AAV2/8-humanFOXP3 gene therapy shows robust anti-atherosclerosis efficacy in LDLR-KO mice on high cholesterol diet.

Inflammation is a key etiologic component in atherogenesis. Previously we demonstrated that adeno-associated virus (AAV) 2/8 gene delivery of Netrin1 inhibited atherosclerosis in the low density lipoprotein receptor knockout mice on high-cholesterol diet (LDLR-KO/HCD). One important finding from this study was that FOXP3 was strongly up-regulated in these Netrin1-treated animals, as FOXP3 is an anti-inflammatory gene, being the master transcription factor of regulatory T cells. These results suggested that the FOXP3 gene might potentially be used, itself, as an agent to limit atherosclerosis. To test this hypothesis AAV2/8 (AAV)/hFOXP3 or AAV/Neo (control) gene therapy virus were tail vein injected into the LDLR-KO/HCD animal model. It was found that hFOXP3 gene delivery was associated with significantly lower HCD-induced atherogenesis, as measured by larger aortic lumen cross sectional area, thinner aortic wall thickness, and lower aortic systolic blood velocity compared with Neo gene-HCD-treated controls. Moreover these measurements taken from the hFOXP3/HCD-treated animals very closely matched those measurements taken from the normal diet (ND) control animals. These data strongly suggest that AAV/hFOXP3 delivery gave a robust anti-atherosclerosis therapeutic effect and further suggest that FOXP3 be examined more stringently as a therapeutic gene for clinical use.

UC blood infection with clinical strains of Mycobacterium tuberculosis: a novel model.

BACKGROUND: Use of unrelated cord blood transplantation (UCBT) is increasing, yet high rates of mortality secondary to infection remain a problem. We investigated the utility of using umbilical cord blood (UCB) as a model to study a naive cell population challenged by Mycobacterium tuberculosis. METHODS: Mononuclear cells were isolated from nine UCB samples and infected with each of four distinct strains of M. tuberculosis. The isolates used were two highly transmissible clinical strains, the virulent laboratory strain H37Rv and a unique strain isolated from only one case (i.e. non-virulent). CFU were assessed at 3 h post-infection (day 0) and at day 7 to generate growth curves. Viability of the mononuclear cells was assessed prior to infection, 3 h post-infection and at days 3, 5 and 7 post-infection. IFN-gamma and TNF-alpha levels were determined at 24 h post-infection. RESULTS: All three of the virulent strains demonstrated rapid growth in UCB cells that was significantly faster than the growth rate observed for the non-virulent unique isolate. There was no significant decrease in UCB cell viability after the 7-day incubation period regardless of infecting isolate. UCB cells secreted IFN-gamma in response to infection, with no significant difference related to infection with different isolates. However, there was a significant increase in the amount of TNF-alpha elicited following infection with the non-virulent isolate compared with the virulent isolates. DISCUSSION: These results show that UCB can be used as a model to study infection, hopefully leading to new therapies for neonates and UCBT recipients.

Activated THP-1 cells: an attractive model for the assessment of intracellular growth rates of Mycobacterium tuberculosis isolates.

Capacity of certain Mycobacterium tuberculosis isolates to grow more rapidly in human macrophages may be indicative of increased virulence. Significant differences were observed in intracellular growth of two isolates from sites of tuberculosis transmission, with an outbreak-associated strain growing faster than a strain causing disease in only one person. Activated THP-1 cells are a suitable alternative to peripheral blood monocyte models.

Immunization with recombinant Pneumocystis carinii p55 antigen provides partial protection against infection: characterization of epitope recognition associated with immunization.

