Host Cell Migration Research Articles

Biosynthetic cellulose induces the formation of a neoduramater following pre-natal correction of meningomyelocele in fetal sheep

The aim of this study was to compare the effectiveness of two dura-mater substitutes, namely human acellular dermal matrix (HADM) and biosynthetic cellulose (BC), in repairing, in utero, surgically-induced meningomyelocele (MMC) in fetal sheep. A neural tube defect was created at 74-77 days gestation in 36 fetal sheep. They were divided into 3 groups, the control group that did not receive pre-natal corrective surgery, and the other two groups that received corrective surgery using HADM (Group A) or BC (Group B). Both materials were used as a dura-mater substitutes between the neural tissue and the sutured skin. Correction was performed at gestation day 100 and the fetuses were maintained in utero until term. Sheep were sacrificed on gestation day 140. The fetal spine was submitted to macro and microscopic analysis. At microscopy, adherence of the material to the skin and neural tissue was analyzed. In the initial phase (pilot), experimentally-induced MMC was performed on 11 fetuses and 4 survived (37%). In the second phase (study), 25 fetuses received surgery and 17 survived (68%). In the study group, 6 fetuses did not undergo repair (control group), 11 cases were submitted to corrective surgery (experimental group) and one fetal loss occurred. Of the surviving cases in the experimental group, 4 constituted Group A and 6 in Group B. Macroscopically, skin and underlying tissues where easily displaced from the BC in all cases it was used; in contrast, HADM adhered to these tissues. To compare the adherence, 4 cases from Group A and 4 in Group B were studied. We observed adherence, host cell migration and vessel proliferation into the HADM all sections from Group A and this aspect was not present in any cases in Group B (p < 0.05). In Group B, we also observed that a new fibroblast layer formed around the BC thus protecting the medulla and constituting a "neoduramater". The use of BC seems to be more adequate as a dura-mater substitute to cover the damaged neural tissue than HADM. It seems promising for use in the in utero correction of MMC because to does not adhere to neural tissue of superficial and deep layers ("tethered spinal cord"). Thus, BC minimizes the mechanical and chemical intrauterine damage to the spinal medulla.

Induction of dendritic cell migration upon Toxoplasma gondii infection potentiates parasite dissemination

The processes leading to systemic dissemination of the obligate intracellular parasite Toxoplasma gondii remain unelucidated. In vitro studies on human and murine dendritic cells (DC) revealed that active invasion of DC by Toxoplasma induces a state of hypermotility in DC, enabling transmigration of infected DC across endothelial cell monolayers in the absence of chemotactic stimuli. Infected DC exhibited upregulation of maturation markers and co-stimulatory molecules. While modulation of cell adhesion molecules CD11/CD18 was similar for Toxoplasma-infected DC and lipopolysaccharide (LPS)-matured DC, Toxoplasma-infected DC did not exhibit upregulation of CD54/ICAM-1. Induction of host cell migration in vitro required live intracellular parasite(s) and was inhibited by uncoupling the Gi-protein signalling pathway with pertussis toxin, but did not depend on CCR5, CCR7 or Toll/interleukin-1 receptor signalling. When migration of Toxoplasma-infected DC was compared with migration of LPS-stimulated DC in vivo, similar or higher numbers of Toxoplasma-infected DC reached the mesenteric lymph nodes and spleen respectively. Adoptive transfer of Toxoplasma-infected DC resulted in more rapid dissemination of parasites to distant organs and in exacerbation of infection compared with inoculation with free parasites. Altogether, these findings show that Toxoplasma is able to subvert the regulation of host cell motility and likely exploits the host's natural pathways of cellular migration for parasite dissemination.

