Cell killing by ultraviolet-inactivated human immunodeficiency virus

Extensive cell killing and cytopathology were observed within 24 hr after exposure of a clonal cell line of human T-4 lymphocytes (RH9) to culture supernatants containing human immunodeficiency virus (HIV). Ultraviolet-irradiated HIV-containing culture fluids were also capable of killing RH9 cells and of inducing specific cytopathic effects which were indistinguishable from those induced by unirradiated virus-containing preparations. The uv-irradiated HIV was incapable of forming proviral DNA using the endogenous virion genomic RNA as a template. The RH9 cells persistently infected with HIV did not release soluble cytotoxic factors to account for the cell killing observed when culture supernatants were added to uninfected RH9 cells. The fraction involved in cell killing had the hydrodynamic properties of a retrovirus. These results suggest that a virion component is responsible for cell killing by HIV.     

Cell proliferative response to vaccinia virus is mediated by VGF

VGF, a polypeptide encoded by vaccinia virus, shares amino acid sequence homology and functional properties with cellular growth factors EGF and TGF-alpha. The availability of a VGF minus (VGF-) virus mutant has enabled us to examine the role of VGF in the replication of virus in vitro and in vivo. Studies in vitro with A431 cells (high EGF receptor density) showed that VGF+ wild-type virus induced the rapid formation of a focus of infection (not a plaque) which could be blocked by a monoclonal antibody to the EGF receptor. In vivo experiments with chicken embryos indicated that VGF+ virus stimulated the growth of ectodermal and entodermal cells of the chorioallantoic membrane. At early times, the majority of proliferating cells contained no detectable virus antigen, indicating that cell growth preceded infection and was a consequence of VGF secretion. Relative to VGF- virus, VGF+ virus produced lesions which contained more proliferating cells, more virus antigen, and increased amounts of infectious progeny. Secretion of VGF thus explains the conundrum of a nontransforming, strongly cytopathic virus inducing a hyperplastic cell response.     

Human T-lymphocyte rosette formation: inhibition by cytochalasin B.

The effects of cytochalasin B, colchicine and vinblastine on the rosette formation of human T lymphocytes with neuraminidase-treated human erythrocytes (nHRBC) and with sheep red blood cells (SRBC) have been studied. Pretreatment of the lymphocytes with cytochalasin B which affects microfilament action reversibly inhibits both nHRBC and SRBC rosette formation. Colchicine and vinblastine known to interact with microtubules causes no major reduction in rosette-forming cells. The results suggest that normally functioning microfilaments are necessary for nHRBC and SRBC rosetting, whereas microtubules are not essential for the blinding of the erythrocytes to the lymphocytes.     

The role of non-specific lymphocytes in antigen-induced proliferative response in vitro

The antigen-induced proliferative response of lymph node cells from immunized mice is proportional to the number of primed lymphocytes in the microtiter wells. Addition of normal lymphocytes did not alter the magnitude of the antigen-specific [³H]TdR uptake of immune lymph node cells. In contrast, normal lymphocytes increased the antigen-specific thymidine incorporation of enriched populations of antigen-specific lymphocytes. This enhancing effect was especially pronounced, and proportional to the number of supplementing unsensitized lymphocytes, with a small cell number of enriched lymphocytes, where the specific responsiveness could barely be detected without addition of normal lymphocytes.Enriched populations derived through adherence to antigen-pulsed macrophages (‘selected’ cultures) as well as those obtained by growing immune lymph node cells with antigen-pulsed macrophages (‘supernatant’ cultures) had an improved proliferative response after addition of normal lymphocytes. However, the proliferative response of the ‘selected’ cultures was extremely dependent on normal lymphocytes as they could express only 10% of their full stimulatory capability in the absence of the latter. This indicates that the blastogenic signal delivered by the activated specific T cells recruits normal lymphocytes which do not adhere to the antigen-pulsed macrophages. Under the same conditions the recruiting signal is incapable of inducing the multiplication of antigen-sensitized cells.The potential recruitable uncommitted lymphocytes are present in excess among immune lymph node cells, while depleted from the ‘selected’ cultures. The enriched ‘supernatant’ cultures seem to contain considerable numbers of recruitable lymphocytes although they are present in quantities less than are required to provide for the full amplifying signal.This meaning of these findings for the evaluation of enrichment of antigen-specific T cells and of antigen-induced response based on [³H]TdR uptake is discussed.     

