Combined implantation of CD34+ and CD14+ cells increases neovascularization through amplified paracrine signalling.

Cell therapy strategies that use adult peripheral blood-derived CD34⁺ progenitor cells are hampered by low cell numbers and the infrequent cellular incorporation into the neovasculature. Hence, the use of CD34⁺ cells to treat ischaemic diseases is under debate. Interaction between CD34⁺ cells and CD14⁺ cells results in superior endothelial differentiation of CD14⁺ cells in vitro, indicating that cell therapy approaches utilizing both CD34⁺ and CD14⁺ cells may be advantageous in therapeutic neovascularization. Here, human CD34⁺ and CD14⁺ cells were isolated from adult peripheral blood and implanted subcutaneously into nude mice, using matrigel as the carrier. Combined implantation of human CD34⁺ and CD14⁺ cells resulted in superior neovascularization, compared to either cell type alone, albeit incorporation of human cells into the murine vasculature was not observed. Human CD34⁺ and CD14⁺ cells produced and secreted a pentad of pro-angiogenic mediators, such as HGF, MCP-1 and IL-8, bFGF and VEGFa in monoculture. The production and secretion of pro-angiogenic mediators by CD14⁺ cells was highly amplified upon incubation with conditioned medium from CD34⁺ cells. In vivo, neovascularization of matrigel implants did not rely on the endothelial differentiation and incorporation of CD34⁺ or CD14⁺ cells, but depended on the paracrine effects of IL-8, MCP-1, HGF, bFGF and VEGFa secreted by implanted cells. Administration of this growth factor/cytokine pentad using matrigel as a carrier results in cell recruitment and microvessel formation equal to progenitor cell-induced neovascularization. These data provide new insights on neovascularization by cell therapy and may contribute to new strategies for the treatment of ischaemic diseases.

Hyaluronic acid-recombinant gelatin gels as a scaffold for soft tissue regeneration.

An array of different types of hyaluronic acid (HA)- and collagen-based products is available for filling soft-tissue defects. A major drawback of the current soft-tissue fillers is their inability to induce cell infiltration and new tissue formation. Our aim is to develop novel biodegradable injectable gels which induce soft tissue regeneration, initially resulting in integration and finally replacement of the gel with new autologous tissue. Two reference gels of pure HA, monophasic HA-1 and micronised HA-2, were used. Furthermore, both gels were mixed with recombinant gelatin (RG) resulting in HA-1+RG and HA-2+RG. All gels were subcutaneously injected on the back of rats and explanted after 4 weeks. Addition of RG to HA-1 resulted in stroma formation (neovascularisation and ECM deposition) which was restricted to the outer rim of the HA-1+RG gel. In contrast, addition of RG to HA-2 induced stroma formation throughout the gel. The RG component of the gel was degraded by macrophages and giant cells and subsequently replaced by new vascularised tissue. Immunohistochemical staining showed that the extracellular matrix components collagen I and III were deposited throughout the gel. In conclusion, this study shows the proof of principle that addition of RG to HA-2 results in a novel injectable gel capable of inducing soft tissue regeneration. In this gel HA has a scaffold function whereas the RG component induces new tissue formation, resulting in proper vascularisation and integration of the HA-2+RG gel with the autologous tissue.

Site-specific tissue inhibitor of metalloproteinase-1 governs the matrix metalloproteinases-dependent degradation of crosslinked collagen scaffolds and is correlated with interleukin-10.

We have previously shown that the foreign body reaction (FBR) against crosslinked collagen type I (Col-I) differs between subcutaneous and epicardial implantation sites; Col-I was quickly degraded epicardially, whereas degradation was attenuated subcutaneously. The current study set out to dissect the nature and regulation of the MMP-based degradation of implanted Col-I in mice during the FBR. Immunohistochemistry showed that MMP-2, MMP-8 and MMP-13 were present in subcutaneous and epicardial implants, whereas only MMP-9 was also present epicardially. Western blotting showed that MMP-8 and MMP-9 were mainly present in their inactive proform. In contrast, collagenase MMP-13 and gelatinase MMP-2 were the predominant active MMPs at both sites. Interestingly, the major MMP inhibitor TIMP-1 was solely observed in subcutaneous implants, which is why MMP-13 and MMP-2 are not able to degrade the collagen scaffold at the subcutaneous implantation site. Interleukin 10 (IL-10), a potent inducer of TIMP-1 expression, was also mainly detected subcutaneously; giant cells were the main source. Therefore, we surmise that IL-10, through regulation of the balance between MMPs and TIMP-1, suppresses the FBR against implanted biomaterials. Together, our findings would provide cues and clues to improve future therapies in regenerative medicine that are based on the tuned regulation of the degradation of biomaterial scaffolds.

