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.

The pattern and level of cytokines secreted by Th1 and Th2 lymphocytes of syphilitic patients correlate to the progression of the disease.

The results of our previous work indicated that cell-mediated immune response, of importance in protection against Treponema pallidum, is distinctly inhibited at certain periods of syphilitic infection. Considering that cytokines, produced by Th1 lymphocytes, take part in this response and that their secretion may be regulated by cytokines of Th2 lymphocytes, we examined if, and in which stages of syphilis, such a regulation may exist. In this study we have examined the ability of cells to produce IL-2, IFN and TNF (Th1 or Th1 like cytokines) as well as IL-6 and IL-10 (Th2 or Th2 like cytokines). It was found that cells of syphilitic patients were able to produce IL-2, IFN, TNF, IL-10 and weakly IL-6 already in primary seronegative syphilis. At the same stage of syphilis, but seropositive, ability of Th1 lymphocytes to produce cytokines reached the highest values, whereas the cells producing IL-10 lost this ability. The cells producing IL-6 and MIF had the highest ability in secondary early syphilis. In this stage of syphilis again slightly increased the ability of cells to secrete IL-10, which reached the highest value in early latent syphilis. The growing ability to produce IL-6 and IL-10 was accompanied with a diminished production of IL-2, IFN and TNF nearly in all stages of syphilis. Only MIF, in contrast to other cytokines, was produced in late syphilis without distinct changes. The greatest suppression of Th1 lymphocytes to produce cytokines and cells to secretion of MIF was found in early latent syphilis when the level of IL-10 in cell culture supernates was the highest. High ability of Th2 lymphocytes to cytokines secretion in late syphilis and low ability of Th1 ones, which are very important for cell-mediated immune response, may be the reason for facilitating T. pallidum multiplication and development of latent stages of disease despite presence of immunologically competent cells.

The ability of peripheral blood mononuclear cells (PBMC) of syphilitic patients to produce IL-2.

The cell-mediated immune response of importance in protection against Treponema pallidum, is distinctly suppressed in some stages of the disease. This may be a result of decreased ability of cells to produce IL-2, or IL-2 absorption by different factors. The experiments were designed to evaluate the ability of peripheral blood mononuclear cells (PBMC) of patients with different stages of syphilis to produce IL-2, and to investigate the causes which could possibly limit its activity. The ability of the PBMC of syphilitic patients to produce IL-2 develops at the beginning of the disease, reaching a maximum in primary seropositive syphilis. In the next stages of the disease this capability is distinctly lowered. The lowest was in malignant syphilis and tabes dorsalis, i.e. during severe disease. Absorption of adherent cells from PBMC increased the ability of lymphocytes to produce IL-2. The highest level of this interleukin was observed at the stages of the disease where suppression was the deepest. Sera of both control and syphilitic patients contained IL-2 inhibitor. Its level was the highest in early and late latent syphilis where no symptoms of disease were present. In all syphilitic sera a distinctly elevated level of soluble IL-2 receptors (sIL-2R) was also found. Its high level was noted in sera of patients in which PBMC had the weakest ability to produce IL-2. These findings suggest that sIL-2R may be bound to IL-2 and in this way would lead to weakening of T cell function and of resistance against Treponema pallidum infection.

The ability of peripheral blood mononuclear cells of rabbits infected with Treponema pallidum to produce IL-2.

It was previously found that the cell-mediated immune response involved in protection against Treponema pallidum is distinctly suppressed during some periods in the course of syphilis infection in rabbits. This may be a result of the weak ability of cells to produce Interleukin-2 (IL-2) as well as of IL-2 absorption. The ability of peripheral blood mononuclear cells (PBMC) of syphilitic rabbits to produce IL-2 develops within the first two weeks after infection reaching a maximum in about the eleventh week. In infection of longer duration, this capability was distinctly lowered. This low level of activity (no higher than in PBMC of normal rabbits) was maintained for 31 weeks. The ability of PBMC to absorb IL-2, in parallel with its production, was found at the same time in the course of syphilis infection (7-11 weeks). In long-lasting syphilis (more than 12 weeks) both abilities seem to be inhibited. Sera of syphilitic rabbits were found to have a higher level of IL-2 inhibitor than those of normal rabbits. Only in syphilis lasting 9 to 11 weeks, when the production of IL-2 was the greatest, was the level of IL-2 inhibitor nearly the same as in normal rabbit sera. In syphilis lasting longer, the increased level of inhibitor was accompanied by a decreased ability of cells to produce IL-2. These findings suggest that IL-2 inhibitor may be bound to IL-2 or IL-2 receptor on T lymphocytes and in this way would lead to weakening of T cell function and resistance against Treponema pallidum infection.

