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Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS that affects around one million people in the United States. Predisposition or protection from this disease is linked with both genetic and environmental factors. In recent years, gut microbiome has emerged as an important environmental factor in the pathobiology of MS. The gut microbiome supports various physiologic functions, including the development and maintenance of the host immune system, the perturbation of which is known as dysbiosis and has been linked with multiple diseases including MS. We and others have shown that people with MS (PwMS) have gut dysbiosis that is characterized by specific gut bacteria being enriched or depleted. Consequently, there is an emphasis on determining the mechanism(s) through which gut bacteria and/or their metabolites alter the course of MS through their ability to provide protection, predispose individuals, or promote disease progression. Improving our understanding of these mechanisms will allow us to harness the enormous potential of the gut microbiome as a diagnostic and/or therapeutic agent. In this chapter, we will discuss current advances in microbiome research in the context of MS, including a review of specific bacteria that are currently linked with this disease, potential mechanisms of disease pathogenesis, and the utility of microbiome-based therapy for PwMS.

As geographical location can impact the gut microbiome, it is important to study region-specific microbiome signatures of various diseases. Therefore, we profiled the gut microbiome of breast cancer (BC) patients of the Midwestern region of the United States. The bacterial component of the gut microbiome was profiled utilizing 16S ribosomal RNA sequencing. Additionally, a gene pathway analysis was performed to assess the functional capabilities of the bacterial microbiome. Alpha diversity was not significantly different between BC and healthy controls (HC), however beta diversity revealed distinct clustering between the two groups at the species and genera level. Wilcoxon Rank Sum test revealed modulation of several gut bacteria in BC specifically reduced abundance of those linked with beneficial effect such as Faecalibacterium prausnitzii. Machine learning analysis confirmed the significance of several of the modulated bacteria found by the univariate analysis. The functional analysis showed a decreased abundance of SCFA (propionate) production in BC compared to HC. In conclusion, we observed gut dysbiosis in BC with the depletion of SCFA-producing gut bacteria suggesting their role in the pathobiology of breast cancer. Mechanistic understanding of gut bacterial dysbiosis in breast cancer could lead to refined prevention and treatment.

The role of the human gut microbiome has been increasingly appreciated in respect to its influence on human health and disease. It has been proposed that dysbiosis in the gut microbiome and subsequent induction of chronic inflammatory state might have an influence on breast cancer development and prognosis. Therefore this study was undertaken to determine the role of gut microbiome in breast cancer. The Breast Molecular Epidemiology Resource (BMER) resources of the Holden Comprehensive Cancer Center (HCCC) were used to recruit 23 women with breast cancer. Fecal samples were used to characterize the gut microbiome of our patient population using 16S rRNA amplification and Illumina sequencing. Reads were then mapped back to amplicon sequence variant using DADA2 pipeline and compared to a cohort of race, age, and gender matched cohort of regional healthy controls. In our preliminary analysis, we observed that breast cancer patients have distinct microbiota compared to age and gender matched healthy controls. We identified depletion of the anti-inflammatory short chain fatty acid producing bacteria specifically Faecalibacterium, Anaerostipes, Parabacteroides and Phascolarctobacterium in feces from BC patients. Additionally, breast cancer cohort showed enrichment of the pro-inflammatory bacteria such as Eubacterium (dolichum), Bifidobacterium, and Blautia. These bacteria have been previously been identified to change with clinical disease stage and/or BMI, as has genus Bifidobacterium (increased in BC cohort). Within BC group, patients with high grade showed lower levels of Eggerthella lenta and Desulfovibrio whereas patients receiving chemotherapy showed depletion of Sulphur reducing Desulfovibrio and methane producing archaea Methanobrevibacter. Further analysis may lead to insights into the interplay between gut microbiome and breast cancer, as well as the feasibility of therapeutic strategies in the form of novel and existing probiotics. Citation Format: Praveen Vikas, Nicole Cady, Jessica Knobbe, Jemmie Hoang, Meeta Yadav, Melissa Curry, Cathernine Cherwin, Sonia Sugg, Sneha Phadke, Edward Filardo, Ashuthosh Mangalam. Breast cancer patients have reduced levels of short chain fatty acid producing beneficial gut bacteria [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-57.

