A Review of the Toxicity and Phytochemistry of Medicinal Plant Species Used by Herbalists in Treating People Living With HIV/AIDS in Uganda.

Introduction: Despite concerns about toxicity, potentially harmful effects and herb-drug interactions, the use of herbal medicines remains widely practiced by people living with HIV/AIDS (PLHIV) in Uganda. Objective: The objective of the paper was to comprehensively review the literature on the toxicity and chemical composition of commonly used medicinal plant species in treating PLHIV in Uganda. Methods: We reviewed relevant articles and books published over the last sixty years on ethnobotany, antiviral/anti-HIV activity, toxicity, phytochemistry of Vachellia hockii, Albizia coriaria, Bridelia micrantha, Cryptolepis sanguinolenta, Erythrina abyssinica, Gardenia ternifolia, Gymnosporia senegalensis, Psorospermum febrifugium, Securidaca longipendunculata, Warburgia ugandensis and Zanthoxylum chalybeum and their synonyms. We searched PubMed, Web of Science, Scopus, Science Direct and Google Scholar. Discussion: Most of the plant species reviewed apart from P. febrifugium, S. longipedunculata and C. sanguinolenta lacked detailed phytochemical analyses as well as the quantification and characterization of their constituents. Crude plant extracts were the most commonly used. However, purified/single component extracts from different plant parts were also used in some studies. The U87 human glioblastoma was the most commonly used cell line. Water, ethanol, methanol and DMSO were the commonest solvents used. In some instances, isolated purified compounds/extracts such as Cryptolepine and Psorospermin were used. Conclusion: Cytotoxicity varied with cell type, solvent and extract type used making it difficult for direct comparison of the plant species. Five of the eleven plant species namely, A. coriaria, C. sanguinolenta, G. ternifolia, P. febrifugium and Z. chalybeum had no cytotoxicity studies in animal models. For the remaining six plant species, the crude aqueous and ethanol extracts were mainly used in acute oral toxicity studies in mice. Herbalists reported only A. coriaria and W. ugandensis to cause toxic side effects in humans. However, selective cytotoxic plant extracts can potentially be beneficial as anticancer or anti-tumour drugs.

Adapting epicutaneous patch testing protocols to assess immediate-type skin reactions.

OBJECTIVE: During the development of cosmetic formulations, in vitro and in vivo methods are essential tools used to reliably assess the skin irritation potential of a product or ingredient. Epicutaneous patch testing (single and/or multiple application protocols) has long been used as an initial in vivo method to screen for possible skin irritation properties of a substance or formulation. To confirm the mildness and dermatological and/or consumer acceptance of a product, use tests are often subsequently conducted. A study was therefore initiated to see how well patch test results correlate with use tests with respect to irritation elicited by skincare (leave-on) products. METHODS/RESULTS: A number of different cosmetic formulations were assessed in both tests. Although the patch test results did not indicate substantial irritation potentials, immediate-type reactions (stinging and redness) were observed in some volunteers which disappeared within approx. 1 h. Although transient, these reactions suggested that consumer acceptance would probably be low and the studies were discontinued. Immediate-type reactions are rare but have been described for some substances used in cosmetics. These unexpected results were nevertheless intriguing and prompted the start of a journey to see if patch test protocols could be modified to assess these reactions. An occlusive short-term patch test protocol with an application period of 20 min was developed. Successful identification of the spontaneous reactions became possible. Furthermore, there was a correlation between the intensity of reactions observed in the short-term patch test and those observed in the controlled in-use studies. Short-term patch testing using the developed protocol can therefore reliably be used as a screening method, for example in the development and optimization of cosmetic formulations containing ingredients that could cause spontaneous reactions, for instance of non-immunological contact urticaria type. CONCLUSION: The lessons learned from this studies indicate that simple modifications of existing test protocols can lead to important insights into skin reactions. These modifications can then be used to create further building blocks in the development and optimization of test strategies for cosmetic formulations which offer reliable study designs for possible reactions product developers may encounter.

