CD4-positive T lymphocytes are required for the generation of the primary but not the secondary CD8-positive cytolytic T lymphocyte response to herpes simplex virus in C57BL/6 mice.

To understand the cellular basis for recovery from HSV infection, it is critical to identify functional interactions between HSV-specific T lymphocyte subpopulations involved in the generation of the optimal response. To this end, the requirement for CD4+ (L3T4+) T lymphocytes in the development of the primary and secondary CD8+ (Lyt-2+) cytolytic T lymphocyte (CTL) response following HSV infection in C57BL/6 mice was investigated. It was found that chronic depletion of CD4+ cells in vivo by treatment with the mAb GK1.5, which resulted in greater than 95% depletion of peripheral CD4+ T lymphocytes in treated animals, caused a profound decrease in the levels of cytolytic activity obtained during the primary response in the draining popliteal lymph nodes of mice responding to infection in the hind footpads. However, treatment did not affect the levels of in vivo secondary CTL activity in the popliteal lymph nodes, nor the in vitro secondary response in the spleen. The decreased CTL activity observed during the primary response was not due to an inability to prime HSV-specific CTL precursors (CTLp), as full cytolytic activity was obtained following culture of lymphocytes in the presence of exogenous IL-2 and antigen, and the response could be reconstituted by treatment with recombinant IL-2 in vivo. Analysis of the secondary CTL response in the spleen indicated that CD4+ cells were not required for either the generation or maintenance of this aspect of the response. However, blockade of IL-2 utilization by CTL using anti-IL-2R antibodies indicated that this lymphokine was absolutely essential for secondary CTL expansion in vitro. Finally, mice that had been infected 12 months previously exhibited a decreased ability to generate secondary HSV-specific CTL in vitro following CD4-depletion in vivo. Taken together, these results suggest two distinct stages of CTL development during the response: an early primary stage dependent upon the presence of CD4+ cells, and a later, CD4-independent stage operative during the secondary response, which decays with time postinfection.     

Pyridoxine deficiency and cytotoxicity of T lymphocytes in vitro.

The effect of pyridoxine deficiency on the proliferation and cytotoxicity of BALB/c mouse lymphocytes stimulated in vitro with irradiated spleen cells from C3H mice was studied. Cytotoxicity was measured by Na⁵¹CrO₄ release from L cells which have the same histocompatibility loci as C3H mouse cells. Pyridoxal-5′-phosphate (PLP) content in the spleen and liver of pyridoxine-deficient animals was determined with Escherichia coli B/1 t7A apotryptophanase. Maintenance of animals on a pyridoxine-deficient diet for 1 to 3 weeks affected neither proliferation of lymphocytes in vitro nor their cytotoxicity. Lymphocytes from mice fed a pyridoxine-deficient diet for 5 to 6 weeks had a reduced capacity to respond to foreign lymphoid cells in vitro. The Cytotoxicity of these lymphocytes was also significantly decreased. PLP, but not pyridoxal, added directly to the medium in vitro partially restored the impaired functions of T lymphocytes.     

Granzymes A and B are targeted to the lytic granules of lymphocytes by the mannose-6-phosphate receptor

To investigate the question of whether lytic granules share a common biogenesis with lysosomes, cloned cytolytic T cell lines were derived from a patient with I-cell disease. The targeting of two soluble lytic granule components, granzymes A and B, was studied in these cells which lack a functional mannose-6-phosphate (Man-6-P) receptor-mediated pathway to lysosomes. Using antibodies and enzymatic substrates to detect the lytic proteins, I-cells were found to constitutively secrete granzymes A and B in contrast to normal cells in which these proteins were stored for regulated secretion. These results suggest that granzymes A and B are normally targeted to the lytic granules of activated lymphocytes by the Man-6-P receptor. In normal cells, the granzymes bear Man-6-P residues, since the oligosaccharide side chains of granzymes A and B, as well as radioactive phosphate on granzyme A from labeled cells, were removed by endoglycosidase H (Endo H). However, in I-cells, granzymes cannot bear Man-6-P and granzyme B acquires complex glycans, becoming Endo H resistant. Although the levels of granzymes A and B in cytolytic I-cell lymphocytes are < 30% of the normal levels, immunolocalization and cell fractionation of granzyme A demonstrated that this reduced amount is correctly localized in the lytic granules. Therefore, a Man-6-P receptor-independent pathway to the lytic granules must also exist. Cathepsin B colocalizes with granzyme A in both normal and I-cells indicating that lysosomal proteins can also use the Man-6-P receptor-independent pathway in these cells. The complete overlap of these lysosomal and lytic markers implies that the lytic granules perform both lysosomal and secretory roles in cytolytic lymphocytes. The secretory role of lytic granules formed by the Man-6-P receptor-independent pathway is intact as assessed by the ability of I-cell lymphocytes to lyse target cells by regulated secretion.     