Trillions of commensal bacteria colonizing humans (microbiome) have emerged as essential player(s) in human health. The alteration of the same has been linked with diseases including autoimmune disorders such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and ankylosing spondylitis. Gut bacteria are separated from the host through a physical barrier such as skin or gut epithelial lining. However, the perturbation in the healthy bacterial community (gut dysbiosis) can compromise gut barrier integrity, resulting in translocation of bacterial contents across the epithelial barrier (leaky gut). Bacterial contents such as lipopolysaccharide and bacterial antigens can induce a systemic inflammatory environment through activation and induction of immune cells. The biggest question in the field is whether inflammation causes gut dysbiosis or dysbiosis leads to disease induction or propagation, i.e., it is inside out or outside in or both. In this review, we first discuss the microbiome profiling studies in various autoimmune disorders, followed by a discussion of potential mechanisms through which microbiome is involved in the pathobiology of diseases. A better understanding of the role of the microbiome in health and disease will help us harness the power of commensal bacteria for the development of novel therapeutic agents to treat autoimmune disorders.

Background Though the gut microbiome has been associated with efficacy of immunotherapy (ICI) in certain cancers, similar findings have not been identified for microbiomes from other body sites and their correlation to treatment response and immune related adverse events (irAEs) in lung cancer (LC) patients receiving ICIs. Methods We designed a prospective cohort study conducted from 2018 to 2020 at a single-center academic institution to assess for correlations between the microbiome in various body sites with treatment response and development of irAEs in LC patients treated with ICIs. Patients must have had measurable disease, ECOG 0–2, and good organ function to be included. Data was collected for analysis from January 2019 to October 2020. Patients with histopathologically confirmed, advanced/metastatic LC planned to undergo immunotherapy-based treatment were enrolled between September 2018 and June 2019. Nasal, buccal and gut microbiome samples were obtained prior to initiation of immunotherapy +/− chemotherapy, at development of adverse events (irAEs), and at improvement of irAEs to grade 1 or less. Results Thirty-seven patients were enrolled, and 34 patients were evaluable for this report. 32 healthy controls (HC) from the same geographic region were included to compare baseline gut microbiota. Compared to HC, LC gut microbiota exhibited significantly lower α-diversity. The gut microbiome of patients who did not suffer irAEs were found to have relative enrichment of Bifidobacterium (p = 0.001) and Desulfovibrio (p = 0.0002). Responders to combined chemoimmunotherapy exhibited increased Clostridiales (p = 0.018) but reduced Rikenellaceae (p = 0.016). In responders to chemoimmunotherapy we also observed enrichment of Finegoldia in nasal microbiome, and increased Megasphaera but reduced Actinobacillus in buccal samples. Longitudinal samples exhibited a trend of α-diversity and certain microbial changes during the development and resolution of irAEs. Conclusions This pilot study identifies significant differences in the gut microbiome between HC and LC patients, and their correlation to treatment response and irAEs in LC. In addition, it suggests potential predictive utility in nasal and buccal microbiomes, warranting further validation with a larger cohort and mechanistic dissection using preclinical models. Trial registration ClinicalTrials.gov, NCT03688347. Retrospectively registered 09/28/2018.

