Flip A. Klatter

In vitro and in vivo antimicrobial activity of covalently coupled quaternary ammonium silane coatings on silicone rubber

Biomaterial-centered infection is a dreaded complication associated with the use of biomedical implants. In this paper, the antimicrobial activity of silicone rubber with a covalently coupled 3-(trimethoxysilyl)-propyldimethyloctadecylammonium chloride (QAS) coating was studied in vitro and in vivo. Gram-positive Staphylococcus aureus ATCC 12600, Staphylococcus epidermidis HBH, 102, and Gram-negative Esherichia coli O2K2 and Pseudomonas aeruginos AK1 were seeded on silicone rubber with and without QAS-coating, in the absence or presence of adsorbed human plasma proteins. The viability of the adherent bacteria was determined using a live/dead fluorescent stain and a confocal laser scanning microscope. The coating reduced the viability of adherent staphylococci from 90% to 0%), and of Gram-negative bacteria from 90% to 25% while the presencc of adsorbed plasma proteins had little influence. The biomaterials were also subcutaneously implanted in rats for 3 or 7 days, while pre- or postoperatively seeded with S. aureus ATCC 12600. Preoperative seeding resulted in infection of 7 out of 8 silicone rubber implants against 1 out of 8 QAS-coated silicone rubber implants. Postoperative seeding resulted in similar infection incidences on both implant types, but the numbers of adhering bacteria were 70% lower on QAS-coated silicone rubber. In conclusion, QAS-coated silicone rubber shows antimicrobial properties against adhering bacteria, both in vitro and in vivo.

Origin of neointimal endothelium and α-actin–positive smooth muscle cells in transplant arteriosclerosis

The development of transplant arteriosclerosis (TA) is todays most important problem in clinical organ transplantation. Histologically, TA is characterized by perivascular inflammation and progressive intimal thickening. Current thought on this process of vascular remodeling assumes that neointimal vascular smooth muscle (VSM) cells and endothelium in TA are graft-derived, holding that medial VSM cells proliferate and migrate into the subendothelial space in response to signals from inflammatory cells and damaged graft endothelium. Using MHC class I haplotype-specific immunohistochemical staining and single-cell PCR analyses, we show that the neointimal alpha-actin-positive VSM cells in rat aortic or cardiac allografts are of recipient and not of donor origin. In aortic but not in cardiac allografts, recipient-derived endothelial cells (ECs) replaced donor endothelium. Cyclosporine treatment prevents neointima formation and preserves the vascular media in aortic allografts. Recipient-derived ECs do not replace graft endothelium after cyclosporine treatment. We propose that, although it progresses beyond the needs of functional repair, TA reflects the activity of a normal healing process that restores vascular wall function following allograft-induced immunological injury.

Bone marrow does not contribute substantially to endothelial-cell replacement in transplant arteriosclerosis

Bone marrow does not contribute substantially to endothelial-cell replacement in transplant arteriosclerosis

Origin of Vascular Smooth Muscle Cells and the Role of Circulating Stem Cells in Transplant Arteriosclerosis

To date, clinical solid-organ transplantation has not achieved its goals as a long-term treatment for patients with end-stage organ failure. Development of so-called chronic transplant dysfunction (CTD) is now recognized as the predominant cause of allograft loss long term (after the first postoperative year) after transplantation. CTD has the remarkable histological feature that the luminal areas of intragraft arteries become obliterated, predominantly with vascular smooth muscle cells (VSMCs) intermingled with some inflammatory cells (transplant arteriosclerosis, or TA). The development of TA is a multifactorial process, and many risk factors have been identified. However, the precise pathogenetic mechanisms leading to TA are largely unknown and, as a result, adequate prevention and treatment protocols are still lacking. This review discusses the origin (donor versus recipient, bone marrow versus nonbone marrow) of the VSMCs in TA lesions. Poorly controlled influx and subsequent proliferative behavior of these VSMCs are considered to be critical elements in the development of TA. Available data show heterogeneity when analyzing the origin of neointimal VSMCs in various transplant models and species, indicating the existence of multiple sites of origin. Based on these findings, a model considering plasticity of VSMC origin in TA in relation to severity and extent of graft damage is proposed.

