Production and properties of immune interferon from spleen cell cultures of Toxoplasma-infected mice

In response to antigenic stimulation, spleen cells from Toxoplasma-infected mice produce a factor showing inhibitory activity against vesicular stomatitis virus infection in L cell cultures. When BALB/C and ICR mice were inoculated intraperitoneally with the low-virulent S-273 strain of T. gondii, such activity was first detected in 4 and 7 days and reached maximum levels at 10 and 14 days respectively, and retained these levels for at least three weeks. However, BALB/C mice, which are considerably more sensitive to Toxoplasma infection than ICR mice, produced significantly smaller amounts of interferon (IF) after challenge with the high virulent strain. The IF produced in this system possessed certain known properties of immune (type II) IF and was not neutralized by rabbit antiserum against mouse type I IF. The immune IF preparation also inhibited multiplication of Toxoplasma within nonphagocytic L cells in an IF-like fashion, whereas Newcastle disease virus-induced (type I) IF had no effect on this parasite. The antiviral and anti-Toxoplasma activity in immune IF preparations could not be distinguished solely on the bases of their molecular weight and isoelectric point. The experiments with anti-theta serum plus complement and with nylon wool column effluent cells strongly suggest that immune IF was produced by T lymphocytes and required the assistance of macrophages.     

Involvement of cystic fibrosis transmembrane conductance regulator in infection-induced edema

Abnormal fluid accumulation in tissues, including the life-threatening cerebral and pulmonary edema, is a severe consequence of bacteria infection. Chlamydia (C.) trachomatis is an obligate intracellular gram-negative human pathogen responsible for a spectrum of diseases, causing tissue fluid accumulation and edema in various organs. However, the underlying mechanism for tissue fluid secretion induced by C. trachomatis and most of other infectious pathogens is not known. Here, we report that in mice C. trachomatis infection models, the expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel, is up regulated together with increased cytokine release and tissue fluid accumulation that can be reversed by treatment with antibiotic specific for C. trachomatis and CFTR channel blocker. However, C. trachomatis infection cannot induce tissue edema in CFTRtm1Unc mutant mice. Administration of exogenous IL-1beta to mice mimics the C. trachomatis infection-induced CFTR upregulation, enhanced CFTR channel activity and fluid accumulation, further confirming the involvement of CFTR in infection-induced tissue fluid secretion.     

Augmentation of mouse natural killer cell activity by interferon and interferon inducers.

Interferon (IF), in addition to its anti-viral capacity, is increasingly being found to be a regulator of cell division, cell surface antigens, and cell function. To determine whether IF also plays a role in the regulation of natural killer (NK) cell activity in mice, the in vivo and in vitro effects of IF and IF inducers on NK activity were studied. We observed that pyran, lipopolysaccharide, and polyinosinicopolycytidylic acid (poly I:C) as well as crude and purified IF preparations significantly elevated splenic NK levels in normal mice within 3 to 24 hr of i.p. administration. Normal spleen cells treated with poly I:C or IF in vitro also had augmented NK activity. Poly I:C and IF were themselves not cytotoxic and their presence was not required during the lytic process, indicating that IF acts on lymphocytes to activate NK function. The addition of anti-IF in the incubation medium completely blocked the boosting of NK activity by poly I:C or IF. The characteristics of the effector cells activated by IF were consistent with those of NK cells rather than macrophages, since the boosted effector cells were not retained by a rayon column or removed by carbonyl iron. Moreover, they were resistant to treatment with anti-Thy 1.2 serum plus complement, which eliminated mature T cells.     

