Ruhangiz T. Kilani

Infection of Placental Trophoblasts by Toxoplasma gondii

How the intracellular parasite Toxoplasma gondii causes placental inflammation and infects the fetus is unknown. By use of a culture model of primary human trophoblasts, we examined the consequences of infection by a virulent strain of T. gondii. Infection fractions (parasitophorous vacuoles per trophoblast nuclei) < or =0.9 were observed 1 day after challenge at an inoculum ratio of T. gondii to nuclei of 10. The culture content of infectious T. gondii increased 45-fold in 48 h. Two days after infection, almost 30% of trophoblast nuclei became apoptotic, and 30%-35% of nuclei were lost. Almost 90% of apoptotic nuclei were not adjacent to a parasitophorous vacuole, suggesting infection protected against apoptosis. However, there was no T. gondii-dependent accumulation of putative cytotoxic factors, such as tumor necrosis factor-alpha, that could mediate paracrine killing. Both mature and immature trophoblasts can be productively infected, and uninfected, but not infected, cells undergo apoptosis.

Immune cell proliferation is suppressed by the interferon‐γ‐induced indoleamine 2,3‐dioxygenase expression of fibroblasts populated in collagen gel (FPCG)

Indoleamine 2,3‐dioxygenase (IDO), a tryptophan‐catabolizing enzyme, is an intracellular enzyme possessing various immunosuppressive properties. Here, we report the possible use of this enzyme to suppress proliferation of immune cells cocultured with IDO‐expressing fibroblasts of an allogenic skin substitute. Fetal skin fibroblasts embedded within bovine collagen were treated with cytokine interferon‐γ (IFN‐γ) to induce expression of IDO mRNA and protein. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by measurement of kynurenine and tryptophan levels in the IFN‐γ untreated and treated fibroblasts. The results of Northern analysis showed a dose‐dependent increase in expression of IDO mRNA in response to various concentrations of IFN‐γ used. The levels of kynurenine and tryptophan measured, as the bioactivity of IDO, were significantly different in the IFN‐γ treated fibroblasts, compared to those of controls (Pu2009<u20090.001). In a lasting effect experiment, the expression of IDO mRNA was gradually reduced to an undetectable level within 32 h of IFN‐γ removal. The results of Western blot analysis, however, revealed a significantly longer (192 h) lasting effect of IFN‐γ on IDO protein level, relative to that of mRNA expression. To demonstrate immunosuppressive effects of IDO on proliferation of immune cells, IDO‐expressing fibroblasts were cocultured with peripheral blood mononuclear cells (PBMC) for a period of 5 days. The results of 3H‐thymidine incorporation showed a significant reduction in proliferation of PBMC when cocultured with IDO‐expressing fibroblasts, compared to those cocultured with non‐IDO‐expressing fibroblasts (Pu2009<u20090.001). Furthermore, addition of IDO‐inhibitor (1‐methyl‐d‐tryptophan) reversed the suppressive effects of IDO on PBMC proliferation in a dose‐dependant fashion. To test the viability of immune cells cocultured with IDO‐expressing fibroblasts, FACS analysis of the PI stained PBMC was conducted and no significant difference was found between these cells and the controls. In another set of experiments, we showed that migration rate and subsequent proliferation of IDO‐expressing fibroblasts are also the same as those of control cells. In conclusion, IDO‐expressing allogenic fibroblasts embedded within collagen gel suppress the proliferation of allogenic immune cells, while they still remain viable in this IDO‐induced tryptophan‐deficient culture environment. J. Cell. Biochem. 90: 206–217, 2003.

Characterization of an immunodominant Giardia lamblia protein antigen related to alpha giardin

The trophozoites ofGiardia lamblia possess several protein antigens, predominant among them a protein of ∼32,000 Da. In the present study, we used monospecific antibodies that recognize this protein to demonstrate its presence on a variety ofG. lamblia isolates from human and animal sources. Immune electron microscopy was used to localize 32-kDa antigen on the trophozoite membrane and disk. Immunofluorescent assays employing monospecific antibodies confirmed the presence of 32-kDa antigen on the membrane and disk and its absence on flagella or nuclei. The N-terminal 17 amino acids of the 32-kDa antigen are identical to α-1-giardin, a protein component of microribbons on the ventral disk. These results suggest that the 32-kDa immunodominant trophozoite antigen is α-1-giardin.

Temperature-sensitive polymer-conjugated IFN-γ induces the expression of IDO mRNA and activity by fibroblasts populated in collagen gel (FPCG)

Indoleamine 2,3‐dioxygenase (IDO) is an intracellular tryptophan‐catabolizing enzyme possessing various immunosuppressive properties. Here, we report the use of this enzyme to suppress the proliferation of peripheral blood mononuclear cells (PBMC) co‐cultured with IDO‐expressing fibroblasts of an allogeneic skin substitute in vitro. Fetal foreskin fibroblasts populated within collagen gel (FPCG) were treated with interferon‐gamma (IFN‐γ) conjugated with a temperature‐sensitive polymer to induce the expression of IDO mRNA and protein. SDS–PAGE showed successful conjugation of IFN‐γ with the temperature‐sensitive polymer. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by the measurement of kynurenine levels. The results of Northern blot analysis showed an induction of IDO mRNA expression when treated with polymer‐conjugated IFN‐γ. Kynurenine levels, as a measure of IDO bioactivity, were significantly higher in IFN‐γ‐treated fibroblasts than in controls (Pu2009<u20090.001). In a lasting effect experiment, the expression of IDO mRNA in FPCG treated with polymer‐conjugated IFN‐γ was significantly longer than in those treated with free (non‐conjugated) IFN‐γ (Pu2009<u20090.001). IFN‐γ radiolabeling showed a prolonged retention of IFN‐γ within collagen gel in its polymer‐conjugated form, compared to its free form. Presence of IDO protein in FPCG was demonstrated by Western analysis even 16 days after removal of the conditioned medium (containing released IFN‐γ). To demonstrate the immunosuppressive effects of IDO on the proliferation of PBMC, IDO‐expressing FPCG treated with polymer‐conjugated IFN‐γ were co‐cultured with PBMC for a period of 5 days. The results showed a significant reduction in proliferation of PBMC co‐cultured with IFN‐γ‐treated IDO‐expressing fibroblasts, compared to those co‐cultured with non‐IDO‐expressing fibroblasts (Pu2009<u20090.001). The addition of an IDO inhibitor (1‐methyl‐D‐tryptophan) reversed the suppressive effects of IDO on PBMC proliferation. In conclusion, IDO expression in FPCG suppresses the proliferation of immune cells in vitro. The use of a temperature‐sensitive polymer further prolongs the effect of IFN‐γ on the expression of IDO. Therefore, modulating IDO levels in situ might be an alternative for prolonging the survival of skin allografts. J. Cell. Physiol. 201: 146–154, 2004.

