The effect of a tissue engineered bilayered living skin analog, over meshed split-thickness autografts on the healing of excised burn wounds. The Apligraf Burn Study Group.

The potential of Apligraf(R) (Graftskin) (Organogenesis Inc., Canton, MA), to improve cosmetic and functional outcomes when applied over meshed split thickness autografts was evaluated in a multicenter, randomized within patient controlled clinical trial. Experimental treatment sites had Apligraf(R) placed over meshed autograft while control sites were treated with meshed autograft covered with meshed allograft, or meshed autograft not covered by a biologic dressing. Forty patients were entered into this study of which 38 were evaluable. There was no difference in the percent take of autograft in the presence or absence of Apligraf(R) or in the median number of days to greater than 75% take of autograft. At the completion of the study 22 (58%) of the Apligraf(R) sites were rated superior to the control sites by the investigators, 10 (26%) were rated equivalent to the control and six (16%) were rated worse than control (p=0. 0037). Pigmentation, in the Apligraf(R) group was significantly better than control and by month 24, 17 (45%) Apligraf(R) sites had normal pigmentation compared with five (13%) control sites (p=0. 0005). Similarly by month 24, 18 (47%) patients had normal vascularity at the Apligraf(R) site compared with six (16%) patients at the control site. Improvements in pliability were observed with Apligraf(R) over control treatment within the first week of treatment. At month 24, 23 (61%) patients had normal height at the Apligraf(R) site compared to 14 (37%) with normal height at the control site (p=0.0117). Vancouver burn scar scores were shown to be statistically better at Apligraf(R) sites compared to control at all time points from week 1 to month 24. These results indicate that Apligraf(R) is a suitable and clinically effective treatment for burn wounds when applied over meshed autografts. Furthermore, cosmetic and functional advantages with Apligraf(R) were demonstrated when applied over meshed autograft compared to the current standard treatments of meshed autografts.

Acute excisional wounds treated with a tissue-engineered skin (Apligraf).

BACKGROUND: Tissue-engineered products are usually composed of living cells and their supporting matrices that have been grown in vitro, using a combination of engineering and life sciences principles. Apligraf is a bilayered product composed of neonatal-derived dermal fibroblasts and keratinocytes, and Type I bovine collagen. OBJECTIVE: To evaluate in a prospective, multicentered open study, the effects of tissue therapy with a tissue-engineered skin (Apligraf) with partial or full-thickness excisional wounds. METHODS: One hundred and seven patients participated in this study. The tissue-engineered skin was applied once, immediately after excisional surgery, usually for skin cancer, and patients were followed for up to one year. RESULTS: The safety results were impressive, with no clinical or laboratory evidence of rejection. Clinically, graft persistence was good to excellent in 77 of 105 (73.3%) of patients at one week, falling to 56.6% and 53.6% at two weeks and one month respectively. CONCLUSION: To date, this is the largest experience with a tissue-engineered skin product in acute wounds, and this study suggests that tissue therapy may be safe and useful.

Photodynamic inactivation of influenza and herpes viruses by hematoporphyrin.

Hematoporphyrin (HP), at concentrations as low as 0.5 microgram/ml, was found to inhibit the in vitro replication of influenza A and herpes simplex viruses, but not of several other viruses. The effect required exposure of the viruses or cells to visible light and was demonstrable when HP was administered shortly before virus inoculation or during the infection. In studies on the mechanism of action of HP, we found that in the presence of light, HP caused decomposition of GMP but not of various other nucleosides. It caused breakdown of yeast tRNA and inhibited polymerization of RNA and DNA by influenza virus and HSV-1-specific polymerases as well as some other polymerases isolated from bacterial and mammalian sources. Protective effects of HP and light were demonstrable in embryonated eggs infected with the WSN and PR8 strains of influenza A virus and in mice infected with the WSN strain. HSV-1-induced keratitis in rabbits and HSV-2-induced dermatitis in mice were not responsive to HP treatment.