The study of cell-mediated immune response in recurrent vulvovaginal candidiasis.

The aim of this work was to examine in vitro the ability of cells from patients with recurrent vulvovaginal candidiasis (RVVC) to cell-mediated immune response. Peripheral blood mononuclear cells (PBMC) and whole blood cells (WBC) of 37 RVVC patients in acute infection and 14 in remission were examined for the ability to proliferation and cytokines production (IFN, TNF, IL-6). As a control, a group of 25 healthy women were examined. The cells were stimulated with Candida antigen (HKCA), LPS and PHA. To indicate the level of cytokines, the following cell-lines were used: A549 for IFN, WEHI 164 for TNF and 7TD1 for IL-6. The proliferation/death of cells was determined by colorimetric test using MTT. Distinct suppression of cell-mediated immune response (CMI) was shown in all patients comparing to the control. Greatest suppression was found in the acute phase of the disease. The ability of cells to proliferate and produce IFN increases only in remission. The data seem to suggest that in this phase of disease, the ability of cell-mediated immune response is restored. It was also indicated that IFN may take part in protection against Candida infection.

The role of protein component of T. pallidum in inducing skin reaction for delayed hypersensitivity.

The protein component of T. pallidum was shown to be an important agent in inducing the skin reaction for delayed type of hypersensitivity (SDH) in syphilis. Treponema preparations which contained T. pallidum deprived of this component by treating the organisms with merthiolate, formalin or heat, were found unable to produce a distinct positive skin reaction in syphilitic rabbits. Using preparations containing treponemes with preserved protein component, it was possible to induce the skin reaction for DH in rabbits as early as 4 weeks after inoculation of T. pallidum and during the subsequent 64 weeks of the infection (and of the observation period). The SDH was fairly well correlated with the results of the MMI test in the whole course of the syphilitic infection; a disagreement of results of these two tests occurred only between the 16th and 26th week of the infection.

Syphilis and AIDS.

Evidence is shown that syphilis has become a major public health problem again. From 1988 to 1995 the permanently growing number of new cases of syphilis in the world was observed. The majority of the syphilitic cases in the patients are difficult for curing. The central nervous system is often involved in early syphilis. Previously the neurosyphilis was very rare. The reason for development of this stage of syphilis, may be an inadequate treatment as well as a weakening of the immunological responses. The latter one very often is caused by additional non-symptomatic infection including human immunodeficiency virus (HIV). Syphilitic ulcers act as a portal of entry for HIV. Analysis of cases with double infection with Treponema pallidum and HIV indicate that HIV infection may accelerate the course of syphilis and the presence of syphilis may also have influence on progression of the chronic HIV infection to AIDS. Taking into account that HIV infection alters the response to the treatment also, one can suggest that all of the patients with syphilis should be examined for the presence of HIV infection.

Identification of cells producing anti-treponemal lymphotoxin (ATL).

Identification of cells producing ATL was carried out on suspensions of cells enriched with lymphocytes T, B and macrophages. The cells were isolated from the spleen of syphilitic rabbits and from their testes when the rabbits were infected intratesticularly. The isolation of lymphocytes and macrophages was performed on different days after infection. It has been found that all examined cells are able to produce ATL but most active are T lymphocytes. These cells isolated from the testes were able to produce ATL already 6 days after infection, whereas lymphocytes B and macrophages acquired this capability not before day 10-13. The ability of cells to produce ATL was followed by marked reduction of treponemes from testes. The isolated treponemes were also immobilized and destroyed. Dependence between capability of cells to produce ATL and disappearance of treponemes from infected organ indicated that ATL kills treponemes also in vivo.

Characterization of anti-treponemal lymphotoxin from lymphocytes of syphilitic rabbits.