OBJECTIF: Lors du développement de formulations cosmétiques, les méthodes in vitro et in vivo sont des outils essentiels utilisés pour évaluer de manière fiable le potentiel d'irritation cutanée d'un produit ou d'un ingrédient. Le test épicutané (protocoles d'application uniques et / ou multiples) est utilisé depuis longtemps comme méthode initiale in vivo pour dépister les éventuelles propriétés d'irritation cutanée d'une substance ou d'une formulation. Afin de confirmer la douceur et l'acceptation dermatologique et / ou consommateur d'un produit, des tests d'usage sont souvent effectués ultérieurement. Une étude a donc été initiée pour voir dans quelle mesure les résultats des tests épicutanés correspondent aux tests d'usage en ce qui concerne l'irritation provoquée par les produits de soin (sans rinçage). MÉTHODES/RÉSULTATS: Un certain nombre de formulations cosmétiques différentes ont été évaluées dans les deux tests. Bien que les résultats du test épicutané n'indiquent pas de potentiels d'irritation substantiels, des réactions de type immédiat (picotements et rougeurs) ont été observées chez certains volontaires. Celles-ci ont disparu en à peu près 1 heure. Bien que transitoires, ces réactions de type 5 suggéraient que l'acceptation du consommateur serait probablement faible et les études ont été interrompues. Les réactions de type immédiat 6 sont rares mais ont été évoquées en relation avec certaines substances utilisées en cosmétique. Ces résultats inattendus étaient néanmoins intrigants et ont incité le lancement d'un processus pour voir si les protocoles de test épicutané pouvaient être modifiés pour évaluer ces réactions. Un protocole de test épicutané à court terme occlusif avec une période d'application de 20 min a été développé, permettant l'identification réussie des réactions spontanées. Il a été de plus constate une corrélation entre l'intensité des réactions observées dans le test épicutané à court terme et celles observées dans les test d'usage contrôlés. Le test épicutané à court terme utilisant le protocole développé peut donc être utilisé de manière fiable comme méthode de dépistage, par exemple dans le développement et l'optimisation de formulations cosmétiques contenant des ingrédients qui pourraient provoquer des réactions spontanées, par exemple de type urticaire de contact non immunologique. CONCLUSION: Les leçons tirées de ces études indiquent que de simples modifications des protocoles de test existants peuvent révéler des informations importantes sur les réactions cutanées. Ces modifications peuvent ensuite être utilisées pour créer d'autres blocs de construction dans le développement et l'optimisation de stratégies de test pour des formulations cosmétiques qui offrent des conceptions d'études fiables pour les réactions possibles que les développeurs de produits peuvent rencontrer.

Internet-based lay person rating of facial photographs to assess effects of a cleansing product and a decent cosmetic foundation on the attractiveness of female faces.

It is well established that decorative cosmetics can enhance female facial attractiveness. In this study, we investigated the effects of a cleanser and a decent foundation on attractiveness of female faces. Comparative rating of a set of facial photographs by a group of lay persons revealed that the cleansing product was significantly reducing the attractiveness of the stimulus persons. Treatment with the foundation increased the attractiveness of the female faces clearly. The authors conclude that even unobtrusive cosmetic treatments like cleansers and light foundations may cause relevant changes of the attractiveness of female faces.

Resolution of HCV infection in a HIV-infected patient under HAART after several hepatitis flare-ups.

Highly active antiretroviral therapy (HAART) has been shown to have a beneficial effect on several opportunistic and other coinfections of HIV infected individuals. The effect of HAART on HCV coinfections is controversial. We describe the case of a patient, in whom a close temporal relationship between changes in HIV viremia, HCV viremia and ALT levels was observed. Longterm suppression of HIV replication by HAART was associated with a normalization of ALT levels and finally clearance of the HCV infection. Our data suggest that improved immune functions due to reductions of the HIV load led to a better control and finally resolution of the HCV infection in this patient.

Analysis of the proliferative responses to peptides in individuals with vigorous Gag protein-specific proliferation.

Proliferative responses to recombinant HIV proteins in infected individuals may represent a correlate of protection from disease progression. In this study, the proliferative responses to HIV p24, p55 and gp120 were evaluated in infected subjects. Whereas, vigorous proliferative responses directed at the Gag proteins were detected in several individuals, Env-specific proliferation was observed in only one subject. Epitope mapping using overlapping peptides demonstrated proliferative responses of PBMC to Gag peptides. Responses were broadly directed at multiple peptides in some subjects. Although several of the peptides that induced proliferative responses also contain CTL epitopes potentially relevant in the particular individuals, many additional Gag T cell epitopes were present in each subject. This finding may be relevant for the design and testing of HIV candidate vaccines.

Phenotypic analysis of the sensitivity of HIV-1 to inhibitors of the reverse transcriptase, protease, and integrase using a self-inactivating virus vector system.

