An AI-based approach for modeling the synergy between radiotherapy and immunotherapy.

Personalized, ultra-fractionated stereotactic adaptive radiotherapy (PULSAR) is designed to administer tumoricidal doses in a pulsed mode with extended intervals, spanning weeks or months. This approach leverages longer intervals to adapt the treatment plan based on tumor changes and enhance immune-modulated effects. In this investigation, we seek to elucidate the potential synergy between combined PULSAR and PD-L1 blockade immunotherapy using experimental data from a Lewis Lung Carcinoma (LLC) syngeneic murine cancer model. Employing a long short-term memory (LSTM) recurrent neural network (RNN) model, we simulated the treatment response by treating irradiation and anti-PD-L1 as external stimuli occurring in a temporal sequence. Our findings demonstrate that: (1) The model can simulate tumor growth by integrating various parameters such as timing and dose, and (2) The model provides mechanistic interpretations of a "causal relationship" in combined treatment, offering a completely novel perspective. The model can be utilized for in-silico modeling, facilitating exploration of innovative treatment combinations to optimize therapeutic outcomes. Advanced modeling techniques, coupled with additional efforts in biomarker identification, may deepen our understanding of the biological mechanisms underlying the combined treatment.

Rare discovery of sacral "ribs": a cadaveric case report.

Supernumerary ribs are a well-documented congenital anomaly that can occur at any point of the vertebral column, most commonly in the cervical or lumbar region. However, accessory ribs found in the sacrococcygeal region are exceptionally rare and may be difficult to distinguish from other bony manifestations of the pelvic girdle. During cadaveric dissection, a pair of sacral "ribs" were found projecting from the left posterolateral sacral region. The bony projections shared a broad base from the posterior sacrum. The projections followed an anteroinferior trajectory, mimicking the thoracic rib structure. Computed tomography (CT) revealed further bony anomalies, including bilateral ossifications embedded in the sacrotuberous ligament, and a blunt bony protrusion extending toward the ischial spine. Most documented supernumerary ribs in the lumbar and sacrococcygeal regions are asymptomatic and are incidental findings in radiographic studies during the exploration of other medical complaints. Correlated symptoms mentioned in the literature include pelvic pain and decreased hip range of motion, with potential obstetric complications. Owing to their asymptomatic nature, sacral ribs may be underreported. The primary aim of this report is to provide a detailed description of these sacral "ribs" in the unique setting of a cadaveric dissection supplemented with medical imaging to enhance visualization.

Absorption and Scattering of UV and Visible Light Through Simulated Martian Regoliths and Rock Samples.

Ultraviolet shielding materials are potential ecological niches for biosignatures. Finding such materials on Mars would narrow the search for potentially habitable regions. A mini-goniometer was built to collect transmission spectra as a function of scattering angle for Mars analog regoliths (JSC Mars-1, basalt, cheto bentonite, and kieserite) and crystalline rock samples from the Haughton impact structure on Devon Island, Nunavut, in the Canadian High Arctic Archipelago. The transmission through the materials was assessed at ultraviolet and visible wavelengths and at different scattering angles. From the results, it is possible to classify the samples into UV transmitters and UV quenchers. UV transmitters are materials that favor transmittance of UV wavelengths compared to photosynthetically active radiation (PAR), while the UV quenchers are materials that effectively block UV radiation from propagating into the subsurface. Additionally, samples that are effective UV quenchers tend to have more isotropic scattering profiles, whereas UV transmitters tend to favor forward scattering profiles. Samples with greater porosity had greater overall transmission. The depths at which radioresistant microorganisms can exist on present-day Mars are estimated by modeling the transmission for regoliths and crystalline rocks under martian insolation. The depth at which LD90 occurs is found to range down to 0.3 mm, while still allowing up to 1000 kJ/m2 of PAR at those depths. Due to the exceptionally protective nature of JSC Mars-1, intimate mixtures of organisms and regolith will result in some organisms experiencing orders of magnitude less UV flux than others, even when protected by only a single grain of simulant.

