Cell-assembled extracellular matrix (CAM): a human biopaper for the biofabrication of pre-vascularized tissues able to connect to the host circulationin vivo.

When considering regenerative approaches, the efficient creation of a functional vasculature, that can support the metabolic needs of bioengineered tissues, is essential for their survival after implantation. However, it is widely recognized that the post-implantation microenvironment of the engineered tissues is often hypoxic due to insufficient vascularization, resulting in ischemia injury and necrosis. This is one of the main limitations of current tissue engineering applications aiming at replacing significant tissue volumes. Here, we have explored the use of a new biomaterial, the cell-assembled extracellular matrix (CAM), as a biopaper to biofabricate a vascular system. CAM sheets are a unique, fully biological and fully human material that has already shown stable long-term implantation in humans. We demonstrated, for the first time, the use of this unprocessed human ECM as a microperforated biopaper. Using microvalve dispensing bioprinting, concentrated human endothelial cells (30 millions ml-1) were deposited in a controlled geometry in CAM sheets and cocultured with HSFs. Following multilayer assembly, thick ECM-based constructs fused and supported the survival and maturation of capillary-like structures for up to 26 d of culture. Following 3 weeks of subcutaneous implantation in a mice model, constructs showed limited degradative response and the pre-formed vasculature successfully connected with the host circulatory system to establish active perfusion.This mechanically resilient tissue equivalent has great potential for the creation of more complex implantable tissues, where rapid anastomosis is sine qua non for cell survival and efficient tissue integration.

PECAM-1 interacts with nitric oxide synthase in human endothelial cells: implication for flow-induced nitric oxide synthase activation.

OBJECTIVE: We have previously shown that fluid shear stress (FSS) triggers endothelial nitric oxide synthase (eNOS) activity in endothelial cells and that the mechanotransduction mechanisms responsible for activation discriminate between rapid changes in FSS and FSS per se. We hypothesized that the particular sublocalization of eNOS at the cell-cell junction would render it responsive to activation by FSS temporal gradients. METHODS AND RESULTS: In human umbilical vein endothelial cells (HUVECs), immunofluorescence revealed strong eNOS membrane staining at the cell-cell junction colocalizing with platelet/endothelial cell adhesion molecule-1 (PECAM-1). In PECAM-1-/- mouse aorta, eNOS junctional localization seen in the wild type was absent. Similarly, junctional staining was lost in wild-type aorta near intercostal artery branches. eNOS/PECAM-1 association in HUVECs was confirmed by coimmunoprecipitation. When HUVECs were subjected to a 0.5s impulse of 12 dynes/cm2, a transient disruption of the eNOS/PECAM-1 complex was observed, accompanied by an increase in eNOS activity (cGMP production). Ramped flow did not trigger complex dissociation or an increase in cGMP production. In a cell-free system, a direct inhibition of eNOS activity by PECAM-1 is shown. CONCLUSIONS: These results suggest that eNOS is complexed with PECAM-1 at the cell-cell junction and is likely involved in the modulation of eNOS activity by FSS temporal gradients but not by FSS itself.

Ribavirin potentiates the efficacy and toxicity of 2',3'- dideoxyinosine in the murine acquired immunodeficiency syndrome model.

The efficacy and toxicity of ribavirin (25 or 125 mg/kg/day), 2',3'-dideoxyinosine (ddI) (200 mg/kg/day) and a combination of both drugs at these doses given for 6 weeks were investigated in the murine acquired immunodeficiency syndrome model. Our results showed a significant protection against splenomegaly, lymphadenopathy and hypergammaglobulinemia in mice treated with ribavirin at 25 mg/kg/day alone or in combination with ddI at 200 mg/kg/day. A good synergistic effect was observed with the drug combination, whereas ddI alone (200 mg/kg/day) did not give any protection. Ribavirin/ddI combination protected against the loss of CD8 T cells in spleen and restored the capacity of splenocytes to proliferate after activation with a mitogenic agent. Moreover, the drug combination resulted in a protection of the spleen and cervical lymph node architectures and a regression of germinal centers. Hematotoxicity appeared at a dose of 125 mg/kg of ribavirin alone and increased when used concomitantly with ddI. In conclusion, ribavirin and ddI at low doses are synergistic and effective in the murine acquired immunodeficiency disease model, but at high doses they are toxic.

Antiviral efficacy and toxicity of ribavirin in murine acquired immunodeficiency syndrome model.

The antiretroviral efficacy and hematotoxicity of ribavirin, a guanosine analogue, have been evaluated in mice infected with the LP-BM5 virus pool [murine acquired immunodeficiency syndrome (MAIDS) model]. Doses ranging from 6.25 to 200 mg/kg/day were injected intraperitoneally twice a day for 6 weeks to infected mice. Drug treatment induced a significant protection against splenomegaly and lymphadenopathy at doses > or = 25 mg/kg. Moreover, doses starting at 50 mg/kg protected against hypergammaglobulinemia, minimized the loss of spleen CD8+ T cells, and reconstituted the capacity of splenocytes to proliferate in response to concanavalin A. The spleen and cervical lymph node architectures were protected, and a reduction in the emergence of germinal centers was observed at 50 mg/kg ribavirin. Hematotoxicity appeared at doses > or = 50 mg/kg ribavirin, and severe anemia was predominant only at doses of 100 and 200 mg/kg. This study shows that ribavirin protects mice against the effects resulting from retrovirus infection at doses of > or = 50 mg/kg in a MAIDS model and induces severe hematotoxicity at doses > or = 100 mg/kg.

