Marco Harmsen

Antiviral activities of lactoferrin

Lactoferrin (LF) is an iron binding glycoprotein that is present in several mucosal secretions. Many biological functions have been ascribed to LF. One of the functions of LF is the transport of metals, but LF is also an important component of the non-specific immune system, since LF has antimicrobial properties against bacteria, fungi and several viruses. This review gives an overview of the present knowledge about the antiviral activities and, when possible, the antiviral modes of action of this protein. Lactoferrin displays antiviral activity against both DNA- and RNA-viruses, including rotavirus, respiratory syncytial virus, herpes viruses and HIV. The antiviral effect of LF lies in the early phase of infection. Lactoferrin prevents entry of virus in the host cell, either by blocking cellular receptors, or by direct binding to the virus particles.

Bone marrow-derived myofibroblasts contribute functionally to scar formation after myocardial infarction.

Myofibroblasts play a major role in scar formation during wound healing after myocardial infarction (MI). Their origin has been thought to be interstitial cardiac fibroblasts. However, the bone marrow (BM) can be a source of myofibroblasts in a number of organs after injury. We have studied the temporal, quantitative and functional role of BM‐derived (BMD) myofibroblasts in myocardial scar formation. MI was induced by permanent coronary artery ligation in mice reconstituted with EGFP or pro‐Col1A2 transgenic BM. In the latter, luciferase and β‐galactosidase transgene expression mirrors that of the endogenous pro‐collagen 1A2 gene, which allows for functional assessment of the recruited cells. After MI, α‐SMA‐positive myofibroblasts and collagen I gradually increased in the infarct area until day 14 and remained constant afterwards. Numerous EGFP‐positive BMD cells were present during the first week post‐MI, and gradually decreased afterwards until day 28. Peak numbers of BMD myofibroblasts, co‐expressing EGFP and α‐SMA, were found on day 7 post‐MI. An average of 21% of the BMD cells in the infarct area were myofibroblasts. These cells constituted up to 24% of all myofibroblasts present. By in vivo IVIS® imaging, BMD myofibroblasts were found to be active for collagen I production and their presence was confined to the infarct area. These results show that BMD myofibroblasts participate actively in scar formation after MI. Copyright

Expression of P2 receptors at sites of chronic inflammation

Extracellular nucleotides have been identified as important signaling molecules. These nucleotides act on the P2 family of receptors that respond by either forming an ion-channel or by activation of a signal transduction cascade, both of which enable a cellular response. Although a role for P2 receptors in inflammation has been implied, the local expression pattern and kinetics of these receptors at sites of inflammation are not known. Therefore, we have studied the expression of the P2 receptors expressed by inflammatory cells or by cells in the vasculature, with special attention to P2X1R, P2X7R, P2Y1R, and P2Y2R. As a suitable model for studying inflammatory reactions, we have employed the foreign body reaction (FBR), a sterile inflammatory reaction induced by implanting degradable cross-linked dermal sheep collagen disks subcutaneously in the rat. We show that, in the vasculature, the expression of P2X7R, P2Y1R, and P2Y2R increase until day 2. The expression of P2X7R and P2Y1R on macrophages and giant cells increased during the course of the inflammatory reaction which was studied for 21 days. The expression of the P2Y2R on macrophages and giant cells inside the foreign body increases with time, whereas the expression on macrophages in the surrounding tissue is maximal at day 5. The expression of P2X1R remains at a constant low level. The upregulation of P2X7R, P2Y1R, and P2Y2R over time suggests a regulatory function for these receptors in inflammation.

CD34 cells augment endothelial cell differentiation of CD14 endothelial progenitor cells in vitro

Neovascularization by endothelial progenitor cells (EPC) for the treatment of ischaemic diseases has been a topic of intense research. The CD34+ cell is often designated as EPC, because it contributes to repair of ischaemic injuries through neovascularization. However, incorporation of CD34+ cells into the neovasculature is limited, suggesting another role which could be paracrine. CD14+ cells can also differentiate into endothelial cells and contribute to neovascularization. However, the low proliferative capacity of CD14+ cell‐derived endothelial cells hampers their use as therapeutic cells. We made the assumption that an interaction between CD34+ and CD14+ cells augments endothelial differentiation of the CD14+ cells. In vitro, the influence of CD34+ cells on the endothelial differentiation capacity of CD14+ cells was investigated. Endothelial differentiation was analysed by expression of endothelial cell markers CD31, CD144, von Willebrand Factor and endothelial Nitric Oxide Synthase. Furthermore, we assessed proliferative capacity and endothelial cell function of the cells in culture. In monocultures, 63% of the CD14+‐derived cells adopted an endothelial cell phenotype, whereas in CD34+/CD14+ co‐cultures 95% of the cells showed endothelial cell differentiation. Proliferation increased up to 12% in the CD34+/CD14+ co‐cultures compared to both monocultures. CD34‐conditioned medium also increased endothelial differentiation of CD14+ cells. This effect was abrogated by hepatocyte growth factor neutralizing antibodies, but not by interleukin‐8 and monocyte chemoattractant protein‐1 neutralizing antibodies. We show that co‐culturing of CD34+ and CD14+ cells results in a proliferating population of functional endothelial cells, which may be suitable for treatment of ischaemic diseases such as myocardial infarction.