Reciprocal upregulation of urokinase plasminogen activator and its inhibitor, PAI-2, by Borrelia burgdorferi affects bacterial penetration and host-inflammatory response

The mammalian plasminogen activation system (PAS) is a complex system involved in multiple physiological and pathological processes. Borrelia burgdorferi interacts with certain components of the PAS. Here we further investigate this interaction to determine its effect on bacterial dissemination and host cell migration in vitro. We show that stimulation of monocytic cells with B. burgdorferi induces the transient production and secretion of urokinase plasminogen activator (uPA), shortly followed by its physiological inhibitor, plasminogen activator inhibitor-2 (PAI-2). Mono Mac 6 (MM6) cells as well as peripheral blood monocytes enhanced transmigration of B. burgdorferi across a barrier coated with fibronectin mediated by uPA. Moreover, the induction of PAI-2 or the addition of recombinant PAI-2 did not have a significant effect on the uPA-potentiated transmigration of B. burgdorferi. In contrast, the induction of PAI-2 by B. burgdorferi resulted in significantly diminished invasion by monocytic cells across a reconstituted basement membrane (matrigel), which could be partially restored by treatment with purified uPA. These results show that the PAS plays a twofold role in the pathogenesis of B. burgdorferi infection, both by enhancing bacterial dissemination and by diminishing host-cell inflammatory migration.

Explant pathology study of decellularized carotid artery vascular grafts.

The purpose of this study was to evaluate the morphologic findings in small-diameter freeze-dried decellularized carotid artery grafts implanted in goats as carotid artery interposition grafts for 6-7 months. Unimplanted decellularized carotid artery grafts did not contain intact cells; however, remnants of smooth muscle cells were present in the media. The extracellular matrix was well preserved. All decellularized grafts were patent at explant, without significant dimensional changes or aneurysm formation. Their luminal surfaces were lined by a thin neointima, consisting of myofibroblasts, collagen, and a discontinuous layer of endothelial cells. Histologic evidence of calcification within the explants was not observed; however, electron microscopy showed calcification of minute remnants of cell membranes. Inflammatory cells were not present in the graft wall. Host cell migration was greatest in the adventitia along the length of the graft. Migration of host cells into the media was more apparent close to the anastomoses, forming cellular nests rich in extracellular proteoglycans, whereas cell migration into areas subjacent to the lumen was minimal. Ingrowth of host blood vessels was not observed. These results demonstrate satisfactory structural and morphologic features of a decellularized carotid artery small-diameter graft implanted for up to 7 months.

Effects of crosslinking degree of an acellular biological tissue on its tissue regeneration pattern

It was reported that acellular biological tissues can provide a natural microenvironment for host cell migration and may be used as a scaffold for tissue regeneration. To reduce antigenicity, biological tissues have to be fixed with a crosslinking agent before implantation. As a tissue-engineering scaffold, it is speculated that the crosslinking degree of an acellular tissue may affect its tissue regeneration pattern. In the study, a cell extraction process was employed to remove the cellular components from bovine pericardia. The acellular tissues then were fixed with genipin at various known concentrations to obtain varying degrees of crosslinking. It was shown in the in vitro degradation study that after fixing with genipin, the resistance against enzymatic degradation of the acellular tissue increased significantly with increasing its crosslinking degree. In the in vivo subcutaneous study, it was found that cells (inflammatory cells, fibroblasts, endothelial cells, and red blood cells) were able to infiltrate into acellular tissues. Generally, the depth of cell infiltration into the acellular tissue decreased with increasing its crosslinking degree. Infiltration of inflammatory cells was accompanied by degradation of the acellular tissue. Due to early degradation, no tissue regeneration was observed within fresh (without crosslinking) and the 30%-degree-crosslinking acellular tissues. This is because the scaffolds provided by these two samples were already completely degraded before the infiltrated cells began to secrete their own extracellular matrix. In contrast, tissue regeneration (fibroblasts, neo-collagen fibrils, and neo-capillaries) was observed for the 60%- and 95%-degree-crosslinking acellular tissues by the histological examination, immunohistological staining, transmission electron microscopy, and denaturation temperature measurement. The 95%-degree-crosslinking acellular tissue was more resistant against enzymatic degradation than its 60%-degree-crosslinking counterpart. Consequently, tissue regeneration was limited in the outer layer of the 95%-degree-crosslinking acellular tissue throughout the entire course of the study (1-year postoperatively), while tissue regeneration was observed within the entire sample for the 60%-degree-crosslinking acellular tissue. In conclusion, the crosslinking degree determines the degradation rate of the acellular tissue and its tissue regeneration pattern.

