Fluoroquinolones (FQ) are powerful broad-spectrum antibiotics whose side effects include renal damage and, strangely, tendinopathies. The pathological mechanisms underlying these toxicities are poorly understood. Here, we show that the FQ drugs norfloxacin, ciprofloxacin, and enrofloxacin are powerful iron chelators comparable with deferoxamine, a clinically useful iron-chelating agent. We show that iron chelation by FQ leads to epigenetic effects through inhibition of α-ketoglutarate-dependent dioxygenases that require iron as a co-factor. Three dioxygenases were examined in HEK293 cells treated with FQ. At sub-millimolar concentrations, these antibiotics inhibited jumonji domain histone demethylases, TET DNA demethylases, and collagen prolyl 4-hydroxylases, leading to accumulation of methylated histones and DNA and inhibition of proline hydroxylation in collagen, respectively. These effects may explain FQ-induced nephrotoxicity and tendinopathy. By the same reasoning, dioxygenase inhibition by FQ was predicted to stabilize transcription factor HIF-1α by inhibition of the oxygen-dependent hypoxia-inducible transcription factor prolyl hydroxylation. In dramatic contrast to this prediction, HIF-1α protein was eliminated by FQ treatment. We explored possible mechanisms for this unexpected effect and show that FQ inhibit HIF-1α mRNA translation. Thus, FQ antibiotics induce global epigenetic changes, inhibit collagen maturation, and block HIF-1α accumulation. We suggest that these mechanisms explain the classic renal toxicities and peculiar tendinopathies associated with FQ antibiotics.

(1) Background: Immigrants represent around 21.9% of the total population in Canada and encounter multifaceted obstacles in accessing and receiving primary healthcare. This literature review explores patient experiences in primary care from the perspective of immigrants and identifies areas for further research and improvement. (2) Methods: A comprehensive search was performed on PubMed, MEDLINE, Embase, SCOPUS, and Google scholar to identify studies published from 2010 to July 2020. Relevant articles were peer-reviewed, in English language, and reported patient experiences in primary healthcare in Canada. (3) Results: Of the 1566 searched articles, 19 articles were included in this review. Overall, the finding from articles were summarized into four major themes: cultural and linguistic differences; socioeconomic challenges; health system factors; patient–provider relationship. (4) Conclusion: Understanding the gaps to accessing and receiving appropriate healthcare is important to shape policies, enhance the quality of services, and deliver more equitable healthcare services. It is therefore pertinent that primary healthcare providers play an active role in bridging these gaps with strong support from policymakers. Understanding and respecting diversity in culture, language, experiences, and systems is crucial in reducing health inequalities and improving access to quality care in a respectful and responsive manner.

Expression of programmed cell death protein 1 (PD-1) on natural killer (NK) cells has been difficult to analyze on human NK cells. By testing commercial clones and novel anti-PD-1 reagents, we found expression of functional PD-1 on resting human NK cells in healthy individuals and reconstituting NK cells early after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Peripheral blood samples from healthy individuals and transplant recipients were stained for PD-1 expression using the commercial anti-PD-1 clone PD1.3.1.3, fluorescein isothiocyanate (FITC)–labeled pembrolizumab, or an FITC-labeled single-chain variable fragment (scFv) reagent made from pembrolizumab. These reagents identified low yet consistent basal PD-1 expression on resting NK cells, a finding verified by finding lower PD-1 transcripts in sorted NK cells compared with those in resting or activated T cells. An increase in PD-1 expression was identified on paired resting NK cells after allo-HSCT. Blockade of PD-1 on resting NK cells from healthy donors with pembrolizumab did not enhance NK function against programmed death-ligand 1 (PD-L1)–expressing tumor lines, but blocking with its scFv derivative resulted in a twofold increase in NK cell degranulation and up to a fourfold increase in cytokine production. In support of this mechanism, PD-L1 overexpression of K562 targets suppressed NK cell function. Interleukin-15 (IL-15) activity was potent and could not be further enhanced by PD-1 blockade. A similar increase in function was observed with scFv PD-1 blockade on resting blood NK cells after allo-HSCT. We identify the functional importance of the PD-1/PD-L1 axis on human NK cells in which blockade or activation to overcome inhibition will enhance NK cell–mediated antitumor control.