Novel polyurethanes with interconnected porous structure induce in vivo tissue remodeling and accompanied vascularization.

Tissue engineering and regenerative medicine have furnished a vast range of modalities to treat either damaged tissue or loss of soft tissue or its function. In most approaches, a temporary porous scaffold is required to support tissue regeneration. The scaffold should be designed such that the turnover synchronizes with tissue remodeling and regeneration at the implant site. Segmented polyester urethanes (PUs) used in this study were based on epsilon-caprolactone (CL) and co-monomers D,L-lactide (D,L-L) and gamma-butyrolactone (BL), and 1,4-butanediisocyanate (BDI). In vitro, the PUs were nontoxic and haemocompatible. To test in vivo biocompatibility, the PUs were further processed into porous structures and subcutaneously implanted in rats for a period up to 21 days. Tissue remodeling and scaffold turnover was associated with a mild tissue response. The tissue response was characterized by extensive vascularization through the interconnected pores, with low numbers of macrophages on the edges and stroma formation inside the pores of the implants. The tissue ingrowth appeared to be related to the extent of microphase separation of the PUs and foam morphology. By day 21, all of the PU implants were highly vascularized, confirming the pores were interconnected. Degradation of P(CL/D,L-L)-PU was observed at this time, whereas the other two PU types remained intact. The robust method reported here of manufacturing and processing, good mechanical properties, and in vivo tissue response of the porous P(CL/D,L-L)-PU and PBCL-PU makes them excellent candidates as biomaterials with an application for soft tissue remodeling, for example, for cardiovascular regeneration.

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.

The downmodulation of the foreign body reaction by cytomegalovirus encoded interleukin-10.

The foreign body reaction (FBR) is of great importance for the function and turnover of biomaterial scaffolds. The development of biological tools that modulate the FBR will augment scaffold functionality and benefit regenerative medicine. The human cytomegalovirus encodes a functional homolog of the potent anti-inflammatory human cytokine interleukin-10 (cmvIL-10). We hypothesized that cmvIL-10 downmodulates the FBR, impairing degradation of biomaterial. We studied the effect of cmvIL-10 on the FBR to subcutaneously implanted hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC) discs in rats. CmvIL-10 impaired macrophage influx, vascularization and ingrowth into the discs up to 21 days. It also impaired the formation of giant cells and the degradation of HDSC. At day 10, deposited fibrin fibers were still present in cmvIL-10 discs. Impaired collagenase activity coincided with the impaired HDSC degradation. These results indicate that cmvIL-10 downmodulates the FBR, impairing the progression of the FBR. This study demonstrates the feasibility of interleukin-10 as a biomolecular tool in biomaterials for regenerative medicine.

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 beta-galactosidase transgene expression mirrors that of the endogenous pro-collagen 1A2 gene, which allows for functional assessment of the recruited cells. After MI, alpha-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 alpha-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.

Material dependent differences in inflammatory gene expression by giant cells during the foreign body reaction.

Multinucleated giant cells (GCs) are often observed in the foreign body reaction against implanted materials. The in vivo function of GCs in this inflammatory process remains to be elucidated. GCs degrade collagen implants in rats and may also orchestrate the inflammatory process via the expression and secretion of modulators, such as cytokines and chemokines. In this study, we show that the gene expression of PMN chemoattractants, CXCL1/KC and CXCL2/MIP-2, is high in GCs micro-dissected from explanted Dacron, cross-linked collagen (HDSC), and bioactive ureido-pyrimidinone functionalized oligocaprolactone (bioactive PCLdiUPy). Conversely, the gene expression levels of TGFbeta and pro-angiogenic mediators VEGF and FGF were found to be low in these GCs as compared with the expression levels in total explants. GCs in bioactive PCLdiUPy displayed high cytokine and angiogenic mediator expression compared with GCs isolated from the two other studied materials, whereas chemokine gene expression in GCs isolated form HDSC was low. Thus, GCs adopt their expression profile in response to the material that is encountered.

Circulating human CD34+ progenitor cells modulate neovascularization and inflammation in a nude mouse model.