Liposomal encapsulation of foscarnet protects against hypocalcemia induced by free foscarnet.

Hypocalcemia and an increase in creatinine level are the most important serious effects associated with foscarnet (PFA) therapy. In an animal model, we have explored the potential protective role of liposome-encapsulated foscarnet (LE-PFA) on these metabolic abnormalities. PFA administered as one bolus injection (0.5 or 1.0 g/kg) caused significant rapid decreases (approximately 20%) in the levels of calcium and phosphorus in serum within a few minutes and up to 30 min after injection. LE-PFA did not induce any of these changes, while peak levels in serum and the half-life of this formulation were much higher than those of the free drug. PFA administered for 2 weeks (340 or 500 mg/kg/day) resulted in no changes in creatinine or blood urea nitrogen levels in serum at the low-dosage level, but at the higher-dosage level, the creatinine level in serum increased by day 5 posttreatment. Furthermore, there was no increase in the creatinine or blood urea nitrogen level after 2 weeks of treatment with LE-PFA at a dosage of 35 mg/kg/day. When the pharmacokinetics of both free PFA and LE-PFA were compared, the plasma half-life of the encapsulated drug was approximately four times longer than that of the free drug. In addition, the systemic clearance of LE-PFA was approximately one-fifth of that of the free drug. In conclusion, free PFA causes hypocalcemia and hypophosphatemia and increases the creatinine level in serum, whereas the LE form of this drug seems to protect against the abnormal changes in calcium and phosphorus levels caused by the free drug. By preventing hypocalcemia and increasing its half-life, LE-PFA can be used at lower doses and at longer intervals. Clinical investigations of these formulations may be worthwhile.

Encapsulation of foscarnet in liposomes modifies drug intracellular accumulation, in vitro anti-HIV-1 activity, tissue distribution and pharmacokinetics.

OBJECTIVE: To improve the in vitro anti-HIV-1 activity, intracellular accumulation in macrophages and in vivo pharmacokinetics and tissue distribution of foscarnet (trisodium phosphonoformate; PFA) by encapsulation in liposomes. METHODS: The accumulation of free and liposome-encapsulated PFA was determined in monocyte-macrophage RAW 264.7 cells and human premonocytoid U937 cells. The antiviral activity was evaluated in U937 cells infected with HIV-1IIIB. Tissue distribution and pharmacokinetics of free and liposomal PFA were determined in female Sprague-Dawley rats following the administration of an intravenous bolus dose (10 mg PFA/kg). RESULTS: The entrapment of PFA in liposomes resulted in a higher drug accumulation in both U937 and RAW 264.7 cells. A slightly greater efficacy against HIV-1IIIB replication into U937 cells was observed upon encapsulation of PFA into liposomes. Improved pharmacokinetics was observed upon entrapment of PFA in liposomes. Much higher drug levels were found in plasma for the liposomal formulation. The systemic clearance of the liposomal drug was 77 times lower than that of free drug. The encapsulation of PFA in liposomes greatly enhanced the drug accumulation in organs of the reticuloendothelial system. CONCLUSION: The encapsulation of PFA in liposomes modified the tissue distribution and plasma pharmacokinetics of the antiviral agent, resulting in a marked improvement of drug accumulation in organs involved in HIV immunopathogenesis and in a greater PFA bioavailability. The antiviral activity of liposomal PFA was slightly greater than that of free drug in HIV-1IIIB-infected U937 cells.

In vitro HIV-1 entry and replication in Langerhans cells may clarify the HIV-1 genome detection by PCR in epidermis of seropositive patients.

Being dendritic antigen-presenting cells in skin and mucous membrane, Langerhans cells (LC) occur in areas at risk for inoculation by human immunodeficiency virus (HIV), and the question whether LC act as a target, reservoir, or vector for transmission of HIV has given rise to much controversy. To address this question, we first analyzed the epidermal compartment of skin from patients seropositive for HIV DNA. Second, we tested the susceptibility of each cell type normally found in this compartment to in vitro infection by HIV-1. A non-denatured DNA was obtained from epidermal sheets after a thermochemical treatment of biopsies (0.5 M ethylenediaminetetraacetic acid (EDTA), pH 7.5 at 60 degrees C for 90 seconds). Optimization of amplification of viral genome was performed with three primer pairs derived from gag, env, and pol sequences. Polymerase chain reaction (PCR) products were analyzed by Southern blot. Viral genome was found in five of 11 HIV-seropositive patients. To control the permissivity of epidermal cell population for HIV, cells isolated from the epidermal sheet of normal skin by trypsinization were co-cultured with HIV-1-carrying promonocytic cells (U937) and observed by electron microscopy. After 3-6 h of co-culture, numerous virions were either tightly bound or apparently engaged in the process of internalization through receptor-mediated endocytosis. At day 4 of co-culture, some infected LC appeared to release mature viral particles through bud formation. The in vitro HIV-1 entry and replication in LC may confirm the presence of the HIV-1 genome by PCR in epidermis of seropositive patients. The consequences of the permissivity of LC for HIV on the antigen-presenting function remain to be determined.