Conventional phenotypic analysis of resistance of the human immunodeficiency virus (HIV) to antiviral therapy is time-consuming and requires culture of infectious virus. Although phenotypic analyses may be desirable, rapid generation of test results and decentralized availability of the test system will be important to achieve utility in the clinical practice. This study describes the design of an alternative phenotypic resistance test using replication incompetent viral vectors. Chimeric HIV vectors containing a marker gene were generated. The env and most of the regulatory and accessory genes of HIV were removed. In addition, the 3'U3 region was deleted to obtain a self-inactivating construct. Cotransfection of the plasmid with a plasmid that provided the vesicular stomatitis virus glycoprotein resulted in the production of replication-incompetent virus vectors. Infection of susceptible cells with the vectors led to marker gene expression. Vector production in the presence of protease (PR) inhibitors, or infection in the presence of reverse transcriptase (RT) or integrase (IN) inhibitors reduced marker gene expression in a dose-dependent manner. Marker gene activity was preserved at higher drug levels if vectors contained RT and PR genes from resistant virus isolates. Sensitivity to nucleoside and non-nucleoside RT inhibitors, protease and integrase inhibitors could be determined in 10 working days. The phenotypic drug resistance test using replication-incompetent HIV vectors significantly speeds up drug resistance measurements and allows testing at reduced biosafety levels. This will make clinical use of phenotypic assessment of antiviral resistance more feasible.

Characterization of HIV-specific proliferative T cell responses in HIV-infected persons.

Virus-specific helper T cell responses are thought to be an important host defense in HIV infection. The proliferative responses to HIV p24, p55, and gp120 were tested in a cohort of 27 HIV-infected subjects. Vigorous proliferative responses directed at the Gag protein with stimulation indices in excess of 6 were detected in 10 of the individuals tested but an Env-specific response was present in only 1 subject. Viral load and proliferative activity to Gag were inversely correlated in untreated individuals. Proliferation was also observed in some individuals treated in the chronic phase of infection, and responses were maintained over time in the absence of detectable viremia. Positive proliferative responses could also occasionally be detected in treated persons with CD4(+) cell counts below 200/microl. Thus, vigorous Gag-specific proliferative responses are present in a minority of HIV-infected individuals and can be detected in individuals receiving highly active antiretroviral therapy at advanced disease stages. Proliferative responses are maintained for an extended time period in the presence of antiviral therapy.

Syncytium formation amplifies apoptotic signals: a new view on apoptosis in HIV infection in vitro.

Infection of CD4+ cells with HIV in vitro causes extensive cytopathology. The mechanism that underlies this process is unclear and conflicting data exist regarding whether cytotoxicity is due to necrosis or apoptosis. It was previously reported and is shown here that the coculture of HIV glycoprotein-expressing cells with CD4+ cells results in apoptosis within several hours. This study demonstrates that apoptosis did not occur in single cells and was mediated neither by CD4 nor by coreceptor signaling, indicating that apoptosis was not induced by intra- or intercellular glycoprotein-receptor interaction. Detection of apoptosis required cell-to-cell fusion and undetectable levels of apoptotic cell death were substantially amplified upon syncytium formation. Similar results were obtained with syncytium-forming cultures of measles virus glycoprotein-expressing cells. These findings indicate that the apoptotic cell death observed in cultures of HIV and other syncytium-forming viruses is primarily due to amplification of background apoptosis in the wake of cell-to-cell fusion.

Activation of cGMP-dependent protein kinase Ibeta inhibits interleukin 2 release and proliferation of T cell receptor-stimulated human peripheral T cells.

