A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens.

The plasma membrane is enriched for receptors and signaling proteins that are accessible from the extracellular space for pharmacological intervention. Here we conducted a series of CRISPR screens using human cell surface proteome and integrin family libraries in multiple cancer models. Our results identified ITGAV (integrin αV) and its heterodimer partner ITGB5 (integrin ß5) as the essential integrin α/ß pair for cancer cell expansion. High-density CRISPR gene tiling further pinpointed the integral pocket within the ß-propeller domain of ITGAV for integrin αVß5 dimerization. Combined with in silico compound docking, we developed a CRISPR-Tiling-Instructed Computer-Aided (CRISPR-TICA) pipeline for drug discovery and identified Cpd_AV2 as a lead inhibitor targeting the ß-propeller central pocket of ITGAV. Cpd_AV2 treatment led to rapid uncoupling of integrin αVß5 and cellular apoptosis, providing a unique class of therapeutic action that eliminates the integrin signaling via heterodimer dissociation. We also foresee the CRISPR-TICA approach to be an accessible method for future drug discovery studies.

Evaluation of two commercial enzyme-linked immunosorbent assay kits for the detection of serum antibodies against Akabane virus in cattle.

In New Zealand, an arbovirus surveillance program has been operating for more than 20 years, which includes testing of cattle with the Akabane virus neutralization test. With the aim to replace this laborious test by an easier-to-perform enzyme-linked immunosorbent assay (ELISA), 2 commercial ELISA kits, ELISA-1 from France (originally from Australia) and ELISA-2 from Japan, were compared, using 334 serum samples from noninfected New Zealand cattle, and 548 serum samples from naturally infected cattle herds in Australia. Diagnostic specificities for the test methods were high, ranging from 99.4% to 100%. The diagnostic sensitivities varied considerably between the test methods and differed from the values reported by the manufacturers (94% for each ELISA). The diagnostic sensitivities relative to the virus neutralization test (n = 378) were 96.0% for ELISA-1 or 98.9% when suspect samples were included, and 78.0% for ELISA-2. Differences in the commercial ELISA kits may be explained by the presence of other Simbu serogroup viruses in Australian cattle herds, causing cross-reactions in ELISA-1. Both commercial ELISA kits would be fit for purpose and could replace the virus neutralization test for Akabane virus surveillance in New Zealand. ELISA-1 may be able to detect other Simbu serogroup viruses, should they be present. The current study shows that despite comparable ELISA test characteristics given by the manufacturers, evaluation on the target population revealed marked differences in the ELISA kits test methods' characteristics.

Introducing samples directly into electrospray ionization mass spectrometers by direct infusion using a nanoelectrospray interface.

Procedures are described for constructing and using a microscale electrospray interface for direct infusion of samples into mass spectrometers. The sensitivity of the nanospray interface is a result of greatly reducing the flow of sample solution while preserving the analyte signal intensity. The described methodology provides a simple and robust way to analyze individual purified peptide and protein samples, i.e., samples that do not require liquid chromatography separation.

Bovine natural killer cells restrict the replication of Mycobacterium bovis in bovine macrophages and enhance IL-12 release by infected macrophages.

In this contribution, the impact of bovine natural killer (NK) cells on resistance to bovine tuberculosis was studied, using a monoclonal antibody against bovine NKp46. NK cells cultured with M. bovis-infected macrophages, but not control uninfected macrophages, proliferated and released IFN-gamma. Blood monocyte-derived macrophages were infected with virulent M. bovis, and growth of intra-macrophage bacteria was monitored by incorporation of tritiated uracil. Co-culturing infected macrophages with autologous NK cells significantly reduced the intracellular bacterial growth. Stimulation of NK cells with interleukin-2 (IL-2) enhanced further the capacity of these cells to reduce M. bovis replication in infected macrophages. NK cells from both BCG vaccinated and unvaccinated animals mediated this intra-macrophage growth restriction at similar levels. The ability of NK cells to reduce bacterial growth was independent of the release of IFN-gamma, as blocking IFN-gamma with an antibody in vitro did not affect intra-macrophage bacterial growth. NK cells reduced bacterial growth and also increased macrophage release of interleukin-12 (IL-12) and nitric oxide (NO) production by M. bovis-infected macrophages. Neutralizing NO production by macrophages in vitro with mono-methyl-l-arginine (MMLA) did not abrogate the ability of NK cells to decrease bacterial growth in infected macrophages. Reduction of mycobacterial intra-macrophage growth by NK cells was dependent on direct contact between NK cells and infected macrophages. Supernatants from NK cells failed to impact significantly on M. bovis replication in infected macrophages. The reduction in bacterial growth in macrophages correlated with the induction of an apoptosis program in infected macrophages. Cell death occurred at a similar rate in infected macrophages, exposed to NK cells or not. We conclude that bovine NK cells are stimulated by and release IFN-gamma in response to infected cells and reduce M. bovis growth in infected macrophages by an unclear mechanism, and are potentially involved in innate resistance of cattle to tuberculosis.