In vitro inhibition of rubella virus by 1-adamantanamine hydrochloride

Summary The effect of 1-adamantanamine hydrochloride (1-AH) on the multiplication of three strains of rubella virus in RK-13 tissue cultures was studied. At a concentration of 25g/ml. the compound depressed the yield of infective virus, reduced the cytopathic end-point titres and inhibited the formation of rubella virus microplaques. The inhibition of microplaques was the most sensitive method of detecting the antiviral activity of 1-AH and concentrations as low as 10g/ml. were effective. Whilst 25g/ml. of 1-AH did not produce obvious toxic effects, 40g/ml. caused cytoplasmic vacuolation and decreased the rate of cell divison of RK-13 cultures.Inhibition of rubella virus CPE by 1-AH was more marked in virus strains poorly adapted to growth in RK-13 cultures.Studies on the mode of action of 1-AH indicate the absence of direct virucidal action and the compound neither decreased the rate of adsorption of virus to RK-13 cells nor inhibited release of intracellular virus from infected cells. The compound appeared to act at an early stage in the growth cycle and was not effective in inhibiting subsequent virus multiplication if added later than 5 hours after infecting tissue cultures with rubella virus.     

Direct inhibition of T-lymphocyte activation by anthrax toxins in vivo

The causative agent of anthrax, Bacillus anthracis, produces two toxins that contribute in part to its virulence. Lethal toxin is a metalloprotease that cleaves upstream mitogen-activated protein kinase kinases. Edema toxin is a calmodulin-dependent adenylate cyclase. Previous studies demonstrated that the anthrax toxins are important immunomodulators that promote immune evasion of the bacterium by suppressing activation of macrophages and dendritic cells. Here we showed that injection of sublethal doses of either lethal or edema toxin into mice directly inhibited the subsequent activation of T lymphocytes by T-cell receptor-mediated stimulation. Lymphocytes were isolated from toxin-injected mice after 1 or 4 days and stimulated with antibodies against CD3 and CD28. Treatment with either toxin inhibited the proliferation of T cells. Injection of lethal toxin also potently inhibited cytokine secretion by stimulated T cells. The effects of edema toxin on cytokine secretion were more complex and were dependent on the length of time between the injection of edema toxin and the isolation of lymphocytes. Treatment with lethal toxin blocked multiple kinase signaling pathways important for T-cell receptor-mediated activation of T cells. Phosphorylation of the extracellular signal-regulated kinase and the stress-activated kinase p38 was significantly decreased. In addition, phosphorylation of the serine/threonine kinase AKT and of glycogen synthase kinase 3 was inhibited in T cells from lethal toxin-injected mice. Thus, anthrax toxins directly act on T lymphocytes in a mouse model. These findings are important for future anthrax vaccine development and treatment.     

The effect of cyclophosphamide on cytotoxic T-lymphocyte responses: inhibition of helper T-cell induction in vitro.

The effects of cyclophosphamide (Cy) on the different cell populations participating in the cytotoxic T lymphocyte (CTL) response against haptenated (trinitrophenyl, TNP) syngeneic cells were studied. Pretreatment of responder cell donor mice with 150 mg/kg Cy decreased the cytotoxicity against TNP-modified syngeneic target cells almost to the background level. When TH cells were added to the culture the cytotoxicity increased significantly. Helper T cells were generated in vivo by priming the mice with TNP-modified syngeneic spleen cells or sensitizing the mice with a reactive hapten (TNCB). However, if the TH cell donor mice were treated with Cy before in vivo priming, the cytotoxicity reached the normal level, which indicated that TH precursors were not destroyed by Cy treatment and TH induction was even more effective after Cy. These data indicate that the decrease of the response by this Cy dose is not due to the sensitivity of CTL or TH precursors. Mice could be primed with male-specific (HY) antigen in spite of Cy pretreatment. However, Cy pretreatment caused a latent period of 2 weeks when effective CTL could not be generated in vitro, but after that the capacity for CTL generation was restored. These experiments confirm that pretreatment of responder cell donor mice with Cy does not destroy CTL or TH precursors, but rather affects their in vitro restimulation probably by destroying a short lived 'inducer' cell that is needed.     

