A.H. Bruggink

The Chemokine and Chemokine Receptor Profile of Infiltrating Cells in the Wall of Arteries With Cardiac Allograft Vasculopathy Is Indicative of a Memory T–Helper 1 Response

Background— Despite improvement in short-term patient survival after heart transplantation (HTx), long-term survival rates have not improved much, mainly because of cardiac allograft vasculopathy (CAV). Cytokines and chemokines are considered to play an important role in CAV development. Methods and Results— We focused on coronary arteries of HTx patients and made an inventory of the infiltrating cells and the expression of cytokines as well as chemokines and chemokine receptors (C+CR) in the different layers of the vessel wall with CAV. Tissue slides were stained for a variety of cell markers (CD3, CD4, CD8, CD20, CD68, CD79a), chemokines (monokine induced by interferon [MIG], interferon-inducible protein 10 [IP-10], interferon-inducible T cell-&agr; chemoattractant [ITAC], RANTES [regulated on activation normal T cell expressed and secreted], and fractalkine), and chemokine receptors (CXCR3, CCR5, and CX3CR1). In reference coronary arteries (not transplanted), almost no infiltrating cells were found, and in transplanted hearts with CAV (HTx+CAV), a large number of T cells were observed (CD4:CD8=2:1), mainly localized in the neointima and adventitia. Most of these T cells appeared to be activated (human leukocyte antigen DR positive). Coronary arteries from transplanted hearts without CAV (HTx−CAV), HTx+CAV, and references were also analyzed for cytokine and C+CR mRNA expression with the use of quantitative polymerase chain reaction. Interferon-&ggr; was highly expressed in HTx+CAV compared with HTx−CAV. Interleukin-4 and interleukin-10 were expressed at the same level in both HTx groups and references. In HTx+CAV, all C+CR, but especially the T–helper 1 (TH1) C+CR, were more abundant than in the HTx−CAV and references. However, TH2 CCR4 expression did not differ significantly between both HTx groups. Conclusions— In coronary arteries with CAV, most T cells are CD4+ and express human leukocyte antigen DR. These activated TH cells are mainly memory TH1 cells on the basis of their C+CR profile and cytokine expression.

Matrix metalloproteinases as profibrotic factors in terminal ileum in Crohn's disease

Background: Returning stenosis in Crohns disease (CD) patients is poorly understood. After resection, newly developed strictures are seen within 10 years in 50% to 70%. Matrix metalloproteinases (MMPs) are involved in matrix‐turnover processes. This study analyzes spatial expression of MMP‐1, MMP‐3, MMP‐9, tissue inhibitor of MMP‐1, and collagen III to get better insight in tissue remodeling of terminal ileum of CD patients. Methods: Expressions were analyzed on mRNA and the protein level (MMP‐1, MMP‐3) in segments from resected terminal ileum from CD and control patients. In CD, macroscopic distinction was made between proximal resection margin, prestenotic, and stenotic tissue. Immunohistochemistry allowed for expression analyses transmurally. Results: MMP‐1 and MMP‐3 gene expression was up‐regulated (P < 0.05) in both prestenotic and stenotic tissue. MMP‐1 protein was significantly up‐regulated in submucosal and muscular tissue of prestenotic parts and in muscular tissue of stenotic Crohn samples. MMP‐3 protein was significantly up‐regulated in all layers of prestenotic and stenotic Crohn samples. Even in submucosa of proximal resection margin tissue, MMP‐3 expression was significantly higher than in controls. Conclusion: Surprisingly, in proximal resection margin tissue up‐regulated MMP‐3 was seen. This suggests that in nonresected terminal ileum, in which anastomosis is made, tissue turnover is present, which may account for the high recurrence of intestinal strictures.

Development of a mRNA profiling multiplex for the inference of organ tissues

Forensic characterisation of organ tissue generally occurs through histological and immunological assays of limited sensitivity. Here, we explore an alternative approach and examine a total of 41 candidate mRNA markers for their ability to differentiate between brain, lung, liver, skeletal muscle, heart, kidney and skin. Various selection rounds are applied involving 85 organ tissues (36 excised autopsy specimens and 49 frozen tissue sections, with at least ten specimens for each organ type), 20 commercially available RNAs from different human tissues and at least two specimens of blood, saliva, semen, vaginal mucosa, menstrual secretion or touch samples. Finally, 14 markers are regarded tissue-specific and included in an endpoint RT-PCR multiplex together with one general muscle, one blood and one housekeeping marker. This 17-plex is successfully used to analyse a blind test set of 20 specimens including mixtures, and samples derived from stabbing of organ tissues. With the blind test set samples, it is shown that an earlier described interpretation strategy for RNA cell typing results [1] is also effective for tissue inference. As organ-typing is embedded in a procedure of combined DNA/RNA extraction and analysis, both donor and organ type information is derived from the same sample. Some autopsy specimens presented DNA profiles characteristic for degraded DNA. Nevertheless, the organ-typing multiplex could generate full RNA profiles, which is probably due to small sizes of the amplicons. This assay provides a novel tool for analysis of samples from violent crimes.

T Cell Apoptosis in Human Heart Allografts : Association with Lack of Co-Stimulation?