IL-2 delivery by engineered mesenchymal stem cells re-invigorates CD8+ T cells to overcome immunotherapy resistance in cancer.

Immune checkpoint blockade (ICB)-based immunotherapy depends on functional tumour-infiltrating lymphocytes (TILs), but essential cytokines are less understood. Here we uncover an essential role of endogenous IL-2 for ICB responsiveness and the correlation between insufficient IL-2 signalling and T-cell exhaustion as tumours progress. To determine if exogenous IL-2 in the tumour microenvironment can overcome ICB resistance, we engineered mesenchymal stem cells (MSCs) to successfully deliver IL-2 mutein dimer (SIL2-EMSC) to TILs. While MSCs have been used to suppress inflammation, SIL2-EMSCs elicit anti-tumour immunity and overcome ICB resistance without toxicity. Mechanistically, SIL2-EMSCs activate and expand pre-existing CD8+ TILs, sufficient for tumour control and induction of systemic anti-tumour effects. Furthermore, engineered MSCs create synergy of innate and adaptive immunity. The therapeutic benefits of SIL2-EMSCs were also observed in humanized mouse models. Overall, engineered MSCs rejuvenate CD8+ TILs and thus potentiate ICB and chemotherapy.

Exogenous signaling repairs defective T cell signaling inside the tumor microenvironment for better immunity.

It is known that tumor-reactive T cells are initially activated in the draining lymph node, but it is not well known whether and how tumor-infiltrating lymphocytes (TILs) are reactivated in the tumor microenvironment (TME). We hypothesize that defective T cell receptor (TCR) signaling and cosignals in the TME limit T cell reactivation. To address this, we designed a mesenchymal stromal cell-based delivery of local membrane-bound anti-CD3 and/or cosignals to explore their contribution to reactivate T cells inside the TME. Combined anti-CD3 and CD40L rather than CD80 led to superior antitumor efficacy compared with either alone. Mechanistically, TCR activation of preexisting CD8+ T cells synergized with CD40L activation of DCs inside the TME for optimum tumor control. Exogenous TCR signals could better reactivate TILs that then exited to attack distal tumors. This study supplies further evidence that TCR signaling for T cell reactivation in the TME is defective but can be rescued by proper exogenous signals.

Concurrent delivery of immune checkpoint blockade modulates T cell dynamics to enhance neoantigen vaccine-generated antitumor immunity.

Neoantigen vaccines aiming to induce tumor-specific T cell responses have achieved promising antitumor effects in early clinical trials. However, the underlying mechanism regarding response or resistance to this treatment is unclear. Here we observe that neoantigen vaccine-generated T cells can synergize with the immune checkpoint blockade for effective tumor control. Specifically, we performed single-cell sequencing on over 100,000 T cells and uncovered that combined therapy induces an antigen-specific CD8 T cell population with active chemokine signaling (Cxcr3+/Ccl5+), lower co-inhibitory receptor expression (Lag3-/Havcr2-) and higher cytotoxicity (Fasl+/Gzma+). Furthermore, generation of neoantigen-specific T cells in the draining lymph node is required for combination treatment. Signature genes of this unique population are associated with T cell clonal frequency and better survival in humans. Our study profiles the dynamics of tumor-infiltrating T cells during neoantigen vaccine and immune checkpoint blockade treatments and high-dimensionally identifies neoantigen-reactive T cell signatures for future development of therapeutic strategies.

Rejuvenation of tumour-specific T cells through bispecific antibodies targeting PD-L1 on dendritic cells.