Defects in tumor cell immunogenicity: lymphokine signals in in vitro cytolytic responses to tumor alloantigens

The B16 melanoma of C57BL/6 mice illustrates a deficiency in immunostimulation which may be important in some host-tumor relationships. B16 immunizes very poorly, even against its own major histocompatibility complex (MHC) antigens. We have compared the anti-MHC cytolytic response induced in vitro by B16 and by other tumors of both lymphoid and nonlymphoid origin. We have also studied the role of indomethacin and exogenous lymphokines in facilitating these responses and examined the relationship of specific and nonspecific effector cells induced. In contrast to normal lymphoid cells and two lymphoid tumor cells (EL4 and WEHI-265), the three nonlymphoid tumors, B16, Lewis lung tumor (3LL), and MC-2 fibrosarcoma, failed to induce primary cytolytic responses by themselves. MC-2 and B16 represented two different defects in immunogenicity. MC-2, which we have shown previously to induce an in vivo cytolytic response, could also immunize in vitro provided that prostaglandin production was blocked with indomethacin. In contrast B16, which is poorly immunogenic in vivo, immunized in vitro only if a concanavalin A-induced lymphokine supernatant (CS) was added as an exogenous source of "signal 2." High concentrations of the interleukin 2-containing Con A-induced spleen cell culture supernatant-induced non-H-2b-specific lymphokine-activated killer (LAK) cells in the absence of B16 stimulator cells. However, lymphokine concentrations too low to induce LAK cells enabled the otherwise nonimmunogenic B16 cells to induce specific cytolytic activity.     

The amount of thiolic antioxidant ingestion needed to improve several immune functions is higher in aged than in adult mice

With aging there is an increase of oxidative stress due to an imbalance between the oxidant production and the antioxidant levels in favor of the former. Since immune cell functions are specially linked to reactive oxygen species (ROS) generation, the oxidant/antioxidant balance is essential for these cells. Although low levels of antioxidants cause a decrease in immune function, very high levels of antioxidant compounds could show prooxidant effects. In the present work, we have studied the effect of diet supplementation, for 4 weeks, with two different doses of two thiolic antioxidants, namely thioproline (TP) and N -acetylcysteine (NAC), at 0.1% (w/w) and 0.3% (w/w, of each antioxidant) on the main immune system cells, i.e.: macrophages, lymphocytes and natural killer (NK) cells of adult (33 λ±λ1 week old) and aged (75 λ±λ1 week old) female Swiss mice. Two groups of animals, adult and aged mice, fed standard diet were used as controls. The results show that the ingestion of 0.1% doses of thiols improves, in the adult mice, several immune functions such as the chemotaxis capacity of both macrophages and lymphocytes, the phagocytosis of macrophages, the lymphoproliferative response to the mitogen Con A and the NK activity. Moreover, no change was observed in adherence capacity of immune cells, and superoxide production was decreased. By contrast, in aged mice the ingestion of these amounts of antioxidants did not change the immune functions studied with the exception of NK activity, which was stimulated. The ingestion of 0.3% of antioxidants by adult mice only increased some immune functions such as adherence and superoxide production, which are markers of oxidative stress. Other functions such as chemotaxis or lymphoproliferative response decreased. However, the ingestion of these very high amounts of thiols by aged animals increased the phagocytosis, the NK activity and specially the lymphoproliferative response to the mitogen, a function that is very depressed with aging.     

