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Resistance to HER2-targeted therapy represents a significant challenge for the successful treatment of patients with breast cancer with HER2-positive tumors. Through a global mass spectrometry–based proteomics approach, we discovered that the expression of the N6-methyladenosine (m6A) demethylase ALKBH5 was significantly upregulated in HER2-targeted therapy-resistant breast cancer cells. Elevated expression of ALKBH5 was sufficient to confer resistance to HER2-targeted therapy, and specific knockdown of ALKBH5 rescued the efficacy of trastuzumab and lapatinib in resistant breast cancer cells. Mechanistically, ALKBH5 promoted m6A demethylation of GLUT4 mRNA and increased GLUT4 mRNA stability in a YTHDF2-dependent manner, resulting in enhanced glycolysis in resistant breast cancer cells. In breast cancer tissues obtained from patients with poor response to HER2-targeted therapy, increased expression of ALKBH5 or GLUT4 was observed and was significantly associated with poor prognosis in the patients. Moreover, suppression of GLUT4 via genetic knockdown or pharmacologic targeting with a specific inhibitor profoundly restored the response of resistant breast cancer cells to trastuzumab and lapatinib, both in vitro and in vivo. In conclusion, ALKBH5-mediated m6A demethylation of GLUT4 mRNA promotes resistance to HER2-targeted therapy, and targeting the ALKBH5/GLUT4 axis has therapeutic potential for treating patients with breast cancer refractory to HER2-targeted therapies. Significance: GLUT4 upregulation by ALKBH5-mediated m6A demethylation induces glycolysis and resistance to HER2-targeted therapy and represents a potential therapeutic target for treating HER2-positive breast cancer.

Resistance to HER2-targeted therapy represents a significant challenge for the successful treatment of patients with breast cancer with HER2-positive tumors. Through a global mass spectrometry–based proteomics approach, we discovered that the expression of the N6-methyladenosine (m6A) demethylase ALKBH5 was significantly upregulated in HER2-targeted therapy-resistant breast cancer cells. Elevated expression of ALKBH5 was sufficient to confer resistance to HER2-targeted therapy, and specific knockdown of ALKBH5 rescued the efficacy of trastuzumab and lapatinib in resistant breast cancer cells. Mechanistically, ALKBH5 promoted m6A demethylation of GLUT4 mRNA and increased GLUT4 mRNA stability in a YTHDF2-dependent manner, resulting in enhanced glycolysis in resistant breast cancer cells. In breast cancer tissues obtained from patients with poor response to HER2-targeted therapy, increased expression of ALKBH5 or GLUT4 was observed and was significantly associated with poor prognosis in the patients. Moreover, suppression of GLUT4 via genetic knockdown or pharmacologic targeting with a specific inhibitor profoundly restored the response of resistant breast cancer cells to trastuzumab and lapatinib, both in vitro and in vivo. In conclusion, ALKBH5-mediated m6A demethylation of GLUT4 mRNA promotes resistance to HER2-targeted therapy, and targeting the ALKBH5/GLUT4 axis has therapeutic potential for treating patients with breast cancer refractory to HER2-targeted therapies.Significance:GLUT4 upregulation by ALKBH5-mediated m6A demethylation induces glycolysis and resistance to HER2-targeted therapy and represents a potential therapeutic target for treating HER2-positive breast cancer.

Supplementary Data from ALKBH5-Mediated m6A Demethylation of GLUT4 mRNA Promotes Glycolysis and Resistance to HER2-Targeted Therapy in Breast Cancer

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows promising antitumor activity in preclinical studies. However, the efficacy of recombinant TRAIL in clinical trials is compromised by its short serum half-life and low in vivo stability. Induction of endogenous TRAIL may overcome the limitations and become a new strategy for cancer treatment. Here, we discovered that metformin increased TRAIL expression and induced apoptosis in triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) cells. Metformin did not alter the expression of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Metformin-upregulated TRAIL was secreted into conditioned medium (CM) and found to be functional, since the CM promoted TNBC cells undergoing apoptosis, which was abrogated by a recombinant TRAIL-R2-Fc chimera. Moreover, blockade of TRAIL binding to DR4/DR5 or specific knockdown of TRAIL expression significantly attenuated metformin-induced apoptosis. Studies with a tumor xenograft model revealed that metformin not only significantly inhibited tumor growth but also elicited apoptosis and enhanced TRAIL expression in vivo. Collectively, we have demonstrated that upregulation of TRAIL and activation of death receptor signaling are pivotal for metformin-induced apoptosis in TNBC and NSCLC cells. Our studies identify a novel mechanism of action of metformin exhibiting potent antitumor activity via induction of endogenous TRAIL., Graphical abstract, The corresponding author and colleagues show that metformin is capable of increasing the levels of a death ligand TRAIL in some breast and lung cancer cells, thereby activating death-receptor-mediated cell death. They discover a novel mechanism of action of metformin exhibiting antitumor activity via induction of internal TRAIL.