Bispecific T-cell engagers (BiTEs) preferentially targeting tumour-associated antigens and stimulating CD3-mediated signalling are being used in patients to treat acute B-cell lymphoblastic leukemia. However, the potency of BiTEs in solid tumours is limited by their short half-life and their severe toxicity at relevant therapeutic doses. Here we report the design and in vivo performance of a bispecific antibody that simultaneously targets the murine T-cell co-receptor CD3ε and the murine immune checkpoint programmed-death ligand 1 (PD-L1). In multiple syngeneic tumour models, the bispecific antibody generated higher antitumour immune responses than conventional BiTEs targeting tumour-associated antigens and CD3ε. We found that the durable antigen-specific T-cell responses resulted from the rejuvenation of CD8 T cells, owing to the blockade of PD-L1 on dendritic cells (but not on tumour cells) and co-stimulation by B7-1&2 (a peripheral membrane protein on dendritic cells). Bispecific T-cell engagers targeting dendritic cells rather than tumour cells may represent a general means of T-cell rejuvenation for durable cancer immunotherapy.

Personalized Ultrafractionated Stereotactic Adaptive Radiotherapy (PULSAR) in Preclinical Models Enhances Single-Agent Immune Checkpoint Blockade.

PURPOSE: Harnessing the immune-stimulatory effects of radiation by combining it with immunotherapy is a promising new treatment strategy. However, more studies characterizing immunotherapy and radiation dose scheduling for the optimal therapeutic effect is essential for designing clinical trials. METHODS AND MATERIALS: A new ablative radiation dosing scheme, personalized ultrafractionated stereotactic adaptive radiation therapy (PULSAR), was tested in combination with α-PD-L1 therapy in immune-activated and resistant syngeneic immunocompetent mouse models of cancer. Specifically, tumor growth curves comparing immunotherapy and radiation therapy dose sequencing were evaluated in immunologically cold and hot tumor models. The response relative to cytotoxic killer T cells was evaluated using an α-CD8 depleting antibody, and immunologic memory was tested by tumor rechallenge of cured mice. RESULTS: We report that both radiation and immunotherapy sequencing, as well as radiation therapy fraction spacing, affect the combination treatment response. Better tumor control was achieved by giving α-PD-L1 therapy during or after radiation, and spacing fractions 10 days apart (PULSAR) achieved better tumor control than traditional daily fractions. We showed that CD8+ depleting antibody abrogated tumor control in the PULSAR combination treatment, and certain treatment schedules induced immunologic memory. CONCLUSIONS: These results illustrate that radiation therapy dosing and scheduling affect tumor control, in combination with checkpoint blockade therapies. PULSAR-style radiation dosing is more complementary in combination with single-agent immunotherapy than traditional daily fractions in this preclinical model. Preclinical investigation could prove helpful in designing clinical trials investigating combination therapy.

Antimicrobial resistance surveillance of Clostridioides difficile in Australia, 2015-18.

BACKGROUND: Clostridioides difficile was listed as an urgent antimicrobial resistance (AMR) threat in a report by the CDC in 2019. AMR drives the evolution of C. difficile and facilitates its emergence and spread. The C. difficile Antimicrobial Resistance Surveillance (CDARS) study is nationwide longitudinal surveillance of C. difficile infection (CDI) in Australia. OBJECTIVES: To determine the antimicrobial susceptibility of C. difficile isolated in Australia between 2015 and 2018. METHODS: A total of 1091 strains of C. difficile were collected over a 3 year period by a network of 10 diagnostic microbiology laboratories in five Australian states. These strains were tested for their susceptibility to nine antimicrobials using the CLSI agar incorporation method. RESULTS: All strains were susceptible to metronidazole, fidaxomicin, rifaximin and amoxicillin/clavulanate and low numbers of resistant strains were observed for meropenem (0.1%; 1/1091), moxifloxacin (3.5%; 38/1091) and vancomycin (5.7%; 62/1091). Resistance to clindamycin was common (85.2%; 929/1091), followed by resistance to ceftriaxone (18.8%; 205/1091). The in vitro activity of fidaxomicin [geometric mean MIC (GM) = 0.101 mg/L] was superior to that of vancomycin (1.700 mg/L) and metronidazole (0.229 mg/L). The prevalence of MDR C. difficile, as defined by resistance to ≥3 antimicrobial classes, was low (1.7%; 19/1091). CONCLUSIONS: The majority of C. difficile isolated in Australia did not show reduced susceptibility to antimicrobials recommended for treatment of CDI (vancomycin, metronidazole and fidaxomicin). Resistance to carbapenems and fluoroquinolones was low and MDR was uncommon; however, clindamycin resistance was frequent. One fluoroquinolone-resistant ribotype 027 strain was detected.