Evidence for the presence of suppressor T lymphocytes in animals treated with cyclosporin A

Mice sensitized with alloantigens and treated with cyclosporin A (CsA) were incapable of generating antigen-specific cytolytic lymphocytes (CL). Lymphocytes from these CsA-treated animals could not be reactivated upon exposure to the same alloantigens in mixed lymphocyte culture (MLC), whereas their response to a third-party antigen remained intact, suggesting a long-lasting and specific effect of CsA. After being irradiated, these lymphocytes from CsA-treated animals were added to normal MLC and were shown to prevent normal lymphocytes from becoming cytolytic in a dose-dependent and antigen-nonspecific fashion. These suppressor cells were not detected in mice receiving CsA only, indicating that CsA did not induce but rather permitted the expression of suppressor cells possibly generated by allosensitization. The suppressor cells appeared to be T lymphocytes, because treatment with anti-Thy-1.2 antibody and C abrogated their suppressive activity. The present results suggest that activation and/or sparing of suppressor cells by CsA may account for the long-lasting unresponsiveness seen in CsA-treated animals.     

Effects of vitamin B6 deficiency on thymic epithelial cells and T lymphocyte differentiation.

Dietary vitamin B6 (pyridoxine) deficiency in young Lewis rats results in a reduction of T lymphocyte numbers and defects of cellular immunocompetence. In vitro studies of thymic epithelial (TE) cells, responsible for inducing T lymphocyte differentiation, revealed that maintenance on a vitamin B6 deficient diet for 2 weeks resulted in a severe defect in TE cell function. When the deficient animals were returned to a normal diet, TE cell function was restored. Exposure of lymphoid precursors from neonatally thymectomized or vitamin B6-deficient donors to normal TE monolayers resulted in their conversion to functional T lymphocytes, as measured by their response in MLR and to mitogens. However, TE monolayers from vitamin B6-deficient animals were unable to effect such a maturation of T lymphocytes. Therefore, it is suggested that the defect in cellular immunocompetence following this dietary deficiency is due, at least in part, to the inability of TE cells to effect the differentiation of T lymphocyte precursors to functional T lymphocytes. The dietary deficiency does not, however, impair lymphoid precursors, which can be stimulated to further differentiation by exposure to normal TE cell monolayers.     

The macrophagal-lymphocytic interaction of heterochronic cells in the induction of delayed hypersensitivity]

The influence of senescence on the functional activity of lymphocytes and macrophages in the induction of sensitivity to tuberculosis has been studied in experiments on 226 CBA mice. The study has revealed that after the injection of BCG old animals exhibit decreased capacity for the formation of delayed hypersensitivity, and their lymphocytes, transplanted to recipients, induce a lower level of hypersensitivity. Joint incubation of lymphocytes and macrophages from animals of different ages has shown that immunological defect appearing with age is localized in lymphocytes, while the antigen-presenting function of macrophages remains basically unchanged.     

Cardiac injury in myocarditis induced by Coxsackievirus group B, type 3 in Balb/c mice is mediated by Lyt 2 + cytolytic lymphocytes

Male Balb/c mice inoculated with a heart-adapted variant of Coxsackievirus, group B, type 3 (CVB3) develop severe myocarditis 7 days later. The lesions are characterized by mononuclear cell inflammation and myocyte necrosis. Infected T-lymphocyte-deficient mice show either minimal or no cardiac injury, although virus concentrations in the hearts of T-cell-deficient and -sufficient animals are similar. Adoptive transfer of 2 X 10(6) CVB3 immune Thy 1+ cells into CVB3-infected T-cell-deficient mice effectively restored myocarditis to levels observed in intact animals. Similar reconstitution with immune Ig+ cells or serum resulted in only a minimal increase in cardiac injury. To determine whether T-lymphocyte-dependent humoral or cellular immunity was responsible for myocarditis. T lymphocytes were obtained from Balb/c mice 6 days after infection with CVB3, separated into Lyt 1+2- (helper) and Lyt 1-2+ (cytolytic/suppressor) cell populations, and 2 X 10(6) of the enriched helper and cytolytic cells were adoptively transfused into infected T-cell-deficient recipients. Animals receiving the immune Lyt2+ cells developed severe myocarditis, had cytolytic T lymphocytes to both CVB3-infected and uninfected myocytes, but lacked a detectable IgG antibody response. Recipients of the Lyt 1+ cells failed to develop either myocarditis or cytolytic T cells but had normal serum IgG antibody titers to the virus. These results demonstrate that cardiac myocarditis is the product of cellular immune mechanisms.     