Background: In recent years, attention has shifted to modification of tumor response to immunotherapy via the host microbiome. The mechanisms of these associations, causative or consequential, remain incompletely understood. We seek to explore this further with a longitudinal study of lung cancer patient microbiomes and immunotherapy-related adverse effects (AEs) preceding and during immunotherapy. Methods: Patients with lung cancer (LC) treated with immunotherapy (anti-PD-1/L1 agents including pembrolizumab, nivolumab, atezolizumab, and durvalumab) with or without chemotherapy at the University of Iowa from November 2018–April 2019 were consented for this ongoing study. Fecal samples and nasal and buccal swabs were obtained prior to therapy. Patients were treated and monitored per respective disease protocol; responses were recorded per RECIST 1.1 and AEs per CTCAE 5.0. Samples underwent DNA extraction followed by 16S rRNA metagenomic analysis and taxonomic profiling using Divisive Amplicon Denoising Algorithm (DADA)-2 pipeline. Microbiomes from all body sites were compared and correlated to treatment response and AEs. In addition, baseline LC gut microbiomes were compared to fecal samples provided by healthy controls (HC) in the same geographic location. This project is registered on clinicaltrials.gov (NCT03688347). Results: Gut microbiota significantly differed compared to oral and buccal microbiota in all patients. Gut microbiota from LC patients was compared to HC samples from the same geographic area. LC patients exhibited drastically different baseline composition, including dramatic increases in Firmicutes, Actinobacteria, and Verrucomicrobia, and significant decreases in Bacteroidetes, Proteobacteria, and Cyanobacteria. We noticed a clear inversion of Firmicutes/Bacteroidetes ratio between HC and LC patients, differences also reflected at the genus level. NSCLC patients experiencing immunotherapy-related adverse events were found to have at baseline markedly fewer Bifidobacterium and Desulfovibrio (p < 0.05 and FDR = PR) exhibited enriched baseline Clostridiales (phylum Firmicutes, p=0.018) but reduced Rikenellaceae (phylum Bacteroidetes, p=0.016). Conclusion: Our study found promising trends in microbiome constitution. Compared to HC, we found significant differences in baseline LC gut microbiome at phylum and genus levels. More importantly, there are notable differences comparing LC patients who suffered AEs to those with none and between immunotherapy responders vs. nonresponders. Our project marks an important first step in a long-term study that could shed new light on the microbiome’s influence on immunotherapy treatment outcomes. Citation Format: Justin J. Chau, Meeta Yadav, Muhammad Furqan, Keri Mercer, Evan Eastman, Shailesh Shahi, Qun Dai, Carlos Chan, George Weiner, Taher Abu Hejleh, Ashutosh Mangalam, Jun Zhang. The microbiome in lung cancer under immunotherapy: Significant compositional differences associated with treatment response and AEs [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B24.

Individuals with the major hemochromatosis (HFE) allele C282Y and iron overload develop hepatocellular and some extrahepatic malignancies at increased rates. No association has been previously reported between the C282Y allele and breast cancer. We hypothesized that due to the pro-oxidant properties of iron, altered iron metabolism in C282Y carriers may promote breast carcinogenesis. Because 1 in 10 Caucasians of Northern European ancestry carries this allele, any impact it may have on breast cancer burden is potentially great. We determined C282Y genotypes in 168 patients who underwent high-dose chemotherapy and blood cell transplantation for cancer: 41 with breast cancer and 127 with predominantly hematological cancers (transplant cohort). Demographic, clinical, and tumor characteristics were reviewed in breast cancer patients. The frequency of C282Y genotypes in breast cancers was compared with the frequency in nonbreast cancers, an outpatient sample from Tennessee (n = 169), and a published United States national sample. The frequency of at least one C282Y allele in breast cancers was higher (36.6%, 5 homozygotes/10 heterozygotes) than frequencies in Tennessee (12.7%, P < 0.001), the general population (12.4%, P < 0.001), and similarly selected nonbreast cancers (17.0%, P = 0.008). The likelihood of breast cancer in the transplant cohort increased with C282Y allele dose (Ptrend = 0.010). These results were supported by the finding in a nontransplant cohort of a higher frequency of C282Y mutations in Caucasian (18.4%, P = 0.039) and African-American (8.5%, P = 0.005) women with breast cancer than race-specific national frequency estimates. A high prevalence of C282Y alleles in women with breast cancer with and without poor risk features suggests that altered iron metabolism in C282Y carriers may promote the development of breast cancer and/or more aggressive forms of the disease.