CD34+ progenitor cells hold promise for therapeutic neovascularization in various settings. In this study, the role of human peripheral blood CD34+ cells in neovascularization and inflammatory cell recruitment was longitudinally studied in vivo. Human CD34+ cells were incorporated in Matrigel, implanted subcutaneously in nude mice, and explanted after 2, 4, 7, or 14 days. Cell-free Matrigels served as controls. Histochemical analyses demonstrated that neovascularization occurred almost exclusively in CD34+ implants. Cellular and capillary density were increased in cell-loaded Matrigels after 2 days and further increased at 14 days. Human CD34+ cells did not incorporate in neovessels, but formed vWF+/CD31+/VEGF+ cell clusters that were present up to day 14. However, CD34+ cells induced host neovascularization, as demonstrated by increased presence of murine CD31+ and vWF+ vasculature from day 7 to 14. Moreover, recruitment of murine monocytes/macrophages was significantly enhanced in CD34+ implants at all time points. Gene expression of chemotactic cytokines MCP-1 and IL-8 was detected on CD34+ cells in vitro and confirmed immunohistochemically in cell-loaded explants at all time points. Our data indicate that human CD34+ cells, implanted in a hypoxic environment, generate an angiogenic niche by secreting chemotactic and angiogenic factors, enabling rapid neovascularization, possibly via recruitment of monocytes/macrophages.

A novel and simple method for endotracheal intubation of mice.

Endotracheal intubation in mice is necessary for experiments involving intratracheal instillation of various substances, repeated pulmonary function assessments and mechanical ventilation. Previously described methods for endotracheal intubation in mice require the use of injection anaesthesia to immobilize the animal during the intubation procedure or the use of a volatile anaesthetic prior to intubation for immobilization. With these methods, the control of anaesthetic depth during the intubation procedure is absent. We describe a method for simple and rapid intratracheal intubation in mice for mechanical ventilation, using a self-built plastic support to facilitate the intubation procedure. General anaesthesia is maintained by means of inhalation through a non-rebreathing circuit connected to the plastic support. This set-up gives the operator control of anaesthetic depth and sufficient time to perform the intubation procedure. A purpose-made laryngoscopic blade is used to facilitate the intubation tube entering the trachea. The blade of the purpose-made laryngoscope is constructed as a retraction guide and is curved for easy handling. Under direct vision, the epiglottis is gently lifted by the laryngoscopic blade while the intubation tube is pushed into the trachea. Following this novel intubation technique, we were able to mechanically ventilate mice for at least 2 h without severely disturbing blood gases. Histological evaluation of the lungs and microscopic evaluation of the trachea and larynx showed no signs of trauma related to the intubation technique or mechanical ventilation.

Signaling factors in stem cell-mediated repair of infarcted myocardium.

Myocardial infarction leads to scar formation and subsequent reduced cardiac performance. The ultimate therapy after myocardial infarction would pursue stem cell-based regeneration. The aim of stem cell-mediated cardiac repair embodies restoration of cardiac function by regeneration of healthy myocardial tissue, which is accomplished by neo-angiogenesis and cardiogenesis. A major reservoir of adult autologous stem cells distal from the heart is the bone marrow. Adequate regulation of signaling between the bone marrow, the peripheral circulation and the infarcted myocardium is important in orchestrating the process of mobilization, homing, incorporation, survival, proliferation and differentiation of stem cells, that leads to myocardial regeneration. In this review, we discuss key signaling factors, including cytokines, chemokines and growth factors, which are involved in orchestrating the stem cell driven repair process. We focus on signaling factors known for their mobilizing and chemotactic abilities (SDF-1, G-CSF, SCF, IL-8, VEGF), signaling factors that are expressed after myocardial infarction involved in the patho-physiological healing process (TNF-alpha, IL-8, IL-10, HIF-1alpha, VEGF, G-CSF) and signaling factors that are involved in cardiogenesis and neo-angiogenesis (VEGF, EPO, TGF-beta, HGF, HIF-1alpha, IL-8). The future therapeutic application and capacity of secreted factors to modulate tissue repair after myocardial infarction relies on the intrinsic potency of factors and on the optimal localization and timing of a combination of signaling factors to stimulate stem cells in their niche to regenerate the infarcted heart.

US28 actions in HCMV infection: lessons from a versatile hijacker.