The effect of various treatments on the activity of anti-treponemal lymphotoxin (ATL) produced by lymphocytes of syphilitic rabbits was studied. Treponema pallidum-killing activity of ATL was slightly reduced after heating at 56 degrees C and completely abolished at 100 degrees C. The significant reduction of the activity was also obtained after exposure of ATL to acidic conditions (pH 1-5) at room temperature, or by treatment with papain and neuraminidase. Activity of ATL was completely resistant to deoxyribonuclease, ribonuclease and trypsin treatment. ATL was eluted from the Sephadex G-100 column together with hemoglobin, that suggested the apparent molecular weight of ATL of about 65,000. The active fraction from the Sephadex G-100 column was further fractionated on DEAE-Sephadex A-50. The activity of ATL was widely spread in the column eluate, indicating the charge heterogeneity. All these data indicate that ATL is a relatively low molecular weight protein. The sensitivity to neuraminidase and heterogeneity of charge suggest that it is a glycosylated protein.

Interleukin 10 and its role in the regulation of the cell-mediated immune response in syphilis.

Data concerning interleukin 10 (IL-10), a cytokine of Th2 lymphocytes, and its inhibition of Th1 lymphocytes from secreting IL-2 and interferon (IFN) are presented. It has been indicated that IL-10 also inhibits other cells from producing IL-12 and nitric oxide (NO). It is known that all these factors take part in the cell-mediated immune response and immunity. This inhibition may facilitate the multiplication of Treponema pallidum and the development of disease despite the presence of immunologically competent cells. It has also been demonstrated that in late latent syphilis, when Th1 lymphocytes are not able to produce IL-2 and IFN, the cells are able to produce only IL-12 and NO. This fact seems to suggest that these factors take over the immune function when cells are stimulated again by treponemes which, after many years of latency, begin to multiply. Thus, a high level of IL-12 and NO seems to be an indicator of the development of the third stage of disease.

Isolation and characterization of low molecular weight antibodies for Treponema pallidum.

A low molecular weight antibody which coated Treponema pallidum without producing any apparent reaction was isolated from human syphilitic serum. Its electrophoretic mobility was that of IgG immunoglobulins, and molecular weight was between 115,000 and 120,000.

Treponemicidal activity of supernatants from cultures of sensitized lymphocytes.

It was found that lymphocytes secured from lymphoid organs or syphilitic rabbits, when cultured with ultrasonicate of T. pallidum (Tp-S) or phytohemagglutinin-P (PHA-P), released to the supernatant fluid a substance (ATL-anti-treponemal lymphotoxin) which exerted a direct killing effect on T. pallidum. Splenic lymphocytes were found much more active than lymphocytes isolated from popliteal and mesenteric lymph nodes, and the peripheral blood. The effect of some factors on release of the ATL by sensitized lymphocytes was established.

Studies on autolymphocytotoxic activity occurring in Treponema pallidum infection.

It was found that in sera of majority of syphilitic rabbits cold lymphocytotoxic activity occurs. This activity appears usually 3 to 10 weeks after infection and it is present for 9 to 10 weeks. Lymphocytotoxic activity was found in undiluted sera or diluted from 1/2 to 1/8. This activity was directed against B lymphocytes only. Lymphocytotoxicity was found only in sera with high level of VDRL antibodies. The role of this activity in syphilis is not known. The killing effect exerted on B lymphocytes producing syphilitic reagins directed not only against T. pallidum, suggests that lymphocytotoxic activity may take part in the regulation of humoral response at the beginning of syphilitic infection.

Effect of serum lymphocytotoxic activity on T and B cells in rabbits infected with Treponema pallidum.

The correlation between the level of cold autolymphocytotoxic activity in the sera of rabbits infected with T. pallidum, and the percentage of B and T cells in the peripheral blood of the same animals was determined. The percentage of cells was estimated by the E and EAC rosette techniques and by the immunofluorescence test on immunoglobulin-bearing (B) cells. It was found that the increase of the autolymphocytotoxic activity was connected with the proportional decrease of B lymphocytes and increase of T lymphocytes. Since the decrease of B cells was significant (p less than 0.05) it is suggested that the autolymphocytotoxic activity may be involved in killing of B lymphocytes also in vivo. The possible role of the complement-dependent autolymphocytotoxic serum activity in regulation of humoral response in syphilis is discussed.

Attempts to isolation and characterization of autolymphocytotoxins from syphilitic sera.