Biological activity of a new Soviet natural inducer, double-stranded RNA

Antiviral activity of a two-spiral RNA (ts RNA), a new natural interferon inductor was studied. It was shown that ts RNA extracted from a phage infected E. coli culture was an active inductor of interferon and resistance to infection with the forestspring encephalitis virus experimental animals. In experiments on 10-12 g mice ts RNA administered in a dose of 50 micrograms/mouse 6 hours after the infection induced up to 1280 units/ml of the serum interferon. When the inductor was administered repeatedly, the experimental animals developed hyporeactivity resulting in a marked decrease in interferon production after the 3rd subsequent injection. The most pronounced effect with respect to the forest-spring encephalitis virus was observed when the inductor was administered intraperitoneally in a dose of 20 micrograms/mouse 4 hours before the infection. The protective effect was less pronounced when the inductor was administered 24 and 48 hours before the infection. A two-fold administration of the inductor did not increase the antiviral effect. When the inductor was administered in a dose of 100 micrograms 14 days before the infection, the animals showed an increase in the nonspecific resistance to the infection resulting in a marked antiviral effect.     

The role of circulating interferon in the modifications of immune responsiveness by mouse hepatitis virus (MHV-3).

MHV-3 modifies the humoral immune response to SRBC. During acute infections timing was critical: infecting mice before antigen administration led to immunodepression. Simultaneous injection with virus and SRBC resulted in immunostimulation. Persistent MHV-3 infections were associated with a chronic immunodepression. The presence of circulating interferon (IF) was well correlated with these modifications. IF peaking before antigen was associated with immunodepression whereas IF secretion after antigen was associated with immunostimulation. Low, permanent levels of IF were associated with chronic immunodepression. Since IF is, up to now, the only product of activated lymphocytes that has been shown to modulate immune responses, our results suggest that induction of IF by MHV-3 may be the main mechanism by which this virus modifies immune responsiveness. Moreover, we have shown that MHV-3 infection in susceptible mice diminishes the secretion of lymphocyte IF in response to Sendai virus. In these animals, the thymus cortex was profoundly depleted although the thymus medulla remained unchanged. The MHV-3 infection may, therefore, interfere with a subpopulation of IF-secreting lymphocytes. The possible physiologic role of such lymphocyte subpopulation in terms of host-virus relationships is discussed.     

Induction of interferon production in mouse spleen cell cultures by corynebacterium parvum.

Spleen cells of CS7BL/6 mice produced considerable amounts of interferon (IF) in vitro when tested 5 to 20 days after injection of killed Corynebacterium parvum. Interferon was also produced when C. parvum was added in vitro to spleen cell cultures of previously untreated mice. High levels were detected after 1 day of culture with some increment during subsequent days. In a number of experiments IF was also produced in untreated control cultures but only after prolonged cultivation and not after 1 day. The highest levels of IF were usually obtained when spleen cells of C. parvum-treated mice were challenged with additional C. parvum in vitro. The IF induced by C. parvum shared certain physicochemical properties with a tested immune IF and was not neutralized by an antiserum raised against a type I IF. Spleen cells of nu/nu mice and spleen cells treated by anti-θ serum plus complement did not differ from their respective controls, indicating that production of IF did not require mature T lymphocytes. Removal of B lymphocytes by nylon wool columns abolished the capacity of spleen cells to produce IF. When spleen cells were freed of adherent cells by the use of plastic surfaces, they no longer produced IF. Peritoneal exudate macrophages (PEC), which by themselves did not produce IF, in small numbers reconstituted nonadherent spleen cells. Nylon column-treated spleen cells, however, could not be restored by PEC. It is concluded that IF upon challenge with C. parvum is produced by B lymphocytes and requires the help of macrophages.     