The effect of pyridoxine deficiency on the metabolism of the aromatic amino acids by isolated rat liver cells

The total activity of three key enzymes and the flux through eight steps of aromatic amino acid metabolism have been determined in liver cells isolated from rats fed either control or pyridoxine-free diet for 5-6 weeks. The pyridoxine-free diet caused a decrease in the catabolism of tyrosine and phenylalanine because of a drop in the flux through tyrosine aminotransferase. This decrease of expressed cellular tyrosine aminotransferase activity can be fully explained in terms of loss of cofactor. Larger decreases in the catabolism of tryptophan were seen after pyridoxine deprivation. The decreased extent of tryptophan catabolism can be solely attributed to loss of cofactor or increased degradation of kynureninase. Inhibition of tryptophan 2,3-dioxygenase was seen in pyridoxine deficiency, probably because of the buildup of the kynurenine metabolites. The control strength of kynureninase, for flux through kynureninase, was calculated to be less than or equal to 0.004, but 0.41 after pyridoxine deprivation. The sensitivity of the three pathways to pyridoxine deprivation is interpreted and discussed in terms of the different affinities for pyridoxal phosphate and the control strengths of the pyridoxal phosphate-dependent enzymes, tyrosine aminotransferase and kynureninase.     

Immune response to a syngeneic mammary adenocarcinoma. III. Development of memory and suppressor functions modulating cellular cytotoxicity.

Measurement of the development of cytolytic activity by mammary tumor primed or unprimed syngeneic spleen cells on in vitro monolayers of the 13762 rat mammary tumor operationally defined several subpopulations of lymphoid cells involved in the cytotoxic response. In vitro sensitization of cells from Fischer 344 animals injected 2 to 10 days earlier with 2 x 10(7) viable tumor cells always resulted in a higher and earlier lytic response than cells from non-inoculated animals. Adoptive transfer of the same in vivo primed cells for 5 days in irradiated syngeneic hosts removed any cytotoxic cells originally present but subsequent in vitro sensitization still resulted in a higher and earlier cytolytic response. We defined such cells as "memory" cells for cytotoxicity. Memory cells were radiosensitive and specific for the immunizing target cell. In contrast to cells from animals inoculated for 3 to 10 days, cells obtained 11 and 12 days after immunization had a lower response than unprimed cells on vitro sensitization. The anamnestic response could be restored either by culturing 12-day primed cells in vitro for 2 days without antigen or by adoptive transfer for 5 days into irradiated syngeneic rats. This suggests that another population of cells is present in spleen and suppresses the conversion of memory to cytotoxic cells. A more direct measurement of suppressor cell function was obtained by coincubating tumor-primed and unprimed cells on monolayers during in vitro sensitization. Cells from animals bearing tumors for 5 to 10 days always caused an increase in the response of the mixed lymphocyte groups, whereas 11- to 13-day tumor primed cells always caused a marked decrease in the cytolytic response. These results suggest the following interpretation of the kinetics of cell-mediated cytotoxicity to syngeneic tumor inoculation. Cytotoxic cells appear about 6 days after immunization, reach peak levels 2 days later, and then decrease rapidly. Memory cells are generated at a faster rate, reach peak levels before maximum cytolytic activity, but are then functionally inhibited from converting into differentiated cytotoxic cells by a new population of suppressor cells which reach peak activity about 12 days after immunization.     

Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus

Porcine T-cell recognition of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP) was tested using in vitro lymphoproliferative responses. Lymphocytes were obtained from outbred pigs experimentally infected with FMDV. Of the different NSP, polypeptides 3A, 3B, and 3C gave the highest stimulations in the in vitro assays. The use of overlapping synthetic peptides allowed the identification of amino acid regions within these proteins that were efficiently recognized by the lymphocytes. The sequences of some of these antigenic peptides were highly conserved among different FMDV serotypes. They elicited major histocompatibility complex-restricted responses with lymphocytes from pigs infected with either a type C virus or reinfected with a heterologous FMDV. A tandem peptide containing the T-cell peptide 3A[21-35] and the B-cell antigenic site VP1[137-156] also efficiently stimulated lymphocytes from infected animals in vitro. Furthermore, this tandem peptide elicited significant levels of serotype-specific antiviral activity, a result consistent with the induction of anti-FMDV antibodies. Thus, inclusion in the peptide formulation of a T-cell epitope derived from the NSP 3A possessing the capacity to induce T helper activity can allow cooperative induction of anti-FMDV antibodies by B cells.     

Effector activity of decidual CD8+ T lymphocytes in early human pregnancy

Although CD8+ T lymphocytes are present in human decidua throughout pregnancy, albeit as a minor population in early pregnancy, their role in normal pregnancy is largely unknown. The present study aimed to characterize their effector phenotype, including cytolytic activity, cytokine profile, and capacity to affect placental invasion. CD8+ lymphocytes were positively selected from normal early pregnancy decidua (7-14 wks gestational age). Decidual CD8+ T lymphocytes were studied using standard and redirected chromium release assays to investigate natural killer cell-sensitive cytotoxicity and cytotoxicity that requires T-cell receptor signal transduction respectively, multiplex cytokine analysis to analyze cytokine production, and a placental explant invasion model to assess the effect of soluble products of decidual CD8+ T lymphocytes on trophoblast invasion. Decidual CD8+ T lymphocytes exhibited cytolytic ability against P815 target cells (mean % Specific Chromium Release at effector:target ratio of 32:1 [SCR(32)] of 32.7 +/- 5.8) and against K562 target cells (mean SCR(32) of 20.3 +/- 0.5). Phytohemagglutinin-P (PHA-P)-stimulated decidual CD8(+) T lymphocytes produced high levels of both interferon gamma and interleukin (IL) 8, and low levels of granulocyte-macrophage colony-stimulating factor (CSF2), IL1B, IL2, IL6, IL10, IL12, and tumor necrosis factor; these did not vary with gestational age. IL4 was undetectable. Decidual CD8+ T lymphocyte supernatants increased the capacity of extravillous trophoblast cells to invade through Matrigel compared with the PHA-P control. These findings suggest that decidual CD8+ T cells can display cytolytic activity, do not evoke a predominant local intrauterine Th2 type cytokine environment, and may act to regulate invasion of extravillous trophoblast cells into the uterus, a crucial process for normal uteroplacental development.     

Enhancement of tumor growth correlates with suppression of the tumor-specific cytolytic T lymphocyte response in mice chronically infected by Trypanosoma cruzi

The immunity of BALB.B mice to syngeneic Gross murine leukemia virus (MuLV)-induced B.GV cells was studied at various times after infection by Trypanosoma cruzi. BALB.B mice chronically infected by the parasite do not develop an effective immune response against B.GV tumor cells, and B.GV tumor growth in vivo is consequently facilitated. The tumor-specific cytolytic T lymphocyte (CTL) compartment in these mice was studied in vitro because CTL are known to participate actively in syngeneic tumor rejection. These analyses showed that: a) CTL differentiation is suppressed in mice with chronic T. cruzi infections; b) suppression is at the level of CTL precursor cell activation; c) suppression is not antigen-specific; and d) suppression is mediated by macrophages and Lyt-2+ T lymphocytes.     