Small molecular drugs reshape tumor microenvironment to synergize with immunotherapy.

Recently, immune checkpoint blockade (ICB), especially anti-programmed death 1 (anti-PD-1) and anti-programmed death-ligand 1 (anti-PD-L1) therapy, has become an increasingly appealing therapeutic strategy for cancer patients. However, only a small portion of patients responds to anti-PD treatment. Therefore, treatment strategies are urgently needed to reverse the ICB-resistant tumor microenvironment (TME). It has become clear that the TME has diminished innate sensing that is critical to activate adaptive immunity. In addition, tumor cells upregulate various immunosuppressive factors to diminish the immune response and resist immunotherapy. In this review, we briefly update the current small molecular drugs that could synergize with immunotherapy, especially anti-PD therapy. We will discuss the modes of action by those drugs including inducing innate sensing and limiting immunosuppressive factors in the TME.

Author Correction: Tumor cells suppress radiation-induced immunity by hijacking caspase 9 signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Laboratory-Based Surveillance of Clostridium difficile Infection in Australian Health Care and Community Settings, 2013 to 2018.

In the early 2000s, a binary toxin (CDT)-producing strain of Clostridium difficile, ribotype 027 (RT027), caused extensive outbreaks of diarrheal disease in North America and Europe. This strain has not become established in Australia, and there is a markedly different repertoire of circulating strains there compared to other regions of the world. The C. difficile Antimicrobial Resistance Surveillance (CDARS) study is a nationwide longitudinal surveillance study of C. difficile infection (CDI) in Australia. Here, we describe the molecular epidemiology of CDI in Australian health care and community settings over the first 5 years of the study, 2013 to 2018. Between 2013 and 2018, 10 diagnostic microbiology laboratories from five states in Australia participated in the CDARS study. From each of five states, one private (representing community) and one public (representing hospitals) laboratory submitted isolates of C. difficile or PCR-positive stool samples during two collection periods per year, February-March (summer/autumn) and August-September (winter/spring). C. difficile was characterized by toxin gene profiling and ribotyping. A total of 1,523 isolates of C. difficile were studied. PCR ribotyping yielded 203 different RTs, the most prevalent being RT014/020 (n = 449; 29.5%). The epidemic CDT+ RT027 (n = 2) and RT078 (n = 6), and the recently described RT251 (n = 10) and RT244 (n = 6) were not common, while RT126 (n = 17) was the most prevalent CDT+ type. A heterogeneous C. difficile population was identified. C. difficile RT014/020 was the most prevalent type found in humans with CDI. Continued surveillance of CDI in Australia remains critical for the detection of emerging strain lineages.

Tumor cells suppress radiation-induced immunity by hijacking caspase 9 signaling.

High-dose radiation activates caspases in tumor cells to produce abundant DNA fragments for DNA sensing in antigen-presenting cells, but the intrinsic DNA sensing in tumor cells after radiation is rather limited. Here we demonstrate that irradiated tumor cells hijack caspase 9 signaling to suppress intrinsic DNA sensing. Instead of apoptotic genomic DNA, tumor-derived mitochondrial DNA triggers intrinsic DNA sensing. Specifically, loss of mitochondrial DNA sensing in Casp9-/- tumors abolishes the enhanced therapeutic effect of radiation. We demonstrated that combining emricasan, a pan-caspase inhibitor, with radiation generates synergistic therapeutic effects. Moreover, loss of CASP9 signaling in tumor cells led to adaptive resistance by upregulating programmed death-ligand 1 (PD-L1) and resulted in tumor relapse. Additional anti-PD-L1 blockade can further overcome this acquired immune resistance. Therefore, combining radiation with a caspase inhibitor and anti-PD-L1 can effectively control tumors by sequentially blocking both intrinsic and extrinsic inhibitory signaling.