Susceptibility of brushtail possums (Trichosurus vulpecula) infected with Mycobacterium bovis is associated with a transient macrophage activation profile.

The Australian brushtail possums are highly susceptible to Mycobacterium bovis and are the principal wildlife reservoir of M. bovis in New Zealand. To better understand the disease process in these animals, brushtail possums were infected by the aerosol route with a virulent strain of M. bovis, and immune parameters measured. M. bovis replicated actively in the lungs of infected animals. Animals began developing macroscopic lung lesions at 4 and 5 weeks following infection, with some lesions appearing in the livers and spleens. Infection determined the emergence of blood lymphocytes which proliferated in response to bovine purified protein derivative from M. bovis (PPD-b) at 3, 4 and 5 weeks. The response to a mitogen (Concanavalin A) waned progressively with time. Infection was associated with a modest increase in the numbers of free lung cells. Nitrite was detectable in the lavage fluids of infected animals at 3 weeks postinfection, but not at 4 and 5 weeks. Macrophage activation in the lungs was evident as alveolar macrophages produced more oxidants, significant levels of nitric oxide (NO), as well as tumor necrosis factor alpha (TNF-alpha) bioactivity at 3 weeks postinfection. However, macrophages from infected animals lost the ability to generate nitrite- and TNF-alpha generation was depressed at 4 and 5 weeks postinfection, the time at which macroscopic lesions in the lungs became apparent. Alveolar macrophages from animals at 3 weeks postinfection blocked the replication of M. bovis in part via a NO-dependent mechanism, and were more refractory for M. bovis growth than cells from naïve animals to bacterial replication. Alveolar macrophages from animals at 4 and 5 weeks postinfection allowed substantial replication of M. bovis, and no NO-dependent bacteriostatic activity was apparent. Introduction of autologous lymphocytes from the blood of infected animals in co-cultures rendered infected macrophages more resistant to M. bovis replication. We conclude that M. bovis infection in brushtail possums is associated with a transient activation of alveolar macrophages, although in vitro exposure to sensitized T cells can enhance this profile.

Oral vaccination with Mycobacterium bovis BCG in a lipid formulation induces resistance to pulmonary tuberculosis in brushtail possums.

A method was developed for formulating Mycobacterium bovis bacille Calmette-Guerin (BCG) for oral vaccination against tuberculosis. Selected lipid-based formulations of BCG were tested in the brushtail possum for their ability to elicit immune responses and protection against bovine tuberculosis. Formulation of BCG in lipid matrices maintained bacteria in a dormant but viable state. Oral delivery of 2 x 10(8) colony forming units of formulated BCG to possums induced strong lymphocyte proliferation responses to bovine purified protein derivative (PPD) in peripheral blood lymphocytes. Oral vaccination of possums also reduced the severity of disease following aerosol challenge with virulent M. bovis compared with animals vaccinated with non-formulated BCG. In a second experiment, levels of protection with lipid-formulated oral BCG were similar to those seen with subcutaneous BCG vaccination. Our data shows that formulated oral BCG is an efficient means of inducing protection against bovine tuberculosis in possums and should be a practical means of vaccinating wildlife against tuberculosis.

A DNA prime-Mycobacterium bovis BCG boost vaccination strategy for cattle induces protection against bovine tuberculosis.

The variable efficacy of bacillus Calmette-Guérin (Mycobacterium bovis BCG) in protecting humans and cattle against tuberculosis has prompted a search for a more effective vaccination regimen. A prime-boost strategy was investigated in cattle naturally sensitized to environmental mycobacteria by using a combination of three DNA vaccines coding for Hsp 65, Hsp 70, and Apa for priming, followed by a boost with BCG prior to experimental challenge with virulent M. bovis. Controls were vaccinated with DNA or BCG alone or were not vaccinated. The immune responses were monitored throughout the study, and protection was assessed based on reductions in the numbers of lesions and viable mycobacteria in lymph node samples. Vaccination with BCG alone or with a DNA prime-BCG boost regimen induced high levels of antigen-specific gamma interferon (IFN-gamma) in whole-blood cultures. In the prime-boost group there were fewer animals with severe lung lesions, fewer lymph nodes with lesions per animal, a smaller proportion of animals with lesions, lower mean lung and lymph node lesion scores, and less M. bovis isolated from retropharyngeal and thoracic lymph nodes compared to the results obtained for the nonvaccinated animals. The prime-boost regimen induced significant enhancement of protection in six parameters, compared with significant enhancement of protection in only two parameters for BCG alone. In addition, following challenge, in vitro IFN-gamma responses against ESAT-6 and CFP-10, as well as bovine tuberculin-induced skin test and in vitro IFN-gamma responses, were identified as immunological markers that predicted protection. The use of the prime-boost strategy suggested that a combination of vaccines may be better than a single vaccine for protection against tuberculosis.