Interferon-beta and recombinant IL 2 can both enhance, but by different pathways, the nonspecific cytolytic potential of T3- natural killer cell-derived clones rather than that of T3+ clones

We studied the enhancement of cytolytic activity of T3- natural killer cell-derived clones, of T3+ T cell activated killer (AK) clones, and of fresh peripheral blood lymphocytes (PBL) by various crude and recombinant interferon (r-IFN) as well as IL 2 preparations. It was found that IFN-beta had the highest cytotoxicity inducing potency as compared to crude or r-IFN-alpha or -gamma preparations. This enhancement was blocked by anti-IFN-beta antibodies but not by anti-IFN-gamma antibodies. IL 2 also strongly enhances cytolytic activity in cloned T3- killer cells that express the IL 2 receptors as determined with the anti-Tac monoclonal antibody (MAb) at concentrations of IL 2 (25 U/ml) which induced one-half of the maximal proliferation capacity in human T cells and murine CTLL cells. For enhancement of cytolytic activity in fresh NK cells, a much higher concentration of IL 2 is required. In addition, the enhancement of cytolytic activity by r-IL 2 but not that by IFN-beta can be reduced by anti-Tac MAb, suggesting that the IL 2 receptor is involved in the enhancement by IL 2, but not by IFN. Both IFN-beta and IL 2 were able to enhance (over threefold) the cytolytic activity of T3- cloned killer cells against a variety of tumor target cell types. Another remarkable observation was that K562 cells, the most commonly used target cell for determining NK cell cytolytic activity, are not the most suitable targets to assess enhancement of nonspecific lytic activity as compared to Daudi or lung tumor-derived cell lines. No enhancement of anti-body-dependent cellular cytotoxicity was observed. Finally, the effects of these biological response modifiers were much more pronounced on "fresh" and cloned T3- natural killer cell-derived than on T3+-activated killer mature T cell-derived clones.     

Alloantigen bound to agarose beads and syngeneic carrier cells are capable of stimulating mouse cytolytic T lymphocytes in vitro.

Bead-bound antigen was prepared by coupling alloantigen covalently to agarose beads. Alloantigen-bearing syngeneic carrier cells were prepared by dilution of detergent solubilized alloantigen in the presence of syngeneic spleen cells. Both types of antigen were compared to spleen cells and reconstituted membrane fragments for the ability to stimulate cytolytic thymus-dependent lymphocytes in vitro. All these types of antigen could stimulate immune but not nonimmune spleen cells to form cytolytic T lymphocytes. The amount of lytic activity obtained with the bead-bound antigen was found to be only dependent upon the amount of H-2 antigen present in the culture and independent of the number of beads.     

Selenium supplementation protects from high fat diet-induced atherogenesis in rats: role of mitogen stimulated lymphocytes and macrophage NO production

The present study was designed to demonstrate the involvement of immune response in experimental atherogenesis. The mitogenic stimulation of lymphocytes and NO production by macrophages in experimental atherogenesis were studied. Further, influence of selenium a potent antioxidant was also studied in the disease process. Three different treatment groups of rats undertaken for study were: group 1, control; group II, high fat diet (HFD) fed group and group III, HFD+Se supplemented group. Atherogenic conditions induced have already been explained earlier [Kang BPS et al. Gen Physiol Biophys, 17 (1998) 71]. Significant increase in 3H-thymidine incorporation was obtained in lymphocytes from HFD fed animals in both presence and absence of mitogen (Con-A). However, these values decreased in group III animals, which were supplemented with selenium. Similarly, NO levels with LPS+ and LPS- macrophages also found to be higher in HFD fed group and decreased in group III. These studies reveal the protective role of selenium in HFD-induced atherogenic process.     