Introduction: Histone deacetylases (HDACs) are dysregulated in human cancers, making them a valuable therapeutic target. The pan-HDAC inhibitor (HDACi) panobinostat is an approved anticancer drug for patients with multiple myeloma. It has been shown that triple negative breast cancer (TNBC), as compared to other breast cancer (BC) subtypes, is more sensitive to panobinostat. Despite the positive data observed in experimental models, to date this HDACi (panobinostat) has not yielded promising results in clinical trials for most solid tumors, including BC. Thus, a better understanding of the molecular mechanisms underlying the action of panobinostat is required to design effective therapeutic strategies against TNBC. Methods: Cell growth MTS assays and LIVE/DEAD cell viability assays were used to determine cell viability. Co-immunoprecipitation (Co-IP) and Western blot were performed to assess the expression, interaction and activation of proteins. Quantitative real-time PCR (qRT-PCR) assays were carried out to measure the expression levels of mRNAs. Lentiviral vectors containing cDNA or specific shRNAs were used to overexpress or knockdown gene expression. Tumor xenograft models via subcutaneous inoculation of MDA-MB-231 cells into nude mice were established to test the in vivo antitumor activity of panobinostat alone or its combination with the EGFR inhibitor gefitinib. Results: Elevated expression of HDAC1/2/3 was observed in BC and significantly associated with poor prognosis in BC patients. In general, all TNBC cells were sensitive to panobinostat-induced growth inhibition and apoptosis. However, in the TNBC cells with HER3-low/undetectable expression, treatment with panobinostat induced upregulation of HER3, and increased the levels of phosphorylated HER3 (p-HER3) and Akt (p-Akt). Specific knockdown of HER3 via shRNAs or inhibition of HER3 by our monoclonal antibody (4A7) sensitized TNBC cells to panobinostat-induced growth inhibition and apoptosis. Further studies revealed an enhanced interaction between HER3 and EGFR due to panobinostat-induced upregulation of HER3. Combinations of panobinostat and the EGFR inhibitor gefitinib exhibited a synergistic effect inhibiting cell growth and promoting apoptosis in TNBC cells. Moreover, in the tumor xenograft models established with MDA-MB-231 cells, panobinostat in combination with gefitinib significantly inhibited tumor growth and induced apoptosis in vivo. Conclusion: While panobinostat exhibits potent anti-proliferative/anti-survival activity in TNBC cells, upregulating HER3 may be an adaptive response compromising the therapeutic potential of panobinostat. Simultaneous targeting of HER3 and its oncogenic dimerization partner, such as EGFR holds promise to combat the compensatory signaling-initiated by HER3. This new therapeutic strategy potentially improves the outcomes of TNBC patients. Citation Format: Hui Lyu, Sanbao Ruan, Congcong Tan, Bolin Liu. Attenuation of HER3-EGFR signaling augments antitumor activity of panobinostat in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3272.

Introduction: HER2-targeted therapies, including the monoclonal antibody (Ab) herceptin and the tyrosine kinase inhibitor (TKI) lapatinib, have been commonly used to treat HER2-positive breast cancer (BC). However, resistance to herceptin and/or lapatinib frequently occurs and currently represents a significant clinical problem. We previously demonstrated that both HER3- and IGF-1 receptor (IGF-1R)-initiated signaling played crucial roles in the development of herceptin resistance. Further studies showed that our herceptin-resistant SKBR3-pool2 and BT474-HR20 sublines, as compared their parental SKBR3 and BT474 cells, respectively, also exhibited refractoriness to lapatinib in vitro. However, the in vivo efficacy of lapatinib against the herceptin-resistant BC models (SKBR3-pool2 and BT474-HR20) remains unclear. Methods: Tumor xenograft models were established via subcutaneous inoculation of SKBR3-pool2 or BT474-HR20 cells into athymic nude mice. Tumor formation was assessed by palpation and measured with fine calipers twice a week. The tumor-bearing mice were treated with vehicle (DMSO) or lapatinib (80 mg/kg) via intraperitoneal injection daily for three weeks. Immunohistochemistry (IHC) assays were performed to assess the expression and activation of proteins. Two individuals independently read and quantified all IHC slides by considering both the intensity of immunostaining and the percentage of positive-staining tumor cells. Results: Treatment with lapatinib profoundly inhibited growth of the tumors established with SKBR3-pool2 cells. In contrast, lapatinib slightly, but not significantly, reduced tumor growth in BT474-HR20 cells-derived xenograft models. IHC analyses of the tumors obtained from both SKBR3-pool2 and BT474-HR20 xenografts showed that lapatinib treatment had little effect on the expression of HER3 and the protein phosphatase PPP3CB. Lapatinib also did not alter the levels of phosphorylated Akt (p-Akt) and FOXO3a (p-FOXO3a) in vivo. Interestingly, in BT474-HR20-derived tumors, lapatinib treatment dramatically enhanced expression of insulin receptor substrate-1 (IRS1) and increased the levels of phosphorylated HER3 (p-HER3). However, such phenomena were not observed in the tumors established with SKBR3-pool2 cells. Conclusion: While the tumors derived from BT474-HR20 cells retain their resistant phenotype to lapatinib, SKBR3-pool2-tumors are highly sensitive to lapatinib treatment. It seemed that lapatinib-induced upregulation of IRS1 and activation of HER3, evidenced by increased p-HER3, contributed to the inefficacy of lapatinib against BT474-HR20-tumors in vivo. Keywords: HER3, IRS1, treatment resistance, HER2-targeted therapy, breast cancer Citation Format: Sanbao Ruan, Hao Liu, Hui Lyu, Congcong Tan, Bolin Liu. Herceptin-resistant breast cancer cells exhibits distinct sensitivity to lapatinib in in vivo tumor xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1790.

Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with high rate of recurrence and refractoriness due to lack of well-defined molecular targets. Human epidermal growth factor receptor 3 (HER3) is differentially expressed in TNBC cells. Numerous studies indicate that elevated expression of HER3, through its dimerization with another receptor, is a major cause of treatment failure in human cancers. To date, there is no FDA-approved HER3-targeted cancer therapy. HDAC inhibitors (HDACis) have been approved for the treatment of refractory or relapsed cutaneous T cell lymphomas and show potency in TNBC cells. However, it remains unclear whether the HDACis may alter HER3 expression to influence the downstream signaling in HER3-overexpressing (HER3high) TNBC. Methods: Colony formation, MTS and LIVE/DEAD cell staining assays were used to detect cell viability. Apoptosis was detected by flow cytometry assays. QRT-PCR, western blots and immunohistochemistry were performed to determine the expression and activation of genes and/or proteins. Co-immunoprecipitation was performed to assess the interaction between proteins. Lentivirus vectors containing cDNA or shRNAs were used to overexpress or knockdown gene expression. Chromatin immunoprecipitation-quantitative PCR and dual luciferase reporter assays were performed to elucidate the regulatory role of gene transcription. Results: Elevated expression of HER3 were observed in approximately half of the TNBC specimens and cell lines tested. HER3 was co-expressed and formed heterodimers with epidermal growth factor receptor (EGFR) to activate the PI-3K/Akt signaling in HER3high-TNBC cells. Panobinostat and romidepsin induced growth inhibition and apoptosis in HER3high-TNBC cells via downregulating HER3 expression and inhibiting PI-3K/Akt signaling. Significantly, HDACis in combination with an EGFR inhibitor (gefitinib) or an Akt inhibitor (Akti1/2) synergistically enhanced the anti-survival effects on HER3high-TNBC cells. Besides, analyses of gene expression profiling datasets revealed a significantly positive correlation between expression of HER3 and transcription factor forkhead box A1 (FOXA1). Further studies discovered that FOXA1 activated HER3 expression through directly binding to HER3 promoter. Moreover, ectopic expression of FOXA1 not only rescued the expression of HER3-downregulated by HDACis, but also attenuated these HDACis-mediated anti-survival effects on HER3high-TNBC cells. Conclusion: HDAC inhibitors exhibits potent inhibitory effects on HER3high-TNBC cells via downregulation of FOXA1-mediated repression of HER3 gene transcription. Our data suggest that epigenetic targeting of FOXA1-HER3/EGFR-PI-3K/Akt signaling axis may be an effective therapeutic strategy for eradication of HER3high-TNBC tumors. Citation Format: Congcong Tan, Hui Lyu, sanbao Ruan, bolin Liu. Histone deacetylase (HDAC) inhibitors exhibit antitumor activity in triple negative breast cancer via suppression of HER3 triggered signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2969.

Pneumocystis pneumonia is a major cause of morbidity and mortality in immunocompromised patients, particularly those infected with HIV. In this study, we evaluated the potential of oral immunization with live Pneumocystis to elicit protection against respiratory infection with Pneumocystis murina. C57BL/6 mice vaccinated with live P. murina using a prime-boost vaccination strategy were protected from a subsequent lung challenge with P. murina at 2, 7, 14, and 28 d postinfection even after CD4+ T cell depletion. Specifically, vaccinated immunocompetent mice had significantly faster clearance than unvaccinated immunocompetent mice and unvaccinated CD4-depleted mice remained persistently infected with P. murina. Vaccination also increased numbers of CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD11b+ macrophages in the lungs following respiratory infection. In addition, levels of lung, serum, and fecal P. murina–specific IgG and IgA were increased in vaccinated animals. Furthermore, administration of serum from vaccinated mice significantly reduced Pneumocystis lung burden in infected animals compared with control serum. We also found that the diversity of the intestinal microbial community was altered by oral immunization with P. murina. To our knowledge, our data demonstrate for the first time that an oral vaccination strategy prevents Pneumocystis infection.

Pneumocystis pneumonia (PCP) is a major cause of morbidity and mortality in patients with HIV infection. CD4 + T lymphocytes are critical for host defense against this infection, but in the absence of CD4 + T lymphocytes, CD8 + T lymphocytes may provide limited host defense. The cytokine interleukin-7 (IL-7) functions to enhance lymphocyte proliferation, survival, and recruitment of immune cells to sites of infection. However, there is little known about the role of IL-7 in PCP or its potential use as an immunotherapeutic agent. We hypothesized that treatment with recombinant human IL-7 (rhIL-7) would augment host defense against Pneumocystis and accelerate pathogen clearance in CD4-depleted mice. Control and CD4-depleted mice were infected with Pneumocystis , and rhIL-7 was administered via intraperitoneal injection. Our studies indicate that endogenous murine IL-7 is part of the normal host response to Pneumocystis murina and that administration of rhIL-7 markedly enhanced clearance of Pneumocystis in CD4-depleted mice. Additionally, we observed increased recruitment of CD8 + T lymphocytes to the lungs and decreased apoptosis of pulmonary CD8 + T lymphocytes in rhIL-7-treated animals compared to those in untreated mice. The antiapoptotic effect of rhIL-7 was associated with increased levels of Bcl-2 protein in T lymphocytes. rhIL-7 immunotherapy in CD4-depleted mice also increased the number of gamma interferon (IFN-γ)-positive CD8 + central memory T lymphocytes in the lungs. We conclude that rhIL-7 has a potent therapeutic effect in the treatment of murine Pneumocystis pneumonia in CD4-depleted mice. This therapeutic effect is mediated through enhanced recruitment of CD8 + T cells and decreased apoptosis of lung T lymphocytes, with a preferential action on central memory CD8 + T lymphocytes.