Insulin-like growth factor (IGF)-I affects parasite growth and host cell migration in experimental cutaneous leishmaniasis.

While the control or progression of leishmaniasis depends on host immune responses, the initial inflammatory process represents a key event. This process involves the participation of several cytokines and growth factors induced during inflammation as well as factors already present at the site of infection such as insulin-like growth factor (IGF)-I. We have previously demonstrated a potential role for IGF-I in experimental cutaneous leishmaniasis based on the significant increase in lesion size seen in mice injected with Leishmania promastigotes preactivated with IGF-I. In the present study we show that preactivation of Leishmania (Leishmania) amazonensis promastigotes with IGF-I induces an increase in the actual number of parasites at the lesion site from seven days postinfection, in addition to a more intense inflammatory infiltrate. There was a higher numerical density of polymorphonuclear neutrophils from 3 to 24 h, and of mononuclear cells from 48 h of infection onward. A higher density of polymorphonuclear neutrophils and mononuclear cells harboring parasites was also observed. The most important observation, however, was that more parasites per cell were present, revealing that IGF-I appears to favour parasite growth within the macrophages. These results strongly suggest an important role for IGF-I in the development of cutaneous leishmaniasis, where it influences both the inflammatory process and parasite growth.

Active cell migration in retransplanted rat cardiac allografts during the course of chronic rejection

BackgroundChronic allograft vasculopathy (CAV) is caused by the infiltration of host immune cells to a graft, but it has been technically difficult to monitor the movements of the cells in graft rejection. MethodsWe used a male-specific gene, SRY, as a marker to investigate the dynamics of host cells in a model of CAV in which immunosuppression was unnecessary and anti-male responses were practically negligible. Fluorescent-based real-time quantitative polymerase chain reaction (PCR) was adapted to estimate the fraction of host cells in a graft by the ratio of SRY to IL-2 gene. Using this technique, we studied the turnover and migration of host cells during the course of CAV progression by retransplanting female allografts from male to female or from female to male rats. ResultsWe detected histologic CAV 60 days after retransplantation in allografts retransplanted to the F1 progeny of donor × recipient on the 5th day, but not in those retransplanted on the 3rd day, regardless of the mismatches in the genders. Most of the initial infiltrating cells disappeared rapidly in both cases. The fraction of migrating cells from the second recipient, however, continuously increased in allografts developing CAV, and 60 days after retransplantation exceeded 50%, whereas it stayed at 5% to 15% in those not developing CAV. ED-1-positive macrophages/monocytes were likely candidates for the migrated cells. ConclusionsWe have developed a simple method to measure the migration of host cells into a graft. This technique was useful, at least in certain rat strains, to investigate the cellular mechanisms of chronic cardiac allograft rejection.

HIV-1 envelope protein gp41 modulates expression of interleukin-10 and chemokine receptors on monocytes, astrocytes and neurones.

To analyse the effect of HIV-1 transmembrane protein gp41 on cytokine production and chemokine receptor expression in blood and brain. Because previous results had demonstrated that recombinant gp41 contributes to HIV-induced dysfunction of blood immune cells we investigated its effect on interleukin (IL)-10 synthesis and expression of the HIV coreceptors CCR5 and CXCR4 in different human brain cells. Astrocytic, microglial and neuronal cell lines were incubated with the extracellular domain of gp41 (aa565-647). Secretion of IL-10 into the medium was measured by ELISA. Chemokine receptor expression was analysed by fluorescence activated cell sorting and by RT-PCR. Incubation of the astrocytic cell line U87 with gp41 induced more than a 10 fold up-regulation of IL-10 secretion. This modulation was shown to be time- and dose-dependent. Use of inhibitors for different signal transduction pathways indicated a similar transduction cascade for the alteration of IL-10 production in astrocytes as in monocytes with participation of cAMP/adenylate cyclase and activation of p70S6 kinase. To a lesser extent IL-10 synthesis was also up-regulated by gp41 in the neuronal cell line SK-N-SH. In all cell types up-regulation of IL-10 paralleled by an enhanced expression of the chemokine receptor and HIV-1 coreceptor CCR5. This up-regulation was driven by IL-10 as shown by use of an IL-10 antibody. Expression of the chemokine receptor CXCR4 was only slightly altered. These findings suggest a role for gp41 in the modulation of brain-specific host defence, cell migration and cell infectivity by HIV.