Structure-activity studies on phosphonoacetate.

Phosphonoacetic acid is a selective antiherpesvirus agent. More than 100 congeners of phosphonoacetic acid were evaluated in vitro and in vivo to understand structure-activity relationships in the hope of designing a superior analog. Results showed that the antiherpesvirus activity had highly specific structural requirements. Neither the carboxylic nor the phosphono groups could be replaced. The distance between these two groups is important. Increase of this distance caused complete loss of activity. However, if this distance was maintained, the addition of groups to the methylene carbon resulted in a reduction, but not loss, of activity. On the other hand, decrease of the carbon chain to formic acid did not deteriorate its antiherpes activity. All analogs tested had lower activity than the parent compound. However, some compounds with decreased activity in vitro appeared to have favorable pharmacological properties in vivo.

Characteristics of herpes simplex virus resistance to disodium phosphonoacetate.

Herpes simplex virus (HSV), which was partially resistant to the inhibitory effect of disodium phosphonoacetate (PAA), could be recovered following four virus passages in the presence of 100 microgram/ml PAA. Resistant strains were isolated from both HSV type 1 and HSV type 2. Virus resistance to PAA was not complete, and in most isolations a significant proportion of the virus stock remained susceptible to the drug. Resistance was shown to be heritable and persisted through virus passage and cloning experiments. PAA inhibited the replication of virus-specific DNA in sensitive strains of HSV but not in resistant strains of HSV. In vitro experiments directly demonstrated that PAA inhibited the activity of the virus-specific DNA polymerase 10 times more effectively in PAA-susceptible HSV than in PAA-resistant HSV. The treatment of HSV-infected mice with high levels of PAA did not induce the formation of resistant virus strains.

Antiviral potential of phosphonoacetic acid.

Phosphonoacetate has been found to inhibit specifically the replication of herpes-viruses. A partial inhibition of vaccinia virus represents the only activity outside the herpesvirus class. The drug was found to be a specific inhibitor of the virus-induced DNA polymerases. Normal cellular polymerases were relatively insensitive to phosphonoacetate, resulting in low cellular toxicity. Our working hypothesis is that the drug binds to the enzyme and that initiation of polynucleotide synthesis occurs in the presence of the drug and the required template, substrates, and cations. However, addition of deoxynucleosides to the elongating nascent chain is prevented by the enzyme-bound drug. Kinetic analyses indicated that phosphonoacetate did not interfere with the binding of DNA template to polymerase; and it did not compete with nucleotide substrate binding. The highly specific inhibitory effects of phosphonoacetate allowed for the selection of partially resistant strains of HSV. Resistance of virus to the drug in cell culture was directly correlated with the same relative resistance of the corresponding cell-free DNA polymerases. Phosphonoacetate was also effective therapeutically in herpesvirus skin and ocular infections in animals. Intraperitoneal administration of the drug reduced death and severity of disease in experimental encephalitis in hamsters. High specificity, low toxicity, and reproducible efficacy in lower animals suggested that phosphonoacetate could be a useful new antiviral drug. Sensitivity to phosphonoacetate also is a useful research tool as a genetic marker for herpesviruses.

Activation of a latent measles virus infection in hamster cells.

The characteristics of infectious measles virus released from latently infected hamster embryo fibroblast cells are described. Low levels of virus were released spontaneously when the cultures were incubated at 37 C; this phenomenon was observed 19 passages after the cells had been exposed to the virus and has continued through cell passage 45. The virus yield could be significantly increased by cocultivation of the hamster cells with BSC-1 cells or incubation of the latently infected cells at 33.5 C rather than at 37 C. Measles virus released after cocultivation demonstrated increased cytopathology in cell culture and reduced temperature sensitivity when compared to the virus released at 33.5 C. After cell passage 45, there was an increase in spontaneous release of virus. However, the viruses recovered by cocultivation or temperature release after cell passage 45 were nearly identical. These observations suggest a possible mechanism for measles virus activation in cells latently infected with this virus.