Alcohol use in persons living with HIV (PLWH) worsens the severity of bacterial pneumonia. However, the exact mechanism(s) by which this occurs remain ill-defined. We hypothesized that alcohol in the setting of HIV infection decreases Streptococcus pneumoniae clearance from the lung through mechanisms mediated by the gut microbiota. Humanized BLT (bone marrow, liver, thymus) mice were infected with 1 × 104 TCID50 of HIV (BAL and JRCSF strains) via intraperitoneal (i.p.) injection. One week post-HIV infection, animals were switched to a Lieber-DeCarli 5% ethanol diet or an isocaloric control diet for 10 days. Alcohol-fed animals were also given two binges of 2 g/kg ethanol on days 5 and 10. Feces were also collected, banked, and the community structures were analyzed. Mice were then infected with 1 × 105 CFU (colony-forming units) of S. pneumoniae and were sacrificed 48 h later. HIV-infected mice had viral loads of ∼2 × 104 copies/mL of blood 1 week post-infection, and exhibited an ∼57% decrease in the number of circulating CD4+ T cells at the time of sacrifice. Fecal microbial community structure was significantly different in each of the feeding groups, as well as with HIV infection. Alcohol-fed mice had a significantly higher burden of S. pneumoniae 48 h post-infection, regardless of HIV status. In follow-up experiments, female C57BL/6 mice were treated with a cocktail of antibiotics daily for 2 weeks and recolonized by gavage with intestinal microbiota from HIV+ ethanol-fed, HIV+ pair-fed, HIV− ethanol-fed, or HIV− pair-fed mice. Recolonized mice were then infected with S. pneumoniae and were sacrificed 48 h later. The intestinal microbiota from alcohol-fed mice (regardless of HIV status) significantly impaired clearance of S. pneumoniae. Collectively, these data indicate that alcohol feeding, as well as alcohol-associated intestinal dysbiosis, compromise pulmonary host defenses against pneumococcal pneumonia. Determining whether HIV infection acts synergistically with alcohol use in impairing pulmonary host defenses will require additional study.

Rationale Pneumocystis pneumonia is a major cause of morbidity and mortality in HIV-infected subjects, cancer patients undergoing chemotherapy and solid organ transplant recipients. No vaccine is currently available. By chemical labeling coupled with proteomic approach, we have identified a putative surface protein (SPD1, Broad Institute gene accession number PNEG_01848) derived from single suspended P. murina cysts. SPD1 was expressed in an insect cell line and tested for vaccine development. Methods Mice were immunized with SPD1 plus adjuvant MF-59 by subcutaneous injection. Three weeks after the last immunization, CD4+ cells were depleted with anti-CD4 antibody GK1.5. The mice were then challenged with 2 × 10 5 Pneumocystis organisms. Mice were sacrificed at 4 and 6 weeks after PC challenge. Spleen/lung cells and serum were harvested. B cells and memory B cells were assessed via flow cytometry. Specific Pneumocystis IgG antibody was measured by ELISA before and after challenge. Infection burden was measured as real-time PCR for P. murina rRNA. Results Normal mice infected with Pneumocystis mounted a serum IgG antibody response to SPD1. Serum from rhesus macaques exposed to Pneumocystis showed a similar serum IgG response to purified SPD1. SPD1 immunization increased B cell and memory B cell absolute cell counts in CD4-depleted Balb/c mice post Pneumocystis challenge in spleen and lung. Immunization with SPD1 significantly increased specific Pneumocystis IgG antibody production before and after challenge. Mice immunized with SPD1 showed significantly decreased P. murina copy number compared with mice that did not receive SPD1 at 6 weeks after challenge. Conclusion Immunization with SPD1 provides protective efficacy against P. murina infection. SPD1 protection against Pneumocystis challenge is associated with enhanced memory B cell production and higher anti– Pneumocystis IgG antibody production. SPD1 is a potential vaccine candidate to prevent or treat pulmonary infection with Pneumocystis .

Background Recent immunological data demonstrated that dendritic cells preferentially recognize advanced glycation end product (AGE)-modified proteins, upregulate expression of the receptor for AGE (RAGE), and consequently bias the immune response toward allergy. Methods Peanut extract was characterized by mass spectrometry (MS) to elucidate the specific residues and specific AGE modifications found in raw and roasted peanuts and on rAra h 1 that was artificially glycated by incubation with glucose or xylose. The binding of the RAGE-V1C1 domain to peanut allergens was assessed by PAGE and Western analysis with anti-Ara h 1, 2, and 3 antibodies. IgE binding to rAra h 1 was also assessed using the same methods. Results AGE modifications were found on Ara h 1 and Ara h 3 in both raw and roasted peanut extract. No AGE modifications were found on Ara h 2. Mass spectrometry and Western blot analysis demonstrated that RAGE binds selectively to Ara h 1 and Ara h 3 derived from peanut extract, whereas the analysis failed to demonstrate Ara h 2 binding to RAGE. rAra h 1 with no AGE modifications did not bind RAGE; however, after AGE modification with xylose, rAra h 1 bound to RAGE. Conclusions AGE modifications to Ara h 1 and Ara h 3 can be found in both raw and roasted peanuts. Receptor for AGE was demonstrated to selectively interact with AGE-modified rAra h 1. If sensitization to peanut allergens occurs in dendritic cells via RAGE interactions, these cells are likely interacting with modified Ara h 1 and Ara h 3, but not Ara h 2.