Recipient origin of neointimal vascular smooth muscle cells in cardiac allografts with transplant arteriosclerosis

BACKGROUND Coronary artery disease is todays most important post-heart transplantation problem after the first perioperative year. Histologically, coronary artery disease is characterized by transplant arteriosclerosis. The current view on this vasculopathy is that vascular smooth muscle (VSM) cells from the media of affected arteries proliferate and migrate into the sub-endothelial space (intima) in response to signals from inflammatory cells and damaged graft endothelium. According to this model, the intimal VSM cells in transplant arteriosclerotic lesions should originate from donor tissue. Using recipient-specific polymerase chain reaction (PCR) analysis of microdissected, single, neointimal VSM nuclei, we recently showed that after allogeneic aorta transplantation the neointimal VSM cells are of recipient and not of donor origin. In this study, we analyzed whether VSM-cell replacement with recipient-derived cells also takes place after allogeneic heart transplantation. METHODS Cardiac allografts, when transplanted from female donors to male immune-modulated recipient rats, eventually developed transplant arteriosclerosis. We microdissected alpha-actin positive neointimal VSM cells from tissue sections and determined the origin (donor or recipient) using recipient-specific (male), single-cell, PCR analysis. RESULTS In total, we analyzed 35 VSM-cell nuclei from 3 allografts, and PCR analysis revealed that 30/35 (86%) of the samples displayed the male-specific 128 base pair DNA fragment. These results indicate that after allogeneic cardiac transplantation, at least 86% of VSM cells in transplant arteriosclerotic lesions are of recipient origin. CONCLUSIONS In contrast to current thought, the neointimal VSM cells in cardiac allografts that show transplant arteriosclerosis are of recipient and not of donor origin.

Positively charged biomaterials exert antimicrobial effects on gram-negative bacilli in rats

Biomaterial-centered infection is a much-dreaded complication associated with the use of biomedical implants. Although positively charged biomaterial surfaces stimulate bacterial adhesion, it has been suggested that surface growth of adhering Gram-negative bacilli is inhibited on positively charged surfaces. In the present paper, we determined the infection rate of differently charged poly(methacrylates) in rats. To this end, 2 x 10(6)/cm(2) Escherichia coli O2K2 or 2 x 10(4)/cm(2) Pseudomonas aeruginosa AK1 were seeded on glass discs coated with three differently charged poly(methacrylates) coatings in a parallel plate flow chamber. Three rats received six subcutaneous discs (two discs of each charge variant) seeded with E. coli, while three other rats received discs seeded with P. aeruginosa. The numbers of viable bacteria on the surfaces were determined 48h after implantation. On 50% of all positively charged discs viable E. coli were absent, while the negatively charged discs were all colonized by E. coli. P. aeruginosa, however, were isolated from both positively and negatively charged discs. Probably, P. aeruginosa can circumvent the antimicrobial effect of the positive charge through the formation of extracellular polysaccharides.

Neonatal oral administration of DiaPep277, combined with hydrolysed casein diet, protects against Type 1 diabetes in BB-DP rats. An experimental study

Aims/hypothesisEnvironmental factors such as diet and bacterial antigens play an important role in the onset of Type 1 diabetes. Different self-antigens are suggested to play a role in the development of diabetes. Antibodies against the 60-kDa heat shock protein 60, which have a high homology to bacterial heat shock protein 65, have been found in the circulation at the onset of diabetes in humans and in pre-diabetic NOD-mice. One of the immunodominant epitopes in autoimmune diabetes is p277, a specific peptide of human heat shock protein 60 corresponding to positions 437–460. In this study we investigated whether neonatal oral administration of DiaPep277 (a synthetic peptide analogue of p277) affected the development of diabetes in the BioBreeding-Diabetes Prone (BB-DP) rat, and whether this could potentiate the effect of a protective hydrolysed casein-diet.MethodsBB-DP rats were orally inoculated once per day with placebo or DiaPep277 at days 4, 5, 6 and 7 of life. At the age of 21 days rats were weaned on to a conventional, cereal-based diet or on to the hydrolysed casein-diet.ResultsThe development of diabetes in animals receiving DiaPep277 in combination with the hydrolysed casein-diet was delayed by 17 days, and a relative reduction of the incidence by 64% was seen. Non-diabetic animals did not show any sign of insulitis.Conclusions/interpretationShort-term neonatal feeding with p277 in early life, combined with diet adaptation, appears to provide a procedure to significantly reduce the development of Type 1 diabetes in later life.