Elderly cancer patients are often excluded from immune-based clinical trials and therapies based on the belief that they respond poorly to tumor antigens. Using melanoma as a model and melanoma related Mart-127–35 epitope specific T cell receptor (TCR) engineered T cells as a tool we compared the T cell responses from young and elderly to the Mart-127–35 epitope, ex vivo. We also compared the natural Treg (nTreg) activities and the expression of a number of genes associated with immune response by quantitative real-time reverse Transcription Polymerase Chain Reaction (qRTPCR) in formalin fixed primary melanomas, in situ. We detected a significant difference in CD8+ T cell response to Flu antigen (influenza matrix peptide Flu MP58–66), but the responses of the two cohorts to melanoma antigen were comparable. nTreg activities in the elderly was significantly compromised. The qPCR analyses of tissues from elderly patients revealed lower levels of Fox-P3 expression but comparable levels of expression of IL-2, IFNγ, TNFα, IL-4, IL-10, IDO, and TGFβ. These findings indicate that elderly patients might be capable of responding to tumor antigens, and need not be excluded from immune-based therapies or clinical trials.

Deficiency of carbamoyl phosphate synthetase I (CPSI) results in hyperammonemia ranging from neonatally lethal to environmentally induced adult-onset disease. Over 24 years, analysis of tissue and DNA samples from 205 unrelated individuals diagnosed with CPSI deficiency (CPSID) detected 192 unique CPS1 gene changes, of which 130 are reported here for the first time. Pooled with the already reported mutations, they constitute a total of 222 changes, including 136 missense, 15 nonsense, 50 changes of other types resulting in enzyme truncation, and 21 other changes causing in-frame alterations. Only ∼10% of the mutations recur in unrelated families, predominantly affecting CpG dinucleotides, further complicating the diagnosis because of the "private" nature of such mutations. Missense changes are unevenly distributed along the gene, highlighting the existence of CPSI regions having greater functional importance than other regions. We exploit the crystal structure of the CPSI allosteric domain to rationalize the effects of mutations affecting it. Comparative modeling is used to create a structural model for the remainder of the enzyme. Missense changes are found to directly correlate, respectively, with the one-residue evolutionary importance and inversely correlate with solvent accessibility of the mutated residue. This is the first large-scale report of CPS1 mutations spanning a wide variety of molecular defects highlighting important regions in this protein.

Several network security and QoS applications require detecting multiple string matches in the packet payload by comparing it against predefined pattern set. This process of pattern matching at line speeds is a memory and computation intensive task. Hence, it requires dedicated hardware algorithms. This paper describes the hardware architecture of a parallel, pipelined pattern matching engine that uses trie based pattern matching algorithmic approach. The algorithm optimizes pattern matching process through two key innovations of parallel pattern matching using incoming content filter and multiple character matching using trie pruning. The hardware implementation is capable of performing at line-speeds and handle traffic rates up to OC-192, the underlying architecture allows for multiple patterns to be detected and for the system to gracefully recover from a failed partial match, the throughput of the system does not degrade with the increase in the number of patterns or the length of the patterns to be matched. The solution described outperforms most current implementations in terms of speed and memory requirement and outperforms TCAM based solutions in terms of power consumption, area, and cost while remaining competitive in terms of throughput and update times. The complete Snort rule set (2005 release) and VoIP RFC were used to validate our performance and achieve a throughput of 12Gbps with 6KBytes of content filter memory and 0.3 MBytes of total memory for Snort and 0.5KBytes of filter memory and 12KBytes of total memory for SIP.