Vertical and Horizontal Heterogeneity of Atmospheric Dust Loading in Northern Gale Crater, Mars.

This paper updates the record of atmospheric dust loading within northern Gale Crater, Mars, by providing line-of-sight extinction (LOS-Ext) measurements of the intervening dust between the rover and the crater rim. These measurements are derived from images taken with the Navigation Cameras (Navcam) onboard the Mars Science Laboratory (MSL) rover, Curiosity. The observations span 2.44 Mars years, from Mars Year (MY) 31 at a solar longitude (L S ) of 208° to t L S = 7° of MY34, sols 100 - 1701 of the MSL surface mission. This work examines the dataset for seasonal trends of the LOS-Ext in addition to horizontal variations and the vertical structure of LOS-Ext. The LOS-Ext has a repetitive pattern with a single peak in the latter half of the Mars year. The atmosphere in the crater is well mixed horizontally but not vertically as larger LOS-Ext is seen nearer the crater floor than at higher altitudes within the crater. The results allow a discussion on whether or not Gale Crater is a sink for atmospheric dust or a source of atmospheric dust in the current era.

Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8+ T Cell Apoptosis.

Increasing evidence demonstrates that interleukin-10 (IL-10), known as an immunosuppressive cytokine, induces antitumor effects depending on CD8+ T cells. However, it remains elusive how immunosuppressive effects of IL-10 contribute to CD8+ T cell-mediated antitumor immunity. We generated Cetuximab-based IL-10 fusion protein (CmAb-(IL10)2) to prolong its half-life and allow tumor-targeted delivery of IL-10. Our results demonstrated potent antitumor effects of CmAb-(IL10)2 with reduced toxicity. Moreover, we revealed a mechanism of CmAb-(IL10)2 preventing dendritic cell (DC)-mediated CD8+ tumor-infiltrating lymphocyte apoptosis through regulating IFN-γ production. When combined with immune checkpoint blockade, CmAb-(IL10)2 significantly improves antitumor effects in mice with advanced tumors. Our findings reveal a DC-regulating role of IL-10 to potentiate CD8+ T cell-mediated antitumor immunity and provide a potential strategy to improve cancer immunotherapy.

Surveillance for antimicrobial resistance in Australian isolates of Clostridium difficile, 2013-14.

OBJECTIVES: The objective of this study was to determine the activity of fidaxomicin and comparator antimicrobials against Clostridium difficile isolated from patients with C. difficile infection (CDI) in Australian hospitals and in the community. METHODS: One private and one public laboratory from five states in Australia submitted a total of 474 isolates/PCR-positive stool samples during three collection periods in August-September 2013 (n = 175), February-March 2014 (n = 134) and August-September 2014 (n = 165). Isolate identification was confirmed by selective culture for C. difficile and a proportion of isolates from each state were characterized by PCR for toxin genes and PCR ribotyping. MICs of fidaxomicin and eight comparator antimicrobials were determined for all isolates using agar methodology. RESULTS: Site collection yielded 440 isolates of C. difficile and PCR revealed a heterogeneous strain population comprising 37 different PCR ribotypes (RTs), 95% of which were positive for tcdA and tcdB (A+B+). The most common RTs were 014 (29.8%) and 002 (15.9%). Epidemic RT 027 was not identified; however, small numbers of virulent RTs 078 and 244 were found. Resistance to vancomycin, metronidazole and fidaxomicin was not detected and resistance to moxifloxacin was very low (3.4%). Fidaxomicin showed potent in vitro activity against all 440 isolates (MIC50/MIC90 0.03/0.12 mg/L) and was superior to metronidazole (MIC50/MIC90 0.25/0.5 mg/L) and vancomycin (MIC50/MIC90 1/2 mg/L). CONCLUSIONS: These data confirm the potent in vitro activity of fidaxomicin against C. difficile. Moreover, this study provides an important baseline for ongoing long-term surveillance of antimicrobial resistance and prospective tracking of prominent and emerging strain types.