Little is known about the role of NK cells or their interplay with other immune cells during opportunistic infections. Using our murine model of Pneumocystis pneumonia, we found that loss of NK cells during immunosuppression results in substantial Pneumocystis lung burden. During early infection of C57B/6 CD4+ T cell–depleted mice, there were significantly fewer NK cells in the lung tissue compared with CD4+ T cell–intact animals, and the NK cells present demonstrated decreased upregulation of the activation marker NKp46 and production of the effector cytokine, IFN-γ. Furthermore, coincubation studies revealed a significant increase in fungal killing when NK cells were combined with CD4+ T cells compared with either cell alone, which was coincident with a significant increase in perforin production by NK cells. Finally, however, we found through adoptive transfer that memory CD4+ T cells are required for significant NK cell upregulation of the activation marker NK group 2D and production of IFN-γ, granzyme B, and perforin during Pneumocystis infection. To the best of our knowledge, this study is the first to demonstrate a role for NK cells in immunity to Pneumocystis pneumonia, as well as to establish a functional relationship between CD4+ T cells and NK cells in the host response to an opportunistic fungal pathogen.

To cite this article: Maleki SJ, Teuber SS, Cheng H, Chen D, Comstock SS, Ruan S, Schein CH. Computationally predicted IgE epitopes of walnut allergens contribute to cross-reactivity with peanuts. Allergy 2011; 66: 1522–1529. Abstract Background: Cross-reactivity between peanuts and tree nuts implies that similar immunoglobulin E (IgE) epitopes are present in their proteins. Objective: To determine whether walnut sequences similar to known peanut IgE-binding sequences, according to the property distance (PD) scale implemented in the Structural Database of Allergenic Proteins, react with IgE from sera of patients with allergy to walnut and/or peanut. Methods: Patient sera were characterized by western blotting for IgE binding to nut protein extracts and to peptides from walnut and peanut allergens, similar to known peanut epitopes as defined by low PD values, synthesized on membranes. Competitive enzyme-linked immunosorbent assay (ELISA) was used to show that peanut and predicted walnut epitope sequences compete with purified Ara h 2 for binding to IgE in serum from a cross-reactive patient. Results: Sequences from the vicilin walnut allergen Jug r 2, which had low PD values to epitopes of the peanut allergen Ara h 2, a 2S albumin, bound to IgE in sera from five patients who reacted to either walnut or peanut or both. A walnut epitope recognized by sera from six patients mapped to a surface-exposed region on a model of the N-terminal pro-region of Jug r 2. This predicted walnut epitope competed for IgE binding to Ara h 2 in serum as well as the known IgE epitope from Ara h 2. Conclusions: Sequences with low PD value (

Pulmonary infection with the fungal pathogen Pneumocystis jirovecii was one of the first recognized complications of human immunodeficiency virus (HIV) infection (35). With antimicrobial prophylaxis and the advent of highly active antiretroviral therapy, the incidence of Pneumocystis pneumonia has declined (40). However, infection with Pneumocystis remains a serious clinical problem among HIV-infected persons and is often the first manifestation of otherwise unrecognized HIV infection (32). Furthermore, recent epidemiologic data indicate an increasing prevalence of Pneumocystis isolates with mutations in genes associated with sulfa antibiotic resistance (25, 38). The clinical relevance of these mutations is unclear, but they correlate with an increased need for mechanical ventilation and mortality from Pneumocystis pneumonia (10). The emergence of Pneumocystis strains potentially resistant to commonly used antibiotics highlights the limitations of conventional antibiotic therapy of infection and underscores the need for new modes of therapy. Our laboratory has a long-standing interest in strategies to augment host defense mechanisms naturally operative against Pneumocystis infection. In a murine model of infection with Pneumocystis carinii f. sp. muris (hereafter referred to as Pneumocystis carinii), we have shown that aerosol delivery of the cytokine and gamma interferon (IFN-γ) (2) or pulmonary delivery of the IFN-γ gene in vivo (30) can restore host defenses against this infection, even in immunosuppressed mice depleted of CD4+ T lymphocytes. The therapeutic effect of cytokine therapy with IFN-γ is associated with increased recruitment into lung tissue of inflammatory cells, particularly CD8+ T lymphocytes (30). Production of IFN-γ is critically dependent upon another cytokine, interleukin-12 (IL-12). IL-12 has been shown to stimulate the development of lymphocytes of the Th1 class (34) and to induce the production of IFN-γ from mouse Th1 clones (21) and from natural killer (NK) cells (31). IL-12 synergizes with IL-18 to enhance production of IFN-γ, in part through increased expression of the IL-18 receptor (41). There is little information available regarding the role of IL-12 in host defense against Pneumocystis. The link between IL-12 and IFN-γ also stimulated our interest in IL-12 as a possible therapy for Pneumocystis infection. Treatment with IL-12 has been employed by other investigators to enhance immune responses to a variety of infectious pathogens (7, 17, 23, 24, 26, 28), not including P. carinii. In this paper, we investigate IL-12 as part of the host response to murine Pneumocystis pneumonia and as a potential immunoadjuvant therapy. Specifically, we asked whether release of IL-12 in lung tissue is part of the normal host response to this infection, whether local treatment with IL-12 can accelerate clearance of infection in normal mice, and whether gene transfer of IL-12 can restore clearance of infection in immunosuppressed mice. (Data were presented in part at the International Conference of the American Thoracic Society, Seattle, WA, May 2003.)

We have previously shown that Tc1 CD8+ T cells have in vitro and in vivo effector activity against Pneumocystis (PC) infection in mice. Because these cells have preferential expression of CXCR3, we investigated whether CXCR3 was required for host defense activity against PC. Mice deficient in CXCR3 but CD4+ T cell intact, showed an initial delay but were able to clear the infectious challenge, indicating that CXCR3 signaling is not essential for clearance of PC. CD4-depleted mice had lower levels of monokine induced by IFN-γ, IFN protein-10 (IP-10), and IFN-inducible T cell α-chemoattractant at day 7 of infection and are permissive to PC infection. Overexpression of IP-10 in the lungs by adenoviral gene transfer did not accelerate clearance of infection in control mice but accelerated clearance by day 28 in mice depleted of CD4+ T cells. This effect was associated with increased recruitment of CD8+ T to the lungs with higher CXCR3+ expression levels and enhanced IFN-γ secretion upon in vitro activation compared with control mice. These results indicate that the CXCR3 chemokines are part of the host defense response to PC, and that IP-10 can direct Tc1 CD8+ T cell recruitment to the lungs and contribute to host defense against PC even in the absence of CD4+ T cells.