Effective inhibition of porcine epidemic diarrhea virus by RNA interference in vitro

Porcine epidemic diarrhea virus (PEDV) is a member of the coronaviridae family, which can cause acute and highly contagious enteric disease of swine characterized by severe entero-pathogenic diarrhea in piglets. Currently, the vaccines of PEDV are only partially effective and there is no specific drug available for treatment of PEDV infection. To exploit the possibility of using RNA interference (RNAi) as a strategy against PEDV infection, five shRNA-expressing plasmids targeting the N, M, and S genes of PEDV were constructed and transfected into Vero cells. The cytopathic effect and MTS assays demonstrated that two shRNAs (pSilencer4.1-M1 and pSilencer4.1-N) were capable of protecting cells against PEDV invasion with very high specificity and efficiency. The two shRNA expression plasmids were also able to inhibit the PEDV replication significantly, as shown by detection of virus titers (TCID₅₀/mL). A real-time quantitative RT-PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with these two plasmids were reduced by 95.0 %. Our results suggest that RNAi might be a promising new strategy against PEDV infection.     

T-lymphocyte rosette inhibition titering of antisera by direct microassay

A 60-sample micro-rosette inhibition assay for determining the 100% rosette inhibitory titers of heterologous antisera is described. The assay is performed in histocompatibility trays, under oil, using frozen-thawed thymocytes or peripheral blood lymphocytes as the rosette forming lymphoid cells. Antisera dilutions are incubated with lymphocytes for an appropriate period, either with or without added complement; SRBC are inoculated into each well, and rosettes formed by centrifugation of the trays at 200 × g. Following centrifugation, glutaraldehyde is added to each well to fix rosettes to the well bottom and the plates inverted to allow unbound SRBC to fall away. One hundred percent rosette inhibition is determined by low power microscopic examination of the inverted wells. Highly reproducible in-assay (±4% average standard error) and between-assay (±9%) inhibition values are obtained which correlated well (r = 0.99) with values calculated by more conventional methodology.     

Selective inhibition of the in vitro murine B lymphocyte response by pentamidine

A study was undertaken on the immunomodulating properties of pentamidine, a diamidine used in the treatment of African trypanosomiasis, pneumocystosis and leishmaniasis. Pentamidine inhibited the ability of mouse splenic lymphocytes to respond to mitogens. In particular inhibition of the B lymphocyte response was observed. At concentrations of 1 microgram/ml (1.7 x 10(-6) M), pentamidine markedly inhibited the response to the B cell mitogen, lipopolysaccharide (LPS), while concentrations of 10 micrograms/ml had to be attained to produce a similar effect on the response to the T cell mitogens phytohaemagglutinin (PHA) and concanavalin A (ConA). Further studies showed that pentamidine was not toxic to either resting or proliferating cells and probably acts by interacting directly with B cells rather than modifying the regulatory cell populations.     

Hapten inhibition of the immune response in vitro

The in vitro primary immune response of mouse spleen cells against a hapten, the 2,4,6-trinitrophenyl (TNP) group, bound to an erythrocyte carrier is inhibited by the polyvalent, but nonimmunogenic TNP₁₇HSA (human serum albumin) when added at the same time as the immunogen. Resistance to the effect of TNP₁₇HSA develops in 24 h after the addition of immunogen. Sensitivity to this inhibition can be quickly re-established after a mild physical dissociation of cell aggregates in the spleen cell cultures even 48 h after the start of a response. This sensitivity is rapidly lost again if the culture are incubated for a few hours before the TNP-HSA is added.     

Malarial immunodepression in vitro: adherent spleen cells are functionally defective as accessory cells in the response to horse erythrocytes

The basis for the depressed response of malarial infected mice to horse red blood cells (HRBC) has been studied in vitro. Results presented show that the adherent spleen cells from infected mice (a) are defective in their ability to allow nonadherent spleen cells of both normal and infected mice to respond to HRBC whereas a response does occur with adherent spleen cells from normal mice (b) do not suppress the response of unfractionated spleen cells from normal mice to HRBC (c) contain phagocytic cells as measured by the uptake of neutral red in numbers which are of the same order of magnitude as in adherent spleen cells from normal mice, but which are unable to take up HRBC. We conclude that a splenic adherent cell, probably the macrophage, is functionally defective as an accessory cell in the response to HRBC of mice infected with Plasmodium berghei yoelii.     

