Exhaustion of cytotoxic effector systems may limit monoclonal antibody-based immunotherapy in cancer patients

The CD20 mAb ofatumumab (OFA) induces complement-mediated lysis of B cells. In an investigator-initiated phase II trial of OFA plus chemotherapy for chronic lymphocytic leukemia (CLL), OFA treatment promoted partial CLL B cell depletion that coincided with reduced complement titers. Remaining CLL B cells circulated with bound OFA and covalently bound complement breakdown product C3d, indicative of ongoing complement activation. Presumably, neither complement- nor effector cell-based mechanisms were sufficiently robust to clear these remaining B cells. Instead, almost all of the bound OFA and CD20 was removed from the cells, in accordance with previous clinical studies that demonstrated comparable loss of CD20 from B cells after treatment of CLL patients with rituximab. In vitro experiments with OFA and rituximab addressing these observations suggest that host effector mechanisms that support mAb-mediated lysis and tumor cell clearance are finite, and they can be saturated or exhausted at high B cell burdens, particularly at high mAb concentrations. Interestingly, only a fraction of available complement was required to kill cells with CD20 mAbs, and killing could be tuned by titrating the mAb concentration. Consequently, maximal B cell killing of an initial and secondary B cell challenge was achieved with intermediate mAb concentrations, whereas high concentrations promoted lower overall killing. Therefore, mAb therapies that rely substantially on effector mechanisms subject to exhaustion, including complement, may benefit from lower, more frequent dosing schemes optimized to sustain and maximize killing by cytotoxic immune effector systems.     

Gene-based vaccination with a mismatched envelope protects against simian immunodeficiency virus infection in nonhuman primates

The RV144 trial demonstrated that an experimental AIDS vaccine can prevent human immunodeficiency virus type 1 (HIV-1) infection in humans. Because of its limited efficacy, further understanding of the mechanisms of preventive AIDS vaccines remains a priority, and nonhuman primate (NHP) models of lentiviral infection provide an opportunity to define immunogens, vectors, and correlates of immunity. In this study, we show that prime-boost vaccination with a mismatched SIV envelope (Env) gene, derived from simian immunodeficiency virus SIVmac239, prevents infection by SIVsmE660 intrarectally. Analysis of different gene-based prime-boost immunization regimens revealed that recombinant adenovirus type 5 (rAd5) prime followed by replication-defective lymphocytic choriomeningitis virus (rLCMV) boost elicited robust CD4 and CD8 T-cell and humoral immune responses. This vaccine protected against infection after repetitive mucosal challenge with efficacies of 82% per exposure and 62% cumulatively. No effect was seen on viremia in infected vaccinated monkeys compared to controls. Protection correlated with the presence of neutralizing antibodies to the challenge viruses tested in peripheral blood mononuclear cells. These data indicate that a vaccine expressing a mismatched Env gene alone can prevent SIV infection in NHPs and identifies an immune correlate that may guide immunogen selection and immune monitoring for clinical efficacy trials.     

Hepatitis C virus infection of human T lymphocytes is mediated by CD5

Hepatitis C virus (HCV) is one of the main causes of chronic liver disease. Although infection of hepatocytes is mainly responsible for manifestations of hepatitis C, the virus also invades the immune system by a yet-to-be-identified mechanism. Using human T cell lines and primary T lymphocytes as targets and patient-derived HCV as inocula, we aimed to identify how HCV gains entry into these cells. HCV replication was determined by detection of the HCV RNA replicative (negative) strand and viral proteins, while specific antibodies, knocking down gene expression and making otherwise-resistant cells prone to HCV, were employed to identify a receptor molecule determining T lymphocyte permissiveness to HCV infection. The results revealed that T cell susceptibility to HCV requires CD5, a lymphocyte-specific glycoprotein belonging to the scavenger receptor cysteine-rich family. Blocking of T cell CD5 with antibody or silencing with specific short hairpin RNA (shRNA) decreased cell susceptibility to HCV, while increasing CD5 expression by mitogen stimulation had the opposite effect. Moreover, transfection of naturally CD5-deficient HEK-293 fibroblasts with CD5 facilitated infection of these otherwise HCV-resistant cells. In contrast to T cells, hepatocytes do not express CD5. The data revealed that CD5 is a molecule important for HCV entry into human T lymphocytes. This finding provides direct insight into the mechanism of HCV lymphotropism and defines a target for potential interventions against HCV propagating in this extrahepatic compartment.     

