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The in vitro effect of nine antibiotic combinations was investigated in Staphylococcus epidermidis biofilms using ATP-bioluminescence for viable bacterial cell quantification. Four slime-producing (SP) strains were used to form biofilms 6, 24 and 48 h old. These biofilms were exposed for 24 h to antibiotics at 4 x, 2 x, 1 x and 0.5 x MIC. Combinations involving tetracycline together with another antibiotic were the most efficient at the biofilm age and concentration range under study. The combination vancomycin-rifampicin produced the highest bactericidal effect on 6 h biofilms at 4 x MIC, but this effect decreased dramatically in older biofilms. To detect possible antibiotic synergy in combinations that had a significant killing effect, antibiotics were studied not only in combination but also individually. Synergic effects were observed in all the combinations tested. Differences between the effect in combination and the sum of individual antibiotic effects (degree of synergy) were significant (mostly P< 0.001) and exceeded 1 log10 cfu/mL in the majority of cases. In 48 h biofilms, antibiotics caused a significant bactericidal effect when applied in combination, but never when used individually. These results indicate that the biofilm test applied allows the detection of synergy between antibiotics and suggests that this assay could be useful in clinical and extensive synergy studies on S. epidermidis biofilms. [ABSTRACT FROM AUTHOR]

Four slime-producing isolates of Staphylococcus aureus were used in an antibiotic susceptibility assay for biofilms developed on 96-well polystyrene tissue culture plates. The study involved 11 antibiotics, two biofilm ages (6 and 48 h), two biofilm growth media (tryptone soy broth (TSB) and delipidated milk) and three antibiotic concentrations (4 x MBC, 100 mg/L and 500 mg/L). ATP-bioluminescence was used for automated bacterial viability determination after a 24 h exposure to antibiotics, to avoid biofilm handling. Under the conditions applied, viability in untreated biofilms (controls) was lower when biofilm growth was attempted in milk rather than in TSB. Various antibiotics had a grater effect on viability when used at higher ( 100 mg/L) antibiotic concentrations and on younger (6 h) biofilms. Increased antibiotic effect was observed in milk-grown rather than TSB-grown biofilms. Phosphomycin and defuroxime, followed by rifampicin, cefazolin, novobiocin, vancomycin, penicillin, ciprofloxacin and tobramycin significantly affected biofilm cell viability at least under some of the conditions tested. Gentamicin and erythromycin had a non-significant effect on cell viability. Transmission electron microscopy revealed that cells at the inner biofilm layers tend to remain intact after antibiotic treatment and that TSB-grown biofilms favoured a uniformity of cell distribution and increased cell density in comparison with milk-grown biofilms. A reduced matrix distribution and enhanced cell density were observed as the biofilm aged. The S. aureus biofilm test discriminated antibiotics requiring shorter (3 h or 6 h) from those requiring longer (24 h) exposure and yielded results which may be complementary to those obtained by conventional tests. [ABSTRACT FROM PUBLISHER]

Three cases of pulmonary hypertension caused by hydatid emboli from the right side of the heart are described; cardiac catheterisation was performed in 2. One case was confirmed at operation and 2 at necropsy. The pulmonary emboli were caused by hydatid vesicles in all 3 cases and in none was there pulmonary thrombosis; free scolices were found in the pulmonary alveoli in 2. In 1 patient with repeated syncopal attacks there was a pedunculated cyst in the right atrium which was though to have intermittently obstructed the tricuspid valve. Gamma radiography, angiocardiography, and necropsy suggested a mechanical cause for the pulmonary hypertenion with no vasoconstrictive element. The surgical patient was alive and well 18 months later. [ABSTRACT FROM PUBLISHER]

Expression of the long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 ( MALAT1 ) correlates with tumor progression and metastasis in many tumor types. However, the impact and mechanism of action by which MALAT1 promotes metastatic disease remain elusive. Here, we used CRISPR activation (CRISPRa) to overexpress MALAT1/Malat1 in patient-derived lung adenocarcinoma (LUAD) cell lines and in the autochthonous K-ras/p53 LUAD mouse model. Malat1 overexpression was sufficient to promote the progression of LUAD to metastatic disease in mice. Overexpression of MALAT1/Malat1 enhanced cell mobility and promoted the recruitment of protumorigenic macrophages to the tumor microenvironment through paracrine secretion of CCL2/Ccl2. Ccl2 up-regulation was the result of increased global chromatin accessibility upon Malat1 overexpression. Macrophage depletion and Ccl2 blockade counteracted the effects of Malat1 overexpression. These data demonstrate that a single lncRNA can drive LUAD metastasis through reprogramming of the tumor microenvironment.