The relationship between tissue inflammation and clearance of the opportunistic pathogenPneumocystis cariniiis poorly understood. We asked whether the anti-inflammatory cytokine interleukin-10 (IL-10) is released during the host response to infection withP. cariniiand whether local delivery of the IL-10 gene could suppress tissue inflammatory responses without compromising clearance of infection. Control and CD4-depleted mice were inoculated withP. carinii, and at serial intervals after inoculation, lung tissue was assayed for IL-10 by enzyme-linked immunosorbent assay. We found that IL-10 was released in lung tissue in control mice and was present in higher concentrations in CD4-depleted mice with progressive infection. Control and CD4-depleted mice were then pretreated with 109PFU of intratracheally administered adenoviral vector containing the viral IL-10 gene or the luciferase gene followed by inoculation withP. carinii. Pretreatment with viral IL-10 did not alter clearance of infection in control mice or severity of infection in CD4-depleted mice but did decrease tissue inflammation. We then asked whether gene transfer of viral IL-10 could decrease tissue inflammation during immune reconstitution. In these experiments, immunodeficientscidmice were inoculated withP. cariniiand were heavily infected after 4 weeks. When these mice are immunologically reconstituted by intravenous administration of spleen cells from normal mice, a hyperinflammatory reaction developed in lung tissue, associated with high mortality. In comparison to control mice, mice treated with viral IL-10 prior to reconstitution showed significantly decreased lung wet weight, bronchoalveolar lavage fluid (BALF) lactate dehydrogenase, and BALF neutrophils. In contrast, infection intensity, as measured by PCR forP. cariniirRNA, was unchanged between the IL-10 and luciferase groups. Survival was also improved in the IL-10-treated mice. We conclude that release of IL-10 is part of the host response to infection withP. cariniiand that gene therapy with viral IL-10 can lessen excessive tissue inflammation without altering pathogen clearance. In the setting of immune reconstitution andP. cariniipneumonia, pretreatment with the viral IL-10 gene decreases excessive tissue inflammation and improves survival. These results are relevant to acute respiratory failure after initiation of antibiotic treatment for humanP. cariniipneumonia and to immune reconstitution syndromes in human immunodeficiency virus-positive patients started on highly active antiretroviral therapy.

Allergic reactions to food are on the rise worldwide and there is a corresponding increase in interest to understand the molecular mechanisms responsible. Peanut allergies are the most problematic because the reaction often persists into adulthood and can be as severe as anaphylaxis and death. The purpose of the work presented here was to develop a reproducible method to produce large quantities of pure recombinant Ara h 1(rAra h 1) that will enable standardization of immunological tests for patients and allow structural and immunological studies on the wild type and mutagenized forms of the protein. Ara h 1 is initially a pre-pro-protein which, following two endoproteolytic cleavages, becomes the mature form found in peanut. The mature form however has flexible regions that make it refractory to some structural studies including crystallography. Therefore, independent purification of the mature and core regions was desirable. Expression constructs were synthesized cDNA clones for each in a pET plasmid vector without tags. Codons were optimized for expression in E. coli. High-level expression was achieved in BL21 strains. Purification to near homogeneity was achieved by a combination of ammonium sulfate precipitation and ion exchange chromatography. The purified rAra h 1 was then compared with natural Ara h 1 for IgE binding. All patients recognized both the folded natural and rAra h 1, but the IgE binding to the rArah1 was significantly reduced in comparison to the natural allergen, which could potentially make it useful for immunotherapeutic purposes.

Background: A link between alcohol abuse and bacterial pneumonia has been recognized for centuries, but mechanisms to explain this relationship are unclarified. Interleukin-17 (IL-17) is a lymphocyte-derived cytokine that is part of the inflammatory cytokine cascade. Previous studies from our laboratory indicated that IL-17 is released in lung tissue in a murine model of bacterial pneumonia caused by Klebsiella pneumoniae. The effects of alcohol consumption on pulmonary release of IL-17 are unknown. Methods: Mice were maintained on 20% ethanol in drinking water or on a control diet without alcohol. After 2 weeks, alcohol and control mice were challenged with intratracheal K. pneumoniae. Mice were followed for survival after bacterial challenge, neutrophil recruitment was assayed as myeloperoxidase, and IL-17 was measured in lung lavage fluid by enzyme-linked immunosorbent assay. In additional experiments, splenocytes from control mice were incubated with ethanol in vitro, and release of IL-17 was measured in culture supernatants. Finally, control and alcohol mice received intrapulmonary gene transfer of E-1–deleted adenovirus containing the murine IL-17 gene. These mice were then challenged with K. pneumoniae and followed for survival and neutrophil recruitment. Results: In these studies, we demonstrate that a 2-week history of ethanol consumption in mice suppresses release of IL-17 into lung tissue, decreases neutrophil recruitment, and increases mortality from experimental K. pneumonia. In vitro experiments confirm a direct suppressive effect of ethanol on the release of IL-17 from splenocytes. In vivo administration of the IL-17 gene in an adenoviral vector to alcohol-consuming mice results in release of IL-17 into lavage fluid and normalizes neutrophil recruitment and mortality after bacterial challenge. Conclusions: The results of these experiments strongly implicate IL-17 as an important pathway for the immunosuppression associated with alcohol abuse and support gene therapeutic approaches to augment immune function in the alcoholic host or to treat infections associated with alcoholism.

