Gut Microbiota and Cancer: From Pathogenesis to Therapy

In the paper

“Any imbalance in this delicate equilibrium may lead to an impaired microbiota, condition called dysbiosis, linked with several human pathologies, including cancer [13].”

What the sources say

• Not checked yetcites [13] Carding, 2015, Dysbiosis of the gut microbiota in disease, Microb. Ecol. Health Dis.

In the paper

“The gut microbiota performs a number of vital functions, including production of vitamins, metabolization of dietary compounds, protection against the expansion and systemic infiltration of gut pathogens [10,11,12].”

What the sources say

• Not checked yetcites [10] Vaishnava, 2008, Paneth cells directly sense gut commensals and maintain homeostasis at the intestinal host-microbial interface, Proc. Natl. Acad. Sci. USA source
• Not checked yetcites [11] Belkaid, 2013, Compartmentalized and systemic control of tissue immunity by commensals, Nat. Immunol. source
• Not checked yetcites [12] Ravcheev, 2015, Systematic genome assessment of B-vitamin biosynthesis suggests co-operation among gut microbes, Front. Genet.

In the paper

“Among a 1000 of different bacterial species within the gut microbiota, the highly represented ones are the Firmicutes and Bacteroidetes phyla [9].”

What the sources say

• Not checked yetcites [9] Greenhalgh, 2016, The human gut microbiome in health: Establishment and resilience of microbiota over a lifetime, Environ. Microbiol. source

In the paper

“For example, the exposure to infectious agents, UV radiation and toxic substances, individual diet and lifestyle, strongly influences cancer rise, although the risk mainly depends on the dose, the duration and the combination of such injuries, coupled with the individual genetic background [5].”

What the sources say

• Not checked yetcites [5] Anand, 2008, Cancer is a preventable disease that requires major lifestyle changes, Pharm. Res. source

In the paper

“Importantly, the gastro-intestinal entero-endocrine cells secrete over 30 different peptide hormones, involved in several functions, such as gastro-intestinal motility, digestive functions and neuromodulation [28].”

What the sources say

• Not checked yetcites [28] Ceranowicz, 2015, Peptidyl hormones of endocrine cells origin in the gut—Their discovery and physiological relevance, J. Physiol. Pharmacol.

In the paper

“Cancer formation is the result of the stochastic intracellular accumulation of spontaneous mutations during DNA replication, combined with the environment exposure and lifestyle habits, both able to significantly influence cancer risk [3,4].”

What the sources say

• Not checked yetcites [3] Tomasetti, 2015, Cancer etiology. Variation in cancer risk among tissues can be explained by the number of stem cell divisions, Science source
• Not checked yetcites [4] Ashford, 2015, Cancer risk: Role of environment, Science source

In the paper

“As a consequence, cancer development may alter the microbiome and, in turn, microbiome changes may affect cancer progression [24].”

What the sources say

• Not checked yetcites [24] Zitvogel, 2017, Anticancer effects of the microbiome and its products, Nat. Rev. Microbiol. source

In the paper

“The gut microbiome, defined as the whole genome of the host’s gut microbiota, encodes 100-fold more genes than the human genome [14].”

What the sources say

• Not checked yetcites [14] Grice, 2012, The human microbiome: Our second genome, Annu. Rev. Genom. Hum. Genet. source

In the paper

“In fact, it has been widely studied that the gut microbiota may produce or transform molecules affecting several aspects of human health, including: host’s metabolism modulation, gut barrier integrity maintenance, xenobiotics and drug metabolism, protection against gastro-intestinal pathogens and host’s immune system modulation [31,32,33,34].”

What the sources say

• Not checked yetcites [31] Gensollen, 2016, How colonization by microbiota in early life shapes the immune system, Science source
• Not checked yetcites [32] Schmidt, 2018, The Human Gut Microbiome: From Association to Modulation, Cell source
• Not checked yetcites [33] Bultman, 2014, Emerging roles of the microbiome in cancer, Carcinogenesis source
• Not checked yetcites [34] Cani, 2018, Human gut microbiome: Hopes, threats and promises, Gut source

In the paper

“With regards to the host’s metabolism, it is known that certain commensal bacteria produce essential micronutrients, such as vitamin K and various components of vitamin B.”