The limited efficacy of currently approved immunotherapies in EGFR-driven lung adenocarcinoma (LUAD) underscores the need to better understand alternative mechanisms governing local immunosuppression to fuel novel therapies. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophage (TA-AM) proliferation which supports tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases proinflammatory immune responses. These results reveal new therapeutic combinations for immunotherapy resistant EGFR-mutant LUADs and demonstrate how cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth.

Acquired resistance to tyrosine kinase inhibitors (TKI), such as osimertinib used to treat EGFR-mutant lung adenocarcinomas, limits long-term efficacy and is frequently caused by non-genetic mechanisms. Here, we define the chromatin accessibility and gene regulatory signatures of osimertinib sensitive and resistant EGFR-mutant cell and patient-derived models and uncover a role for mammalian SWI/SNF chromatin remodeling complexes in TKI resistance. By profiling mSWI/SNF genome-wide localization, we identify both shared and cancer cell line-specific gene targets underlying the resistant state. Importantly, genetic and pharmacologic disruption of the SMARCA4/SMARCA2 mSWI/SNF ATPases re-sensitizes a subset of resistant models to osimertinib via inhibition of mSWI/SNF-mediated regulation of cellular programs governing cell proliferation, epithelial-to-mesenchymal transition, epithelial cell differentiation, and NRF2 signaling. These data highlight the role of mSWI/SNF complexes in supporting TKI resistance and suggest potential utility of mSWI/SNF inhibitors in TKI-resistant lung cancers., Competing Interests: Declaration of interests C.K. is the Scientific Founder, Scientific Advisor to the Board of Directors, Scientific Advisory Board member, shareholder, and consultant for Foghorn Therapeutics, Inc. (Cambridge, MA), serves on the Scientific Advisory Boards of Nereid Therapeutics, Nested Therapeutics, Accent Therapeutics, and Fibrogen, Inc. and is a consultant for Cell Signaling Technologies and Google Ventures. C.K. is also a member of the Molecular Cell and Cell Chemical Biology Editorial Boards. D.L. Rimm reports grants and personal fees from Amgen, Astra Zeneca, Cepheid, Konica – Minolta, Lilly, NextCure and personal fees from Cell Signaling Technology, Danaher, Fluidigm, GSK, Merck, Monopteros, NanoString, Odonate, Paige.AI, Regeneron, Roche, Sanofi, Ventana, Verily. K. Politi reports grants from the NCI/NIH; grants and personal fees from AstraZeneca; grants from Kolltan, Roche/Genentech, Boehringer Ingelheim, D2G Oncology and Symphogen; and personal fees from Janssen, Dynamo Therapeutics, Halda, Maverick Therapeutics, and Tocagen; and a patent for EGFR(T790M) mutation testing issued, licensed, and with royalties paid from Molecular Diagnostics/Memorial Sloan Kettering Cancer Center. P.A.J. is an equity owner in Gatekeeper Pharmaceuticals; consults for AstraZeneca, Boehringer Ingelheim, Pfizer, Roche/Genentech, Chugai Pharmaceuticals, Eli Lilly Pharmaceuticals, Araxes Pharmaceuticals, SFJ Pharmaceuticals, Voronoi, Daiichi Sankyo, Biocartis, Novartis, Sanofi, Takeda Oncology, Mirati Therapeutics, Transcenta, Silicon Therapeutics, Syndax, Nuvalent, Bayer, Esai, Allorion Therapeutics, Accutar Biotech, and Abbvie; receives research support from AstraZeneca, Daiichi Sankyo, PUMA, Eli Lilly, Boehringer Ingelheim, Revolution Medicines, and Takeda Oncology and is a co-inventor and receives postmarketing royalties on a DFCI owned patent on EGFR mutations licensed to LabCorp. Q.Y. reports grants and personal fees from AstraZeneca, and is a Scientific Advisory Board member of AccuraGen Inc. D.X.N received research funding from AstraZeneca. The other authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Immunotherapy resistance in non-small cell lung cancer (NSCLC) may be mediated by an immunosuppressive microenvironment, which can be shaped by the mutational landscape of the tumor. Here, we observed genetic alterations in the PTEN/PI3K/AKT/mTOR pathway and/or loss of PTEN expression in >25% of patients with NSCLC, with higher frequency in lung squamous carcinomas (LUSC). Patients with PTEN-low tumors had higher levels of PD-L1 and PD-L2 and showed worse progression-free survival when treated with immunotherapy. Development of a Pten-null LUSC mouse model revealed that tumors with PTEN loss were refractory to antiprogrammed cell death protein 1 (anti-PD-1), highly metastatic and fibrotic, and secreted TGFβ/CXCL10 to promote conversion of CD4+ lymphocytes into regulatory T cells (Treg). Human and mouse PTEN-low tumors were enriched in Tregs and expressed higher levels of immunosuppressive genes. Importantly, treatment of mice bearing Pten-null tumors with TLR agonists and anti-TGFβ antibody aimed to alter this immunosuppressive microenvironment and led to tumor rejection and immunologic memory in 100% of mice. These results demonstrate that lack of PTEN causes immunotherapy resistance in LUSCs by establishing an immunosuppressive tumor microenvironment that can be reversed therapeutically., Significance: PTEN loss leads to the development of an immunosuppressive microenvironment in lung cancer that confers resistance to anti-PD-1 therapy, which can be overcome by targeting PTEN loss-mediated immunosuppression., (©2023 American Association for Cancer Research.)