Virulence factors of Actinobacillus actinomycetemcomitans.

A. actinomycetemcomitans has clearly adapted well to its environs; its armamentarium of virulence factors (Table 2) ensures its survival in the oral cavity and enables it to promote disease. Factors that promote A. actinomycetemcomitans colonization and persistence in the oral cavity include adhesins, bacteriocins, invasins and antibiotic resistance. It can interact with and adhere to all components of the oral cavity (the tooth surface, other oral bacteria, epithelial cells or the extracellular matrix). The adherence is mediated by a number of distinct adhesins that are elements of the cell surface (outer membrane proteins, vesicles, fimbriae or amorphous material). A. actinomycetemcomitans enhances its chance of colonization by producing actinobacillin, an antibiotic that is active against both streptococci and Actinomyces, primary colonizers of the tooth surface. The fact that A. actinomycetemcomitans resistance to tetracyclines, a drug often used in the treatment of periodontal disease, is on the rise is an added weapon. Periodontal pathogens or their pathogenic products must be able to pass through the epithelial cell barrier in order to reach and cause destruction to underlying tissues (the gingiva, cementum, periodontal ligament and alveolar bone). A. actinomycetemcomitans is able to elicit its own uptake into epithelial cells and its spread to adjacent cells by usurping normal epithelial cell function. A. actinomycetemcomitans may utilize these remarkable mechanisms for host cell infection and migration to deeper tissues. A. actinomycetemcomitans also orchestrates its own survival by elaborating factors that interfere with the host's defense system (such as factors that kill phagocytes and impair lymphocyte activity, inhibit phagocytosis and phagocyte chemotaxis or interfere with antibody production). Once the organisms are firmly established in the gingiva, the host responds to the bacterial onslaught, especially to the bacterial lipopolysaccharide, by a marked and continual inflammatory response, which results in the destruction of the periodontal tissues. A. actinomycetemcomitans has at least three individual factors that cause bone resorption (lipopolysaccharide, proteolysis-sensitive factor and GroEL), as well as a number of activities (collagenase, fibroblast cytotoxin, etc.) that elicit detrimental effects on connective tissue and the extracellular matrix. It is of considerable interest to know that A. actinomycetemcomitans possesses so many virulence factors but unfortunate that only a few have been extensively studied. If we hope to understand and eradicate this pathogen, it is critical that in-depth investigations into the biochemistry, genetic expression, regulation and mechanisms of action of these factors be initiated.

Adsorption of basic fibroblast growth factor onto Dacron vascular prosthesis and its biological efficacy.

Adsorption of basic fibroblast growth factor (bFGF) onto a plain fabric Dacron vascular prosthesis (Micron, InterVascular Co., Ltd., Clearwater, FL, U.S.A.) and its release properties from the graft were examined using labeled bFGF, and its biological efficacy was evaluated in an animal study. In an in vitro study, 6 pieces of a Dacron graft were soaked in [(125)I]-bFGF solution for 30 min. Then these pieces were soaked in 500 ml saline solution for 15 days, and the radioactivity of each piece was counted at various times. The initial amount of adsorbed bFGF was 2.48 ng/cm2. At 24 h, 41% of the adsorbed bFGF was released and 59% remained. On the third day 55% remained, on the seventh day 52%, and on the 15th day 50% remained on the Dacron surface. Using the atmospheric glow discharge (APG) plasma treatment to render hydrophilic properties, no significant difference between plasma-treated grafts and non-plasma-treated grafts regarding the adsorption of bFGF was observed. In an in vivo study, 6 Dacron pieces adsorbed with bFGF were implanted in the subcutaneous layer of 2 dogs and removed 5 days after implantation. Six Dacron pieces without bFGF were also implanted as a control into the 2 dogs. Fibroblast migration with capillary ingrowth was observed in the specimens with bFGF compared to the controls. These results indicate that the simple adsorption of bFGF onto Dacron fabric is a useful method for the acceleration of host cell migration and capillary ingrowth into Dacron fabric vascular prostheses.