Checkpoint blockade has become a promising immunotherapy for the treatment of a variety of malignancies. In particular, the receptor programmed death-1 (PD-1) has become a focus of intense study due to its expression on and negative regulation of T-cell function. The ligand for PD-1, PD-L1, is upregulated on many tumors and, as a result, can suppress antigen-specific T-cells thereby limiting their anti-tumor response. Pharmacological PD-1/PD-L1 axis disruption can occur with either Pembrolizumab and Nivolumab (PD-1 antagonists) and Avelumab and Atezolizumab (PD-L1 antagonists). These antibodies (mAbs) are being used to treat melanoma, non-small cell lung cancer, kidney, bladder and head and neck cancer with varying degrees of success. Like T-cells, natural killer cells (NK) also have potent antitumor cytolytic properties. The expression and functional effects of PD-1 on NK cells remain unclear due to difficulties in receptor detection and efficacy of receptor blockade by available commercial reagents. While some studies have been unable to detect PD-1 on resting NK cells, others have identified PD-1 expression only on specific NK populations under certain conditions (e.g. Cytokine stimulation or virus infection). Here, we identify PD-1 expression on peripheral blood NK cells. Using commercial reagents (Figure 1A) and a FITC-labeled clinical mAb (Pembrolizumab, Pembro), we detect low yet consistent PD-1 expression on all circulating, resting NK cells. Since FITC-Pembro mean fluorescent intensity was low and a high proportion of FITC labeled NK cells overlapped with the isotype control (Figure 1B), we designed a short-chain variable fragment (scFv) of the mAb to determine whether the smaller scFv molecule has better binding and functional activity than the intact mAb. The Pembro scFv bound to resting NK cells with a distinct fluorescent peak compared to the native Prembro from which the scFv was derived (Figure 1B). Compared to intact Prembro, use of the Pembro scFv as a PD-1 antagonist resulted in a 2-fold increase of NK cell cytolytic activity and a 3-4 fold increase in cytokine production against the PD-L1 expressing CML target, K562 (Figure 1C-D) and the AML target, THP-1 (Figure 1E-F). While PD-1 blockade enhanced NK cell degranulation and target cell killing, a greater functional enhancement was seen for interferon-γ production. PD-1 signaling inhibits PI3K induced pAkt and NK function. PD-1/PD-1 ligand blockade by the Pembro scFv resulted in increased NK cell pAKT in the presence of PD-L1 and NK activating NKG2D-ligand-expressing THP-1 cells. In addition to natural cytotoxicity, NK-mediated ADCC was also enhanced with PD-1 blockade. CD33 mAb immunoconjugates have been used to treat AML. Combined anti-CD33 mAb and PD-1 blockade against THP-1 cells resulted in a small but significant increase in NK cell degranulation and a 4-fold increase in cytokine production compared to anti-CD33 mAb without PD-1 blockade (Figure 1G-H). Since stimulation with IL-15, a cytokine that effectively lowers the NK activation threshold, abrogated the benefits of Pembro scFv in diminishing PD-1 inhibitory effects on NK cells, PD-1 control of NK function appears limited to be mostly relevant to resting NK cells. To understand the physiologic expression of PD-1 in vivo, we studied samples taken from AML patients receiving matched sibling donor transplantation at the University of Minnesota. Increased PD-1 on reconstituting NK cells in BMT recipients up to day 100 post-transplant was shown by both flow-cytometric (Figure 2A) and mass-cytometric (CyTOF) analyses (Figure 2B). Blockade of PD-1 on these cells significantly enhanced both NK degranulation (Figure 2C) and cytokine production (Figure 2D) against K562 targets. A similar increase in NK function was observed with PD-1 blockade in AML patients receiving umbilical cord transplants (not shown). These data indicate that PD-1 is present on human NK cells and PD-1 ligation negatively regulates NK function against PD-L1 expressing tumor targets. The observation that functional PD-1 is expressed on NK cells under resting conditions strongly suggests that the use of a PD-1 antagonist, in combination with NK cell therapy, should be clinically effective for treatment of cancer. Disclosures Felices: GT Biopharma.: Other: consulting funds, Research Funding. Blazar:Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Leukemia and Lymphoma Society: Research Funding; Abbvie Inc: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees. Vallera:GT Biopharma, Inc.: Consultancy, Research Funding. Miller:Fate Therapeutics, Inc: Consultancy, Research Funding; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Keytruda. PD-1 blockade on NK cells for tumor immunotherapy