Many therapeutic options exist for the treatment of Pneumocystis carinii pneumonia, a common fungal opportunistic pulmonary pathogen, but treatment is often complicated by side effects and toxicity and, more recently, markers of drug resistance have been described. The development of immunotherapetic modalities such as active immunization or passive immunotherapy may play an increasing important role in the prevention and treatment of infection. Passive immunotherapy with polyclonal anti-P. carinii reagents, such as serum or T cells, and monospecific reagents reactive with the major surface glycoprotein (MSG or gpA), such as monoclonal antibodies or MSG primed T cells, reduce the severity or eradicate infection. Active immunization with whole P. carinii, P. carinii extracts or MSG has afforded partial protection against the subsequent development of P. carinii pneumonia in some animal models. Identification of additional antigens with protective benefits will aid in the development of vaccines or other reagents. The p55 antigen of rat-derived P. carinii is well recognized by animals following natural exposure to the organism. This 414 amino acid residue antigen found within the cell wall of P. carinii contains 7 repeats of a glutamic acid-rich motif in the carboxyl portion of the molecule. Both humoral and cellular immune responses reactive with this repeated domain are present following natural infection while, the amino terminal portion of the molecule is immunologically silent. In this study, immunization with recombinant p55 elicited significant humoral and cellular immune responses which persisted during 10 weeks of immunosupression in corticosteroid treated rats; rp55 immunization resulted in a significant reduction in organism burden, improved histological score, lower lung weight to body weight ratio (a marker of infection or lung inflammation) and improved survival (P < 0.01). Greater protection was afforded by immunization with a peptide containing amino acid residues 1-200, than by the entire rp55 molecule. Epitope recognition by serum from animals immunized with rp55 differed from that of naturally exposed animals with oligoclonal responses to residues 22-92 and residues 196-218. This study demonstrates that protection against P. carinii can be afforded by immunization with antigen preparations other than whole extracts of P. carinii or the major surface antigen, MSG. This antigen moiety will likely be most useful as a vaccine candidate in combination with other immunogens which provide similar partial protection.

Immunization with the major surface glycoprotein of Pneumocystis carinii elicits a protective response.

Pneumocystis carinii, a leading opportunistic pulmonary pathogen, contains a major surface glycoprotein (MSG) which plays a central role in its interaction with the host. Naive Lewis rats were immunized with varying concentrations of purified native MSG and a recombinant form of the protein (MSG-B), placed in a conventional rat colony with exposure to P. carinii, and immunosuppressed with corticosteroids for 10 weeks to induce the development of pneumocystosis. Immunization elicited humoral and cellular immune responses to MSG which persisted throughout the experiment. Compared with animals immunized with ovalbumin or adjuvant alone, the MSG-immunized rats had improved survival (29 vs 66%, p < 0.001), lowered organism burden (log10 9.03 +/- 0.33/lung vs 7.51 +/- 0.38/lung, p < 0.001), less alveolar involvement as assessed by lung histologic score (3.54 +/- 0.42 vs 2.50 +/- 0.42, p < 0.01) and lung weight:body weight ratio (18.2 +/- 1.4 vs 14.6 +/- 1.7, p < 0.01). Animals immunized with MSG-B also showed a significantly lower organism burden, lung histologic score and lung weight:body weight ratio than control rats. Thus, MSG is the first P. carinii antigen which can elicit a protective response in the immunosuppressed rat model of pneumocystosis and this finding supports the rationale of developing a P. carinii vaccine.

Cell wall antigens of Pneumocystis carinii trophozoites and cysts: purification and carbohydrate analysis of these glycoproteins.

We have purified and biochemically analyzed individual cell wall glycoproteins of Pneumocystis carinii. Our results show that corresponding core glycoproteins constitute the cell wall antigens in both trophozoites and cysts, and glycosylation of these glycoproteins does not appear to be significantly altered during development. Cysts and trophozoites in rat-derived organism preparations were separated from each other by counterflow centrifugal elutriation, then treated with Zymolyase to obtain the cell wall fractions. Gel electrophoresis patterns of these fractions from both life-cycle stages were qualitatively similar. Ten major antigenic glycoproteins in these fractions were purified by preparative continuous elution gel electrophoresis. All ten glycoproteins from cysts and trophozoites contained mannose, glucose, galactose, and N-acetylglucosamine, and some contained traces of fucose. The glycoproteins of cysts had more mannose than their trophozoite counterparts. The trophozoite glycoproteins differed from those of the cyst by the presence of xylose. To examine the species-specificity of glycoprotein glycosylation, preparations of human-derived P. carinii (comprised of mixed life-cycle stages) were also examined and found to contain the same sugars as those found in rat-derived organisms. Most of the purified rat-derived glycoproteins bound Concanvalin A, which was abolished by treatment with N-glycanase. This suggested that the majority of the oligosaccharides were N-linked to the proteins, but attempts to identify carbohydrate linkage sites by amino acid sequencing were hampered by apparent modifications of residues. The peptides derived by cyanogen bromide cleavage revealed distinct size patterns for each glycoprotein, suggesting that they were distinct proteins. Most of the glycoproteins reacted with monoclonal antibodies which recognize a highly conserved epitope on rat P. carinii. Four of the individually purified glycoprotein preparations elicited in vitro cellular immune responses, implicating their involvement in the recognition of P. carinii by host T cells. The identification and characterization of P. carinii cell wall proteins will be helpful in analyzing the relationship of the organism to its mammalian host.