Interaction of human epidermal Langerhans cells with HIV-1 viral envelope proteins (gp 120 and gp 160s) involves a receptor-mediated endocytosis independent of the CD4 T4A epitope.

The CD4 molecule is known to be the preferential receptor for the HIV-1 envelope glycoprotein. Epidermal Langerhans cells are dendritic cells which express several surface antigens, among them CD4 antigens. To clarify the exact role of CD4 molecules in Langerhans cell infection induced by HIV-1, we investigated the possible involvement of the interactions between HIV-1 gp 120 or HIV-1 gp 160s (soluble gp 160) and Langerhans cell surface. We also assessed the expression of CD4 molecules on Langerhans cell membranes dissociated by means of trypsin from their neighbouring keratinocytes. The cellular phenotype was monitored using flow cytometry and quantitative immunoelectron microscopy. We reported that human Langerhans cells can bind the viral envelope proteins (gp 120 or gp 160s), and that this binding does not depend on CD4 protein expression. This binding is not blocked by anti-CD4 monoclonal antibodies. We show that a proportion of gp 120/gp 160s-receptor complexes enters Langerhans cells by a process identified as a receptor-mediated endocytosis. The amount of surface bound gp 120/gp 160s is not consistent with the amount of CD4 antigens present on Langerhans cell membranes. Gp 120/gp 160s binding sites on Langerhans cell suspensions appeared to be trypsin resistant, while CD4 antigens (at least the epitopes known to bind the HIV-1) are trypsin sensitive. A burst of gp 120 receptor expression was detected on 1-day cultured Langerhans cells while CD4 antigens disappeared. These findings lead to the most logical conclusion that binding of gp 120/gp 160s is due to the presence of a Langerhans cell surface molecule different from CD4 antigens.

Trypsin-resistant gp120 receptors are upregulated on short-term cultured human epidermal Langerhans cells.

The CD4 molecule is known to be the preferential receptor for the HIV1 envelope glycoprotein. Epidermal Langerhans cells (LC) are dendritic cells which express several surface antigens, among them the CD4 antigens. LC infection was suggested when these cells were seen to present buddings coincident with membrane thickening of roughly 100 nm in size. These buddings were similar in ultrastructural aspect to HIV buddings on in vitro infected promonocytic cells (U937). To clarify the exact role of CD4 molecules in LC infection induced by HIV1, we investigated the possible involvement of between native and recombinant HIV1 gp120 and the LC surface. We also assessed the expression of CD4 molecules on LC membranes dissociated by means of trypsin from their neighbouring keratinocytes. The cellular phenotype was monitored using flow cytometry. We show that human LC can bind the viral envelope protein and that this binding does not depend on CD4 protein expression. The amount of surface bound gp120 was not consistent with the amount of CD4 antigens present on LC membranes. The gp120-binding sites on LC in suspension appear to be typsin-resistant while the CD4 antigens (at least the epitopes known to bind HIV1) are trypsin-sensitive. A burst of gp120 receptor expression was detected on 1-day cultured LC while the CD4 antigens disappeared. These findings lead to the logical conclusion that the binding of gp120 is due to the presence of a LC surface molecule which is different from CD4 antigens.

Cyclosporin A inhibits DNA synthesis by epidermal Langerhans cells.

Cyclosporin A, a potent immunosuppressive drug currently used in organ transplant recipients, has been shown to exert in vitro a direct antiproliferative effect on a number of cell types present in the skin, including keratinocytes, fibroblasts, and endothelial cells. Although in vitro studies suggest that cyclosporin A may interfere with the functional capacities of epidermal Langerhans cells, there is no evidence that the treatment influences the distribution or number of Langerhans cells in vivo. We used a model of normal human skin graft to "nude" mice, which is free of the human systemic control mechanisms, for studies on the DNA synthesis of human Langerhans cells under the influence of cyclosporin A. The grafted animals were given daily subcutaneous (50 mg/kg) or intraperitoneal (5, 12.5, and 25 mg/kg) drug injections during three weeks, which resulted in mean blood levels comparable to those observed in treated patients with organ transplants or psoriasis, respectively. BrdU administered during the last week of the experiment was incorporated by all cells synthesizing DNA, including those passing through S-phase. Langerhans cells were detected on deparaffinized or frozen tissue sections of xenografts with anti-CD1a and anti-HLA DR monoclonal antibodies, and the number of BrdU-positive cells was determined by double labeling. Our results indicate that the Langerhans cell DNA synthesis is impaired by therapeutic levels of cyclosporin A.