes to the origin and slow enlargement of the lipid core. The cause of macrophage death is not yet certain, but is under investigation.

Foam cell apoptosis and the development of the lipid core of human atherosclerosis

A characteristic feature of the advanced atherosclerotic lesion is the acellular lipid core, which appears to result at least partly from the death of macrophage foam cells. This study shows that foam cell death at the edge of the lipid core includes both necrosis and apoptosis and that remnants of apoptotic nuclei are present within the lipid core. Apoptotic cells were identified by transmission electron microscopy and by nick end‐labelling using terminal deoxynucleotidyl transferase (TUNEL). Some TUNEL‐positive cells also expressed proliferating cell nuclear antigen (PCNA). The cause of foam cell death in atherogenesis is unknown, but oxidized low‐density lipoprotein (LDL) can cause macrophage apoptosis in vitro and might therefore play a role in the formation and enlargement of the lipid core.

Tissue expression of human complement inhibitor, decay-accelerating factor, in transgenic pigs. A potential approach for preventing xenograft rejection.

Since complement-mediated hyperacute rejection of xenografts prevents the use of pigs as organ donors to man, the development of transgenic animals expressing species-specific complement inhibitors could provide a strategy for overcoming hyperacute rejection. The complement inhibitor, human decay-accelerating factor (hDAF), prevents the assembly of C3 and C5 convertases. In this article, the first histologic analysis of hDAF expression in pig tissues, specifically expression in endothelial cells of pigs transgenic for hDAF, is described. Twenty-seven transgenic pigs were categorized into 4 groups based on the expression patterns in endothelial, vascular smooth muscle, and squamous epithelial cells of skin biopsy specimens. Skin biopsy specimens permitted evaluation of the pigs without the need to kill them or to perform invasive procedures. Sixteen cases demonstrated endothelial cell staining. Complete necropsy evaluation, available in 14 of the 27 pigs, correlated with the skin biopsy specimen expression of hDAF. The immunoperoxidase data matched identically with the presence of the mRNA transcript in 25 of the 26 cases where RNA data were available. Also, the staining patterns of 6 transgenic pig founders and their 9 offspring (total of 9 founder-offspring pairs) correlated. Since transgenes are variably expressed in different cell types and since tissue lysates represent a melange of cell types, histologic evaluation for protein expression in tissues from transgenic animals will be critical if they are to be bred to become clinical organ donors. In addition to endothelial expression of hDAF, its expression on vascular smooth muscle cells may be important in preventing tissue damage when breaks in the endothelium occur.

Changes in elemental concentrations are associated with early stages of apoptosis in human monocyte-macrophages exposed to oxidized low-density lipoprotein: an X-ray microanalytical study.

This study examines ion homeostasis in monocyte–macrophages committed to death by apoptosis. X‐ray microanalysis has been used to demonstrate that intracellular concentrations of potassium decreased whilst those of sodium increased following 3 h of exposure to 100 µg/ml of oxidized low‐density lipoprotein (LDL) in vitro. In contrast, the maximal incidence of cell death, as determined by the inability to exclude trypan blue, was not seen until 24 h of exposure. At 12 h, less than 1 per cent of cells were stained using terminal transferase‐mediated DNA nick‐end labelling, which is generally accepted as a marker of late stages in the apoptotic pathway. This is the first demonstration of early perturbations of ion homeostasis in monocyte–macrophages exposed to concentrations of oxidized LDL known to cause apoptosis. Copyright

