Baicalin from the extract of Scutellaria baicalensis affects the innate immunity and apoptosis in leukocytes of children with acute lymphocytic leukemia.

Scutellariae Radix (root of Scutellaria baicalensis) has a long history of application in traditional and in modern herbal medications. The major components of Scutellariae Radix are baicalin, baicalein, wogonoside and wogonin. Accumulating evidence demonstrates that Scutellaria has immunomodulatory effects and possesses compelling anticancer potential. Treatment of peripheral blood leukocytes (PBLs) with Scutellaria extract (SBE) enriched in baicalin, reduced viability of PBLs obtained from patients with acute lymphoblastic leukemia (ALL). SBE had no impact on the survival of healthy, control leukocytes. The immune system modulation by SBE resulted in increased production of IFNγ in PBLs, and reduced TNFα and IL-10 production in bone marrow cells (BMC), in ALL patients. SBE stimulated the nonspecific antiviral immunity, assessed by resistance of PBLs and BMC to vesicular stomatitis virus (VSV) infection. SBE showed pro-apoptotic activity in NALM-6 cell line (B-type human leukemia). The number of cells expressing annexin V increased from 6% in control cultures to 29% and 52% after treatment with 100 µg/ml and 200 µg/ml respectively. Increased percentage of apoptotic cells was observed when cells were treated with corresponding concentration of baicalin. SBE enhanced apoptosis of PBLs in BMC of leukemic children. The percentage of PBLs that underwent apoptosis and mean annexin V expression increased from 11% in the control to 17% and 24% for the doses of 100 µg/ml and 200 µg/ml respectively. Importantly, SBE did not induce apoptosis of PBLs in the healthy, control group.

Innate antiviral immunity is not impaired in patients with chronic renal failure on hemodialysis.

UNLABELLED: Although it is well documented that chronic renal failure patients are susceptible to infectious diseases, the reason for this has not been clarified. The aim of the study was to assess the antiviral natural (innate) immunity of peripheral leukocytes in 37 hemodialysis patients and compare it with that of a of control group (70 blood donors). We investigated 16 patients with anti-hepatitis C virus (HCV) antibodies, anti-HCV(+) and 21 patients without anti-HCV antibodies, anti-HCV(-). METHODS: Innate immunity was measured using the method of direct infection of peripheral blood leukocytes with indicatory VS virus. The VS virus did not replicate in leukocytes with strong innate immunity, whereas by impaired immunity the virus multiplied to high titer. RESULTS: Patients on hemodialysis expressed the same levels of nonspecific antiviral immunity as the control group. We found complete, partial or deficient of innate immunity respectively in 33, 52.5, 14.5% of anti-HCV(-) patients, 43, 43 and 14% of anti-HCV(+) patients, and 44, 40 and 16% of controls. CONCLUSIONS: Innate antiviral immunity was not impaired (disturbed) in chronic HD patients. The categories of innate immunity disposition in dialysis patients and the healthy population did not differ.

Effect of cyclosporine A on the non-specific, innate antiviral immunity of mice.

Different infections are the most common complication of immunosuppressive therapy. In this context, the effect of cyclosporine A (CsA) on the innate antiviral immunity of mice was studied. The presence of immunity was shown by infection of resident peritoneal cells (RPC) of BALB/c mice with herpes simplex virus type 1 (HSV-1) and vesicular stomatitis virus (VSV). While the cells infected immediately after isolation were resistant to the viruses, the cells cultured for several days before infection lost the immunity. The lack of activity to neutralize HSV-1 and VSV in the sera of the mice excluded a participation of specific antibodies in the resistance. To study the effect of CsA on the innate immunity, BALB/c mice were intraperitoneally (i.p.) injected with cyclosporine (20 or 100 microg/mouse, twice a day) for 3 days. The other group of animals was injected in the same way with PBS only. Then the peritoneal cells were isolated and infected with VSV immediately after cell isolation. The kinetics of viral replication in the control and CsA-treated groups was compared. While in the cells from the control group VSV did not multiply, in the cells from the CsA-treated mice the virus reached considerable titers. The cyclosporine effect on VSV replication was dose dependent and statistically significant. We conclude that innate antiviral immunity was suppressed in the cyclosporine-treated mice and that this mechanism may be involved in the high susceptibility of patients to viral infections during immunosuppressive therapy.

Study on risk factors for transplacental viral infections; effect of bacterial factors and double viral infections on virus replication in placenta and amniotic membranes.