Uninfected and cytomegalic endothelial cells in blood during cytomegalovirus infection: effect of acute rejection.

After transplantation, human cytomegalovirus (HCMV) infections can cause vascular damage to both the graft and the host. To study a possible relationship between the degree of vascular injury, clinical symptoms of HCMV infection, and transplant rejection, the appearance and numbers of endothelial cells (ECs) in blood of 54 kidney transplant recipients were investigated in a prospective clinical study. Two types of endothelial cells were identified: cytomegalic ECs (CECs) were detected in patients with moderate or high HCMV antigenemia, and uninfected ECs were observed in patients with and without HCMV infection. The incidence of either CECs, ECs, or the combination of both was associated with HCMV-related clinical symptoms (P<.01). Remarkably, the occurrence of rejection episodes before HCMV infection was an important risk factor for the occurrence of ECs in blood (ECs, CECs, or both) during HCMV infection (P<.001).

Viral Load in Breast Milk Correlates with Transmission of Human Cytomegalovirus to Preterm Neonates, but Lactoferrin Concentrations Do Not

ABSTRACT In vitro, lactoferrin (LF) strongly inhibits human cytomegalovirus (HCMV), which led us to hypothesize that in vivo HCMV might also be inhibited in secretions with high LF concentrations. In breast milk, high viral loads observed as high viral DNA titers tended to coincide with higher LF levels. However, the LF levels did not correlate to virus transmission to preterm infants. The viral load in the transmitting group was highest compared to the nontransmitting group. We conclude that viral load in breast milk is an important factor for transmission of the virus.

Pulmonary involvement during cytomegalovirus infection in immunosuppressed patients

Abstract: Although cytomegalovirus (CMV) pulmonary involvement after solid organ transplantation is infrequently seen nowadays, CMV pneumonitis is still a potential lethal complication. Introduction of the pp65 antigenemia assay enabled early and rapid diagnosis of CMV viremia in transplant patients prior to symptoms. Also, in asymptomatic patients with CMV viremia, a decreased pulmonary diffusion capacity could be demonstrated. In this review, we discuss clinical and subclinical pulmonary involvement of CMV infection in the immunocompromised host with an emphasis on transplant recipients. The clinical course, diagnosis, therapy, prophylaxis, and pathophysiology of CMV pneumonitis are discussed.

Cytomegalovirus infection increases the expression and activity of ecto-ATPase (CD39) and ecto-5 ' nucleotidase (CD73) on endothelial cells

We describe enhanced expression and enzymatic activity of ecto‐ATPase and ecto‐5′nucleotidase on CMV infected endothelial cells as compared to uninfected cells. These ectoenzymes play a major role in modulation of platelet activation and aggregation. Furthermore, adenosine has a modulatory effect upon inflammation. Addition of ATP, ADP or AMP to cultures of CMV infected or uninfected endothelial cells revealed increased turnover of AMP in CMV infected endothelial cells. In addition, the superoxide production by stimulated polymorphonuclear cells was inhibited in the presence of CMV infected endothelial cells as compared to uninfected cells, probably due to the enhanced activity of ecto‐5′nucleotidase and associated to production of adenosine.

Dissemination of rat cytomegalovirus through infected granulocytes and monocytes in vitro and in vivo

ABSTRACT The role of leukocytes in the in vivo dissemination of cytomegalovirus was studied in this experiment. Rat cytomegalovirus (RCMV) could be transferred to rat granulocytes and monocytes by cocultivation with RCMV-infected fibroblasts in vitro. Intravenous injection of purified infected granulocytes or monocytes resulted in a systemic infection in rats, indicating that our model is a powerful tool to gain further insight into CMV dissemination and the development of new antivirals.

Donor-derived circulating endothelial cells after kidney transplantation.

Background. In solid-organ transplantation, the allograft vasculature, in particular the endothelium, is prone to injury inflicted by peritransplantational and posttransplantational factors. Previously, we have shown that circulating endothelial cells (cEC) can be detected in the peripheral blood of kidney allograft recipients and are often associated with acute rejection and active infections with human cytomegalovirus. In the present study we hypothesized that cEC after kidney transplantation are of donor origin, thus reflecting transplantation-related damage to the allograft endothelium. Methods. Using hydraulic micromanipulation equipment, we isolated single cEC (n=153) from the peripheral blood of nine kidney allograft recipients at various time points after transplantation. We demonstrated the origin of these cells (donor or recipient) by typing their HLA-DRB alleles by single-cell, genomic, nested polymerase chain reaction. Results. The majority (71.8%) of cEC were of donor origin and could be detected up to 141 days after onset of acute rejection episodes. Although less frequent (28.2%), recipient-type cEC were detected in the same time course as donor-type cEC. Conclusion. We conclude that posttransplantational injury to the allograft endothelium is reflected by the presence of donor-derived cEC in the blood.