Chlamydia trachomatis infection alters the development of memory CD8+ T cells

The obligate intracellular bacterium Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States and the leading cause of preventable blindness worldwide. Prior exposure to C. trachomatis has been shown to provide incomplete protection against subsequent infection. One possible explanation for the limited immunity afforded by prior C. trachomatis infection is poor activation of Chlamydia-specific memory CD8+ T cells. In this study, we examined the development of CD8+ memory T cell responses specific for the Chlamydia Ag CrpA. The percentage of CrpA63-71-specific T cells expressing an effector memory T cell phenotype (IL-7R+ CD62low) was dramatically diminished in mice immunized with C. trachomatis, compared with mice immunized with vaccinia virus expressing the CrpA protein. These alterations in memory T cell development were correlated with a significant reduction in the capacity of convalescent mice to mount an enhanced recall response to Chlamydia Ags, compared with the primary response. CrpA-specific memory T cells primed during VacCrpA infection also failed to respond to a challenge with Chlamydia. We therefore investigated whether C. trachomatis infection might have a global inhibitory effect on CD8+ T cell activation by coinfecting mice with C. trachomatis and Listeria monocytogenes and we found that the activation of Listeria-specific naive and memory CD8+ T cells was reduced in the presence of C. trachomatis. Together, these results suggest that Chlamydia is able to alter the development of CD8+ T cell responses during both primary and secondary infection, perhaps accounting for the incomplete protection provided by prior Chlamydia infection.     

Toll-like receptor-2, but not Toll-like receptor-4, is essential for development of oviduct pathology in chlamydial genital tract infection

The roles of Toll-like receptor (TLR) 2 and TLR4 in the host inflammatory response to infection caused by Chlamydia trachomatis have not been elucidated. We examined production of TNF-alpha and IL-6 in wild-type TLR2 knockout (KO), and TLR4 KO murine peritoneal macrophages infected with the mouse pneumonitis strain of C. trachomatis. Furthermore, we compared the outcomes of genital tract infection in control, TLR2 KO, and TLR4 KO mice. Macrophages lacking TLR2 produced significantly less TNF-alpha and IL6 in response to active infection. In contrast, macrophages from TLR4 KO mice consistently produced higher TNF-alpha and IL-6 responses than those from normal mice on in vitro infection. Infected TLR2-deficient fibroblasts had less mRNA for IL-1, IL-6, and macrophage-inflammatory protein-2, but TLR4-deficient cells had increased mRNA levels for these cytokines compared with controls, suggesting that ligation of TLR4 by whole chlamydiae may down-modulate signaling by other TLRs. In TLR2 KO mice, although the course of genital tract infection was not different from that of controls, significantly lower levels of TNF-alpha and macrophage-inflammatory protein-2 were detected in genital tract secretions during the first week of infection, and there was a significant reduction in oviduct and mesosalpinx pathology at late time points. TLR4 KO mice responded to in vivo infection similarly to wild-type controls and developed similar pathology. TLR2 is an important mediator in the innate immune response to C. trachomatis infection and appears to play a role in both early production of inflammatory mediators and development of chronic inflammatory pathology.     

Chlamydia trachomatis pulmonary infection induces greater inflammatory pathology in immunoglobulin A deficient mice

Chlamydia trachomatis is an intracellular bacterial pathogen that primarily infects via mucosal surfaces. Using mice with a targeted disruption in IgA gene expression (IgA(-/-) mice), we have studied the contribution of IgA, the principal mucosal antibody isotype, in primary immune defenses against pulmonary C. trachomatis infection. Bacterial burden was comparable between IgA(-/-) and IgA(+/+) animals following C. trachomatis challenge. Serum and pulmonary anti-Chlamydia antibody levels were higher in IgA(-/-) animals, with the exception of IgA. Lung sections of challenged IgA(-/-) mice showed more extensive immunopathology than corresponding IgA(+/+) animals. Real-time PCR analysis demonstrated significantly greater IFN-gamma and TGF-beta mRNA expression in IgA(-/-) as compared to IgA(+/+) animals. Together, these results suggest that IgA may not be necessary for clearance of primary C. trachomatis infection. However, IgA(-/-) mice displayed exaggerated lung histopathology and altered cytokine production, indicating an important role for IgA in regulating C. trachomatis induced pulmonary inflammation and maintenance of mucosal homeostasis.     