Among risk factors for vertical transmission of HIV there are listed concomitant viral and bacterial infections. Therefore the influence on the viruses replication in human placenta and amniotic membrane cultures of double viral infection with two unrelated viruses - encephalomyocarditis (EMCV) and vesicular stomatitis virus (VSV) - was studied and compared with the replication of the viruses in single virus infection (EMCV or VSV) in the same organ cultures. Additionally effect of bacterial factors - lipopolysaccharide (LPS) Escherichia coli and sonicated Treponema pallidum antigens (Tpa) - on VSV replication in the same culture system was studied and compared with VSV replication in untreated explants. Two effects were observed in double-virus infected cultures and also in bacterial factors treated cultures: inhibition and stimulation of virus replication. The kind of effect in the both cases was dependent on the presence or absence of innate antiviral immunity. In virus-sensitive organs double infected or treated with LPS or Tpa, inhibition of virus titer (2-5 log TCID(50)/ml) was observed. In the organs expressing the innate immunity, stimulation (1-4 log TCID(50)/ml) of virus replication was noticed. Contribution of endogenous TNFalpha in both reactions (stimulation and inhibition) was confirmed using antibodies against the TNF.

Effect of proline rich polypeptide from ovine colostrum on virus replication in human placenta and amniotic membrane at term; possible role of endogenous tumour necrosis factor alpha.

Freshly prepared organ cultures of human placentae and amniotic membranes at term show different sensitivity to vesicular stomatitis virus (VSV) infection. In six of 16 amniotic membranes and seven of 17 placentae VSV replicated to relatively high titres (10(3)-10(6)TCID(50)/ml). The others were partially or completely resistant to virus infection (<10(1)-10(2)TCID(50)/ml). Addition of the immunomodulating agent, proline-rich-polypeptide (PRP) from ovine colostrum to explants freshly obtained from the organs, influenced VSV replication in a manner dependent on the innate immune state of the organ culture. In cultures resistant to the virus, PRP at a concentration of 10 microg/ml increased 10-10 000 times the VSV titre. In contrast, treatment of highly sensitive cultures by PRP hardly influenced viral replication at all. The effect of virus stimulation by PRP was abolished by specific anti-TNF antibodies. The results indicate that endogenous TNF may be one of the mediators of virus stimulation by PRP. Antibodies against TNFalpha, added to VSV infected organ cultures sensitive to the virus reduced viral replication. The antibodies caused stimulation of virus replication in VSV-infected resistant organ cultures. The results indicate the double role of endogenous TNF in viral replication in placenta and the amniotic membrane.

[Innate nonspecific antiviral immunity]. / Wrodzona nieswoista przeciwwirusowa odpornosc.

Participation of macrophages, NK cells, gamma delta T and alpha beta T lymphocytes to creation of innate antiviral defence is described in the review. Antiviral activity of interferons, members of tumour necrosis factor family, interleukins, chemokines and their possible role in formation of nonspecific immunity is reviewed. The hypothesis on the nature and function of receptors important for innate immunity and its interdependence with acquired immunity is discussed.

Antiviral effect of proline-rich polypeptide in murine resident peritoneal cells.

It is known that resident peritoneal (RP) cells from BALB/c female mice express a constitutive non-specific antiviral immunity which is progressively reduced during several days of cultivation in vitro. In this report, we have studied the effect of a proline-rich polypeptide (PRP) isolated from ovine colostrum on the kinetics of vesicular stomatitis virus (VSV) replication in freshly isolated and one-day cultured RP cells. The polypeptide was added to the cells immediately after virus adsorption or one day before or after viral infection. Independently on time of PRP addition, an inhibition of VSV replication (virus titres reduced by up to 4 log units) was observed. Occasionally, however, a weak stimulation of VSV replication by PRP (virus titres increased by 1-2 log units) was noticed in RP cells constitutively resistant to the infection.

Constitutive and induced cytokine production by human placenta and amniotic membrane at term.