Conditional ablation of defined cell populations in vivo can be achieved using genetically engineered mice in which the human diphtheria toxin (DT) receptor (DTR) is placed under control of a murine tissue-specific promotor, such that delivery of DT selectively ablates cells expressing this high-affinity human DTR; cells expressing only the endogenous low-affinity mouse DTR are assumed to be unaffected. Surprisingly, we found that systemic administration of DT induced rapid regression of murine lung adenocarcinomas that express human mutant EGFR in the absence of a transgenic allele containing human DTR. DT enzymatic activity was required for tumor regression, and mutant EGFR-expressing tumor cells were the primary target of DT toxicity. In FVB mice, EGFR-mutant tumors upregulated expression of HBEGF, which is the DTR in mice and humans. HBEGF blockade with the enzymatically inactive DT mutant CRM197 partially abrogated tumor regression induced by DT. These results suggest that elevated expression of murine HBEGF, i.e. the low-affinity DTR, confers sensitivity to DT in EGFR-mutant tumors, demonstrating a biological effect of DT in mice lacking transgenic DTR alleles and highlighting a unique vulnerability of EGFR-mutant lung cancers., Competing Interests: Competing interests S.M.K. is Academic Editor at the Journal of Experimental Medicine and has a consultant/advisory board relationship with Celsius Therapeutics and Evolveimmune. K.P. reports receiving commercial research grants (to the Institution) from AstraZeneca, Boehringer Ingelheim and Roche/Genentech; has an ownership interest (including patents) in MSKCC/Molecular MD; and has an unpaid consultant/advisory board relationship with the Lung Cancer Research Foundation. No competing interests are declared by other authors., (© 2021. Published by The Company of Biologists Ltd.)

Patient-derived xenografts are a useful tool in cancer immunology, as they allow researchers to study human cancers in vivo when starting with a relatively small amount of human tumor tissue. These models make it possible to study tumor cell-intrinsic changes that occur in response to external stimuli including cytokines like interferon gamma (IFNγ) that are important for effective anti-tumor immune responses. IFNγ responsiveness can be measured by assessing surface expression of MHC class I on tumor cells, the molecule on which tumor antigens are presented to cytotoxic T cells in the tumor microenvironment. Low levels of MHC class I and lack of responsiveness have been associated with resistance to T-cell directed therapies like immune checkpoint inhibitors. In this chapter, we present a protocol for an assay to screen patient-derived xenografts for their responsiveness to IFNγ. The results of this assay can be used as a starting point for uncovering cancer cell-intrinsic mechanisms of resistance to immunotherapies in patient tumors., (© 2020 Elsevier Inc. All rights reserved.)

Background: Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) like erlotinib are effective for treating patients with EGFR mutant lung cancer; however, drug resistance inevitably emerges. Approaches to combine immunotherapies and targeted therapies to overcome or delay drug resistance have been hindered by limited knowledge of the effect of erlotinib on tumor-infiltrating immune cells., Methods: Using mouse models, we studied the immunological profile of mutant EGFR-driven lung tumors before and after erlotinib treatment., Results: We found that erlotinib triggered the recruitment of inflammatory T cells into the lungs and increased maturation of alveolar macrophages. Interestingly, this phenotype could be recapitulated by tumor regression mediated by deprivation of the EGFR oncogene indicating that tumor regression alone was sufficient for these immunostimulatory effects. We also found that further efforts to boost the function and abundance of inflammatory cells, by combining erlotinib treatment with anti-PD-1 and/or a CD40 agonist, did not improve survival in an EGFR-driven mouse model., Conclusions: Our findings lay the foundation for understanding the effects of TKIs on the tumor microenvironment and highlight the importance of investigating targeted and immuno-therapy combination strategies to treat EGFR mutant lung cancer.

