Preclinical Analysis of JAA-F11, a Specific Anti-Thomsen-Friedenreich Antibody via Immunohistochemistry and In Vivo Imaging.

The tumor specificity of JAA-F11, a novel monoclonal antibody specific for the Thomsen-Friedenreich cancer antigen (TF-Ag-alpha linked), has been comprehensively studied by in vitro immunohistochemical (IHC) staining of human tumor and normal tissue microarrays and in vivo biodistribution and imaging by micro-positron emission tomography imaging in breast and lung tumor models in mice. The IHC analysis detailed herein is the comprehensive biological analysis of the tumor specificity of JAA-F11 antibody performed as JAA-F11 is progressing towards preclinical safety testing and clinical trials. Wide tumor reactivity of JAA-F11, relative to the matched mouse IgG3 (control), was observed in 85% of 1269 cases of breast, lung, prostate, colon, bladder, and ovarian cancer. Staining on tissues from breast cancer cases was similar regardless of hormonal or Her2 status, and this is particularly important in finding a target on the currently untargetable triple-negative breast cancer subtype. Humanization of JAA-F11 was recently carried out as explained in a companion paper "Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis" (Neoplasia 19: 716-733, 2017), and it was confirmed that humanization did not affect chemical specificity. IHC studies with humanized JAA-F11 showed similar binding to human breast tumor tissues. In vivo imaging and biodistribution studies in a mouse syngeneic breast cancer model and in a mouse-human xenograft lung cancer model with humanized 124I- JAA-F11 construct confirmed in vitro tumor reactivity and specificity. In conclusion, the tumor reactivity of JAA-F11 supports the continued development of JAA-F11 as a targeted cancer therapeutic for multiple cancers, including those with unmet need

Physiological Studies on Candida albicans

Candida albicans is a common opportunistic, dimorphic human fungal pathogen. One of its virulence factors is the morphological switch between yeasts and hyphal or pseudohyphal forms, which can invade tissues and cause damage. Our studies focus on factors regulating pseudohyphae and epigenetic modifications of C. albicans. Regulating factors of pseudohyphae are aromatic alcohols and high phosphate. At low concentrations, exogenous aromatic alcohols induced pseudohyphae, as did high phosphate. For addressing the pathways involved in inducing pseudohyphae by aromatic alcohols or high phosphate, we used mutants defective in cAMP dependent PKA pathway (efg1/efg1), MAP kinase pathway (cph1/cph1), or both (cph1/cph1/efg1/efg1). These mutants failed to produce either hyphae or pseudohyphae in the presence of aromatic alcohols; but high phosphate still stimulated pseudohyphae. Gcn4, a transcription activator of more than 500 amino acid related genes, is turned-on in response to amino acid starvation. The accumulation of aromatic alcohols sends nitrogen starvation signals, which inhibit eIF2B, which in turn derepresses Gcn4p. High phosphate also induces pseudohyphae by derepressing Gcn4p, although the pathways involved are still unknown. In sum, aromatic alcohols and high phosphate induce pseudohyphae by derepressing Gcn4. In this study we found a novel posttranslational histone modification in C. albicans, which is biotinylation. Western blot and Mass spectrometry techniques were used to find that Histones H2B and H4 were biotinylated at every condition tested such as yeast vs. hyphae, aerobic growth vs. anaerobic growth, rich medium vs. defined medium. In C. albicans lysines K8, K11 in histone H4 and lysines K17, K18, K31 in histone H2B are biotin attachment sites as found using mass spectrometry. Biotin was also found to enhance the germ tube formation of C. albicans. Germ tube formation assays with biotin-starved cells as inoculum showed low percent of germ tubes (1-5%). Addition of biotin to the media showed 100% germ tubes. Biotinylation of histones were not detected from biotin-starved cells. Appendix-A details work related to Farnesol quantification assays in several strains of C.albicans and Ceratocystis ulmi, and growth studies of class E VPS strains of Saccharomyces Cerevisiae. Adviser: Kenneth W. Nickerso