Cytomegalovirus Infection Modulates Cellular Immunity in an Experimental Model for Autoimmune Diabetes

Background: Viral infections are thought to play a role in the development of autoimmune diseases like type 1 diabetes. In this study we investigated the effect of Rat Cytomegalovirus (RCMV) infection on cellular immunity in a well-defined animal model for diabetes, the Biobreeding (BB) rat. Methods: Diabetes prone (DP)- and Diabetes resistant (DR)-BB rats were infected with 2 × 106 plaque forming units (pfu) RCMV. Diabetes development was monitored by frequent blood-glucose analysis. Effects of RCMV on CD4+, CD8+ and Vβ-TCR+ T-cell subsets were measured in vivo, and in vitro after restimulation with RCMV-infected fibroblasts. Proliferative capacity was determined by 3H-Thymidine incorporation. Results: RCMV-infection resulted in a significant acceleration of diabetes onset in DP-BB rats ( p=0.003). Percentages CD4+ and CD8+ T-cells were not affected in vivo. In vitro, RCMV-restimulation resulted in a decreased CD4+/CD8+ blastoid T-cell ratio compared to ConA ( p=0.00028). Furthermore, RCMV-restimulation resulted in a strong RCMV-specific proliferation, which comprises about 50% of the response triggered by ConA. Vβ-TCR percentages did not change upon RCMV-infection or RCMV-restimulation. Interpretation: RCMV-restimulation of splenic T-cells in vitro resulted in a strong RCMV-specific proliferation, probably also including autoreactive T-cells. In vivo, this polyclonal response might be involved in the observed accelerated diabetes development in DP-BB rats upon RCMV-infection.

Timing of pentoxifylline treatment determines its protective effect on diabetes development in the Bio Breeding rat

Diabetes-prone Bio Breeding (DP-BB) rats spontaneously develop diabetes between 60 and 120 days of age. Diabetes-resistant (DR)-BB rats can be induced to develop diabetes by poly(I:C) and anti-RT6. Here, we studied the effect of pentoxifylline, a potent anti-inflammatory agent, on diabetes development in both BB rat models of insulin-dependent diabetes mellitus and investigated whether these effects were related to differential modulation of tumour necrosis factor (TNF)-alpha and interleukin-10. When DP-BB rats received pentoxifylline from day 60 onwards, diabetes development was delayed and reduced. The other treatment protocols had no effect. In DR-BB rats, pentoxifylline treatment resulted only in a delay of diabetes development. In both BB rat models, in vivo pentoxifylline treatment potently suppressed TNF-alpha, but only moderately affected interleukin-10 production in vitro. These results show that timing of pentoxifylline treatment determines its protective effect on diabetes development in DP-BB rats. The observed pentoxifylline-induced increase of the interleukin-10/TNF-alpha ratio might be a mechanism for protection or delay of the diabetes development.

Intrathymic immune modulation prevents acute rejection but not the development of graft arteriosclerosis (chronic rejection)

Background. We showed previously that our intrathymic immune modulation protocol induces virtually permanent graft survival of simultaneously transplanted cardiac allografts in MHC-incompatible rat strain combinations. It is, however, unknown whether this procedure prevents the development of graft arterial disease (GAD). Methods. Male AO recipient rats were intrathymically inoculated with 2.5×107 PVG splenocytes immediately followed by heterotopic transplantation of a PVG cardiac allograft (day 0). Immunosuppression consisted of 1 ml of antilymphocyte serum i.p. (day 0) and cyclosporine i.m. (15 mg/kg body weight) on days 1, 2, and 3 posttransplantation. Histological analysis, mixed lymphocyte reactions, and intragraft cytokine mRNA expression were performed at several time points after engraftment. Results. Histological analysis revealed that GAD was already present 14 days after transplantation. At 200 days, virtually all vessels were affected and over 80% of the vessels showed severe intimal lesions. Infiltrate analysis displayed massive parenchymatous infiltrates (CD8+ cells and ED1+ macrophages) 2 weeks after transplantation. At later time points, infiltrates became epicardial and/or blood vessel associated and mainly consisted of CD4+, CD8+, and B cells. Mixed lymphocyte reactions showed nonspecifically decreased responses at 60 days but complete restoration of these responses at later time points (120 to 280 days). Intragraft cytokine mRNA expression showed decreased interleukin-2/interferon-&ggr; and sustained interleukin-10 expression 2 weeks after transplantation. Transforming growth factor-&bgr; mRNA expression was increased >200 days after transplantation. Conclusions. Intrathymic immune modulation does not abolish alloreactivity, and despite induction of long-lasting graft survival, this procedure does not prevent and may even facilitate the development of GAD.