Results of our previous study on the immunity of human placenta and amniotic membranes revealed that in majority of cases these organs present constitutive non-specific antiviral immunity in the organ culture (OC) system. It is possible that interferons (IFNs), tumour necrosis factors (TNFs) and interleukin 6 (IL-6) may be responsible for the antiviral effect. Here, the constitutive and lipopolysaccharide (LPS)-induced production of these cytokines and, additionally, interleukin 10 (IL-10) were determined in OC of chorionic villi, decidua and amniotic membranes. Significant amounts of constitutive TNF-alpha (2-64 U/ml), IL-6 (200-12,000 U/ml) and IL-10 (1-70 ng/ml) were detected in the maternal decidua and chorionic villi of placenta. Amniotic membranes produced lower concentrations of the cytokines. LPS increased the production of cytokines from two- to eightfold. In contrast, activity of IFN released spontaneously was found only in four of 50 placentae and amniotic membranes. LPS and Newcastle disease virus (NDV) induced IFN production in the OC system. However, the increase of IFN after induction was also very small (up to 32 U/ml). Individual differentiation in the cytokines production was observed among placentas and amniotic membranes. TNF was identified as type alpha with addition TNF-beta, IFN as type alpha, beta and gamma.

Stimulatory effect of ovine colostrinine (a proline-rich polypeptide) on interferons and tumor necrosis factor production by murine resident peritoneal cells.

We describe effects of ovine colostrinine (proline-rich polypeptide--PRP) isolated from ovine colostrum and nonapeptide fragment of PRP on interferon (IFN) and tumor necrosis factor (TNF) production by murine resident peritoneal cells (RPC). The cells from several mouse strains have been found to produce small amounts of IFN-beta and TNF-alpha constitutively. The colostrinine at concentrations of 1-100 micrograms per one ml of cell suspension containing 1 x 10(6) RPC isolated from BALB/c mice, enhanced the IFN and TNF production by 3-30 folds. Upregulation of TNF and IFN production has been observed in the RPC cultures that produced spontaneously less than 16 units of the cytokines only. Synthetic nonapeptide fragment of the colostrinine (Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro) at concentration of 1-100 micrograms/ml stimulated TNF synthesis but not IFN production. In 1996 Inglot et al. suggested that the colostrinines may be classified as cytokines produced by the mammary gland of mammals. In this paper we have found that the ovine colostrinine at low concentrations modulate the production of other cytokines (IFN-beta and TNF-alpha) in mouse cells that means that it may function in the cytokine network.

Effect of viral infections on the ability of human endothelium for interferon, tumor necrosis factor and interleukin 6 production.

We have reported that cultured human umbilical cord vein endothelial cells (HUVEC) differ from endothelium present on vein surface of organ culture (OC) in production of cytokines and susceptibility to viral infections. In this paper we present the effect of viral infections on interferon (IFN), tumor necrosis factor (TNF), and interleukin 6 (IL-6) production in two culture systems: HUVEC and OC. Infection of 24-48 h HUVEC with herpes simplex type 1 (HSV-1) and vesicular stomatitis virus (VSV) reduced the amounts of IL-6 and TNF produced in comparison to those released spontaneously by uninfected cells. No IFN was detected in media from infected and uninfected HUVEC. Limited viral infections of 3-h-HUVEC and OC usually diminished their efficiency of IL-6 and TNF production. In the case of IL-6 synthesis by OC, effect of viral infection depended, however, on the constitutive synthesis of the cytokine. When spontaneous production was high (> 800 U/ml), VSV and HSV-1 infection reduced IL-6 level by 2-50 times; in the case of low production (< 150 U/ml) the stimulation effect (2-4 fold) was observed. OC released spontaneously some IFN activity (2-32 U/ml). HSV-1 infection of OC reduced IFN level, while VSV in single cases slightly upregulated IFN synthesis.

Antiviral nonspecific immunity of human placenta at term: possible role of endogenous tumor necrosis factors and interferons.

The antiviral immunity of human placenta and amniotic membrane in an organ culture (OC) system was studied. Freshly isolated explants of most of the placentas at term and the amniotic membranes were found to be relatively resistant to herpes simplex virus type 1 (HSV-1), encephalomyocarditis virus (EMCV), and vesicular stomatitis virus (VSV) infections. After in vitro aging, however, the OC acquired the sensitivity to the viruses. In about 66%-90% of placentas, resistance of freshly isolated explants to the infection was observed. This indicates that the placentas displayed a constitutive immunity against the viruses. To study the role of endogenous cytokines in antiviral immunity, we added specific antibodies neutralizing IFN and TNF activities to VSV-infected OC and checked their influence on viral replication. Increases of 10-fold to 100-fold of VSV replication in the OC treated with anti-TNF-alpha, anti-IFN-alpha, anti-IFN-gamma or anti-IFN-beta sera were observed. The results indicate the importance of the endogenous cytokines in placental and amniotic membrane immunity. However, we did not observe a simple correlation between the spontaneous IFN and TNF production and the level of resistance against viruses. In view of the results, the participation of TNF and IFN in the constitutively expressed immunity of human placenta is of a more complex nature.