Several major functions of type I cGMP-dependent protein kinase (cGK I) have been established in smooth muscle cells, platelets, endothelial cells, and cardiac myocytes. Here we demonstrate that cGK Ibeta is endogenously expressed in freshly purified human peripheral blood T lymphocytes and inhibits their proliferation and interleukin 2 release. Incubation of human T cells with the NO donor, sodium nitroprusside, or the membrane-permeant cGMP analogs PET-cGMP and 8-pCPT-cGMP, activated cGK I and produced (i) a distinct pattern of phosphorylation of vasodilator-stimulated phosphoprotein, (ii) stimulation of the mitogen-activated protein kinases ERK1/2 and p38 kinase, and, upon anti-CD3 stimulation, (iii) inhibition of interleukin 2 release and (iv) inhibition of cell proliferation. cGK I was lost during in vitro culturing of primary T cells and was not detectable in transformed T cell lines. The proliferation of these cGK I-deficient cells was not inhibited by even high cGMP concentrations indicating that cGK I, but not cGMP-regulated phosphodiesterases or channels, cAMP-dependent protein kinase, or other potential cGMP mediators, was responsible for inhibition of T cell proliferation. Consistent with this, overexpression of cGK Ibeta, but not an inactive cGK Ibeta mutant, restored cGMP-dependent inhibition of cell proliferation of Jurkat cells. Thus, the NO/cGMP/cGK signaling system is a negative regulator of T cell activation and proliferation and of potential significance for counteracting inflammatory or lymphoproliferative processes.

Treatment of cells with detergent activates caspases and induces apoptotic cell death.

Due to their amphiphilic properties, detergents readily disrupt cellular membranes and cause rapid cytolysis. In this study we demonstrate that treatment of cells with sublytic concentrations of detergents such as Triton X-100, Nonidet P-40, n-octylglucoside and the bile salt sodium deoxycholate induce typical signs of apoptosis including DNA fragmentation and cleavage of poly(ADP-ribose) polymerase molecules. The detergent concentration required for apoptosis was below the critical micellar concentration. Induction of apoptosis was not restricted to human cells but similarly occurred in a variety of other vertebrate cell lines. Unstimulated peripheral blood mononuclear cells were susceptible to apoptosis induction by detergent suggesting that apoptosis in this circumstance is not mediated by CD95. Cell death was not due to influx of calcium from the medium. Apoptosis was blocked and cytolysis prevented by treatment with peptide inhibitors of caspases. These findings suggest a process of apoptosis that is initiated upon nonspecific alterations at the cell membrane level. Physiologic correlates of this process still have to be defined.

Influenza virus-induced NF-kappaB-dependent gene expression is mediated by overexpression of viral proteins and involves oxidative radicals and activation of IkappaB kinase.

Influenza A viruses are capable of inducing the expression of a variety of cytokine and proapoptotic genes in infected cells. The promoter regions of most of these genes harbor binding sites for the transcription factor NF-kappaB which is an important mediator of immune and inflammatory responses. Our present study is based on an observation that influenza A virus infection of cells stimulates transcriptional activation of the HIV-1 long terminal repeat (LTR) which harbors two regulatory NF-kappaB elements, and is aimed at identifying the molecular mechanisms involved in this process. We found that the expression of influenza virus hemagglutinin (HA), matrix protein (M), and nucleoprotein (NP), as single factors is sufficient to transcriptionally activate the HIV-1 LTR. This process is mediated by oxidative radicals because treatment of cells with pyrrolidine dithiocarbamate, a scavenger of such radicals, abolished the transactivating ability. Expression of different influenza proteins induces activation of NF-kappaB-dependent gene expression but not transcriptional activation of an AP-1/Ets-dependent promoter, indicating a selectivity for NF-kappaB transactivation. Furthermore, influenza protein expression induces activation of IkappaB kinase (IKK). Accordingly coexpression of a catalytically inactive mutant of IKK abolishes influenza protein induced activation of NF-kappaB as well as HIV-1 LTR-dependent reporter gene expression, suggesting that IKK is an important intermediate within this signaling process. Taken together, our results show that various influenza virus proteins act as viral transactivators to modulate transcriptional activity of kappaB-element harboring promoters such as the HIV-LTR.

Dopamine activates HIV in chronically infected T lymphoblasts.

HIV infection is associated with a marked vulnerability of the dopaminergic system. We found recently that dopaminergic substances increase brain pathology in the simian model of HIV infection. In the current study we used the chronically HIV-infected T-lymphoblasts ACH-2 to elucidate the effects of dopamine (DA) on HIV infection. Cells were exposed to various concentrations of DA for 24 hours. Flow cytometry measurements demonstrated that DA induced a concentration-dependent HIV activation. To study the mechanism of action of DA, cells were treated besides DA with glutathione, one of the main components of cellular defense mechanisms against oxidative stress as well as its indirect precursor N-acetylcysteine. Treatment with these antioxidants attenuated DA-induced-HIV activation indicating that changes in cellular redox states might have been the causative factor for the observed effect. Our data suggest that HIV activation is tightly linked to intracellular oxidant/antioxidant levels and that excessive DA exposure may modulate cellular vulnerability to HIV.