Control of Electron Transfer and Catalysis in Neuronal Nitric-oxide Synthase (nNOS) by a Hinge Connecting Its FMN and FAD-NADPH Domains

In nitric-oxide synthases (NOSs), two flexible hinges connect the FMN domain to the rest of the enzyme and may guide its interactions with partner domains for electron transfer and catalysis. We investigated the role of the FMN-FAD/NADPH hinge in rat neuronal NOS (nNOS) by constructing mutants that either shortened or lengthened this hinge by 2, 4, and 6 residues. Shortening the hinge progressively inhibited electron flux through the calmodulin (CaM)-free and CaM-bound nNOS to cytochrome c, whereas hinge lengthening relieved repression of electron flux in CaM-free nNOS and had no impact or slowed electron flux through CaM-bound nNOS to cytochrome c. How hinge length influenced heme reduction depended on whether enzyme flavins were pre-reduced with NADPH prior to triggering heme reduction. Without pre-reduction, changing the hinge length was deleterious; with pre-reduction, the hinge shortening was deleterious, and hinge lengthening increased heme reduction rates beyond wild type. Flavin fluorescence and stopped-flow kinetic studies on CaM-bound enzymes suggested hinge lengthening slowed the domain-domain interaction needed for FMN reduction. All hinge length changes lowered NO synthesis activity and increased uncoupled NADPH consumption. We conclude that several aspects of catalysis are sensitive to FMN-FAD/NADPH hinge length and that the native hinge allows a best compromise among the FMN domain interactions and associated electron transfer events to maximize NO synthesis and minimize uncoupled NADPH consumption.     

Platelet-activating factor receptor agonists mediate xeroderma pigmentosum A photosensitivity

To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.     

BRCA2 protein deficiency exaggerates doxorubicin-induced cardiomyocyte apoptosis and cardiac failure

The tumor suppressor breast cancer susceptibility gene 2 (BRCA2) plays an important role in the repair of DNA damage, and loss of BRCA2 predisposes carriers to breast and ovarian cancers. Doxorubicin (DOX) remains the cornerstone of chemotherapy in such individuals. However, it is often associated with cardiac failure, which once manifests carries a poor prognosis. Because BRCA2 regulates genome-wide stability and facilitates DNA damage repair, we hypothesized that loss of BRCA2 may increase susceptibility to DOX-induced cardiac failure. To this aim, we generated cardiomyocyte-specific BRCA2 knock-out (CM-BRCA2(-/-)) mice using the Cre-loxP technology and evaluated their basal and post-DOX treatment phenotypes. Although CM-BRCA2(-/-) mice exhibited no basal cardiac phenotype, DOX treatment resulted in markedly greater cardiac dysfunction and mortality in CM-BRCA2(-/-) mice compared with control mice. Apoptosis in left ventricular (LV) sections from CM-BRCA2(-/-) mice compared with that in corresponding sections from wild-type (WT) littermate controls was also significantly enhanced after DOX treatment. Microscopic examination of LV sections from DOX-treated CM-BRCA2(-/-) mice revealed a greater number of DNA double-stranded breaks and the absence of RAD51 focus formation, an essential marker of double-stranded break repair. The levels of p53 and the p53-related proapoptotic proteins p53-up-regulated modulator of apoptosis (PUMA) and Bax were significantly increased in samples from CM-BRCA2(-/-) mice. This corresponded with increased Bax to Bcl-2 ratios and elevated cytochrome c release in the LV sections of DOX-treated CM-BRCA2(-/-) mice. Taken together, these data suggest a critical and previously unrecognized role of BRCA2 as a gatekeeper of DOX-induced cardiomyocyte apoptosis and susceptibility to overt cardiac failure. Pharmacogenomic studies evaluating cardiac function in BRCA2 mutation carriers treated with doxorubicin are encouraged.     