To solve problem if autolymphocytotoxins (AL) present in syphilitic sera are not circulating immune complexes (CIC), sera containing strong AL activity and weak CIC were precipitated with polyethylene glycol (PEG). Received PEG-precipitates were filtrated on Bio-Gel A-1.5 column to separate CIC from AL. The Al were found in the first three after void volume fractions of the column. The fractions were examined on content of protein, sugar and sialic acid. The only correlation seen refers to the sugar and protein. The fraction which display the AL activity have the ratios of sugar to protein higher than non active fractions. The data indicated besides that AL is not CIC because it is not immunoglobulin nor Treponema pallidum antigen. For further biochemical characterization of these factors more material is needed.

Cell-mediated immunity in experimental syphilis in rabbits.

The development of cell-mediated immunity was studied in rabbits: a) experimentally injected with T. pallidum, and b) artificially immunized with nonviable T. pallidum. The macrophage migration inhibition test (MMI test) using ultrasonicate of T. pallidum as antigen was employed to demonstrate this type of immunologic response. Lymphocytes of syphilitic rabbits were found to exert a pronounced inhibitory effect upon macrophage migration as early as one month after infection; between the fourth and sixth month, a transient decrease of this capacity occurred, followed by a slow but steady increase during the next two years, i.e. until the end of the observation period. In rabbits in which the infection had been controlled by penicillin treatment in the fifth month, no increase but gradual diminution of this capacity was observed. In the group of rabbits artificially immunized with nonviable T. pallidum, the MMI response appeared very shortly after commencement of the immunization and persisted for about nine months after completion of the immunization procedure. Only a crude correlation could be established between levels of the MMI response of the immunized animals and their resistance to infection with T. pallidum.

The protective function of cell-mediated immunity in syphilis.

Experimental evidence has been obtained to show that the cell-mediated response in syphilis is protective. The use of chemical immunosuppressors (cyclophosphamide, 5-fluorouracil) resulted in the abolition of the cell-mediated response measured by the macrophage migration inhibition test (MMI test), and was accompanied by an enhancement in the severity of the lesions during the course of experimental syphilis in the rabbit. Successful adoptive transfer of resistance to syphilitic infection from immune donor rabbits to normal recipient rabbits employing lymph node lymphocytes was accomplished.

Development of immunological responsiveness and resistance to infection with Treponema pallidum in rabbits given immune lymphocyte preparations.

Three groups of normal rabbits were administered: 1. viable lymphocytic cells; 2. lysates of lymphocytes obtained by freezing and thawing, and 3. low molecular weight dialyzable transfer factor (TF). The lymphocytes were derived from peripheral blood, lymph nodes, and spleens of rabbits: 1. cured of longlasting syphilitic infection by penicillin treatment, and 2. immunized with nonviable T. pallidum, exhibiting a good reactivity by tests for humoral and cell-mediated responses. All the three lymphocyte preparations were found to be able to transfer cell-mediated response as measured by the Macrophage Migration Inhibition Test (MMI) and the Skin Test for Delayed Hypersenitivity to Treponemal Antigens (SDH), from a reactive donor to the nonreactive recipient rabbit; the intensity of the response found in the recipient was roughly the same as in the donor. None of the lymphocyte preparations transferred humoral response, as measured by the TPI and VDRL tests. Only whole lymphocytic cells were found to be able to confer a stat of resistance to infection with T. pallidum on normal recipients.

Effect of immunosuppressive agents on the course of experimental syphilis in rabbits.

Rabbits infected intradermally with T. pallidum at four sites on the clipped back were treated with cyclophosphamide, 1 mg/kg body weight, twice before and every two days after inoculation for five months, or 5-fluorouracil 2.5 mg/kg body weight, according to the same regimen. Humoral response, as measured repeatedly by VDRL, FTA, and hemagglutination tests, was not affected by the application of the immunosuppressors. In contrast, the cell-mediated response, as determined by the Macrophage Migration Inhibition test was completely abolished in the treated rabbits. In the group of rabbits which were given immunosuppressors, syphilitic lesions appeared not only at the challenge sites, as usual, but also multiple multiform lesions developed over the entire dorsum after healing of the primary lesions. Taking into account the fact that abolishment of the cell-mediated response led to an aggravated course of the syphilitic infection, the results of these experiments were interpreted as indicating the protective role of the cell mediated response in syphilis.