Effect of exogenous tumor necrosis factor alpha, interleukin 6 and interferons on vesicular stomatitis virus replication in human placenta and amniotic membrane organ cultures.

Effects of exogenous cytokines on replication of vesicular stomatitis virus (VSV) in amniotic membrane and placental organ cultures (OC) were studied. We compared the effects observed in OC and established human carcinoma cell lines: A549 and HEp-2. Recombinant human tumor necrosis factor alpha (rHuTNF-alpha), added to amniotic membrane, villous, or decidual OC at concentrations of 30 to 3000 U/ml, potentiated VSV replication by 10-1000 fold. Addition of 5 to 10000 U/ml of recombinant human interleukin 6 (rHuIL-6) to OC from 5 placentas was without effect on VSV growth, except one culture in which enhanced VSV replication has been observed. rHuTNF-alpha was found to have no effect on VSV growth in HEp-2 and A549 cell cultures. In contrast, the placental OC were sensitive to antiviral activity of natural interferons (IFNs): alpha, beta and recombinant IFN-gamma, although A549 cells were 5 to 10 fold more responsive to the cytokines.

Acquisition of susceptibility to vesicular stomatitis virus infection by murine resident peritoneal cells during culturing in vitro.

We have studied the effect of culturing in vitro of BALB/c--mouse resident peritoneal cells (RPC) on their ability to replicate vesicular stomatitis virus (VSV). Results of 36 experiments performed on RPC, freshly isolated from individual female mice, showed that over 70% of them expressed constitutive antiviral immunity against VSV infection. The virus multiplied to low titers (< or = 10(3) TCID50/10(6) cells) only in 10 out of 36 cases. After several days in culture, the RPC were found to lose the resistance and VSV multiplied in the cells to high titers (up to 10(7) TCID50/10(6) cells). The time period, required for the cultures to reach a full ability to replicate the virus, was individually differentiated and ranged from 24 h to over 96 h.

[The importance of endothelium as a target for virus infection]. / Znaczenie sródblonka naczyn jako tkanki docelowej w wirusowych zakazeniach.

The review concerns the susceptibility of human endothelial cells to several viruses, especially CMV and HIV. Viral infections of endothelium in vivo develop usually in immuno-compromised transplant recipients and in patients with AIDS. Endothelium of noninfected persons presents endogenous, nonspecific immunity against viruses. The constitutive production of cytokines with antiviral activities such as IFN, TNF and IL-6, seems to be responsible for the immunity of endothelium. The consequence of viral infections of endothelium (especially CMV) may be potentiation of allograft rejection and also development of arteriosclerosis. In the article the possible mechanisms and contribution of viruses to the processes are discussed.

Production of cytokines with antiviral activity by endothelial cells.

We compared the replication of VSV in human umbilical cord vein organ cultures (OC) with that in endothelial cells detached from the vein and infected immediately or infected after 24 h in culture. In four experiments of five, the virus titers obtained in the endothelial cells infected after 24 h in culture were 10-100 times higher than in the OC and 100-1000 times higher than in the freshly detached endothelial cells. To determine whether production of IFN, TNF, and IL-6 in the various cultures correlated with the data for virus yields, we measured the amounts of these cytokines formed. The OC produced considerably larger amounts of all of these. The endothelial cells produced very little IFN and TNF. Overall, there was no clear correlation between virus production and the amounts of these cytokines formed.

Does physiological interferon play a role in Mengo virus infection of mice?

NZB, BALB/c and C3H mice, differing in their production of physiological interferon (IFN), were used as models to investigate its role in their resistance to Mengo virus infection. These mouse strains also differ in their susceptibility to the infection. NZB and BALB/c mice, producing higher titers of physiological IFN than C3H mice, proved to be less resistant to the infection than the latter mouse strain. This suggests that physiological IFN does not play protective role against Mengo virus infection in NZB and BALB/c mice. Instead, the higher level of in vitro spontaneously released physiological macrophage IFN correlated with the higher death rate in Mengo virus infected mice (NZB > BALB/c > C3H). Our results suggest a reverse correlation between the amount of detectable physiological IFN and resistance to the infection.