The TF-antigen binding lectin from Sclerotium rolfsii inhibits growth of human colon cancer cells by inducing apoptosis in vitro and suppresses tumor growth in vivo

Glycan array analysis of Sclerotium rolfsii lectin (SRL) revealed its exquisite binding specificity to the oncofetal Thomsen-Friedenreich (Galβ1-3GalNAcα-O-Ser/Thr, T or TF) antigen and its derivatives. This study shows that SRL strongly inhibits the growth of human colon cancer HT29 and DLD-1 cells by binding to cell surface glycans and induction of apoptosis through both the caspase-8 and -9 mediated signaling. SRL showed no or very weak binding to normal human colon tissues but strong binding to cancerous and metastatic tissues. Intratumor injection of SRL at subtoxic concentrations in NOD-SCID mice bearing HT29 xenografts resulted in total tumor regression in 9 days and no subsequent tumor recurrence. As the increased expression of TF-associated glycans is commonly seen in human cancers, SRL has the potential to be developed as a therapeutic agent for cancer.     

Indole RSK inhibitors. Part 2: optimization of cell potency and kinase selectivity

A series of inhibitors for the 90 kDa ribosomal S6 kinase (RSK) based on an 1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a]indole-8-carboxamide scaffold were optimized for cellular potency and kinase selectivity. This led to the identification of compound 24, BIX 02565, an attractive candidate for use in vitro and in vivo to explore the role of RSK as a target for the treatment heart failure.     

The antimicrobial activity of the appetite peptide hormone ghrelin

The present study examined the antimicrobial activity of the peptide ghrelin. Both major forms of ghrelin, acylated ghrelin (AG) and desacylated ghrelin (DAG), demonstrated the same degree of bactericidal activity against Gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), while bactericidal effects against Gram-positive Staphylococcus aureus (S. aureus) and Enterococcus faecalis (E. faecalis) were minimal or absent, respectively. To elucidate the bactericidal mechanism of AG and DAG against bacteria, we monitored the effect of the cationic peptides on the zeta potential of E. coli. Our results show that AG and DAG similarly quenched the negative surface charge of E. coli, suggesting that ghrelin-mediated bactericidal effects are influenced by charge-dependent binding and not by acyl modification. Like most cationic antimicrobial peptides (CAMPs), we also found that the antibacterial activity of AG was attenuated in physiological NaCl concentration (150mM). Nonetheless, these findings indicate that both AG and DAG can act as CAMPs against Gram-negative bacteria.     

A novel functional assay for fungal histidine kinases group III reveals the role of HAMP domains for fungicide sensitivity

Signal transduction systems comprising histidine kinases are suggested as new molecular targets of antibiotics. The important human fungal pathogen Candida albicans possesses three histidine kinases, one of which is the type III histidine kinase CaNik1, which activates the MAP kinase Hog1. We established a screening system for inhibitors of this class of histidine kinases by functional expression of the CaNIK1 gene in S. cerevisiae. This transformant was susceptible to fungicides to which the wild type strain was resistant, such as fludioxonil and ambruticin. Growth inhibition correlated with phosphorylation of Hog1 and was dependent on an intact Hog1 pathway. At the N-terminus the histidine kinase CaNik1 has four amino acid repeats of 92 amino acids each and one truncated repeat of 72 amino acids. Within these repeats we identified 9 HAMP domains with a paired structure. We constructed mutants in which one or two pairs of these domains were deleted. S. cerevisiae transformants expressing the full-length CaNIK1 showed the highest sensitivity to the fungicides, any truncation reduced the susceptibility of the transformants to the fungicides. This indicates that the HAMP domains are decisive for the mode of action of the antifungal compounds.