Senescence is an important p53-controlled tumor suppressor program that not only opposes the proliferation of cancer cells but also promotes their immune-mediated clearance in certain contexts. In hepatocellular cancer, p53 induction promotes an innate immune cell-mediated clearance of senescent cells wherein natural killer (NK) cells seem to play the primary sentinel role. Whether NK cells also surveil cancer cells in other tumor types when p53 is activated to promote a senescence response is unknown. To identify the role that NK and other innate immune cell types have on the surveillance and destruction of lung adenocarcinoma cells, we developed an orthotopic transplantation model where p53 gene function could be restored to induce senescence after successful engraftment of tumor cells in the mouse lung. Contrary to precedent, we found that NK cells actually limited the efficient clearance of tumor cells from the mouse lung after p53 restoration. Instead, activation of p53 induced the infiltration of monocytes, neutrophils, and interstitial macrophages. Loss of NK cells further promoted expansion of these inflammatory cell types and tumor clearance after p53 restoration. These observations suggest that NK cell responses to p53 activation in lung adenocarcinoma is distinct from those found in other tumor types and that diverse innate immune cell populations may play context-dependent roles during tumor immune surveillance. Further, our data provide an impetus to understand the broader mechanisms that regulate cancer cell destruction by multiple cell types of the innate immune system and distinct cancer contexts.

Synthetic biological tools that enable precise regulation of gene function within in vivo systems have enormous potential to discern gene function in diverse physiological settings. Here we report the development and characterization of a synthetic gene switch that, when targeted in the mouse germline, enables conditional inactivation, reports gene expression and allows inducible restoration of the targeted gene. Gene inactivation and reporter expression is achieved through Cre-mediated stable inversion of an integrated gene-trap reporter, whereas inducible gene restoration is afforded by Flp-dependent deletion of the inverted gene trap. We validate our approach by targeting the p53 and Rb genes and establishing cell line and in vivo cancer model systems, to study the impact of p53 or Rb inactivation and restoration. We term this allele system XTR, to denote each of the allelic states and the associated expression patterns of the targeted gene: eXpressed (XTR), Trapped (TR) and Restored (R).

We investigated whether TCRs restricted to the more ubiquitously expressed MHC class I molecules could be used to redirect human regulatory T cells (Tregs). Using a series of HLA-A2-restricted TCRs that recognize the same peptide-MHC class I complex (pMHC) with affinities varying up to 3500 fold, we observed that TCR affinity had no effect on the ability of the introduced TCRs to confer potent Ag-specific suppressive activity. Surprisingly, we found a naturally occurring, low-affinity MHC class I-restricted TCR specific for an NY-ESO-1 epitope that was unable to redirect a functional CD4 T-effector cell response could confer potent antigen-specific suppressive activity when expressed in Tregs and severely impair the expansion of highly functional HIV-1(GAG)-specific CD8 T cells expressing a high-affinity TCR. This suppressive activity was only observed when both Ags were presented by the same cell, and no suppression was observed when the target Ags were put in distinct cells. These studies underscore the clinical utility of using MHC class I-restricted TCRs to endow Tregs with specificity to control autoimmune disease and highlight the conditions in which this approach would have most therapeutic benefit.

The in vitro killing effect of widely used antibiotics (cephalothin, clindamycin, erythromycin, ofloxacin, rifampicin, teicoplanin, tetracycline, phosphomycin and vancomycin) was comparatively analyzed in this study on 24-h biofilms of 64 Staphylococcus epidermidis clinical isolates. This effect was assessed at the expected antibiotic concentration reached in serum, using ATP-bioluminescence. Erythromycin, rifampicin, tetracycline and phosphomycin presented generally a higher killing effect than vancomycin, clindamycin, cephalothin, teicoplanin and ofloxacin in these biofilms. Differences in the resistance profiles obtained in classical assays (broth microdilution and diffusion) did not help to predict differences in the killing effect of the antibiotics in biofilms. Only some antibiotics (vancomycin but not rifampicin or tetracycline) highly decreased their killing effect as the biofilm age increased (from 6 to 24 or 48 h). These studies underline the relevance of biofilm susceptibility testing and the potential danger of the indiscriminate use of vancomycin monotherapy as the ultimate resource against infections involving aged biofilms.