It is unclear whether the intracardial immune reactivity after heart transplantation influences the peripheral immunological status (activation or nonresponsiveness) of the patient. Co-stimulation and activation-induced cell death (AICD) or apoptosis play an important role in determining the balance between lymphocyte reactivity and nonreactivity. Therefore, we studied the expression of co-stimulatory molecules and the process of apoptosis in biopsies of human heart allografts, using immunohistochemistry. Although a normal expression of co-stimulatory molecules on antigen-presenting cells was observed, the expression of their counter-structures on T cells was absent. This may be due to chronic T cell activation, which can lead to the induction of apoptosis via the Fas/Fas ligand pathway. In the infiltrates, a considerable percentage of the lymphocytes, but not the macrophages, were apoptotic. Apoptosis was confirmed by DNA fragmentation analysis. Increased numbers of Bax-expressing versus decreased numbers of Bcl2-expressing lymphocytes in comparison with normal lymphoid tissue confirmed a imbalance in favor of apoptosis. Apoptosis was biased towards CD4+ T cells (65.7% versus 26.6% in CD8+ T cells). Fas was expressed on most of the infiltrating cells. Fas ligand expression was also observed, not only on most of the T cells but also on all macrophages. Because macrophages were often detected in close contact with T cells, they may play a role in T cell regulation via the Fas/Fas ligand pathway. This study indicates that, during rejection, not only is tissue damage induced by infiltrating T cells, but also the infiltrating lymphocytes themselves are actively down-regulated (eg, AICD) by one another and by macrophages in the infiltrate. This regulatory process may affect the immunological status of the patient after heart transplantation.

Type IV collagen degradation in the myocardial basement membrane after unloading of the failing heart by a left ventricular assist device

After left ventricular assist device (LVAD) support in patients with end-stage cardiomyopathy, cardiomyocytes decrease in size. We hypothesized that during this process, known as reverse remodeling, the basement membrane (BM), which is closely connected to, and forms the interface between the cardiomyocytes and the extracellular matrix, will be severely affected. Therefore, the changes in the myocardial BM in patients with end-stage heart failure before and after LVAD support were studied. The role of MMP-2 in this process was also investigated. Transmission electron microscopy showed that the BM thickness decreased post-LVAD compared to pre-LVAD. Immunohistochemistry indicated a reduced immunoreactivity for type IV collagen in the BM after LVAD support. Quantitative PCR showed a similar mRNA expression for type IV collagen pre- and post-LVAD. MMP-2 mRNA almost doubled post-LVAD (P<0.01). In addition, active MMP-2 protein as identified by gelatin zymography and confirmed by Western blot analysis was detected after LVAD support and in controls, but not before LVAD support. Active MMP was localized in the BM of the cardiomyocyte, as detected by type IV collagen in situ zymography. Furthermore, in situ hybridization/immunohistochemical double staining showed that MMP-2 mRNA was expressed in cardiomyocytes, macrophages, T-cells and endothelial cells. Taken together, these findings show reduced type IV collagen content in the BM of cardiomyocytes after LVAD support. This reduction is at least in part the result of increased MMP-2 activity and not due to reduced synthesis of type IV collagen.

Stem cell-derived cardiomyocytes after bone marrow and heart transplantation

Cardiomyocytes are a stable cell population with only limited potential for renewal after injury. Tissue regeneration may be due to infiltration of stem cells, which differentiate into cardiomyocytes. We have analysed the influx of stem cells in the heart of patients who received either a gender-mismatched BMT (male donor to female recipient) or a gender-mismatched cardiac transplant (HTX; female donor to male recipient). The proportion of infiltrating cells was determined by Y-chromosome in situ hybridization combined with immunohistochemical cell characterization. In BM transplanted patients and in cardiac allotransplant recipients, cardiomyocytes of apparent BM origin were detected. The proportions were similar in both groups and amounted up to 1% of all cardiomyocytes. The number of stem cell-derived cardiomyocytes did not alter significantly in time, but were relatively high in cases where large numbers of BM-derived Y-chromosome-positive infiltrating inflammatory cells were present. The number of Y-chromosome-positive endothelial cells was small and present only in small blood vessels. The number of BM-derived cardiomyocytes in both BMT and HTX is not significantly different between the two types of transplantation and is at most 1%.

IL-4 promotor gene polymorphism in heart transplantation

test. Results: Overall survival was borderline significantly different between all groups (OKT3: 52.4%, F-ATG: 44.3 %, THG: 67.5% and BT563: 51.8%; p 0.095). Causes of death were different between the four groups. OKT3 treated patients had the highest rate of death from graftsclerosis (40%) and BT 563 the highest rate in death from acute rejection (23%). In the THG group there was the lowest incidence of acute rejections (57.3%, 29.5 %, 18% and 50%; p 0.002). There was no difference in overall freedom from severe infections (90.5%, 81.8 %, 92.4% and 88.9%; p 0.232) and CMV-disease (80.9%, 79.6 %, 83.6% and 77.8%; p 0.822). The incidence of cancer was similar in all groups (14.3 %, 22.8 %, 11.7% and 11.1%; p 0.146), whereas there was a clear trend of lower incidence of graftsclerosis in THG treated patients (52.2 %, 38.0 %, 25.7% and 41.2%; p 0.053). Conclusions: Different Antibody induction protocols seem to be dissimilar in their immunmodulating function. Although all three studies were powered to detect differences in early rejection, long-term data show different incidence of development of graftsclerosis and on survival. Long-term adverse events were comparable and tolerable. These data clearly show that early immunmodulating influence of induction antibodies have longrange effects on the immune system.