Hepatic veno-occlusive disease (HVOD) is a serious complication of hematopoietic stem cell transplantation (HSCT). Since the liver is a major site of iron deposition in HFE-associated hemochromatosis, and iron has oxidative toxicity, we hypothesized that HFE genotype might influence the risk of HVOD after myeloablative HSCT. We determined HFE genotypes in 166 HSCT recipients who were evaluated prospectively for HVOD. We also tested whether a common variant of the rate-limiting urea cycle enzyme, carbamyl-phosphate synthetase (CPS), previously observed to protect against HVOD in this cohort, modified the effect of HFE genotype. Risk of HVOD was significantly higher in carriers of at least one C282Y allele (RR=3.7, 95% CI 1.2–12.1) and increased progressively with C282Y allelic dose (RR=1.7, 95% CI 0.4–6.8 in heterozygotes; RR=8.6, 95% CI 1.5–48.5 in homozygotes). The CPS A allele, which encodes a more efficient urea cycle enzyme, reduced the risk of HVOD associated with HFE C282Y. We conclude that HFE C282Y is a risk factor for HVOD and that CPS polymorphisms may counteract its adverse effects. Knowledge of these genotypes and monitoring of iron stores may facilitate risk-stratification and testing of strategies to prevent HVOD, such as iron chelation and pharmacologic support of the urea cycle.

Carbamyl phosphate synthetase I (CPSI) determines the rate-limiting entry of free ammonia into the urea cycle. Disruption of CPSI affects the liver's ability to remove waste nitrogen and produce arginine, citrulline, and urea. Arginine is the necessary precursor for the critical biomolecule, nitric oxide (NO). We have studied the classic model of CPSI deficiency, which results in severe hyperammonemia, and identified a large number of molecular defects. A number of CPSI polymorphisms have been found that appear to result in functional consequences. We have examined the association of these polymorphisms with various environmental stress conditions and found that certain CPSI alleles are associated with clinical outcome. We refer to these associations as environmentally determined genetic expression (EDGE) affects. In addition to studies of classic CPSI deficiency, we have developed data for the EDGE concept in post-cardiac surgery-related pulmonary hypertension, hepatic veno-occlusive disease after bone marrow transplantation, and persistent pulmonary hypertension of the newborn. We have linked these outcomes and genotypes to the availability of the urea cycle intermediates, citrulline and arginine, and their role in NO synthesis. We hypothesize that these polymorphisms affect the functional efficiency of CPSI and thus the entire urea cycle and as such, the availability of the NO substrates. By piecing together the various functional aspects of the urea cycle changes we have seen, we can better understand the clinical vulnerabilities of patients in environmentally stressful situations. This knowledge should allow us to design intervention strategies to either predict or modify the associated adverse outcomes.

Introduction: The incidence of melanoma is rising at a rapid rate in elderly population. Melanoma is an immunogenic tumor but T lymphocyte in the elderly persons are believed to be hypo/dysfunctional. Methods: HLA-A2 positive eleven elderly and eight young patients (median age 82 Y and 48 Y respectively) participated in the IRB approved study following informed consent. Peripheral blood (PB) derived CD8+ cytotoxic T lymphocytes (CTL), CD4+ helper T lymphocytes (TL) and CD4+CD25+ natural regulatory (nTreg) were used. Mart-1 and flu peptide was presented through PB derived antigen presenting cells (APC) to respective autologous CD8+ T cells to generate antigen specific CTL. The respective CD8+ and CD4+ cells from six elderly and 5 young patients, were engineered to express MHC class I restricted Mart-1 specific TCR and tested in functional assays. nT reg cells were tested for suppressive effect upon anti-CD3 mediated proliferation of autologous CD4+CD25- cells. Results: We found no significant differences in transduction efficiencies of Mart-1 specific TCR between young and old patients. Both Mart-1 and Flu specific CTL could be generated from all the 11 elderly and eight young patients. In vitro expanded antigen specific T effector cell responses were also comparable with inherent and engineered TCR between the two age groups. The elderly group, however, exhibited a significantly lower suppressive nTreg activity (94.4 % in young vs.16.7% suppression in elderly of CD4+TL stimulated by anti CD3 antibody). Conclusion: Our studies show that the CTL function is comparable between the two groups of patients but the nTreg activity in elderly patients is significantly reduced. More critical studies will be needed in larger number of patients to develop a comprehensive understanding of the T cell-mediated function that is important for immune based treatment of elderly melanoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5017. doi:10.1158/1538-7445.AM2011-5017