What the sources say

• Not checked yetcites [35] Mohajeri, 2018, The role of the microbiome for human health: From basic science to clinical applications, Eur. J. Nutr. source

In the paper

“The gut is bidirectionally connected with the nervous system through the so-called “gut-brain axis” (GBA), which includes the central nervous system (CNS), the autonomic nervous system (ANS), the enteric nervous system (ENS), the hypothalamic pituitary adrenal (HPA) axis and the entero-endocrine system (EES).”

What the sources say

• Not checked yetcites [25] Carabotti, 2015, The gut-brain axis: Interactions between enteric microbiota, central and enteric nervous systems, Ann. Gastroenterol.

In the paper

“Also, members of the Bacterioides family are able to synthetize the anti-diabetic compound linoleic acid, to catabolize secondary bile acids and to break down phenolic compounds.”

What the sources say

• Not checked yetcites [35] Mohajeri, 2018, The role of the microbiome for human health: From basic science to clinical applications, Eur. J. Nutr. source

In the paper

“For example, the widely studied bacterial lipopolysaccharide (LPS), a major component of the outer membrane in gram-negative bacteria, activates the host’s cell surface receptor toll-like receptor 4 (TLR4), belonging to the family of pattern recognition receptors (PRRs), thus activating immune T cell-mediated response against cancer cells [40].”

What the sources say

• Not checked yetcites [40] Paulos, 2007, Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling, J. Clin. Investig. source

In the paper

“Microbial pathogens are known to drive the 20% of tumorigenesis and a larger number of malignancies are associated with microbial commensal imbalance, or dysbiosis [53].”

What the sources say

• Not checked yetcites [53] Bhatt, 2017, The role of the microbiome in cancer development and therapy, CA Cancer J. Clin. source

In the paper

“Enterococcus faecalis produces extracellular superoxide and derivative oxygen species is capable to diffuse into host’s cells. In turn, the increase in the oxidative milieu enhances the possibility of host’s cellular DNA mutations [79].”

What the sources say

• Not checked yetcites [79] Huycke, 2001, Extracellular superoxide production by Enterococcus faecalis requires demethylmenaquinone and is attenuated by functional terminal quinol oxidases, Mol. Microbiol. source

In the paper

“It has been also reported in several studies that Lactobacilli may stimulate host’s immune cells such as NK cells or dendritic cells (DC) or TH1 response, which, in turn, leads to the elimination of cancerous or precancerous cells, although the exact bacterial bioproduct mediating such stimulatory effect still needs to be identified [45,46,47,48].”

What the sources say

• Not checked yetcites [45] Lenoir, 2016, Lactobacillus casei BL23 regulates Treg and Th17 T-cell populations and reduces DMH-associated colorectal cancer, J. Gastroenterol. source
• Not checked yetcites [46] Lee, 2004, Immunomodulatory and antitumor effects in vivo by the cytoplasmic fraction of Lactobacillus casei and Bifidobacterium longum, J. Vet. Sci. source
• Not checked yetcites [47] Baldwin, 2010, Probiotic Lactobacillus acidophilus and L. casei mix sensitize colorectal tumoral cells to 5-fluorouracil-induced apoptosis, Nutr. Cancer source
• Not checked yetcites [48] Takagi, 2008, Relationship between the in vitro response of dendritic cells to Lactobacillus and prevention of tumorigenesis in the mouse, J. Gastroenterol. source

In the paper

“For example, Fusobacterium nucleatum inhibits for its own advantage host’s Natural Killer (NK) cells, in order to recruit at the site of the infection myeloid suppressor cells, therefore indirectly helping cancer genesis.”

What the sources say

• Not checked yetcites [80] Gur, 2015, Binding of the Fap2 protein of Fusobacterium nucleatum to human inhibitory receptor TIGIT protects tumors from immune cell attack, Immunity source

In the paper

“Moreover, the very same anti-cancer treatment might shape the patient’s microbiome and, at the same time, host’s specific microbiome can deeply affect patient’s response to therapy [19].”