Electric field pulses can induce apoptosis.

Injection of electric field pulses of high intensity (kV/cm) and short duration (microsecond range) into a cell suspension results in a temporary increase of the membrane permeability due to a reversible electric breakdown of the cell membrane. Here we demonstrate that application of supercritical field pulses between 4. 5 and 8.1 kV/cm strength and 40 microsec duration induce typical features of apoptosis in Jurkat T-lymphoblasts and in HL-60 cells including DNA fragmentation and cleavage of the poly(ADP ribose) polymerase. Apoptosis induction did not depend on the presence of any particular electrolyte in the extracellular medium. However, no apoptosis was observed in solutions without a minimum amount of salt. Apoptotic DNA fragmentation was prevented by the caspase inhibitor zVAD.

Lipoprotein creams: utilization of multifunctional ingredients for the preparation of cosmetic emulsions with excellent skin compatibility.

Lipoproteins are plant-derived surface-active biopolymers, which act as emulsifying as well as viscosity-enhancing agents in oil-in-water emulsions. Depending on the degree of hydrolization, lipoproteins are dispersible or even soluble in water. In the presence of low to medium polar oils, lipoproteins are adsorbed and align at the oil-water interface, whereas in mixtures with high polar oils the lipoproteins are repelled from the oil-water interface. The water-dispersible lipoproteins show higher interfacial activity than the hydrolysates. Lipoproteins bear a negative electric charge in aqueous dispersions at pH 6.5, which is probably the reason for the stabilization of oil droplets against coalescence. Lipoprotein creams were characterized in terms of particle size, rheology, and emulsion stability against sedimentation, which was evaluated by a near-infrared sedimentometer. After topical application, emulsion stability breaks down and an emulsion film is formed on the skin surface. Lipoprotein creams cause a distinct increase in skin pliability and skin moisture and show excellent skin compatibility. In a home use test the panelists appreciated the cosmetic and caring properties of the lipoprotein cream.

Plasma membrane-targeted Raf kinase activates NF-kappaB and human immunodeficiency virus type 1 replication in T lymphocytes.

Increasing evidence points to a role of the mitogenic Ras/Raf/MEK/ERK signaling cascade in regulation of human immunodeficiency virus type 1 (HIV-1) gene expression. Stimulation of elements of this pathway leads to transactivation of the HIV-1 promoter. In particular, the NF-kappaB motif in the HIV long terminal repeat (LTR) represents a Raf-responsive element in fibroblasts. Regulation of the Raf kinase in T cells differs from findings with a variety of cell lines that the catalytic domain of Raf (Raf(delta26-303)) shows no activity. In this study, we restored the activity of the kinase in T cells by fusing its catalytic domain to the CAAX motif (-Cx) of Ras, thus targeting the enzyme to the plasma membrane. Constitutive activity of Raf was demonstrated by phosphorylation of mitogen-activated protein kinase kinase (MEK) and endogenous mitogen-activated protein kinase 1/2 (ERK1/2) in A3.01 T cells transfected with Raf(delta26-303)-Cx. Membrane-targeted Raf also stimulates NF-kappaB, as judged by kappaB-dependent reporter assays and enhanced NF-kappaB p65 binding on band shift analysis. Moreover, we found that active Raf transactivates the HIV(NL4-3) LTR in A3.01 T lymphocytes and that dominant negative Raf (C4) blocked 12-O-tetradecanoylphorbol-13-acetate induced transactivation. When cotransfected with infectious HIV(NL4-3) DNA, membrane-targeted Raf induces viral replication up to 10-fold over basal levels, as determined by the release of newly synthesized p24gag protein. Our study clearly demonstrates that the activity of the catalytic domain of Raf in A3.01 T cells is dependent on its cellular localization. The functional consequences of active Raf in T lymphocytes include not only NF-kappaB activation and transactivation of the HIV(NL4-3) LTR but also synthesis and release of HIV particles.

Rapid and selective depletion of CD4+ T lymphocytes and preferential loss of memory cells on interaction of mononuclear cells with HIV-1 glycoprotein-expressing cells.