Production of tumor necrosis factor alpha by resident peritoneal cells of mice.

Freshly isolated resident peritoneal cells (RPC) of three mouse strains: BALB/c, NZB and C3H, release spontaneously small amounts of tumor necrosis factor (TNF) when cultured in vitro. RPC of BALB/c mice produced higher TNF levels than the cells isolated from two other strains (BALB/c > NZB > C3H). Lipopolysaccharide (LPS) potentiated the TNF production. Kinetics of the TNF production depended on temperature of incubation of the cells. At 37 degrees C TNF was produced earlier (with a peak at 7 h) than at 26 degrees C (a peak at 20 h). Adherent cell fraction of RPC produced TNF at 26 degrees C as well as at 37 degrees C. Nonadherent cells released TNF only at 37 degrees C. The neutralization assay with media containing TNF and polyclonal rabbit anti-mouse TNF-alpha serum revealed that in every case, independently on the incubation temperature and cell fraction origin, only TNF-alpha was detected. Antibodies against human monoclonal or polyclonal TNF-alpha did not neutralize mouse TNF.

Spontaneous and lipopolysaccharide-induced tumor necrosis factor alpha and interferon beta production by resident peritoneal cells of mice: effect of sex, age and strain.

BALB/c and NZB mice differ in incidence of autoimmunological disorders. We have studied the dependence of sex and age of these mouse strains on their capacity to produce interferon beta (IFN-beta) and tumor necrosis factor alpha (TNF-alpha). Short term cultures of the mouse resident peritoneal cells (RPC) were used. Three to six week-old female and male BALB/c mice, in contrast to adult mice, did not produce spontaneous IFN-beta (< 2 units/ml) and only low level of TNF-alpha (2-4 units/ml). The levels of lipopolysaccharide (LPS)--induced IFN-beta and TNF-alpha increased progressively with age of BALB/c mice. Female BALB/c mice however, were found to produce approximately two-fold higher levels of IFN-beta and TNF-alpha than male BALB/c mice. Sex dependent differences in IFN-beta production were much more expressed when NZB mice were used in the experiments. RPC of young (3-5 week-old) female NZB mice produced relatively high levels of IFN-beta. The spontaneous IFN-beta production by RPC of older female mice (6-8 week-old) declined. In contrast, RPC of young NZB male mice did not produce spontaneous IFN-beta, while RPC of adult male mice were able to release some amounts of IFN-beta. The levels of spontaneous and LPS induced TNF-alpha and LPS induced IFN-beta were apparently not so correlated with sex and age of NZB mice as spontaneous IFN-beta.

Production of cytokines by mouse peritoneal cells treated with Tolpa Torf Preparation (TTP): dependence on age and strain of mice.

Production of two cytokines: Interferon beta (IFN-beta) and tumor necrosis factor alpha (TNF-alpha) by freshly isolated resident mouse peritoneal cells (RPC) treated with immunostimulating drug Tolpa* Torf Preparation (TTP) was investigated. Nontoxic concentrations of TTP (10 and 100 micrograms/ml) potentiated the cytokine production by RPC of BALB/c mice. The observed effects were dose-dependent. The optimal cytokine-inducing concentration of TTP was 100 micrograms/ml. The effect of TTP depended on the age of mice. The high potentiation (16-fold) of IFN-beta and TNF-alpha production by TTP was observed when 5 and 8 week-old mice were used. However, in RPC isolated from one year old mice, only two (fold) potentiation of IFN and TNF was observed. The activities of the same commercial preparations of TTP were compared in the RPC cultures isolated from mice of the three different strains: BALB/c, C3H/HeJ and NZB. The significant stimulation (16-32-fold) of TNF and IFN was observed in the cells isolated from BALB/c mice. In contrast, only slight stimulation of IFN and TNF (1-3-fold) was observed in cells of NZB mice. Peritoneal cells of C3H mice did not respond to stimulation with TTP.

[Latent virus infections of cells in the immune system]. / Latentne infekcje wirusowe komórek ukladu odpornosciowego.

The article concerns the latent infections of cells of immune system caused by herpesviruses and retroviruses. Molecular basis of latency maintained by Epstein-Barr virus in B lymphocytes as well as by human immunodeficiency virus in TCD4 lymphocytes is described. The switch between latency and replication of the viruses and immune surveillance is discussed.