TB research at UT-Houston--a review of cord factor: new approaches to drugs, vaccines and the pathogenesis of tuberculosis.

Tuberculosis remains a major threat as drug resistance continues to increase. Pulmonary tuberculosis in adults is responsible for 80% of clinical cases and nearly 100% of transmission of infection. Unfortunately, since we have no animal models of adult type pulmonary tuberculosis, the most important type of disease remains largely out of reach of modern science and many fundamental questions remain unanswered. This paper reviews research dating back to the 1950's providing compelling evidence that cord factor (trehalose 6,6 dimycolate [TDM]) is essential for understanding tuberculosis. However, the original papers by Bloch and Noll were too far ahead of their time to have immediate impact. We can now recognize that the physical and biologic properties of cord factor are unprecedented in science, especially its ability to switch between two sets of biologic activities with changes in conformation. While TDM remains on organisms, it protects them from killing within macrophages, reduces antibiotic effectiveness and inhibits the stimulation of protective immune responses. If it comes off organisms and associates with lipid, TDM becomes a driver of tissue damage and necrosis. Studies emanating from cord factor research have produced (1) a rationale for improving vaccines, (2) an approach to new drugs that overcome natural resistance to antibiotics, (3) models of caseating granulomas that reproduce multiple manifestations of human tuberculosis. (4) evidence that TDM is a key T cell antigen in destructive lesions of tuberculosis, and (5) a new understanding of the pathology and pathogenesis of postprimary tuberculosis that can guide more informative studies of long standing mysteries of tuberculosis.

EFFICACY AND MECHANISM OF â-DEFENSIN2 FUSED ANTIGEN PROTEIN VACCINES

Vaccines which use the strategy of fusing adjuvant murine â-defensin2 (mBD2) to an antigen in order to elicit stronger anti-antigen immune responses are referred to as murine â-defensin2 (mBD2) vaccines. Previous studies have validated the potential of mBD2 vaccines, thus in this study we focus on increasing vaccine efficacy as well as mechanism elucidation. Initially, we demonstrate superior IFN-ã release levels by antigen specific effector T cells when antigen is crosspresented by dendritic cells (DC) which absorbed mBD2 vaccine (mBD2 fused antigen protein) over antigen alone. We move unto an in vivo model and note significant increases in the expansion of antigen specific class I T cells but not class II T cells when receiving mBD2 vaccine over antigen alone. Further, knowing mBD2’s link with CC chemokine receptor 6 (CCR6) and Toll-like receptor 4 (TLR4) we note that this enhanced class I T cell expansion is CCR6 independent but TLR4 dependent. With anti-tumor responses desired, we demonstrate in tumor protection experiments with mice, compelling tumor protection when combining adoptive T cell therapy and mBD2 vaccine immunization. We further note that mBD2 vaccines are not limited by the antigen and characterize a viable strategy for enhancing tumor antigen immunogenicity.

Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+ metastatic breast cancer in mice.

INTRODUCTION: Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models. METHODS: The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad of mice as a model of solid tumor growth or was injected intravenously as a model of lung metastasis. SINCP-neu, a plasmid containing Sindbis virus genes and the gene for rat HER2/neu, and Adeno-neu, an E1,E2a-deleted adenovirus also containing the gene for rat HER2/neu, were tested as preventive and therapeutic vaccines. RESULTS: Vaccination with SINCP-neu or Adeno-neu before tumor challenge with A2L2 cells significantly inhibited the growth of the cells injected into the mammary fat or intravenously. Vaccination 2 days after tumor challenge with either vaccine was ineffective in both tumor models. However, therapeutic vaccination in a prime-boost protocol with SINCP-neu followed by Adeno-neu significantly prolonged the overall survival rate of mice injected intravenously with the tumor cells. Naive mice vaccinated using the same prime-boost protocol demonstrated a strong serum immunoglobulin G response and p185-specific cellular immunity, as shown by the results of ELISPOT (enzyme-linked immunospot) analysis for IFNgamma. CONCLUSION: We report herein that vaccination of mice with a plasmid gene vaccine and an adenovirus gene vaccine, each containing the gene for HER2/neu, prevented growth of a HER2/neu-expressing breast cancer cell line injected into the mammary fat pad or intravenously. Sequential administration of the vaccines in a prime-boost protocol was therapeutically effective when tumor cells were injected intravenously before the vaccination. The vaccines induced high levels of both cellular and humoral immunity as determined by in vitro assessment. These findings indicate that clinical evaluation of these vaccines, particularly when used sequentially in a prime-boost protocol, is justified.

Regulation of mucin gene expression by CREB via a nonclassical retinoic acid signaling pathway.