Contact of HIV glycoprotein-expressing cells with CD4+ T lymphocytes in vitro causes cell-cell fusion and/or cytopathogenicity. The question of whether this process similarly underlies the death of helper T cells in vivo has not yet been resolved. To investigate the loss of uninfected CD4+ T cells in an environment that may reflect the in vivo situation, unfractionated, unstimulated peripheral blood mononuclear cells were cocultured with HIV-1 glycoprotein-expressing cells, and early alterations of T-cell numbers were quantitated using a newly developed quantitative flow cytometric assay. The results demonstrate that a large fraction of normal-sized, regular CD4+ T cells disappeared immediately on cocultivation with envelope glycoprotein-expressing cells. In contrast, CD8+ T lymphocytes remained unaffected. Significant loss of uninfected T-helper cells required the presence of less than 1% infected cells. Moreover, memory T cells (CD45RO+, CD29 hi+) were depleted more rapidly than naive cells (CD45RO-, CD29 lo+). The observation that a large fraction of intact primary T-helper cells disappeared on contact with HIV glycoprotein-expressing cells suggests that a similar process may occur in vivo and contribute to the loss of T-helper cells in the infected individual. In addition, the preferential loss of memory cells may account for the early loss of immune functions in the course of HIV infection.

CD46 expression does not overcome the intracellular block of measles virus replication in transgenic rats.

The study of measles pathogenesis and the testing of improved vaccine candidates is hampered by the lack of a small animal model which is susceptible to infection by the intranasal route. With the identification of CD46 as a measles virus (MV) receptor, it was feasible to generate transgenic rats to overcome this problem. Although there was widespread expression of CD46 in the transgenic Sprague-Dawley rats, no measles-like disease could be induced after various routes of infection. The expressed transgenic protein was functionally intact since it mediated MV fusion and was downregulated by contact with MV hemagglutinin. In vitro studies revealed that CD46-expressing rat fibroblasts take up MV but do not allow viral replication, which explains the nonpermissiveness of the transgenic rats for in vivo infection.

Human immunodeficiency virus glycoprotein-specific CD4+ cytotoxic T lymphocytes are involved in two types of cytotoxicity: antigen-specific and cell-cell fusion-related cell lysis.

Human immunodeficiency virus (HIV) glycoprotein-specific CD4+ cytotoxic T lymphocytes (CTLs) lyse target cells in an MHC-restricted calcium-dependent fashion similar to the mechanism used by CD8+ CTLs. However, contact of unprimed peripheral blood CD4+ T cells with HIV glycoprotein-expressing cells has been shown to cause, in addition to cell-cell fusion, rapid cytolysis that may resemble antigen-specific cytotoxicity in the chromium release assay. In this study, the ability of glycoprotein-specific CD4+ CTLs to undergo similar fusion-related cytolysis was examined. The data obtained demonstrate that in addition to antigen-specific calcium-dependent cytotoxicity, envelope-specific CD4+ CTLs are involved in fusion-related, calcium-independent cytolysis.

Vaccinia virus serpin B13R (SPI-2) inhibits interleukin-1beta-converting enzyme and protects virus-infected cells from TNF- and Fas-mediated apoptosis, but does not prevent IL-1beta-induced fever.

The vaccinia virus (VV) strain Western Reserve B13R gene encodes a 38.5 kDa intracellular polypeptide that is non-essential for virus replication in vitro and does not affect virus virulence in a murine intranasal model. The protein has 92% amino acid identity with the cowpox virus cytokine response modifier A (crmA) protein which inhibits the interleukin (IL)-1beta converting enzyme (ICE). Here, we show that extracts from THP-1 cells infected with VV strains expressing B13R prevent the cleavage of in vitro transcribed and translated pro-IL-1beta into mature IL-1beta. Similarly, THP-1 cells infected with VVs expressing B13R process pro-IL-1beta into mature IL-1beta inefficiently in situ. Despite its inhibition of ICE, B13R does not prevent fever in infected mice, a systemic effect mediated by IL-1beta. Instead, fever is controlled by the VV IL-1beta receptor, encoded by gene B15R, and deletion of both the B13R and B15R genes did not increase the febrile response compared to deletion of B15R alone. The B13R protein does, however, block apoptosis mediated by anti-Fas antibodies or by tumour necrosis factor (TNF) and cycloheximide. Using DNA fragmentation, chromium release and microscopic analyses it was shown that cells infected with wild-type VV strain WR, or a revertant virus in which the B13R gene had been re-inserted into the B13R deletion mutant, are more resistant than uninfected cells or deletion mutant-infected cells to apoptosis mediated by anti-Fas and TNF.