Compensatory T cell responses in IRG-deficient mice prevent sustained Chlamydia trachomatis infections

The obligate intracellular pathogen Chlamydia trachomatis is the most common cause of bacterial sexually transmitted diseases in the United States. In women C. trachomatis can establish persistent genital infections that lead to pelvic inflammatory disease and sterility. In contrast to natural infections in humans, experimentally induced infections with C. trachomatis in mice are rapidly cleared. The cytokine interferon-γ (IFNγ) plays a critical role in the clearance of C. trachomatis infections in mice. Because IFNγ induces an antimicrobial defense system in mice but not in humans that is composed of a large family of Immunity Related GTPases (IRGs), we questioned whether mice deficient in IRG immunity would develop persistent infections with C. trachomatis as observed in human patients. We found that IRG-deficient Irgm1/m3((-/-)) mice transiently develop high bacterial burden post intrauterine infection, but subsequently clear the infection more efficiently than wildtype mice. We show that the delayed but highly effective clearance of intrauterine C. trachomatis infections in Irgm1/m3((-/-)) mice is dependent on an exacerbated CD4(+) T cell response. These findings indicate that the absence of the predominant murine innate effector mechanism restricting C. trachomatis growth inside epithelial cells results in a compensatory adaptive immune response, which is at least in part driven by CD4(+) T cells and prevents the establishment of a persistent infection in mice.     

In vitro production of immune interferon by spleen cells of mice immunized with herpes simplex virus.

In Vitro production of Immune Interferon (IF) in response to Herpes Simplex Virus (HSV) antigen by sensitized spleen cells from C57B1/6 (B6) mice could be detected as early as 3 and for at least 20 days after ip infection of HSV. Maximal levels of IF were produced after 10 hr of culture, but there was no decay of activity when supernatants were sampled during the subsequent 3 days. The IF produced shared certain known properties of immune IF and was not neutralized by an antiserum against viral-induced (type I) IF. DBA/2 (D2) mice which are considerably more sensitive in vivo to HSV infection than B6 mice produced significantly lower amounts of immune IF in the in vitro test system regardless whether high or low doses of virus were injected. The same pattern of results was observed when resistant B6D2F1 hybrid mice were compared with $\text{A}\text{J}$ and Balb/c mice which are about as sensible to ip infection with HSV as DBA/2 mice in our laboratory. These results demonstrate a remarkable defect of in vitro cellular immunity in mice susceptible to a virus infection when compared with resistant mice. Conceivably, a similar defect may be of in vivo relevance.     

Effect of double-stranded RNA and interferon type I on reproduction of cytomegalovirus in human fibroblasts]

Double-stranded RNAs (dsRNA) - larifan and ridostin, and recombinant interferons-alpha-2 and beta-1, manufactured in the USSR, inhibited the reproduction of CMV in cell culture. Antiviral effect depended on concentration of preparations, time of administration and m. o. i. DEAE-dextran enhances antiviral effect dsRNA. DsRNA and interferons added to cells infected with CMV at high m. o. i. enhance the viral reproduction. Among studied preparations highest antiviral effect was shown by natural leucocyte interferon (Egis). It was 10 times more active, than recombinant IFNs and in concentration of 1000 U/ml was comparable to activity of larifan (200 mg/ml).     

Interferonogenic and antiviral activity of the double-stranded replicative form of phage f2 RNA in tissue culture and on animals]

Biological activity of a new natural interferon inductor, the replicative RNA form of phage f2 (RFf2) was studied. A possibility of using RFf2 for production of highly active interferon under conditions of superinduction providing an increase in the interferon yield by to 256--512 times as compared to the control samples was shown. The protective interferonogenic and antiviral effect of RFf2 in mice infected with Semliki forest virus (SFV) and tickborne encephalitis virus (TBEV) was studied on administration of the inductor by various routes. It was found that intraperitoneal administration of RFf2 in a dose of 10 gamma per a mouse protected the infected animals from death. It was accompanied by production of up to 1280 units/ml of interferon in the blood serum of the animals. Maximum protection of the animals from death under conditions of the experiment (80 per cent on infection with SFV and 65 per cent on infection with TBEV) was observed when the preparation was administered twice: 4 hours after the infection. Combined use of RFf2 with chemotherapeutics (rimantadine) increased the protective effect both in the tissue culture and in vivo.     