Treponema pallidum, lipoproteins, and synthetic lipoprotein analogues induce human immunodeficiency virus type 1 gene expression in monocytes via NF-kappaB activation.

Syphilitic genital ulcers are cofactors for the bidirectional transmission of human immunodeficiency virus (HIV). U937 human promonocytic cells chronically infected with HIV-1 (U1 cells) or transiently transfected with wild type or mutant HIV long terminal repeat (LTR) reporter constructs were used to examine mechanisms that likely underlie Treponema pallidum-induced immune cell activation and consequent induction of HIV. Virulent T. pallidum, a representative native treponemal lipoprotein (NTp47), or synthetic lipoprotein analogues (lipopeptides) all induced HIV replication in U1 cells. These stimuli also induced HIV gene expression from a wild type HIV LTR. HIV gene expression correlated with the translocation of NF-kappaB, and mutations within the NF-kappaB binding sites of the HIV LTR abrogated HIV gene expression. This study implicates treponemal lipoproteins as key mediators of immune cell activation and provides insights into the cellular and molecular bases for enhanced HIV transmission in syphilitic persons.

Proliferative and cytokine responses of human T lymphocytes isolated from human immunodeficiency virus-infected patients to the major surface glycoprotein of Pneumocystis carinii.

The current study examined the proliferative capacity and cytokine secretion pattern of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus type 1 (HIV-1)-infected patients in response to the major surface glycoprotein (MSG) of Pneumocystis carinii. PBMC from AIDS patients with <200 CD4 cells/mL had significantly less proliferative responses to MSG than did healthy controls. Cytokine analysis indicated that interferon-gamma secreted in response to MSG was also significantly less. There was no significant difference in interleukin-4 levels following incubation with MSG between any of the groups; however, all the HIV-infected persons had slightly elevated levels. When the CDC class C3 patients who had a previous episode of P. carinii pneumonia were compared with those who had not had a previous episode, there was a significant increase in the proliferative response to MSG and in interleukin-4 secretion. CDC class C3 patients who had a previous episode of P. carinii pneumonia showed a predominately Th2 response to MSG.

Cytokine responses to the native and recombinant forms of the major surface glycoprotein of Pneumocystis carinii.

Pneumocystis carinii is a major opportunistic pathogen and leading cause of morbidity in patients with AIDS. The major surface glycoprotein (MSG) of P. carinii, represented by a family of related proteins encoded by unique genes, is highly immunogenic and contains T cell-protective epitopes. We undertook the present study to define the CD4 T helper (Th) response by cytokine secretion to native MSG and a recombinant form of the protein, MSG-B. Spleen cells were collected from Lewis rats and restimulated with both native MSG and MSG-B. Within 24 h, the CD4 cells secreted high levels of interferon-gamma (IFN-gamma) in response to both types of antigen, indicative of a Th1 response; however, after 72h of incubation, only the native MSG stimulated secretion of IL-4 (Th2 response) from the cells. We then investigated whether the presence of IL-4 could alter the predominant Th1 phenotype by the CD4 cells in response to MSG and MSG-B. Cells cultured with native MSG and IL-4 produced low levels of IFN-gamma and elevated levels of IL-4. Interestingly, cells incubated with MSG-B and IL-4 reduced production of IFN-gamma, but were not stimulated to produce increased levels of IL-4. The presence of anti-IFN-gamma antibody in the MSG- or MSG-B-stimulated cultures did not effect the expression of IFN-gamma mRNA, suggesting that the generation of Th1 cells in response to MSG or MSG-B was not dependent on IFN-gamma. We conclude that native MSG, which contains multiple forms of this antigen, and recombinant MSG elicit different cytokine responses in vitro. These data are not only important to studies of MSG, but may also be relevant to the role of MSG in the immunopathogenesis of P. carinii infection in vivo.