Vitamin A and its metabolite retinoic acid (RA) are essential elements for normal lung development and the differentiation of lung epithelial cells. We previously showed that RA rapidly activated cyclic AMP response element-binding protein (CREB) in a nonclassical manner in normal human tracheobronchial epithelial (NHTBE) cells. In the present study, we further demonstrated that this nonclassical signaling of RA on the activation of CREB plays a critical role in regulating the expression of airway epithelial cell differentiation markers, the MUC2, MUC5AC, and MUC5B genes. We found that RA rapidly activates the protein kinase Calpha isozyme and transmits the activation signal to CREB via the Raf/MEK/extracellular signal-regulated kinase/p90 ribosomal S6 kinase (RSK) pathway. Activated RSK translocated from the cytoplasm to the nucleus, where it phosphorylates CREB. Activated CREB then binds to a cis-acting replication element motif on the promoter (at nucleotides [nt] -878 to -871) of the MUC5AC gene. The depletion of CREB using small interfering RNA abolished not only the RA-induced MUC5AC but also RA-induced MUC2 and MUC5B. Taken together, our findings demonstrate that CREB activation via this nonclassical RA signaling pathway may play an important role in regulating the expression of mucin genes and mediating the early biological effects of RA during normal mucous differentiation in NHTBE cells.

CA125/MUC16 is dispensable for mouse development and reproduction.

Cancer antigen 125 (CA125) is a blood biomarker that is routinely used to monitor the progression of human epithelial ovarian cancer (EOC) and is encoded by MUC16, a member of the mucin gene family. The biological function of CA125/MUC16 and its potential role in EOC are poorly understood. Here we report the targeted disruption of the of the Muc16 gene in the mouse. To generate Muc16 knockout mice, 6.0 kb was deleted that included the majority of exon 3 and a portion of intron 3 and replaced with a lacZ reporter cassette. Loss of Muc16 protein expression suggests that Muc16 homozygous mutant mice are null mutants. Muc16 homozygous mutant mice are viable, fertile, and develop normally. Histological analysis shows that Muc16 homozygous mutant tissues are normal. By the age of 1 year, Muc16 homozygous mutant mice appear normal. Downregulation of transcripts from another mucin gene (Muc1) was detected in the Muc16 homozygous mutant uterus. Lack of any prominent abnormal phenotype in these Muc16 knockout mice suggests that CA125/MUC16 is not required for normal development or reproduction. These knockout mice provide a unique platform for future studies to identify the role of CA125/MUC16 in organ homeostasis and ovarian cancer.

Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid.

Linezolid, which targets the ribosome, is a new synthetic antibiotic that is used for treatment of infections caused by Gram-positive pathogens. Clinical resistance to linezolid, so far, has been developing only slowly and has involved exclusively target site mutations. We have discovered that linezolid resistance in a methicillin-resistant Staphylococcus aureus hospital strain from Colombia is determined by the presence of the cfr gene whose product, Cfr methyltransferase, modifies adenosine at position 2503 in 23S rRNA in the large ribosomal subunit. The molecular model of the linezolid-ribosome complex reveals localization of A2503 within the drug binding site. The natural function of cfr likely involves protection against natural antibiotics whose site of action overlaps that of linezolid. In the chromosome of the clinical strain, cfr is linked to ermB, a gene responsible for dimethylation of A2058 in 23S rRNA. Coexpression of these two genes confers resistance to all the clinically relevant antibiotics that target the large ribosomal subunit. The association of the ermB/cfr operon with transposon and plasmid genetic elements indicates its possible mobile nature. This is the first example of clinical resistance to the synthetic drug linezolid which involves a natural resistance gene with the capability of disseminating among Gram-positive pathogenic strains.

Immune responses against major histocompatibility complex and interleukin-2 gene transfected K1735 murine melanoma: Potential vaccines for the immunotherapy of cancer