First human case of fatal Halicephalobus gingivalis meningoencephalitis in Australia.

Halicephalobus gingivalis (previously Micronema deletrix) is a free-living nematode known to cause opportunistic infections, mainly in horses. Human infections are very rare, but all cases described to date involved fatal meningoencephalitis. Here we report the first case of H. gingivalis infection in an Australian human patient, confirmed by nematode morphology and sequencing of ribosomal DNA. The implications of this case are discussed, particularly, the need to evaluate real-time PCR as a diagnostic tool.

Impact-generated endolithic habitat within crystalline rocks of the Haughton impact structure, Devon Island, Canada.

The colonization of rocks by endolithic communities is an advantageous trait, especially in environments such as hot or cold deserts, where large temperature ranges, low water availability, and high-intensity ultraviolet radiation pose a significant challenge to survival and growth. On Mars, similar conditions (albeit more extreme) prevail. In these environments, meteorite impact structures could provide refuge for endolithic organisms. Though initially detrimental to biology, an impact event into a rocky body can favorably change the availability and habitability of a substrate for endolithic organisms, which are then able to (re)colonize microfractures and pore spaces created during the impact. Here, we show how shocked gneisses from the Haughton impact structure, Devon Island, Canada, offer significant refuge for endolithic communities. A total of 28 gneiss samples representing a range of shock states were analyzed, collected from in situ, stable field locations. For each sample, the top centimeter of rock was examined with confocal scanning laser microscopy, scanning electron microscopy, and bright-field microscopy to investigate the relationship of biomass with shock level, which was found to correlate generally with increased shock state and particularly with increased porosity. We found that gneisses, which experienced pressures between 35 and 60 GPa, provide the most ideal habitat for endolithic organisms.

Copper complexation screen reveals compounds with potent antibiotic properties against methicillin-resistant Staphylococcus aureus.

Macrophages take advantage of the antibacterial properties of copper ions in the killing of bacterial intruders. However, despite the importance of copper for innate immune functions, coordinated efforts to exploit copper ions for therapeutic interventions against bacterial infections are not yet in place. Here we report a novel high-throughput screening platform specifically developed for the discovery and characterization of compounds with copper-dependent antibacterial properties toward methicillin-resistant Staphylococcus aureus (MRSA). We detail how one of the identified compounds, glyoxal-bis(N4-methylthiosemicarbazone) (GTSM), exerts its potent strictly copper-dependent antibacterial properties on MRSA. Our data indicate that the activity of the GTSM-copper complex goes beyond the general antibacterial effects of accumulated copper ions and suggest that, in contrast to prevailing opinion, copper complexes can indeed exhibit species- and target-specific activities. Based on experimental evidence, we propose that copper ions impose structural changes upon binding to the otherwise inactive GTSM ligand and transfer antibacterial properties to the chelate. In turn, GTSM determines target specificity and utilizes a redox-sensitive release mechanism through which copper ions are deployed at or in close proximity to a putative target. According to our proof-of-concept screen, copper activation is not a rare event and even extends to already established drugs. Thus, copper-activated compounds could define a novel class of anti-MRSA agents that amplify copper-dependent innate immune functions of the host. To this end, we provide a blueprint for a high-throughput drug screening campaign which considers the antibacterial properties of copper ions at the host-pathogen interface.