Liposome associated interferon-alpha-2b functions as an anti-fibrogenic factor in dermal wounds in the guinea pig

We have previously reported that interferon-alpha-2b (IFN-α-2b) can be encapsulated in liposomes without compromising its anti-fibrogenic effects on dermal fibroblasts in vitro. This study was conducted to determine whether this preparation applied topically to guinea pig wounds can affect their healing. The rationale for this approach is that systemic administration of IFN-α-2b by injection for treatment of dermal fibrosis is uncomfortable, requires a large quantity of the cytokine and cannot be easily used in children. Liposomes are potentially useful vehicles for the topical delivery of drugs. Empty sonicated liposome vesicles were mixed with various concentrations of IFN-α-2b and then dried and rehydrated. An enzyme-linked immunosorbent assay (ELISA) was used to determine the efficiency of encapsulation and the stability of the preparation under experimental conditions. A total of 36 full thickness skin wounds (6/animal, 3 on each side) were made with an 8 mm disposable punch. Each wound on the right side received cream (100 mg/wound) containing 3000 units of liposome-encapsulated IFN-α-2b, while wounds on the left side received cream containing empty liposomes. There was a significant reduction in rate of contraction of wounds treated with IFN-α-2b as early as 5 days after wounding. This reduction remained significant up to 10 days. Northern analysis, used to evaluate the expression of mRNAs for type I and type III collagens in response to IFN-α-2b showed a marked reduction in abundance of the transcripts for the pro-α1(I) chain of type 1 collagen on days 11 and 14 after wounding. Similarly, the level of mRNA for type III procollagen was markedly reduced as early as day 7 and remained depressed up to day 14. These findings were consistent with results obtained for the total collagen content in tissue samples. Cellularity of the IFN-α-2b-treated wounds, assessed by vimentin content, was also markedly reduced at day 7 and remained depressed up to day 14. Liposome associated IFN-α-2b applied 5 days after completion of epithelialization reduced mRNA for the pro-α1(I) chain of type 1 collagen., confirming its transepidermal penetration and effectiveness. The activity of liposome-associated IFN-α-2b in vivo supports the concept of the topical use of this anti-fibrogenic agent for treatment of fibroproliferative disorders.

Fluorescent-activated cell-sorting analysis of intracellular interferon-γ and interleukin-4 in fresh and frozen human peripheral blood T-helper cells

T‐helper (Th) cells can be classified into at least three subsets based on their cytokine profiles: Th0, Th1, and Th2. The functional significance of each subset of Th cells can be determined in isolated human peripheral blood mononuclear cells (PBMC). Using two‐ or three‐color cytometric detection of intracellular cytokines. These analyses have been limited by the requirement for fresh cells making sequential samples and longitudinal studies difficult. Cryopreservation of PBMC in liquid nitrogen for up to 1 year was evaluated to determine whether the Th1/Th2 ratio remained unchanged in cryopreserved lymphocytes. Aliquots of human PBMC from normal volunteers analyzed for activation using phorbol myristate acetate and evaluated using morphology showed that the surface marker expression was unchanged in fresh and frozen cells. Cytokine expression was measured using intracellular cytokine staining and three‐color flow cytometric analysis. The percentages of cells producing interferon (IFN)‐γ or interleukin (IL)‐4 were determined after 16u2003hours of phorbol myristate acetate and ionomycin stimulation in the presence of brefeldin A. No significant difference was found in cytokine production between fresh and frozen cells. The percentage of IFN‐γ and IL‐4 producing CD3‐positive fresh T cells was 19.2u2003±u20035.8 percent and 0.9u2003±u20030.4 percent vs. 17.6u2003±u20030.75 percent and 0.9u2003±u20030.3 percent, respectively, for frozen PBMC. The effects of thermal injury on the Th1/Th2 cytokine ratio and the development of hypertrophic scarring were then determined. Twelve burn patients examined 4 weeks postburn showed a significant shift in the Th1/Th2 ratio, compared with 13 normal human volunteers used as controls. IL‐4 levels in the patient group were significantly higher than controls at 1 month postburn (12.7u2003±u20032.6 percent vs. 3.9u2003±u20030.5 percent, pu2003<u20030.01) and IFN‐γ levels were significantly lower (9.3u2003±u20031.7 percent vs. 15.3u2003±u20032.3 percent, pu2003<u20030.05). Thus, PBMC can be cryopreserved for up to 1 year, enabling investigation of chronologic changes in Th1/Th2 profiles. It is suggested that a “locked on” Th2 profile may contribute to the development of hypertrophic scarring after burn injury.