Tumor specific immunity is mediated by cytotoxic T lymphocytes (CTL) that recognize peptide antigen (Ag) in the context of major histocompatibility complex (MHC) class I molecules and by helper T (Th) lymphocytes that recognize peptide Ag in the context of MHC class II molecules. The purpose of this study is (1) to induce or augment the immunogenicity of nonimmunogenic or weakly immunogenic tumors by genetic modification of tumor cells, and (2) to use these genetically altered cells in cancer immunotherapy. To study this, I transfected a highly tumorigenic murine melanoma cell line (K1735) that did not express constitutively either MHC class I or II molecules with syngeneic cloned MHC class I and/or class II genes, and then determined the tumorigenicity of transfected cells in normal C3H mice. K1735 transfectants expressing either $\rm K\sp{k}$ or $\rm A\sp{k}$ molecules alone produced tumors in normal C3H mice, whereas most transfectants that expressed both molecules were rejected in normal C3H mice but produced tumors in nude mice. The rejection of K1735 transfectants expressing $\rm K\sp{k}$ and $\rm A\sp{k}$ Ag in normal C3H mice required both $\rm CD4\sp+$ and $\rm CD8\sp+$ T cells. Interestingly, the $\rm A\sp{k}$ requirement can be substituted by IL-2 because transfection of $\rm K\sp{k}$-positive/A$\sp{\rm k}$-negative K1735 cells with the IL-2 gene also resulted in abrogation of tumorigenicity in normal C3H mice but not in nude mice. In addition, 1735 $(\rm I\sp+II\sp+)$ transfected cells can function as antigen presenting cells (APC) since they could process and present native hen egg lysozyme (HEL) to HEL specific T cell hybridomas. Furthermore, the transplantation immunity induced by K1735 transfectants expressing both $\rm K\sp{k}$ and $\rm A\sp{k}$ molecules completely cross-protected mice against challenge with $\rm K\sp{k}$-positive transfectants but weakly protected them against challenge with parental K1735 cells or $\rm A\sp{k}$-positive transfectants. Finally, I demonstrated that MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells can function as anti-cancer vaccines since they can abrogate the growth of established tumors and metastasis.^ In summary, my results indicate that expression of either MHC class I or II molecule alone is insufficient to cause the rejection of K1735 melanoma in syngeneic hosts and that both molecules are necessary. In addition, my data suggest that the failure of $\rm K\sp{k}$-positive K1735 cells to induce a primary tumor-rejection response in normal C3H mice may be due to their inability to induce the helper arm of the anti-tumor immune response. Finally, the ability of MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells to prevent growth of established tumors or metastasis suggests that these cell lines can serve as potential vaccines for the immunotherapy of cancer. (Abstract shortened by UMI.) ^

cDNA cloning and expression of a novel cell surface-expressed \b heparan sulfate/heparin \b interacting \b protein (HIP) from human cell lines and functional studies of a synthetic HIP peptide

Heparan sulfate proteoglycans and their corresponding binding sites have been suggested to play an important role during the initial attachment of blastocysts to uterine epithelium and human trophoblastic cell lines to uterine epithelial cell lines. Previous studies on RL95 cells, a human uterine epithelial cell line, characterized a single class of cell surface heparin/heparan sulfate (HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were isolated from RL95 cell surfaces by tryptic digestion and partial amino-terminal amino acid sequence from each peptide fragment was obtained. In the current study, using the approaches of reverse transcription-polymerase chain reaction and cDNA library screening, a novel cell surface $\rm\underline{H}$P/HS $\rm\underline{i}$nteracting $\rm\underline{p}$rotein (HIP) has been isolated from RL95 cells. The full-length cDNA of HIP encodes a protein of 259 amino acids with a calculated molecular weight of 17,754 Da and pI of 11.75. Transfection of HIP cDNA into NIH-3T3 cells demonstrated cell surface expression and a size similar to that of HIP expressed by human cells. Predicted amino acid sequence indicates that HIP lacks a membrane spanning region and has no consensus sites for glycosylation. Northern blot analysis detected a single transcript of 1.3 kb in both total RNA and poly(A$\sp+$) RNA. Examination of human cell lines and normal tissues using both Northern blot and Western blot analysis revealed that HIP is differentially expressed in a variety of human cell lines and normal tissues, but absent in some cell lines examined. HIP has about 80% homology, at the level of both mRNA and protein, to a rodent protein, designated as ribosomal protein L29. Thus, members of the L29 family may be displayed on cell surfaces where they participate in HP/HS binding events. Studies on a synthetic peptide derived from HIP demonstrate that HIP peptide binds HS/HP with high selectivity and has high affinity (Kd = 10 nM) for a subset of polysaccharides found in commercial HIP preparations. Moreover, HIP peptide also binds certain forms of cell surface, but not secreted or intracellular. HS expressed by RL95 and JAR cells. This peptide supports the attachment of several human trophoblastic cell lines and a variety of mammalian adherent cell lines in a HS-dependent fashion. Furthermore, studies on the subset of HP specifically recognized by HIP peptide indicate that this high-affinity HP (HA-HP) has a larger median MW and a greater negative charge density than bulk HP. The minimum size of oligosaccharide required to bind to HIP peptide with high affinity is a septa- or octasaccharide. HA-HP also quantitatively binds to antithrombin-III (AT-III) with high affinity, indicating that HIP peptide and AT-III may recognize the same or similar oligosaccharide structure(s). Furthermore, HIP peptide antagonizes HP action and promotes blood coagulation in both factor Xa- and thrombin-dependent assays. Finally, HA-HP recognized by HP peptide is highly enriched with anticoagulant activity relative to bulk HP. Collectively, these results demonstrate that HIP may play a role in the HP/HS-involved cell-cell and cell-matrix interactions and recognizes a motif in HP similar or identical to that recognized by AT-III and therefore, may modulate blood coagulation. ^

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro.

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.

Analogues of polyamine alkaloids and their synthetic advantages

Several polyamine derivatives were synthesized in order to produce novel antagonists of muscular nicotinic acetylcholine receptors. Their affinities were compared with those of philanthotoxin PhTX-343.