Histone biotinylation in \u3ci\u3eCandida albicans\u3c/i\u3e

Candida albicans is an opportunistic fungal pathogen in humans. It is a polymorphic fungus: it can live as yeasts, hyphae, or pseudohyphae. Biotin is required for cell growth and fatty acid metabolism because it is used as a cofactor for carboxylases such as acetyl-CoA carboxylase, and pyruvate carboxylase. In addition, we have discovered that biotin is used to modify histones in C. albicans. Biotinylation was detected by Western blots using a monoclonal antibiotin HRP-conjugated antibody as well as with qTOF and LC/MS/MS mass spectrometry. As a precaution, the antibiotin antibody was dialyzed against neutravidin prior to use. During this study, we observed that three histones, H2A, H2B, and H4, were biotinylated at many lysine residues in an apparently nonsite-specific manner. Roughly, equivalent levels of acetylation, methylation, and phosphorylation were found in histones from biotin-replete and biotin-starved cells, but histone biotinylation was only observed for cells grown in excess biotin. The function of histone biotinylation in C. albicans is still unknown but, because C. albicans is a natural biotin auxotroph, a storage reservoir for biotin is attractive. Techniques used to detect histone biotinylation in C. albicans did not detect any histone biotinylation in Saccharomyces cerevisiae

\u3ci\u3eCandida albicans\u3c/i\u3e cellwall components and farnesol stimulate the expression of both inflammatory and regulatory cytokines in the murine RAW264.7 macrophage cell line

Candida albicans causes candidiasis, secretes farnesol, and switches from yeast to hyphae to escape from macrophages after phagocytosis. However, before escape, macrophages may respond to C. albicans’ pathogen-associated molecular patterns (PAMPs) through toll-like receptor 2 (TLR2) and dectin-1 receptors by expressing cytokines involved in adaptive immunity, inflammation, and immune regulation. Therefore, macrophages and the RAW264.7 macrophage line were challenged with C. albicans preparations of live wild-type cells, heat-killed cells, a live mutant defective in hyphae formation, a live mutant producing less farnesol, or an isolate producing farnesoic acid instead of farnesol. Interleukin-6 (IL-6), and IL-1b, IL- 10, and tumor necrosis factor-a (TNF-a) expression were evaluated by ELISA and/ or qRT-PCR within 6 h after challenge. All viable strains producing farnesol, regardless of hyphae phenotype, induced IL-6, IL-1b, IL-10, and TNF-a. To determine which components of C. albicans induced IL-6, RAW264.7 cells were incubated with farnesol, farnesoic acid, with or without zymosan, a yeast cell wall preparation that contains PAMPs recognized by TLR2 and dectin-1. The highest expression of IL-6, TLR2, and dectin-1 occurred when RAW264.7 cells were stimulated with zymosan and farnesol together. Our results suggest that the rapid expression of cytokines from macrophages challenged with C. albicans is due to cell-wall PAMPs combined with farnesol

Biotin Auxotrophy and Biotin Enhanced Germ Tube Formation in \u3ci\u3eCandida albicans\u3c/i\u3e

Due to the increased number of immunocompromised patients, infections with the pathogen Candida albicans have significantly increased in recent years. C. albicans transition from yeast to germ tubes is one of the essential factors for virulence. In this study we noted that Lee’s medium, commonly used to induce filamentation, contained 500-fold more biotin than needed for growth and 40-fold more biotin than is typically added to growth media. Thus, we investigated the effects of excess biotin on growth rate and filamentation by C. albicans in different media. At 37 ˚C, excess biotin (4 µM) enhanced germ tube formation (GTF) ca. 10-fold in both Lee’s medium and a defined glucose-proline medium, and ca. 4-fold in 1% serum. Two biotin precursors, desthiobiotin and 7-keto-8-aminopelargonic acid (KAPA), also stimulated GTF. During these studies we also noted an inverse correlation between the number of times the inoculum had been washed and the concentration of serum needed to stimulate GTF. C. albicans cells that had been washed eight times achieved 80% GTF with only 0.1% sheep serum. The mechanism by which 1–4 µM biotin enhances GTF is still unknown except to note that equivalent levels of biotin are needed to create an internal supply of stored biotin and biotinylated histones. Biotin did not restore filamentation for any of the four known filamentation defective mutants tested. C. albicans is auxotrophic for biotin and this biotin auxotrophy was fulfilled by biotin, desthiobiotin, or KAPA. However, biotin auxotrophy is not temperature dependent or influenced by the presence of 5% CO2. Biotin starvation upregulated the biotin biosynthetic genes BIO2, BIO3, and BIO4 by 11-, 1500-, and 150-fold, respectively, and BIO2p is predicted to be mitochondrion-localized. Based on our findings, we suggest that biotin has two roles in the physiology of C. albicans, one as an enzymatic cofactor and another as a morphological regulator. Finally, we found no evidence supporting prior claims that C. albicans only forms hyphae at very low biotin (0.1 nM) growth conditions