What the sources say

• Not checked yetcites [19] Gopalakrishnan, 2018, The Influence of the Gut Microbiome on Cancer, Immunity, and Cancer Immunotherapy, Cancer Cell source

In the paper

“In particular, CagA from Helicobacter pylori can bind many host’s proteins intracellularly, including the protein tyrosine phosphatase SHP-2. CagA-SHP-2 complex formation deregulates the phosphatase activity of SHP-2, which, in turn, promotes Ras/MAPK signaling activation [73].”

What the sources say

• Not checked yetcites [73] Matozaki, 2009, Protein tyrosine phosphatase SHP-2: A proto-oncogene product that promotes Ras activation, Cancer Sci. source

In the paper

“Accordingly, this augmented intake of estrogen hormones is linked with an increased risk of developing breast cancer, supporting the finding that the gut microbiota composition of women with breast cancer differs from that from healthy controls, and suggesting that several gut bacteria, which could be over-expressed during dysbiosis, may be linked with breast cancer development [83].”

What the sources say

• Not checked yetcites [83] Fernández, M.F., Reina-Pérez, I., Astorga, J.M., Rodríguez-Carrillo, A., Plaza-Díaz, J., and Fontana, L. (2018). Breast Cancer and Its Relationship with the Microbiota. Int. J. Environ. Res. Public Health, 15. source

In the paper

“In animal models, LGG administered with food attenuates 5-FU-mediated as well as radiation-mediated gut epithelial injury, therefore helping the preservation of the gut microbiota balance and the intestinal epithelial barrier functionality [91,92,93].”

What the sources say

• Not checked yetcites [91] Chang, 2018, Variety, Front. Microbiol. source
• Not checked yetcites [92] Riehl, T.E., Alvarado, D., Ee, X., Zuckerman, A., Foster, L., Kapoor, V., Thotala, D., Ciorba, M.A., and Stenson, W.F. (2018). GG protects the intestinal epithelium from radiation injury through release of lipoteichoic acid, macrophage activation and the migration of mesenchymal stem cells. Gut. source
• Not checked yetcites [93] Zhang, 2017, GG Affects Microbiota and Suppresses Autophagy in the Intestines of Pigs Challenged with, Front. Microbiol. source

In the paper

“In addition, LGG prevents polyps’ formation in a colorectal APC/min mouse cancer model, and reduces colitis-associated cancer in mice [108,109].”

What the sources say

• Not checked yetcites [108] Ni, 2017, A metagenomic study of the preventive effect of Lactobacillus rhamnosus GG on intestinal polyp formation in APC, J. Appl. Microbiol. source
• Not checked yetcites [109] Mendes, 2018, Microbiota modification by probiotic supplementation reduces colitis associated colon cancer in mice, World J. Gastroenterol. source

In the paper

“Additionally, host’s DC exposed to LGG induce TH1 immune cells polarization and, in turn, antitumor immune-response potentiation [111].”

What the sources say

• Not checked yetcites [111] Cai, 2016, Lactobacillus rhamnosus GG Activation of Dendritic Cells and Neutrophils Depends on the Dose and Time of Exposure, J. Immunol. Res. source

In the paper

“The gut microbial population plays a key role in training, functioning and stimulating the host immune system, with the final effect of developing tolerance towards beneficial microbiota and eliciting immune response against gut pathogens [76].”

What the sources say

• Not checked yetcites [76] Belkaid, 2014, Role of the microbiota in immunity and inflammation, Cell source

In the paper

“LGG triggers the immune response also within the normal not transformed gut epithelium, thus protecting towards inflammation, which can support the formation of a cancer-favorable milieu [112].”

What the sources say

• Not checked yetcites [112] Suzuki, C., Aoki-Yoshida, A., Aoki, R., Sasaki, K., Takayama, Y., and Mizumachi, K. (2017). The distinct effects of orally administered Lactobacillus rhamnosus GG and Lactococcus lactis subsp. lactis C59 on gene expression in the murine small intestine. PLoS ONE, 12. source

In the paper

“Among these probiotic species, LGG is one of the first studied specifically in oncology [87].”

What the sources say

• Not checked yetcites [87] Goldin, 1996, The effect of Lactobacillus GG on the initiation and promotion of DMH-induced intestinal tumors in the rat, Nutr. Cancer source

In the paper

“The mechanism involves the induction of pro-apoptotic genes within the tumor mass and the enhancement of host’s immune response [166].”