Axonal Growth into Tubes Implanted within Lesions in the Spinal Cords of Adult Rats

Cultured Schwann cells were implanted into the thoracic spinal cords of adult rats inside thin tubes made of polycarbonate film coated with poly-l-lysine. Additional control tubes were implanted which did not contain cultured Schwann cells. Some of the control tubes were coated with poly-l-lysine and others were not. One week to 2 months later the animals were perfused with fixatives and the tubes were prepared for light or electron microscopy. Immunocytochemical studies of the Schwann cell tubes reveal that they contain axons. Most of these axons are grouped in fascicles that run longitudinally through the tubes. The distribution of these axons matches precisely the distribution of basal lamina within the tubes as displayed by immunolabeling with an antibody to laminin. Surprisingly, the same patterns of labeling are seen in the control tubes, although they contain fewer axons. Control tubes lacking poly-l-lysine contain the fewest. Electron microscopy verifies that the tubes, including control tubes, contain Schwann cells and axons of different diameters. Furthermore, the Schwann cells ensheathe and myelinate the axons. These results strengthen the hypothesis that Schwann cells can support axonal growth in the spinal cords of adult animals. They also demonstrate that these Schwann cells can be implanted or they can be derived from the host animal. This finding raises the possibility that therapies could be devised for bridging spinal cord lesions that are based on maximizing migration of endogenous host cells into the sites of lesions.

Cell Viability and Migration in Nerve Isografts and Allografts

Even though autogenous nerve grafts are used frequently, there is little information concerning cell survival rates and migration patterns, following peripheral nerve grafting. Labeling techniques with a vital fluorescent stain (PKH-26, Zynaxis Cell Science, Malvern, PA) allow cell migrations from both the nerve graft and host nerve to be tracked for up to 45 days from the time of nerve transplantation. With this labeling technique, two phases of nerve graft incorporation were identified, early and late, in an animal model using inbred Lewis and Brown-Norway rats. In genetically identical Lewis rats, isografts were performed as a means of modeling the autografts used clinically. At approximately 3 days after isogeneic transplantation, with the proximal host nerve end labeled, there was an early migration of host cells from the proximal nerve end into the epineural tissue of the nerve graft. At 25 days, a late phase was evident, with fluorescent labeling of host cells into the perineural and endoneural tissues. When the nerve grafts were labeled, the label persisted for up to 45 days, indicating viability of the graft. Cells migrated from the labeled nerve graft into the distal host nerve segment. Cellular migration from peripheral nerve tissue, following allograft transplantation, was initially similar to the isograft studies. But after 25 days, with the proximal host nerve end labeled, a significant decrease in the labeled host cells migrating into the graft was noted (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis and fate of proteoglycans in cartilage and bone during development and mineralization

Subcutaneous implantation of demineralized bone matrix in rats induces migration of host cells into the site and results in the sequential development of cartilage and bone. The biosynthesis and metabolic fate of proteoglycans in the plaques at the bone matrix implantation site were investigated by [ 35S]sulfate labeling in vivo. 35S-Labeled proteoglycans were extracted with 4 m guanidine HCl and purified by DEAE-Sephacel chromatography. Analysis of proteoglycans on Sepharose CL-2B chromatography showed two major peaks at K d = 0.28 and 0.68 (peaks I and II, respectively). Peak I proteoglycan has a high buoyant density and contains chondroitin sulfate chains of average M r = 20,000. Peak II proteoglycan has a lower average buoyant density and contains dermatan sulfate chains of average M r = 33,000. Throughout the endochondral bone development sequence, peak II proteoglycan predominates. Peak I was low on Day 3, became prominent on Day 7 (approximately 30% of the total radioactivity), and declined after Day 9. The calculated half-lives of peak I and II proteoglycans labeled on Day 7 were about 1.8 and 2.8 days, respectively. After the initiation of osteogenesis, a species of mineral-associated proteoglycan was extracted with a 4 m guanidine HCl solvent containing 0.5 m EDTA. This proteoglycan has a small hydrodynamic size ( K d = 0.38 on Sepharose CL-6B chromatography) and shows a long half-life, about 6 days.