A potential role for sterol 27-hydroxylase in atherogenesis

OBJECTIVE 27-hydroxycholesterol is the product of the mitochondrial cytochrome P450 sterol 27-hydroxylase, a key enzyme in cholesterol metabolism present in most tissues of the body. 27-hydroxycholesterol increases in abundance with progression of human atherosclerotic lesions, therefore the aim of this study was to determine the pattern of sterol 27-hydroxylase gene expression in normal and diseased arteries and to identify the cell types responsible for its expression. METHODS Reverse transcription-polymerase chain reaction (RT-PCR) analysis and in situ hybridisation, utilising a sterol 27-hydroxylase cDNA probe, and immunohistochemistry, utilising an antibody to sterol 27-hydroxylase, together with an antibody to smooth muscle cell alpha-actin and an antibody to CD68, a marker for macrophages, were used to study expression of 27-hydroxylase in arterial specimens. In addition, RT-PCR was used to study expression of 27-hydroxylase in cultured macrophages and smooth muscle cells. RESULTS Semi-quantitative RT-PCR analysis of normal and atherosclerotic human aortas showed that 27-hydroxylase is constitutively expressed in the normal artery wall, and is substantially up-regulated in atherosclerosis. RT-PCR analysis of 27-hydroxylase expression in vitro demonstrated that macrophages constitutively express high levels throughout their differentiation in culture whilst de-differentiated vascular smooth muscle cells express very low levels. In situ hybridisation revealed that in normal artery and fatty streaks, expression of mRNA for 27-hydroxylase was low in the media, but higher in intimal smooth muscle cells. The macrophages of fatty streaks expressed low or undetectable levels of 27-hydroxylase. However in advanced lesions the highest expression of 27-hydroxylase was detectable in macrophages. Immunohistochemistry demonstrated that high levels of 27-hydroxylase protein occurred in macrophages near the shoulder region of plaques, at the edge of the lipid core. CONCLUSIONS 27-hydroxylase may constitute a protective mechanism for removing cholesterol from macrophages and smooth muscle cells. Genetic heterogeneity resulting in differences in sterol 27-hydroxylase activity between individuals may affect their ability to deal with accumulated cholesterol in the arterial intima, and hence their relative degree of predisposition to atherosclerosis.

Human leukocyte antigen compatibility in heart transplantation: evidence for a differential role of HLA matching on short- and medium-term patient survival.

BACKGROUND Studies of the influence of human leukocyte antigen (HLA) matching on cardiac transplant outcome have proved inconclusive, mainly due to the lack of well-matched grafts. However, a growing number of studies report improved clinical course and patient survival in cases with increased HLA compatibility. Opelz et al. believe these benefits justify the introduction of prospective HLA-matching strategies. METHODS We performed univariate and multivariate analyses to examine the short- and medium-term influence of HLA matching on 556 consecutive primary heart transplants performed at a single center between 1983 and 1994. Overall graft survival at 1, 3, and 5 years was 80%, 74%, and 67% respectively. Sixteen (2.9%) grafts failed within 5 days and were not considered in the analysis of the HLA matching and graft survival data. RESULTS Complete HLA-A, -B, and -DR typing data were available on 477 transplant pairs. The results demonstrate a 12% 1-year survival advantage for 31 patients with zero to two HLA antigen mismatches compared with three to six mismatches. The influence of each individual locus was 6.1%, 8.4%, and 5.4% for zero HLA-A, -B, and -DR mismatches, respectively, compared with two mismatches. However, when outcome from 1 to 5 years was considered, analysis of the role of each locus revealed marked differences. HLAA-matched grafts (n=45) had a 24% lower survival rate compared with two-antigen-mismatched grafts (n=148; 88% [SE 3.1] vs. 64% [SE 8.2], respectively; P=0.009). Furthermore, 34% of HLA-A-matched grafts failed between 1 and 5 years, compared with only 5% of HLA-B-matched grafts (P=0.013). CONCLUSIONS These data suggest that although HLA matching is effective at reducing acute graft loss, in the longer term, HLA-A matching may impair survival. HLA-A may serve as a restriction element for indirect presentation of allopeptides or tissue-specific minor histocompatibility antigens, facilitating chronic graft loss. Therefore, we advocate a differential role for HLA matching over two epochs. A blanket approach to prospective matching for heart transplants may be premature for optimal long-term survival.

bcl‐2 PROTEIN IS STRONGLY EXPRESSED IN POST‐TRANSPLANT LYMPHOPROLIFERATIVE DISORDERS

The aim of this study was to examine a series of Epstein–Barr virus (EBV)‐driven post‐transplant lymphoproliferative disorders (PTLDs), in order to ascertain the level of bcl‐2 immunostaining; to explore the relationship between bcl‐2 and p53 protein expression; and to see if any correlation exists between bcl‐2 and EBV‐latent membrane protein 1 (LMP‐1). Seventeen renal and 11 heart/heart–lung PTLD cases were stained with antibodies to EBV‐LMP‐1, bcl‐2 and p53, using paraffin‐embedded tissue. All cases of PTLD strongly co‐expressed bcl‐2 and EBV‐LMP. Positive staining was present in small lymphoid and larger immunoblastic cells. These two antibodies showed parallel staining intensity. p53 expression was noted in 13 of 17 renal PTLDs, but in ten of the positive cases only 5–10 per cent of cells were stained. Seven of the 11 heart/heart–lung cases showed 50–60 per cent of cells to be p53‐positive; in the remaining four cases, 10–20 per cent of cells were positive. bcl‐2 protein, as detected by immunohistochemistry, is markedly overexpressed in all cases of PTLD. This study also demonstrates a strongly positive correlation between bcl‐2 expression and EBV‐LMP‐1 detection in PTLDs. An inverse pattern of p53 and bcl‐2 immunoexpression is noted in PTLDs with ‘high grade’ histology: these show marked expression of bcl‐2, while p53 is downregulated. A Fishers exact test yielded a P value of 0·12 when comparing p53‐positive renal PTLDs with p53‐positive heart/heart–lung PTLDs, indicating that any difference seen is not statistically significant. The postulated mechanism for the positive correlation between bcl‐2 and EBV‐LMP‐1 is that EBV upregulates bcl‐2, either directly or indirectly, thus promoting cell survival and ultimately successful viral replication.