Biphasic in vivo immune function after low- versus high-dose alcohol consumption.

A series of experiments was performed to assess the alterations in immune status in vivo that are associated with differences in the amount and duration of ethanol intake. Using a nonspecific delayed cutaneous hypersensitivity-like response to the intradermal injection of phytohemagglutinin, the area of induration (skin test response) was significantly enhanced (p = 0.008) after low-dose ethanol (0.5 g/kg) administered daily by gastric gavage for 5 days. High-dose ethanol (6.0 g/kg) significantly diminished this response (p = 0.03). Using an experimental model of Mycobacterium bovis hepatitis, the host immune response was also altered in a biphasic manner after chronic, 28-day ethanol consumption. With this model 0.43 +/- 0.03 g/kg/day (mean +/- SEM) of ethanol (low dose) was associated with a 40% improvement in the removal of the organisms from liver tissue (p = 0.002). High dose (12.1 +/- 0.5 g/kg/day) impaired removal, resulting in a 55% increase in the number of viable organisms (p = 0.001). The levels of three cytokines, MIF, TNF-alpha, and IL-2, known to be involved in the modulation of the host response to mycobacterial infections, were measured in sera after the infection. The serum levels of these cytokines in response to infection did not correlate with this biphasic response to different alcohol dose levels.

Characterization of rat CD4 T cell clones specific for the major surface glycoprotein of Pneumocystis carinii.

Pneumocystis carinii are coated by abundant heterogenous major surface glycoproteins (MSGs), which facilitate interaction with the host. We have produced MSG-specific T-cell clones from the spleens of P. carinii-exposed Lewis rats and analyzed five for antigen specificity to native MSG and a recombinant form of MSG, cell surface markers, and cytokine profiles. All five of the clones were CD4+. All of the clones proliferated specifically to both the native MSG and the recombinant MSG only in the presence of antigen presenting cells demonstrating that the response is antigen/driven rather than mitogen/driven. All five of the clones secreted IL-2 and IFN-gamma, although in differing amounts, implicating a Th1 response. Only one of the clones produced any detectable IL-4. This is the first report of T cell clones responsive to a specific antigen of P. carinii, MSG. We conclude that the T cell clones will be helpful in mapping protective epitopes present in MSG and in functional studies of MSG.

Adoptive transfer of lymphocytes sensitized to the major surface glycoprotein of Pneumocystis carinii confers protection in the rat.

Pneumocystis carinii is a major opportunistic pathogen and a leading cause of morbidity in patients with AIDS. CD4+ cells have been shown to be important in host defenses against P. carinii, but the antigen(s) involved with this response have not been identified. We undertook the present study to determine whether the major surface glycoprotein (MSG) of P. carinii contains epitopes that can elicit a protective cellular immune response. Spleen cells and purified CD4+ cells isolated from Lewis rats, pulsed 1-4 d with MSG, and injected into corticosteroid-treated Lewis rats with pneumocystosis resulted in significant reduction in the P. carinii burden, as judged by organism quantitation and lung histology. The protective response demonstrated by the donor cells was dependent on previous exposure to P. carinii, cell concentration, and time of incubation with MSG. In addition, reconstitution with MSG-specific CD4+ cells resulted in an early hyperinflammatory response within the lungs of these animals with a high percentage of mortality. Thus, in this model, MSG can elicit an immune response mediated by CD4+ cells, which has a harmful as well as helpful effect on the host, and these responses occur despite the presence of corticosteroids.