Introduction: The outcome of cases of near-drowning and initially poor prognostic signs are usually discouraging because of the severity of the consequent encephalopathy in most survivors. However, good recovery has been described, in spite of bad prognostic factors initially. It is difficult to establish the predictors of poor outcome which would enable one to decide when to establish and maintain advanced cardio-pulmonary resuscitation measures (CPR), since each case of near-drowning is different., Clinical Case: A four year old boy survived near-drowning in cold water without sequelas but with initial signs of very poor prognosis, including prolonged immersion time, coma, severe metabolic acidosis, hyperglycemia and persistent bilateral arreactive mydriasis., Discussion: The beneficial effect of hypothermia is well known, and explains (at least partially) survival in cases of apparently irreversible near-drowning. Potential benefits are reduced metabolic demand which prevents the adverse effects of hypoxia and the 'diving reflex' which short-circuits the blood supply to vital organs such as the brain and heart. We consider that the persistently arreactive pupils were not due to hypoxia, but rather to bilateral uncal compression of the third cranial nerves due to cerebral edema secondary to initial hypoxia and water intoxication., Conclusion: This observation is yet another argument for the establishment and maintenance of aggressive manoeuvers of CPR and treatment in all children who have nearly-drowned, independently of the apparent seriousness or irreversibility.

A rat experimental osteomyelitis model was used to study the efficiency of antibiotics on biofilm bacteria adhered to implants in relation to the efficiency obtained in vitro. In the osteomyelitis model, 10(4) bacteria of the strain variant used for the in vitro studies (a slime-producing variant of Staphylococcus aureus) were inoculated into the rat tibia at surgery, after implanting a stainless steel canula precolonized for 12 h with this strain. After 5 weeks, a 21-day antibiotic treatment was applied (using cefuroxime, vancomycin, or tobramycin). Subsequently, implant and tibia were studied for presence of bacteria. In this osteomyelitis model, cefuroxime inhibited bone colonization and reduced the number of bacteria in metal and bone at a higher degree (P < 0.05) than vancomycin and trobramycin (the latter antibiotic did not have this reduction effect). The in vitro assay was applied using three concentrations of each antibiotic (8, 100, and 500 microg/ml) and 6-, 24-, and 48-h biofilms. Bacterial viability was evaluated by ATP-bioluminescence after 24 h of antibiotic treatment. In this in vitro assay, cefuroxime significantly (P < 0.05) reduced in all cases the number of viable bacteria in biofilms, tobramycin did not affect viability, and vancomycin affected viability except at the lowest concentration used (8 microg/ml, i.e., 8x the minimal bactericidal concentration of this antibiotic) when facing the oldest (48 h) biofilm. These results demonstrate the usefulness of the osteomyelitis model applied in providing evidence for a close correlation between the in vitro and in vivo findings on the effect of three antibiotics under study., (Copyright 1998 Academic Press.)

The risk of bacterial infection through allogeneic bone transplantation is one of the problems facing tissue banks. The purpose of this study is to report the contamination rate in 987 grafts obtained under strictly aseptic conditions, between 1989 and 1992. The grafts were stored at -80 degrees C (cortical bone and tendons) and -40 degrees C (cancellous bone). The overall contamination rate was 6.6%, with Gram-positive bacteria responsible for 80% of the positive cultures. We discuss the sources of contamination, the most frequently isolated bacteria and the steps in the donation and transplantation procedures that help to reduce the risk of contamination. We conclude that the methods of procurement, processing and storage of tissues are effective in making sterile allografts available.

A clinical case characterized by renal amyloidosis, Crohn's disease of the colon and rectum, multiple chronic anal fistulae and ankylosing spondylitis is reported. This association has been infrequently recognized. In this patient Crohn's disease became manifest at 20 years of age and underwent a chronic and relapsing course. Proteinuria and renal amyloidosis were detected after eight years of evolution. Panproctocolectomy was performed. The patient progressed satisfactorily and six months later he remains asymptomatic, free from medication and with normal creatinine clearance. The authors recommend early surgical treatment on the diseased intestinal segment once the association has been diagnosed and before serious impairment of renal function has occurred.