Histatin 5 resistance of Candida glabrata can be reversed by insertion of Candida albicans polyamine transporter-encoding genes DUR3 and DUR31.

Candida albicans and Candida glabrata are predominant fungi associated with oral candidiasis. Histatin 5 (Hst 5) is a small cationic human salivary peptide with high fungicidal activity against C. albicans, however many strains of C. glabrata are resistant. Since Hst 5 requires fungal binding to cell wall components prior to intracellular translocation, reduced Hst 5 binding to C. glabrata may be the reason for its insensitivity. C. glabrata has higher surface levels of β-1,3-glucans as compared with C. albicans; however these differences did not account for reduced Hst 5 uptake and killing in C. glabrata. Similarly, the biofilm matrix of C. glabrata contained significantly higher levels of β-1,3-glucans compared with C. albicans, but it did not reduce the percentage of Hst 5 positive fungal cells in the biofilm. Hst 5 enters C. albicans cell through polyamine transporters Dur3p and Dur31p that are uncharacterized in C. glabrata. C. glabrata strains expressing CaDur3 and CaDur31 had two-fold higher killing and uptake of Hst 5. Thus, neither C. glabrata cell surface or biofilm matrix β-1,3-glucan levels affected Hst 5 toxicity; rather the crucial rate limiting step is reduced uptake that can be overcome by expression of C. albicans Dur proteins in C. glabrata

Biotin Auxotrophy and Biotin Enhanced Germ Tube Formation in Candida albicans

Due to the increased number of immunocompromised patients, infections with the pathogen Candida albicans have significantly increased in recent years. C. albicans transition from yeast to germ tubes is one of the essential factors for virulence. In this study we noted that Lee’s medium, commonly used to induce filamentation, contained 500-fold more biotin than needed for growth and 40-fold more biotin than is typically added to growth media. Thus, we investigated the effects of excess biotin on growth rate and filamentation by C. albicans in different media. At 37 °C, excess biotin (4 µM) enhanced germ tube formation (GTF) ca. 10-fold in both Lee’s medium and a defined glucose-proline medium, and ca. 4-fold in 1% serum. Two biotin precursors, desthiobiotin and 7-keto-8-aminopelargonic acid (KAPA), also stimulated GTF. During these studies we also noted an inverse correlation between the number of times the inoculum had been washed and the concentration of serum needed to stimulate GTF. C. albicans cells that had been washed eight times achieved 80% GTF with only 0.1% sheep serum. The mechanism by which 1–4 µM biotin enhances GTF is still unknown except to note that equivalent levels of biotin are needed to create an internal supply of stored biotin and biotinylated histones. Biotin did not restore filamentation for any of the four known filamentation defective mutants tested. C. albicans is auxotrophic for biotin and this biotin auxotrophy was fulfilled by biotin, desthiobiotin, or KAPA. However, biotin auxotrophy is not temperature dependent or influenced by the presence of 5% CO2. Biotin starvation upregulated the biotin biosynthetic genes BIO2, BIO3, and BIO4 by 11-, 1500-, and 150-fold, respectively, and BIO2p is predicted to be mitochondrion-localized. Based on our findings, we suggest that biotin has two roles in the physiology of C. albicans, one as an enzymatic cofactor and another as a morphological regulator. Finally, we found no evidence supporting prior claims that C. albicans only forms hyphae at very low biotin (0.1 nM) growth conditions

Secreted aspartic protease cleavage of Candida albicans Msb2 activates Cek1 MAPK signaling affecting biofilm formation and oropharyngeal candidiasis.

Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence

Strains used in this study.

<p>Strains used in this study.</p