In vitro andin vivo inhibition of virus multiplication by microwave hyperthermia

Summary The effects of microwave hyperthermia (41° and 43° C) on virus multiplication have been exploredin vitro (HSV-1 infected primary rabbit kidney cultures) andin vivo (mice infected with HSV-1 or vaccinia). In vitro the cells were inoculated with HSV-1 and heated to 41° or 43° C either before or after infection. Virus yields were significantly decreased when the cells were exposed to hyperthermia within the first few hours after infection, while hyperthermia was without effect when applied before infection or with several hours delay after infection.In mice inoculated intranasally with HSV-1, mortality due to herpes encephalitis was significantly reduced upon daily exposure to microwave hyperthermia from the day of infection onward.In mice inoculated intravenously with vaccinia, a significant decrease in the number of specific tail lesions was observed if the animals were exposed to microwave hyperthermia within the first three days after infection, while irradiation prior to infection or delayed until several days after infection did not exhibit an appreciable effect.Our data suggest that microwave hyperthermia interferes directly with the virus multiplication cycle bothin vitro andin vivo.With 3 Figures     

Immune response and immunodepression inHymenolepis diminuta infection in rats

Maximum size range ofHymenolepis diminuta was found in rats 22 days postinfection (p.i.) with single or several (3–6) tapeworms. Immunologic reactions of the host were connected with the growth and development of parasites. Positive results of macrophage migration inhibition test (MMI) were recorded from 7 until 22 days p.i. in infection with a single and to 16 days p.i. in those with 3–6H. diminuta. The negative results of the MMI test observed at later postinfection times are considered as a manifestation of immunosuppression caused by the presence ofH. diminuta. The expulsion of tapeworm by anthelmintic treatment with Yomesan ® restored for a short time cell-mediated immunity, as demonstrated by MMI test. The level of antibodies, as determined by passive hemagglutination test (HA), varied during the infection; it was highest at 22 days p.i. The removal of tapeworms as a results of drug treatment caused a rise in titers.     

Silybin inhibition of human T-lymphocyte activation

Silybin, a 3-oxyflavone occurring in the thistle Silybum marianum, displays a dose-dependent inhibition of in-vitro lymphocyte blastogenesis induced by lectins (phytohaemagglutinin, Concanavalin A and pokeweed) and by anti-CD3 monoclonal antibody. The drug has no effect on cell viability and spontaneous 3H-thymidine incorporation, suggesting that the inhibitory activity is not due to aspecific toxicity. Since all the T-cell responses investigated require cell-membrane-associated events, the effect of silybin is probably at the level of the cell membrane, as for other flavonoids. Addition of CuSO4 prevents the inhibitory activity of silybin on PHA-induced proliferative response, indicating that the drug could exert its activity also by virtue of a chelation mechanism.     

Effect of Ebola virus antigen on proliferative response of human lymphocytes in vitro: imbalance in production of tumor necrosis factor alpha and interleukin-1

An in vitro model infection caused by Ebola virus (EV) showed a high production of tumor necrosis factor-alpha by human peripheral lymphocytes concurrently with a simultaneous reduction in the synthesis of interleukin-1 in response EV antigen stimulation. This may be an important factor in that VE suppresses the body's immunological resistance, which in turn causes unterferon deficiency and suppresses the formation of T helper cells.     

Human immunodeficiency virus (HIV) infects human arterial smooth muscle cells in vivo and in vitro: implications for the pathogenesis of HIV-mediated vascular disease

Human immunodeficiency virus (HIV) infection is associated with accelerated atherosclerosis and vasculopathy, although the mechanisms underlying these findings have not been determined. Hypotheses for these observations include: 1) an increase in the prevalence of established cardiac risk factors observed in HIV-infected individuals who are currently experiencing longer life expectancies; 2) the dyslipidemia reported with certain HIV anti-retroviral therapies; and/or 3) the proinflammatory effects of infiltrating HIV-infected monocytes/macrophages. An unexplored possibility is whether HIV itself can infect vascular smooth muscle cells (SMCs) and, by doing so, whether SMCs can accelerate vascular disease. Our studies demonstrate that human SMCs can be infected with HIV both in vivo and in vitro. The HIV protein p24 was detected by fluorescence confocal microscopy in SMCs from tissue sections of human atherosclerotic plaques obtained from HIV-infected individuals. Human SMCs could also be infected in vitro with HIV by a mechanism dependent on CD4, the chemokine receptors CXCR4 or CCR5, and endocytosis, resulting in a marked increase in SMC secretion of the chemokine CCL2/MCP-1, which has been previously shown to be a critical mediator of atherosclerosis. In addition, SMC proliferation appeared concentric to the vessel lumen, and minimal inflammation was detected, unlike typical atherosclerosis. Our data suggest that direct infection of human arterial SMCs by HIV represents a potential mechanism in a multifactorial paradigm to explain the exacerbated atherosclerosis and vasculopathy reported in individuals infected with HIV.