Mimicking host proteins is a strategy adopted by several herpesviruses to exploit the host cell for their own benefit. In this respect the human cytomegalovirus (HCMV) chemokine receptor homologue US28, has been extensively studied. Molecular pirates such as US28 can teach us about crucial events in HCMV infection and may either offer a potential target for antiviral therapy or provide an alternative strategy to immune suppression. Despite elaborate research into the chemokine binding affinity, signalling properties, intracellular trafficking and expression kinetics of US28, a solid hypothesis about the role of US28 in HCMV infection has not yet been proposed. It appears that US28 may behave as a molecular pirate that employs smart strategies for cell entry, host gene regulation and immune evasion. This review will elaborate on these aspects of US28 biology and discuss possible implications for HCMV infection.

Tissue response of defined collagen-elastin scaffolds in young and adult rats with special attention to calcification.

Collagen-elastin scaffolds may be valuable biomaterials for tissue engineering because they combine tensile strength with elasticity. In this study, the tissue response to and the calcification of these scaffolds were evaluated. In particular, the hypothesis was tested that calcification, a common phenomenon in biomaterials, may be due to microfibrils within the elastic fibre, and that these microfibrils might generate a tissue response. Four scaffolds were subcutaneously implanted, viz. collagen, collagen + pure elastin, collagen+microfibril-containing, and collagen + pulverised elastic ligament (the source for elastin). Explants were evaluated at day 3, 7 and 21. In young Sprague Dawley rats, collagen + ligament calcified substantially, whereas collagen + elastin (with and without microfibrils) calcified less, and collagen did not. Calcification started at elastic fibres. In both Sprague Dawley and Wistar adult rats, however, none of the scaffolds calcified. Mononuclear cell infiltration was prominent in young and adult Sprague Dawley rats. In adult Wistar rats, this infiltration was associated with the presence of microfibrils. Degradation of scaffolds and new matrix formation were related with cellular influx and degree of vascularisation. In conclusion, absence of microfibrils from the elastic fibre does not prevent calcification in young Sprague Dawley rats, but does reduce the tissue response in adult Wistar rats. Cellular response and calcification differs with age and strain and therefore the choice of animal model is of key importance in biomaterial evaluation.

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 of P2X(1), P2X(7)R, P2Y(1)R, and P2Y(2)R. 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 express of P2X(7)R, P2Y(1)R and P2Y(2)R increase until day 2. The expression of P2X(7)R and P2Y(1)R on macrophages and giant cells increased during the course of the inflammatory reaction which was studied for 21 days. The expression of the P2Y(2)R 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 P2X(1)R remains at a constant low level. The upregulation of P2X(7)R, P2Y(1)R, and P2Y(2)R over time suggests a regulatory function for these receptors in inflammation.

A hyaluronan-based nerve guide: in vitro cytotoxicity, subcutaneous tissue reactions, and degradation in the rat.

We investigated possible cytotoxic effects, biocompatibility, and degradation of a hyaluronan-based conduit for peripheral nerve repair. We subjected the conduits to an in vitro fibroblast cytotoxicity test and concluded that the conduits were not cytotoxic. Subsequently, we implanted the conduits subcutaneously in rats, in order to investigate tissue reactions and biodegradation. Initially, a fibrin matrix was formed around the material, while the surroundings were relatively quiet. Macrophages (MØ) migrated to the conduits and formed giant cells next to the material after 5 days. The maximum presence of MØ was found after 3-6 weeks. The appearance of MHC class II cells showed a similar pattern. Highest numbers of giants reached a maximum after 6-12 weeks. Angiogenesis was started in the surroundings of the hyaluronan-based conduit within a few days. Massive ingrowth of blood vessels into the biomaterial was found after 6 weeks as well as cellular ingrowth into the lumen of the tube. At that time the tubular structure of the conduit was lost and loose biomaterial fibers were observed. The results show that a hyaluronan-based conduit is not cytotoxic and shows good biocompatibility. Such a conduit may be suitable as a guide in peripheral nerve repair.

Preparation of degradable porous structures based on 1,3-trimethylene carbonate and D,L-lactide (co)polymers for heart tissue engineering.