The Antibacterial Activity of Haemocyte Clumps in the Gills of the Shore Crab,Carcinus Maenas

INTRODUCTIONThe shore crab,Carcinus maenas(L.), rapidly clears bacteria from the circulation to the tissues by virtue of the phagocytic and clumping capacity of circulating haemocytes and fixed phagocytic cells (Smith &amp; Ratcliffe, 1978, 1980a,b; White &amp; Ratcliffe, 1982). If re-infection resulting from the re-entry of sequestered micro-organisms into the circulation is to be prevented then the host must eliminate or incapacitate the foreign particles in some way. The migration of laden host cells to the exterior does not appear to be an important means of disposal of segregated non-self material in the Crustacea (Merril, Mongeon &amp; Fisher, 1979; White &amp; Ratcliffe, 1982), so that the ability to resist infection must reside with the cellular killing capacity, perhaps augmented by humoral antibacterial activity. Indeed, our earlier observations on the disappearance of bacteria fromC. maenasgills seem to indicate that their loss resulted from the lytic action of host haemocytes (White &amp; Ratcliffe, 1982), as does the elimination of micro-organisms from the hepatopancreas of the crayfish,Parachaeraps bicarinatus(McKay &amp; Jenkin, 1970a).

Effect of mouse phagocytes on Candida albicans in in vivo chambers.

Mouse phagocytic cells in in vivo diffusion chambers had either candidacidal or candidastatic activity depending on the kind of phagocyte studied, the type of mouse whence the phagocytes came, and whether the chamber inoculum of Candida albicans consisted primarily of yeasts or hyphae. Killing of C. albicans occurred when yeasts were placed into chambers with membranes with 3.0-micrometer pores and implanted intraperitoneally into normal mice or thymus-deficient (nude) mice. Although C. albicans remains in chambers with 3.0-micrometer pores, host phagocytic cells can migrate into the chambers. Killing also occurred when yeasts were combined with normal or nude mouse neutrophils in chambers made with membranes with 0.45-micrometer pores, which restrict migration of host cells, but not diffusion of soluble factors. Populations of cells rich in macrophages were candidastatic for yeasts when the phagocytes came from normal mice but candidacidal when obtained from nude mice. Results of gradient fractionations of peritoneal exudates indicated that more than one cell type may be responsible for candidacidal activity by nude mouse macrophage-rich cells. Hyphal-phase cells of C. albicans appear to be more resistant than yeast-phase cells to killing by normal and nude mouse phagocytic cells.

Studies on the internal defense mechanisms of sponges: III. Cellular reactions in Terpios zeteki to implanted heterologous biological materials

Cellular responses in Terpios zeteki to implanted heterologous biological materials consisting of human erythrocytes, trematode rediae and cercariae, and molluscau muscles, were examined at 6, 12, 24, and 48 hr postimplantation. It was determined that erythrocytes were either phagocytized or encapsulated by sponge archaeocytes and were gradually eliminated from the mesoglea via the migration of host cells into excurrent canals. The mesoglea was essentially free of implanted erythrocytes by the 48th hr. Implants of rediae only evoked slight encapsulation involving archaeocytes and collencytes, with some of the latter forming syncytia. Freed cercariae were enveloped by a relatively thick syncytial capsule of archaeocytes and collencytes. This capsule, however, disintegrated by the 48th hr when the cercarial tissues decomposed. Simultaneously, a large number of archaeocytes including amorphous materials, most probably fragments of disintegrated implanted tissues, were found in the lumina of excurrent canals. Response to muscle implants consisted primarily of an irregular distribution of collencytes and archaeocytes on the surfaces, but with no evidence of true capsule formation. Thus, incomplete or complete encapsulation of heterografts has been demonstrated in sponges for the first time. Incision sites in sponges of the control series revealed a gradual concentration of parenchymal cells along the cut surfaces. No fusion of these surfaces was observed.