Atomically accurate de novo design of single-domain antibodies.

Despite the central role that antibodies play in modern medicine, there is currently no way to rationally design novel antibodies to bind a specific epitope on a target. Instead, antibody discovery currently involves time-consuming immunization of an animal or library screening approaches. Here we demonstrate that a fine-tuned RFdiffusion network is capable of designing de novo antibody variable heavy chains (VHH's) that bind user-specified epitopes. We experimentally confirm binders to four disease-relevant epitopes, and the cryo-EM structure of a designed VHH bound to influenza hemagglutinin is nearly identical to the design model both in the configuration of the CDR loops and the overall binding pose.

Extracellular Delivery of Functional Mitochondria Rescues the Dysfunction of CD4+ T Cells in Aging.

Mitochondrial dysfunction alters cellular metabolism, increases tissue oxidative stress, and may be principal to the dysregulated signaling and function of CD4+ T lymphocytes in the elderly. In this proof of principle study, it is investigated whether the transfer of functional mitochondria into CD4+ T cells that are isolated from old mice (aged CD4+ T cells), can abrogate aging-associated mitochondrial dysfunction, and improve the aged CD4+ T cell functionality. The results show that the delivery of exogenous mitochondria to aged non-activated CD4+ T cells led to significant mitochondrial proteome alterations highlighted by improved aerobic metabolism and decreased cellular mitoROS. Additionally, mito-transferred aged CD4+ T cells showed improvements in activation-induced TCR-signaling kinetics displaying markers of activation (CD25), increased IL-2 production, enhanced proliferation ex vivo. Importantly, immune deficient mouse models (RAG-KO) showed that adoptive transfer of mito-transferred naive aged CD4+ T cells, protected recipient mice from influenza A and Mycobacterium tuberculosis infections. These findings support mitochondria as targets of therapeutic intervention in aging.

Selective delipidation of Mycobacterium bovis BCG retains antitumor efficacy against non-muscle invasive bladder cancer.

PURPOSE: Repeated instillations of bacillus Calmette et Guérin (BCG) are the gold standard immunotherapeutic treatment for reducing recurrence for patients with high-grade papillary non-muscle invasive bladder cancer (NMIBC) and for eradicating bladder carcinoma-in situ. Unfortunately, some patients are unable to tolerate BCG due to treatment-associated toxicity and bladder removal is sometimes performed for BCG-intolerance. Prior studies suggest that selectively delipidated BCG (dBCG) improves tolerability of intrapulmonary delivery reducing tissue damage and increasing efficacy in preventing Mycobacterium tuberculosis infection in mice. To address the lack of treatment options for NMIBC with BCG-intolerance, we examined if selective delipidation would compromise BCG's antitumor efficacy and at the same time increase tolerability to the treatment. MATERIALS AND METHODS: Murine syngeneic MB49 bladder cancer models and in vitro human innate effector cell cytotoxicity assays were used to evaluate efficacy and immune impact of selective delipidation in Tokyo and TICE BCG strains. RESULTS: Both dBCG-Tokyo and dBCG-TICE effectively treated subcutaneous MB49 tumors in mice and enhanced tumor-infiltrating CD8+ T and natural killer cells, similar to conventional BCG. However, when compared to conventional BCG, only dBCG-Tokyo retained a significant effect on intratumoral tumor-specific CD8+ and γδ T cells by increasing their frequencies in tumor tissue and their production of antitumoral function-related cytokines, i.e., IFN-γ and granzyme B. Further, dBCG-Tokyo but not dBCG-TICE enhanced the function and cytotoxicity of innate effector cells against human bladder cancer T24 in vitro. CONCLUSIONS: These data support clinical investigation of dBCG-Tokyo as a treatment for patients with BCG-intolerant NMIBC.

Blood RNA signatures predict recent tuberculosis exposure in mice, macaques and humans.

Tuberculosis (TB) is the leading cause of death due to a single infectious disease. Knowing when a person was infected with Mycobacterium tuberculosis (M.tb) is critical as recent infection is the strongest clinical risk factor for progression to TB disease in immunocompetent individuals. However, time since M.tb infection is challenging to determine in routine clinical practice. To define a biomarker for recent TB exposure, we determined whether gene expression patterns in blood RNA correlated with time since M.tb infection or exposure. First, we found RNA signatures that accurately discriminated early and late time periods after experimental infection in mice and cynomolgus macaques. Next, we found a 6-gene blood RNA signature that identified recently exposed individuals in two independent human cohorts, including adult household contacts of TB cases and adolescents who recently acquired M.tb infection. Our work supports the need for future longitudinal studies of recent TB contacts to determine whether biomarkers of recent infection can provide prognostic information of TB disease risk in individuals and help map recent transmission in communities.

Communication pitfalls in interpreted genetic counseling sessions.