What the sources say

• Not checked yetcites [166] Gui, 2015, Well-balanced commensal microbiota contributes to anti-cancer response in a lung cancer mouse model, Genet. Mol. Res. source

In the paper

“With reference to immunotherapy, the administration of CpG oligodeoxynucleotides, synthetic molecules mimicking bacterial DNA, strongly stimulate the immune system, therefore showing anti-tumor activity in several cancers [169].”

What the sources say

• Not checked yetcites [169] Weiner, 2008, CpG oligodeoxynucleotides as immunotherapy in cancer, Update Cancer Ther. source

In the paper

“Specifically, interventions on microbiome may be pivotal to ameliorate anti-cancer therapy-related toxicity, as well as to improve anti-cancer therapy efficacy [151,152].”

What the sources say

• Not checked yetcites [151] Nayak, R.R., and Turnbaugh, P.J. (2016). Mirror, mirror on the wall: Which microbiomes will help heal them all?. BMC Med., 14. source
• Not checked yetcites [152] Fessler, 2017, The Microbiota: A New Variable Impacting Cancer Treatment Outcomes, Clin. Cancer Res. source

In the paper

“These two effects combined together may explain the strong involvement of the patient’s microbiome composition in affecting the efficacy of both chemotherapy and immunotherapy [19].”

What the sources say

• Not checked yetcites [19] Gopalakrishnan, 2018, The Influence of the Gut Microbiome on Cancer, Immunity, and Cancer Immunotherapy, Cancer Cell source

In the paper

“The microbiota, when affected by dysbiosis, can deeply influence both cancer pathogenesis (as reported in Section 2) and its therapeutic outcome.”

In the paper

“Along this line, the intra-tumoral injection of CpG oligodeoxynucleotides administered together with an anti-interleukin-10 receptor (IL-10R) antibody, induce TNF production from tumor infiltrating myeloid cells and, in turn, reduce the growth several types of tumors in mice [165].”

What the sources say

• Not checked yetcites [165] Iida, 2013, Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment, Science source

In the paper

“Back in 1890 two heat-inactivated microorganisms (Streptococci) were injected intratumorally for the very first time in humans as an attempt to cure cancer [153,154].”

What the sources say

• Not checked yetcites [153] McCarthy, 2006, The toxins of William B. Coley and the treatment of bone and soft-tissue sarcomas, IOWA Orthop. J.
• Not checked yetcites [154] Nauts, 1946, The treatment of malignant tumors by bacterial toxins as developed by the late William B. Coley, M.D., reviewed in the light of modern research, Cancer Res.

In the paper

“Because almost every available anti-cancer treatment is toxic also towards normal cells, their use may be coupled with side effects, some of which can compromise the overall survival of the patients [140].”

What the sources say

• Not checked yetcites [140] Dy, 2013, Understanding, recognizing, and managing toxicities of targeted anticancer therapies, CA Cancer J. Clin. source

In the paper

“In particular, the regulation of such therapeutic outcome is tightly connected with the ability of the gut microbiota to metabolize anti-tumoral compounds, as well as to modulate host’s immune response and inflammation pathways [164].”

What the sources say

• Not checked yetcites [164] Schwabe, 2013, The microbiome and cancer, Nat. Rev. Cancer source

In the paper

“Consistently, mice bearing lung tumors treated with cisplatin coupled with antibiotics, survive less and develop bigger tumors.”

What the sources say

• Not checked yetcites [166] Gui, 2015, Well-balanced commensal microbiota contributes to anti-cancer response in a lung cancer mouse model, Genet. Mol. Res. source

In the paper

“In fact, radiotherapy, chemotherapy and immunotherapy treatments can all modify patients’ microbiome and, at the same time, microbiome composition can deeply affect patients’ response to such therapies [150].”

What the sources say

• Not checked yetcites [150] Roy, 2017, Microbiota: A key orchestrator of cancer therapy, Nat. Rev. Cancer source

In the paper

“Regarding the correlation between inflammasomes, microbiome and cancer, NRPL6 plays a key role in colorectal carcinogenesis and, in particular, NLRP6 regulates susceptibility to intestinal inflammation through its microbiome-modulatory activity [139].”