Vascular renin-like activity in aorta and mesenteric artery of the rat

Vascular renin-like enzymatic activity (VRLA) has been measured in the artery wall of control and experimental rats. The following groups have been studied: 1-normal salt diet; 2-high salt diet; 3-low salt diet; 4-bilateral nephrectomy (Nx); 5-sham operated for Nx. VRLA was evaluated in the aorta (ARLA) and in the mesenteric arteries (MRLA). Blood samples were obtained for plasma renin activity (PRA) determination. High salt diet decreased PRA, ARLA and MRLA whereas low salt diet increased PRA, did not change ARLA and decreased MRLA. PRA was almost undetectable in Nx animals while ARLA showed a 40% reduction and MRLA was unchanged in these animals. These results would indicate that the changes in PRA induced different variations in the renin-like content of the aorta and the mesenteric artery. The differences could be mainly due to two factors: 1) the capacity of the vascular tissue to bind circulating renin and 2) the capacity of each tissue to synthetize renin-like material in situ.     

In vitro and in vivo effects of some inhibitors of signal cascades on cytokines and signal proteins production in raw 264.7 macrophage cells and in mouse lymphocytes

In vitro and in vivo effects of some inhibitors of the activity of signal cascades NF-κB and SAPK/JNK, and the TLR4 receptor on the immune cells activity were studied. To evaluate in vitro effects, the macrophage-like RAW 264.7 cells were cultured with each of the inhibitors, namely IKK inhibitor XII, SP600125, CLI-095, and OxPAPK (the first two are the inhibitors of NF-κB, SAPK/JNK cascades, and the last two compounds are the inhibitors of the TLR4 receptor activity). On the whole, all of the used inhibitors did not induce pro-inflammatory response in RAW 264.7 cells. On the contrary, the inhibitor of SAPK/JNK cascade, and, especially, the inhibitor of NF-κB cascade significantly decreased production of the TNF-α, IL-1, IL-6, IFN-γ, and IL-10 in RAW 264.7 cells. In these cells, the inhibitors substantially decreased “back-ground stress response” of macrophages, differently reducing a production of heat shock proteins, HSP72 and HSP90-α, and diminishing phosphorylation of signal proteins from NF-κB and SAPK/JNK cascades. Results of in vitro experiments suggest that the inhibitor of NF-κB activity was the most effective. It was this inhibitor that was intraperitonealy injected in Balb/C male mice in the in vivo experiments in order to study its effect on the activity of immune cells. Results showed that IKK Inhibitor XII applied in vivo did not induce pro-inflammatory response in mice, but decreased the activity of NF-κB cascade, and lowered HSP90-α expression in mouse splenic lymphocytes. So, among the studied compounds, IKK Inhibitor XII seems to be a very effective inhibitor that may be used to decrease cytokine and stress response in various pathologies.     

Immune responses to Herpesvirus sylvilagus infection in cottontail rabbits

Immunologic changes produced by Herpesvirus sylvilagus infection of cottontail rabbits were investigated to evaluate this virus infection system as an animal model for EBV infection in humans. H. sylvilagus neutralizing antibodies appeared as early as 7 days after infection, peaked 2 to 4 wk postinfection and decreased to low levels by 8 to 10 wk postinfection. Complement-dependent antibodies mediating the protection of in vitro infection of monocytes and Con A-stimulated lymphoblasts with H. sylvilagus were observed as were complement-dependent cytotoxic antibodies against H. sylvilagus-infected cells. No cytolytic activity was present in sera taken either before or 3 days after infection; cytolysis was first observed 7 days after infection. The development of cytolytic antibodies appeared to be biphasic during an infection course of 12 to 16 wk. In vivo induction of a primary cytotoxic lymphocyte response to H. sylvilagus was also investigated. Splenic lymphocytes from infected animals lysed H. sylvilagus-infected skin fibroblasts; however, similar activity was not observed when PBMC or mesenteric lymph node lymphocytes were used as effector cells. H. sylvilagus-infected autologous skin fibroblasts were preferentially lysed as compared to heterologous skin fibroblasts. This virus-specific cytotoxic activity appeared 5 days postinfection and peaked 7 days postinfection. By 28 days postinfection, only low levels of cytotoxic activity were detected in spleen cells. Herpesvirus sylvilagus infection of cottontail rabbits provides an animal model for the study of lymphoproliferative disorders induced by herpesviruses.