Error rates with which endomyocardial biopsy specimens are graded for rejection after cardiac transplantation

Control of the immune response to the transplanted organ is fundamental to the success of transplantation. Endomyocardial biopsy to diagnose and grade rejection is the mainstay in achieving this control. As rejection tends to be a patchy process, accurate diagnosis depends on adequate sampling from the myocardium. This study estimates the error rates with which biopsy specimens are graded. The results of 459 biopsy sets, in which at least 4 fragments were graded, were analyzed. Combinations of grades observed at the same biopsy session were used to estimate error rates. An E-M algorithm was used to estimate error rates. Predictive probabilities of true grades, given a set of 4 graded fragments, were calculated using Bayes theorem. If 4 fragments at a biopsy session were negative there was a 0.02% chance of missing clinically significant rejection (moderate or severe). Similarly, if minimal rejection was the highest grade observed, the probability of missing moderate-severe rejection was negligible, between 0.06 and 0.09%. However, where mild rejection is the highest observed on the 4 fragments, there is between a 2% (1 mild fragment) and 28% (4 mild fragments) chance of moderate-severe rejection being the underlying grade. This study concludes that 4 fragments are adequate as a minimum in most cases. However, if only 4 fragments are available, and greater than or equal to 3 are graded mild, the risk of missing moderate-severe rejection is unacceptably high, and repeat biopsy or treatment may be indicated.

EBV latent membrane protein (LMP-1) and bcl-2 protein expression in Reed-Sternberg-like cells in post-transplant lymphoproliferative disorders

An inconsistent association exists between EBV‐LMP‐1 and bcl‐2 protein expression in Reed‐Sternberg cells seen in Hodgkin’s disease. In fact, many studies have concluded that there is no correlation between EBV‐LMP and bcl‐2 expression in Hodgkins disease. We undertook an analysis of post‐transplant lymphoproliferative disorders to explore the relationship between EBV‐LMP and bcl‐2 in Reed‐Sternberg‐like cells found in this condition, given the strong association between this disorder and EBV. Reed‐Sternberg‐like cells were found histologically in 11 of 28 cases of renal, heart and heart‐lung post‐transplant lymphoproliferative disorders. Formalin‐fixed, paraffin‐embedded sections were stained with monoclonal antibodies to EBV‐LMP‐1 and bcl‐2 proteins. Reed‐Sternberg‐like cells in all 11 cases co‐expressed EBV‐LMP and bcl‐2. A similar relationship was noted with large, mononuclear cells and occasional small lymphoid cells. The staining pattern seen with both antibodies was of similar intensity and both displayed cytoplasmic Golgi accentuation. In the setting of post‐transplant lymphoproliferative disorders, Reed‐Sternberg‐like cells exhibit strong co‐expression of EBV‐LMP‐1 and bcl‐2 proteins, supporting a positive correlation between them. This is in contrast to the findings in Hodgkins disease. The reason for this discrepancy may be due to the iatrogenic immunosuppression and resultant severe EBV infection, together with other cellular events.

Evaluation of the International Society for Heart Transplantation (ISHT) grading of pulmonary rejection in 100 consecutive biopsies

Heart-lung and lung transplantation are accepted treatments for patients with end-stage pulmonary vascular disease or parenchymal lung disease. Survival rates for heart-lung and lung transplantation are lower than those for heart transplantation alone. The 5-year actuarial survival for heart-lung transplantation has been 41% largely due to rejection and infection remaining as the limiting factors for long-term survival. A standardized nomenclature for the histological grading of pulmonary rejection was formulated by the International Society for Heart Transplantation (ISHT) in July 1990. Infection, however, is a major problem in the histological assessment of lung recipient biopsies, potentially limiting the usefulness of such a classification. In this study, 100 consecutive transbronchial biopsies (TBBs) from lung transplant recipients were analysed, together with microbiological and serological data, in order to evaluate the proposed ISHT grading system for pulmonary rejection and the importance of concomitant infections in the histological interpretation of TBBs.