What the sources say

• Not checked yetcites [139] Elinav, 2018, NLRP6 and Dysbiosis: Avoiding the Luring Attraction of Over-Simplification, Immunity source

In the paper

“In CRC inflammasomes activation has a protective role, on the contrary in breast and skin cancers their triggering leads to detrimental outcomes [119].”

What the sources say

• Not checked yetcites [119] Kantono, 2017, Inflammasomes and Cancer: The Dynamic Role of the Inflammasome in Tumor Development, Front. Immunol. source

In the paper

“NLRP6 KO mice show dysbiosis and increased incidence of inflammation-associated CRC [135,136,137].”

What the sources say

• Not checked yetcites [135] Elinav, 2011, NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis, Cell source
• Not checked yetcites [136] Levy, 2015, Microbiota-Modulated Metabolites Shape the Intestinal Microenvironment by Regulating NLRP6 Inflammasome Signaling, Cell source
• Not checked yetcites [137] Levy, 2017, NLRP6: A Multifaceted Innate Immune Sensor, Trends Immunol. source

In the paper

“Importantly, this intra-tumoral variety is tightly linked with the development of the resistance to therapy, considered the first cause of failure of the available anti-cancer treatments, as well as subsequent tumor relapses [146].”

What the sources say

• Not checked yetcites [146] McGranahan, 2015, Biological and therapeutic impact of intratumor heterogeneity in cancer evolution, Cancer Cell source

In the paper

“Although the immune checkpoint inhibitors have success in treating various malignancies, there is still a significant number of patients that can use such therapy only for a limited amount of time, given the occurrence of strong toxic side effects, including gut inflammation, due to the subsequent immune-dysregulation (i.e., autoimmunity) [177].”

What the sources say

• Not checked yetcites [177] Larkin, 2015, Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma, N. Engl. J. Med. source

In the paper

“A mixture of two probiotic species Bifidobacterium longum (BB536) and Lactobacillus johnsonii (La1) was administered to the patients in the double-blind study, finding that one of that, La1, was able to adhere to the colonic mucosa, thereby reducing the concentration of gut pathogens and modulating the local immunity [95].”

What the sources say

• Not checked yetcites [95] Gianotti, 2010, A randomized double-blind trial on perioperative administration of probiotics in colorectal cancer patients, World J. Gastroenterol. source

In the paper

“Functional studies performed with FMT in germ free mice further demonstrated how treating mice with the identified bacteria, along with the anti-PD1 therapy, enhanced the anti-cancer effects and reduces the melanoma growth [171].”

What the sources say

• Not checked yetcites [171] Gopalakrishnan, 2018, Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients, Science source

In the paper

“Immune checkpoint inhibition consists in the administration of therapeutic agents able to block the immune-inhibitory pathway, thus modulating T cell activation against tumor target cells.”

What the sources say

• Not checked yetcites [173] Chen, 2018, Delivery Strategies for Immune Checkpoint Blockade, Adv. Healthc. Mater. source

In the paper

“Performing FMT from patients to germ free mice, the authors found that Akkermansia muciniphila (alone or in combination with Enterococcus hirae) was able to increase intra-tumoral cytotoxic T cell infiltrates, thus increasing the PD-1 blockade response in mice [170].”

What the sources say

• Not checked yetcites [170] Routy, 2018, Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors, Science source

In the paper

“Moreover, the administration of a specific bacteria, Alistipes shahii, to antibiotic-treated tumor bearing mice, restores TNF production with a notable improvement in the therapeutic outcome [165].”

What the sources say

• Not checked yetcites [165] Iida, 2013, Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment, Science source

In the paper

“Moreover, according to the result of a trial published in 2017, the randomized administration of Bifidobacterium lactis and Lactobacillus acidophilus to CRC patients, can change the epigenetic patterns of tumor tissue from its baseline, with potential therapeutic benefits in CRC by manipulation of the gut microbiota [100].”