+ cells that secreted interferon (IFN)-g (Th1 cytokine) and interleukin (IL)-4 (Th2 cytokine) after concanavalin A stimulation. An ELISA was used to assay culture supernatants for cytokine concentrations. Precursor frequency of both IFN-g- and IL-4-secreting cells was increased in lymph nodes at 1 week, whereas increases in Th1 and Th2 cells in lung tissue were delayed 3 weeks before declining. The frequency of IL-4-secreting cells always was greater than the frequency of IFN- g secreting cells. These results demonstrate an early T lymphocyte response in draining lymph nodes, followed by later recruitment of Th1 and Th2 lymphocytes into lung tissue. The overall CD4 + T cell response to P. carinii involves both Th1 and Th2 subsets, but the response is Th2 dominant in both lymph node and lung tissue. Pulmonary infection with Pneumocystis carinii is the most common severe opportunistic infection that complicates human immunodeficiency virus (HIV) infection (1, 2). Host defense mechanisms against P. carinii are poorly understood. Human infection with P. carinii is associated more with defects in cell- mediated immunity, such as HIV infection, than with neutrophil dysfunction. As a result, P. carinii infections have become a particular clinical problem associated with HIV infection. The risk of an HIV-infected subject's acquiring P. carinii pneumonia shows a linear and inverse correlation with the number of cir- culating CD4 1 lymphocytes (3). The importance of the CD4 1

In this study, we examined the effects of radiation and ara-C on induction of apoptosis and on the apoptosis-promoting genes p53, Bax and Fas/APO-1, in BV173 human leukemia cells, which harbor the wild-type p53 gene. It has been reported that p53 upregulates Fas/APO-1 and Bax expression. Both irradiation and ara-C treatment resulted in apoptosis and induction of p53 proteins within hours. The Bax gene was activated in irradiated and ara-C-treated BV173 cells, but Fas/APO-1 was induced only in irradiated BV173 cells. Radiation and ara-C treatment did not induce Bax or Fas/APO-1 protein expression in p53-null HL60 cells. Radiation weakly induced Fas/APO-1 expression in KBM-7 cells, which harbor a partially defective p53 gene. Both HL60 and KBM-7 cells are more resistant to radiation- and ara-C-induced apoptosis than BV173 cells. These results suggest that functional p53 is necessary for the activation of Bax and Fas/APO-1 expression. However, elevated p53 protein is not sufficient to activate Fas/APO-1 gene expression in ara-C-treated cells. Using two-dimensional gel electrophoresis, we found that the p53 proteins in irradiated and ara-C-treated BV173 cells have different isoelectric points; they converged to a single isoelectric point after in vitro treatment with phosphatase. These results suggest that different genotoxic treatments cause different phosphorylations of p53, which may account for the different levels of activation of Fas/APO-1 expression.

The p21WAF1/Cip1 (p21) protein, a negative regulator of G1 checkpoint control, was overexpressed in the majority of human gliomas. To investigate whether p21 expression in brain metastases from various systemic origins is similar to that in gliomas and whether p21 expression is regulated differently in brain metastases and in corresponding primary tumors, we used immunohistochemical staining to examine the expression of p21 in paraffin-embedded sections prepared from primary colon and breast carcinomas and from metastatic brain tumors that originated from colon, breast, lung, and kidney cancers and from melanoma. Our results showed that 56% (28 of 50) of the brain metastases samples have more than 1% p21-positive staining cells compared with 87% of primary gliomas reported previously. Among the samples analyzed, p21 expression in brain metastases from breast carcinomas was much higher than in primary breast carcinomas. In contrast, p21 expression in brain metastases from colon carcinomas was less than primary colon carcinomas. The results from this pilot study suggest that p21 expression is regulated differently in metastatic and primary tumors.

Allergic reactions to peanuts and tree nuts are major causes of anaphylaxis in the United States. We compare different properties of natural and recombinant versions of Ara h 1, a major peanut allergen, through structural, immunologic, and bioinformatics analyses. Small angle x-ray scattering studies show that natural Ara h 1 forms higher molecular weight aggregates in solution. In contrast, the full-length recombinant protein is partially unfolded and exists as a monomer. The crystal structure of the Ara h 1 core (residues 170–586) shows that the central part of the allergen has a bicupin fold, which is in agreement with our bioinformatics analysis. In its crystalline state, the core region of Ara h 1 forms trimeric assemblies, while in solution the protein exists as higher molecular weight assemblies. This finding reveals that the residues forming the core region of the protein are sufficient for formation of Ara h 1 trimers and higher order oligomers. Natural and recombinant variants of proteins tested in in vitro gastric and duodenal digestion assays show that the natural protein is the most stable form, followed by the recombinant Ara h 1 core fragment and the full-length recombinant protein. Additionally, IgE binding studies reveal that the natural and recombinant allergens have different patterns of interaction with IgE antibodies. The molecular basis of cross-reactivity between vicilin allergens is also elucidated.