The impact of language interpretation on interactive communication in genetic counseling sessions is not well studied. We sought to determine whether interpretation affects communication in genetic counseling sessions using communication analysis. With a sample of pregnant patients of advanced maternal age, we audiotaped, analyzed, and compared seven Spanish-speaking patients with limited-English proficiency to seven English-speaking patients on length of session, English words spoken, and number of questions asked. An analysis of verbal listening cues, including back-channels and questions asked was performed to evaluate the level of engagement by both provider and patient. Session length did not differ between groups (p > 0.05), however, English-speaking sessions involved significantly more words (mean: 4,798 vs. 2,524) exchanged over the course of the conversation than interpreter-mediated sessions (p < 0.001). The number of back-channeling responses was significantly greater in English-speaking compared to Spanish-speaking sessions. We found the same information was covered, but there was less provider-patient interaction when the session was interpreter-mediated. The patient asked fewer questions and the genetic counselor spoke in condensed ideas. Overall, our observations suggest diminished levels of interactive communication in interpreter-mediated sessions. Our work highlights the need for further evaluation in how genetic counselors communicate during interpreter-mediated sessions.

The Lung Mucosa Environment in the Elderly Increases Host Susceptibility to Mycobacterium tuberculosis Infection.

As we age, there is an increased risk for the development of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection. Few studies consider that age-associated changes in the alveolar lining fluid (ALF) may increase susceptibility by altering soluble mediators of innate immunity. We assessed the impact of adult or elderly human ALF during Mtb infection in vitro and in vivo. We identified amplification of pro-oxidative and proinflammatory pathways in elderly ALF and decreased binding capability of surfactant-associated surfactant protein A (SP-A) and surfactant protein D (SP-D) to Mtb. Human macrophages infected with elderly ALF-exposed Mtb had reduced control and fewer phagosome-lysosome fusion events, which was reversed when elderly ALF was replenished with functional SP-A/SP-D. In vivo, exposure to elderly ALF exacerbated Mtb infection in young mice. Our studies demonstrate how the pulmonary environment changes as we age and suggest that Mtb may benefit from declining host defenses in the lung mucosa of the elderly.

Altered monocyte phenotypes but not impaired peripheral T cell immunity may explain susceptibility of the elderly to develop tuberculosis.

Tuberculosis (TB) is the leading killer due to a single infectious disease worldwide. With the aging of the global population, the case rate and deaths due to TB are highest in the elderly population. While general immunosenescence associated with old age is thought to contribute to the susceptibility of the elderly to develop active TB disease, very few studies of immune function in elderly individuals with Mycobacterium tuberculosis (M.tb) infection or disease have been performed. In particular, impaired adaptive T cell immunity to M.tb is one proposed mechanism for the elderly's increased susceptibility primarily on the basis of the decreased delayed type hypersensitivity response to tuberculin-purified protein derivative in the skin of elderly individuals. To investigate immunological reasons why the elderly are susceptible to develop active TB disease, we performed a cross-sectional observational study over a five year period (2012-2016) enrolling participants from 2 age groups (adults: 25-44 years; elderly: 65 and older) and 3 M.tb infection statuses (active TB, latent TB infection, and healthy controls without history of M.tb infection). We hypothesized that impaired peripheral T cell immunity plays a role in the biological susceptibility of the elderly to TB. Contrary to our hypothesis, we observed no evidence of impaired M.tb specific T cell frequency or altered production of cytokines implicated in M.tb control (IFN-γ, IL-10) in peripheral blood in the elderly. Instead, we observed alterations in monocyte proportion and phenotype with age and M.tb infection that suggest their potential role in the susceptibility of the elderly to develop active TB. Our results suggest a potential link between the known widespread low-grade systemic inflammation of old age, termed "inflammaging," with the elderly's specific susceptibility to developing active TB. Moreover, our results highlight the need for further research into the biological reasons why the elderly are more susceptible to disease and death from TB, so that public health systems can be better equipped to face the present and future problem of TB in an aging global population.

A comprehensive catalog of predicted functional upstream open reading frames in humans.

Upstream open reading frames (uORFs) latent in mRNA transcripts are thought to modify translation of coding sequences by altering ribosome activity. Not all uORFs are thought to be active in such a process. To estimate the impact of uORFs on the regulation of translation in humans, we first circumscribed the universe of all possible uORFs based on coding gene sequence motifs and identified 1.3 million unique uORFs. To determine which of these are likely to be biologically relevant, we built a simple Bayesian classifier using 89 attributes of uORFs labeled as active in ribosome profiling experiments. This allowed us to extrapolate to a comprehensive catalog of likely functional uORFs. We validated our predictions using in vivo protein levels and ribosome occupancy from 46 individuals. This is a substantially larger catalog of functional uORFs than has previously been reported. Our ranked list of likely active uORFs allows researchers to test their hypotheses regarding the role of uORFs in health and disease. We demonstrate several examples of biological interest through the application of our catalog to somatic mutations in cancer and disease-associated germline variants in humans.