What the sources say

• Not checked yetcites [100] Hibberd, 2017, Intestinal microbiota is altered in patients with colon cancer and modified by probiotic intervention, BMJ Open Gastroenterol. source

In the paper

“probiotics containing Lactobacillus species are suggested be used to prevent diarrhea in patients receiving chemotherapy and/or radiation therapy for a pelvic malignancy (Level of evidence III) [193,194].”

What the sources say

• Not checked yetcites [193] Peterson, 2015, Management of oral and gastrointestinal mucosal injury: ESMO Clinical Practice Guidelines for diagnosis, treatment, and follow-up, Ann. Oncol. source
• Not checked yetcites [194] Lalla, 2014, MASCC/ISOO clinical practice guidelines for the management of mucositis secondary to cancer therapy, Cancer source

In the paper

“Although probiotics are generally regarded as safe, the main concerns of administering them to immunocompromised cancer patients are both the potential risk of opportunistic infection development and the transfer of antibiotics resistance [190,191].”

What the sources say

• Not checked yetcites [190] Vanderhoof, 2008, Probiotics in the United States, Clin. Infect. Dis. source
• Not checked yetcites [191] Redman, 2014, The efficacy and safety of probiotics in people with cancer: A systematic review, Ann. Oncol. source

In the paper

“While CTLA-4 regulates T cells proliferation early in the immune response within the lymph nodes, PD-1 suppresses T-cell activation later, within the body periphery [174].”

What the sources say

• Not checked yetcites [174] Buchbinder, 2016, CTLA-4 and PD-1 Pathways: Similarities, Differences, and Implications of Their Inhibition, Am. J. Clin. Oncol. source

In the paper

“In fact, they demonstrated that oral administration of a cocktail of Bifidobacterium species combined with the anti-PD-L1 antibody, specifically boosts T cell response and blocks the melanoma growth [176].”

What the sources say

• Not checked yetcites [176] Sivan, 2015, Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy, Science source

In the paper

“Despite of that, probiotics administration in multiple trials has shown beneficial effects on ameliorating diarrhea and other gut-related damages following anti-cancer therapy, thus re-establishing a healthy intestinal microbiota composition [192].”

What the sources say

• Not checked yetcites [192] Mego, 2013, Probiotic bacteria in cancer patients undergoing chemotherapy and radiation therapy, Complement. Ther. Med. source

In the paper

“Importantly, metagenomics analysis of patients’ fecal gut microbiome showed a difference in the composition of their gut microbiome.”

What the sources say

• Not checked yetcites [170] Routy, 2018, Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors, Science source

In the paper

“Altogether, these data provide a strong evidence of the role of gut microbiota composition in modulating the effect of both immunotherapy response and toxicity.”

In the paper

“LGG is a very good example of a probiotic well studied in cancer, often administered as complementary therapeutic to cure dysbiosis.”

In the paper

“Even if the last decade witnessed massive advances in unveiling the role of gut microbiome in cancer and other diseases, there are still many obstacles for translating basic microbiome research into therapeutic applications.”

In the paper

“Regarding anti-tumor therapeutic applications, preclinical studies performed in mice demonstrated the efficacy of FMT in reducing colon tumorigenesis, although the efficacy in clinical trials still needs to be further proven [198].”

What the sources say

• Not checked yetcites [198] Bel, 2014, Reprogrammed and transmissible intestinal microbiota confer diminished susceptibility to induced colitis in TMF−/− mice, Proc. Natl. Acad. Sci. USA source

In the paper

“For example, FMT has been used to cure recurrent Clostridium difficile duodenal infection [195,196].”

What the sources say

• Not checked yetcites [195] Dijkgraaf, 2013, Duodenal infusion of feces for recurrent Clostridium difficile, N. Engl. J. Med.
• Not checked yetcites [196] Khoruts, 2016, Inflammatory Bowel Disease Affects the Outcome of Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection, Clin. Gastroenterol. Hepatol. source

In the paper

“Some bacterial subpopulations are able to rise during gut dysbiosis and, in turn, to trigger the formation of an inflammatory and pro-cancerogenic environment.”

In the paper

“On the other hand, many gut derived probiotics are able to protect the host, re-establishing the conditions of a healthy intestinal microbiota within dysbiotic patients, including cancer patients.”