The translation efficiency of ovalbumin mRNA is determined in part by a 5'-end hairpin structure.

We have investigated the role in translation of the secondary structure in the noncoding leader (NCL) sequence of ovalbumin mRNA. Deletion of a stem-loop structure from the mRNA 5'-end (hairpin-1) produced a 2.5-fold decrease in mRNA translation rate in both rabbit reticulocyte wheat germ cell-free systems. A corresponding 2-fold reduction in mRNA binding affinity for reticulocyte eucaryotic initiation factor 2 (eIF-2) was also observed. These effects were independent of mRNA capping. Both translation rate and eIF-2 binding affinity were restored by addition to the mRNA of a sequence containing a hairpin-1-like structure. The positive correlation between cell free translation rate and mRNA binding to eIF-2 suggests that hairpin-1 is both an initiation signal and part of a specific eIF-2 recognition site. Methylation interference indicated a direct interaction between eIF-2 and hairpin-1 of ovalbumin mRNA.

Cellular responses to a 55-kilodalton recombinant Pneumocystis carinii antigen.

The host-parasite interaction in Pneumocystis carinii pneumonia is poorly understood. In recent years, two major groups of P. carinii antigens have been identified. One class of antigens is characterized by a broad band of immunoreactivity between 45 and 55 kDa in P. carinii derived from rats. This antigen complex is the P. carinii antigen most commonly found in respiratory tract specimens and most frequently recognized by the host immune response. The availability of a recombinant antigen has permitted studies focusing on the cellular and humoral responses to a single antigen within this class, p55. In this study, we have demonstrated that the p55 antigen elicits a cell-mediated immune response in animals previously exposed to P. carinii. Under conditions of natural exposure, the 5' portion of the molecule, p55(1-200), appears immunologically silent, failing to elicit lymphocyte proliferation or cytokine secretion. Following active immunization, the 5' portion is capable of stimulating lymphocyte proliferation. The 3' portion, p55(268-414), has at least one immunodominant region which contains a 7-amino-acid repeat motif. The cells responding to p55 include a CD4+ T cell which secretes a Th1 cytokine pattern. A detailed understanding of the host-parasite interaction will facilitate the development of immunoprophylaxis and immunotherapy for P. carinii infection.

Selective myelotoxicity of propanil.

Propanil, a commonly used herbicide, has been previously shown to be immunotoxic for selected immune functions as well as specific cell types, such as the macrophage. Propanil has also been shown to cause a methemoglobulinemia and anemia through direct action on the erythrocyte. Demonstrated toxicity to both macrophages and erythrocytes raised concern for the possible myelotoxicity of propanil which could contribute to the observed effects of exposure. Therefore, the effect of propanil on several stem and progenitor cell types was assessed 7 days after acute propanil exposure. The results described herein show that propanil, at doses of 50-200 mg/kg body wt, resulted in reduction in the number of myeloid stem cells and early myeloid and erythroid progenitor cells. No reduction in the numbers of more differentiated myeloid and erythroid progenitor cells was noted at even the highest dose used (200 mg/kg). In addition, no statistically significant difference in number of leukocytes per femur was noted. These data suggest that propanil is myelotoxic to early hemapoietic stem cells, but that this reduction is apparently compensated by proliferation of more differentiated progenitor cells for the myeloid and erythroid lineages. It remains unknown whether chronic exposure leads to progressive depletion of additional myeloid and erythroid cells.

Cellular immune response in Pneumocystis carinii infection.

Pneumocystis carinii is an important pulmonary pathogen causing disease in immunocompromised individuals. The majority o f conditions predisposing to Pneumocystis pneumonia are associated with profound defects in cellular immunity. Although our understanding o f the host response to the organism is still limited, advances in antigen preparation and the availability o f animal models have permitted an improved understanding of some aspects o f the cell-mediated immune response to Pneumocystis. In this review, George Smulian and Sue Theus will highlight recent advances in our knowledge regarding the role of macrophages, T cells and cytokines in the response to the organism.