沙眼衣原体感染诱导小鼠生殖道局部促炎性细胞因子的表达情况

建立小鼠生殖道沙眼衣原体感染模型,观察小鼠生殖道局部促炎性细胞因子的表达。将小鼠生物型沙眼衣原体C. muridarum 1＆#215;104 IFU阴道接种于C57B6背景雌性小鼠,取感染后阴道拭子做沙眼衣原体培养,计算IFU,监测小鼠感染和病原体清除情况;80 d后处死小鼠,检测子宫输卵管病理改变;ELISA检测感染过程中小鼠生殖道促炎性细胞因子IL-1α、IL-6、MIP-2和TNF-α产生情况。小鼠感染在第3至第15天维持较高水平,然后病原体被逐渐清除,整个病程约3~5周;病理检测显示子宫输卵有严重炎症、管腔扩张积水,狭窄等;于感染后第3天检测到局部IL-1α、IL-6、MIP-2分泌,第7天达高峰,然后逐渐下降至正常水平（ IL-6于11 d恢复正常,IL-1α和 MIP-2于15 d恢复正常）。 TNF-α仅在第7天检测到高水平表达。相对于TNF-α和IL-6,IL-1α和MIP-2维持时间较长。成功建立沙眼衣原体感染小鼠生殖道模型,沙眼衣原体急性感染可诱导小鼠生殖道局部分泌IL-1α、IL-6、MIP-2和TNF-α。     

The two-step leukocyte migration inhibition factor (LIF) assay. Its use in evaluation of cellular immune function in patients with immunodeficiency diseases.

Administration of interferon (IF) inducers to mice enhances the uptake of antibody-coated erythrocytes (EA) by peritoneal macrophages. To evaluate the role of induced IF in macrophage activation, serum IF titers and phagocytosis of EA by macrophages were determined in recombinant inbred (RI) mice inoculated with Newcastle disease virus (NDV). RI strains carry either a “high” or a “low” response allele for a gene that controls their IF titers induced by NDV. C × BH and C × BK strains, both high responders for IF induction, were also found to be high responders for enhancement of phagocytosis by NDV. Conversely, strains C × BD, C × BI and C × BJ, low responders for IF induction, were also shown to be low responders for phagocytosis of EA by macrophages. In contrast, phagocytosis of EA by macrophages from high responder C × BH mice and low responder C × BD mice was similarly enhanced by the administration of a lipopolysaccharide. When data from all NDV-inoculated mice were analysed, a significant correlation was obtained between serum IF titers and the percentage of macrophages that ingested four or more EA. The results are compatible with two main possibilities: (i) IF induced by NDV enhances phagocytosis of EA by macrophages; or (ii) a macrophage-activating factor different from IF is released together with IF in response to NDV and the activity of this factor correlates with serum IF titers.     