BackgroundApoptosis of lymphocytes is important in the termination of an immune response to infection but has also been shown to have detrimental effects in animal models of systemic infection and sepsis. We sought to characterize lymphocyte apoptosis in an animal model of pneumonia due toPneumocystis murina, an infection localized to the lungs.MethodsControl mice and mice depleted of CD4+ lymphocytes were inoculated withPneumocystis. Apoptosis of lung and spleen lymphocytes was assayed by flow cytometry and PCR assay of apoptotic proteins.ResultsIn control mice, apoptosis of lung lymphocytes was maximal just after the infection was cleared from lung tissue and then declined. However, in CD4-depleted mice, apoptosis was also upregulated in recruited lymphocytes in spite of progressive infection. In splenic lymphocytes, apoptosis was observed early at 1 week after inoculation and then declined. Apoptosis of lung lymphocytes in control mice was associated with a decrease in mRNA for Bcl-2 and an increase in mRNA for Bim. In CD4-depleted mice, lavaged CD8+ cells did change intracellular Bcl-2 but showed increased mRNA for Bim.ConclusionApoptosis of both pulmonary and extrapulmonary lymphocytes is part of the normal host response toPneumocystisbut is also triggered in CD4-deficient animals with progressive infection. In normal mice apoptosis of pulmonary lymphocytes may serve to terminate the immune response in lung tissue. Apoptosis of lung lymphocytes takes place via both the intrinsic and extrinsic apoptotic pathways and is associated with changes in both pro- and anti-apoptotic proteins.

Strategies to augment host defense against pulmonary infection run the risk of inducing excess pulmonary inflammation and tissue injury. To address this problem, we investigated conditional expression in lung tissue of the murine interferon-gamma (IFN-gamma) transgene. A recombinant adenoviral vector (AdTetIFN) was constructed by placing a murine IFN-gamma cDNA downstream of a tetracycline (Tet)-responsive promoter, inserted into a replication-defective adenoviral vector. Co-infection of target cells with AdTetIFN and a second vector encoding a reverse tetracycline controlled transactivator allowed doxycycline (Dox)-regulated IFN-gamma production. We then administered 10(8) plaque-forming units (PFU) of AdTetIFN to mice by intratracheal injection. When the mice were provided with Dox in drinking water (0.5mg/ml in 5% sucrose), there was significant release of IFN-gamma in lavage fluid by ELISA in comparison to mice on water/sucrose alone (399+/-74 pg/ml vs undetectable, p

The effectiveness of chemotherapy for human cancers is limited by pharmacokinetic parameters such as variation in metabolism and is determined by the cellular response. In this work, we aimed to gain a more holistic understanding of the molecular basis of glioma response to the DNA-alkylating agent 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) by using a systematic approach: we investigated the expression of 588 genes with various cellular functions in a BCNU-resistant glioblastoma cell line and a BCNU-sensitive subline before and after treatment with BCNU. Our gene expression profiling revealed major differences in gene expression between these two cell lines, especially after treatment with BCNU. One striking example was that BCNU decreased the expression of six DNA-repair genes in sensitive but not in resistant cells. In sensitive cells, BCNU treatment resulted in the induction of two MAP kinase genes; this finding suggests that the specific response to BCNU in sensitive cells may involve the Jun kinase signal transduction pathway. After BCNU treatment, marked induction of tumor necrosis factor was detected only in sensitive cells, suggesting that tumor necrosis factor is a mediator of BCNU-induced cell death. Bcl-2 family members were not altered by BCNU in sensitive cells, suggesting that BCNU-induced cell death may be independent of the bcl-2 pathway. Results of the present study demonstrate that gene expression profiling may facilitate identification of cellular pathways associated with specific responses to chemotherapeutic agents and contribute to an understanding of the molecular basis of drug action.

In most normal somatic cells the telomeres of human chromosomes shorten with each cell division because of low expression or lack of telomerase activity. Telomerase, a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends, is reactivated or upregulated in tumor cells and maintains the stability of telomere length. We previously showed that treatment of HL60 leukemia cells with differentiation-inducing agents resulted in inhibition of telomerase activity. In the present study, we found that the decrease in telomerase activity did not temporally correlate with the expression of a differentiation marker, CD11b, on the cell surface. Mixing of protein extracts from telomerase-negative differentiated HL60 cells with those from parental HL60 cells did not result in inhibition of telomerase activity, suggesting that a diffusible cellular telomerase inhibitor was not produced in the differentiated cells. However, a decrease in telomerase activity correlated with a selective decrease of telomerase RNA expression, a decrease in the levels of total cellular RNA, and an increase in cells at G(0) phase.

Alcohol binge-drinking (acute ethanol consumption) is immunosuppressive and alters both the innate and adaptive arms of the immune system. Antigen presentation by macrophages (and other antigen presenting cells) represents an important function of the innate immune system that, in part, determines the outcome of the host immune response. Ethanol has been shown to suppress antigen presentation in antigen presenting cells though mechanisms of this impairment are not well understood. The constitutive and immunoproteasomes are important components of the cellular proteolytic machinery responsible for the initial steps critical to the generation of MHC Class I peptides for antigen presentation. In this study, we used an in-vitro cell culture model of acute alcohol exposure to study the effect of ethanol on the proteasome function in RAW 264.7 cells. Additionally, primary murine peritoneal macrophages obtained by peritoneal lavage from C57BL/6 mice were used to confirm our cell culture findings. We demonstrate that ethanol impairs proteasome function in peritoneal macrophages through suppression of chymotrypsin-like (Cht-L) proteasome activity as well as composition of the immunoproteasome subunit LMP7. Using primary murine peritoneal macrophages, we have further demonstrated that, ethanol-induced impairment of the proteasome function suppresses processing of antigenic proteins and peptides by the macrophage and in turn suppresses the presentation of these antigens to cells of adaptive immunity. The results of this study provide an important mechanism to explain the immunosuppressive effects of acute ethanol exposure.