Biodegradable porous scaffolds for heart tissue engineering were prepared from amorphous elastomeric (co)polymers of 1,3-trimethylene carbonate (TMC) and D,L-lactide (DLLA). Leaching of salt from compression-molded polymer-salt composites allowed the preparation of highly porous structures in a reproducible fashion. By adjusting the salt particle size and the polymer-to-particle weight ratio in the polymer-salt composite preparation the pore size and porosity of the scaffolds could be precisely controlled. The thermal properties of the polymers used for scaffold preparation had a strong effect on the morphology, mechanical properties and dimensional stability of the scaffolds under physiological conditions. Interconnected highly porous structures (porosity, 94%; average pore size, 100 microm) based on a TMC-DLLA copolymer (19:81, mol%) had suitable mechanical properties and displayed adequate cell-material interactions to serve as scaffolds for cardiac cells. This copolymer is noncytotoxic and allows the adhesion and proliferation of cardiomyocytes. During incubation in phosphate-buffered saline at 37 degrees C, these scaffolds were dimensionally stable and the number average molecular weight (Mn) of the polymer decreased gradually from 2.0 x 10(5) to 0.3 x 10(5) in a period up to 4 months. The first signs of mass loss (5%) were detected after 4 months of incubation. The degradation behavior of the porous structures was similar to that of nonporous films with similar composition and can be described by autocatalyzed bulk hydrolysis.

In vivo behavior of poly(1,3-trimethylene carbonate) and copolymers of 1,3-trimethylene carbonate with D,L-lactide or epsilon-caprolactone: Degradation and tissue response.

The degradation and the tissue response evoked by poly(1,3-trimethylene carbonate) [poly(TMC)] and copolymers of TMC with either 52 mol % D,L-lactide (DLLA) or 89 mol % epsilon-caprolactone (CL) were evaluated in vivo by subcutaneous implantation of polymer films in rats for periods up to one year. Poly(TMC) specimens were extensively degraded after 3 weeks and, as confirmed by histology, totally resorbed in less than a year. A fast linear decrease in thickness and mass without a change in molecular weight was observed. Initially an acute sterile inflammatory tissue reaction, caused by the implantation procedure, was observed, followed by a mild macrophage-mediated foreign body reaction that lasted during the resorption period of the polymer. It is concluded that in vivo, poly(TMC) is degraded via surface erosion involving cellular-mediated processes. The degradation of the copolymers was slower than that of poly(TMC), taking place via autocatalyzed bulk hydrolysis, preferentially of ester bonds. The TMC-DLLA copolymer degraded 20 times faster than the TMC-CL one. In both cases, the tissue reaction upon implantation resembled a sterile inflammatory reaction followed by a foreign body reaction that led to the polymer encapsulation. Significant mass loss was only observed for the TMC-DLLA copolymer, which underwent 96% mass loss in 1 year. When extensive mass loss started, a mild-to-moderate secondary foreign body reaction, related to clearance of the polymer fragments, was triggered. The results presented in this study demonstrate that poly(TMC) and both TMC copolymers are biodegradable and biocompatible materials, making these polymers attractive for the preparation of short- and long-term degradable devices for soft tissue engineering.

IL-2 loaded dextran microspheres with attractive histocompatibility properties for local IL-2 cancer therapy.

Biodegradable dextran microspheres (MS) were developed as a slow-release system for interleukin-2 (IL-2) to apply them for local IL-2 therapy of cancer. We describe the tissue reactions induced by these MS without or with IL-2 in rats. Dextran MS stain bright red-purple with the periodic acid Schiff (PAS), visualising the exact spot of IL-2 release and its relation to the histological reaction pattern. Subcutaneously injected MS always form a well-circumscribed deposit. In the first 2 days there is a PMN inflammation within the MS-deposit, but the surroundings show only a scanty inflammatory reaction. The PMN reaction is replaced by an abundant macrophage reaction in particular in the MS-deposit. At day 21 a fibrous capsule of about 50 mum surrounds the deposit. The effect of IL-2 administered in its free form is mainly vascular, with vascular dilatation, vascular leakage and oedema. It is remarkable that lymphocytes are present in the injection area already at day 2. When IL-2 releasing MS were used, the various reactions induced by IL-2 and MS were amplified leading to local necrosis. We conclude that neither placebo MS nor IL-2 leads to necrosis after subcutaneous injection in rats. In contrast, when IL-2 was released from MS, then massive necrosis was induced. This might be due to increased phagocytosis or changes in the micro-niche due to the release of humoral factors by the infiltrating cells. This is probably fortuitous for local IL-2 therapy of cancer, as massive necrosis of tumour cells can be expected to lead to an increased antitumour reaction.

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

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.