NK T cell activation promotes Chlamydia trachomatis infection in vivo

We used two approaches to examine the role of NK T cells (NKT) in an intracellular bacterial (Chlamydia trachomatis mouse pneumonitis (C. muridarum)) infection. One is to use CD1 gene knockout (KO) mice, which lack NKT, and the other is to activate NKT using alpha-galactosylceramide (alpha-GalCer), a natural ligand of these cells. The data showed a promoting effect of NKT activation on Chlamydia lung infection. Specifically, CD1 KO mice exhibited significantly lower levels of body weight loss, less severe pathological change and lower chlamydial in vivo growth than wild-type mice. Immunological analysis showed that CD1 KO mice exhibited significantly lower C. muridarum-specific IL-4 and serum IgE Ab responses as well as more pronounced delayed-type hypersensitivity response compared with wild-type controls. In line with the finding in KO mice, the in vivo stimulation of NKT using alpha-GalCer enhanced chlamydial growth in vivo, which were correlated with reduced delayed-type hypersensitivity response and increased C. muridarum-driven IL-4/IgE production. Moreover, neutralization of IL-4 activity in the alpha-GalCer-treated BALB/c mice significantly reduced the promoting effect of alpha-GalCer treatment on chlamydial growth in vivo. These data provide in vivo evidence for the involvement of NKT in a bacterial pathogenesis and its role in promoting Th2 responses during infection.     

Chlamydia trachomatis infection of human fallopian tube organ cultures

The pathogenic events that precede Chlamydia trachomatis salpingitis in the human fallopian tube have not been fully described. We used a model of human fallopian tubes in organ culture (HFTOC) infected with strain E/UW-5/CX of C. trachomatis to study these events. The model supported sustained C. trachomatis infection as demonstrated by recovery of viable C. trachomatis from medium and tissue over 5-7 d. However, the level of infectivity was low. Maximal infection occurred at 72 h after initial inoculation. In contrast to gonococcal infection of the HFTOC, C. trachomatis did not damage overall ciliary function of HFTOC. However, a local direct cytotoxic effect characterized by loss of microvilli and disruption of cell junctions was noted when multiple chlamydial elementary bodies attached to mucosal cells. Beginning at 24 h, and continuing throughout the course of C. trachomatis infection of HFTOC, ruptured epithelial cells releasing elementary bodies were noted. Chlamydial inclusions were seen in the mucosa by 72 h in approximately 6% of both ciliated and nonciliated epithelial cells. Mucosal inclusions contained all forms of the C. trachomatis developmental cycle. These data suggest that factors present in the human fallopian tube may limit susceptibility to chlamydial infection but support the use of the HFTOC model in the study of the pathogenesis of C. trachomatis salpingitis.     

Immunology of Chlamydia infection: implications for a Chlamydia trachomatis vaccine

Sexually transmitted Chlamydia trachomatis infections are a serious public-health problem. With more than 90 million new cases occurring annually, C. trachomatis is the most common cause of bacterial sexually transmitted disease worldwide. Recent progress in elucidating the immunobiology of Chlamydia muridarum infection of mice has helped to guide the interpretation of immunological findings in studies of human C. trachomatis infection and has led to the development of a common model of immunity. In this review, we describe our current understanding of the immune response to infection with Chlamydia spp. and how this information is improving the prospects for development of a vaccine against infection with C. trachomatis.     

Polymerase chain reaction for detection of endocervical Chlamydia trachomatis infection in women attending a gynecology outpatient department in India

OBJECTIVES: To detect Chlamydia trachomatis infection by polymerase chain reaction (PCR) in symptomatic women attending a gynecology clinic in a city hospital and in randomly selected slum dwellers. STUDY DESIGN: Endocervical specimens were collected from 350 women with genitourinary complaints (group I) and 53 slum dwellers (group II). Samples were analyzed by PCR, direct fluorescence assay (DFA) and Giemsa stain cytology for detection of C trachomatis and compared for their sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). RESULTS: The prevalence of endocervical C trachomatis infection was 43.1% and 24.5% in groups I and II, respectively. The sensitivity, specificity, PPV and NPV of PCR were 80.0%, 75.0%, 66.6% and 85.7%, respectively, when DFA was considered true positive. The percent increment in detection of C trachomatis by PCR was 15.3%. CONCLUSION: Giemsa stain cytology has low sensitivity and specificity; hence, it cannot be recommended for use as a diagnostic technique. It appears that PCR can be used routinely in Chlamydia diagnosis and in screening selected populations. The high positivity of C trachomatis infection in urban slum dwellers is cause for concern.