Sexual transmission of urogenital bacteria: whole metagenome sequencing evidence from a sexual network study.

Sexual transmission of the urogenital microbiota may contribute to adverse sexual and reproductive health outcomes. The extent of sexual transmission of the urogenital microbiota is unclear as prior studies largely investigated specific pathogens. We used epidemiologic data and whole metagenome sequencing to characterize urogenital microbiota strain concordance between participants of a sexual network study. Individuals who screened positive for genital Chlamydia trachomatis were enrolled and referred their sexual contacts from the prior 60-180 days. Snowball recruitment of sexual contacts continued for up to four waves. Vaginal swabs and penile urethral swabs were collected for whole metagenome sequencing. We evaluated bacterial strain concordance using inStrain and network analysis. We defined concordance as ≥99.99% average nucleotide identity over ≥50% shared coverage; we defined putative sexual transmission as concordance between sexual contacts with <5 single-nucleotide polymorphisms per megabase. Of 138 participants, 74 (54%) were female; 120 (87%) had genital chlamydia; and 43 (31%) were recruited contacts. We identified 115 strain-concordance events among 54 participants representing 25 bacterial species. Seven events (6%) were between sexual contacts including putative heterosexual transmission of Fannyhessea vaginae, Gardnerella leopoldii, Prevotella amnii, Sneathia sanguinegens, and Sneathia vaginalis (one strain each), and putative sexual transmission of Lactobacillus iners between female contacts. Most concordance events (108, 94%) were between non-contacts, including eight female participants connected through 18 Lactobacillus crispatus and 3 Lactobacillus jensenii concordant strains, and 14 female and 2 male participants densely interconnected through 52 Gardnerella swidsinskii concordance events.IMPORTANCEEpidemiologic evidence consistently indicates bacterial vaginosis (BV) is sexually associated and may be sexually transmitted, though sexual transmission remains subject to debate. This study is not capable of demonstrating BV sexual transmission; however, we do provide strain-level metagenomic evidence that strongly supports heterosexual transmission of BV-associated species. These findings strengthen the evidence base that supports ongoing investigations of concurrent male partner treatment for reducing BV recurrence. Our data suggest that measuring the impact of male partner treatment on F. vaginae, G. leopoldii, P. amnii, S. sanguinegens, and S. vaginalis may provide insight into why a regimen does or does not perform well. We also observed a high degree of strain concordance between non-sexual-contact female participants. We posit that this may reflect limited dispersal capacity of vaginal bacteria coupled with individuals' comembership in regional transmission networks where transmission may occur between parent and child at birth, cohabiting individuals, or sexual contacts.

Lactobacillus iners and genital health: molecular clues to an enigmatic vaginal species.

Purpose of review: Vaginal lactobacilli are recognized as important drivers of genital health including protection against bacterial vaginosis and sexually transmitted infections. Lactobacillus iners is distinct from L. crispatus, L. gasseri, and L. jensenii by its high global prevalence in vaginal microbiomes, relatively small genome, production of only L-lactic acid, and inconsistent associations with genital health outcomes. In this review, we summarize our current understanding of the role of L. iners in the vaginal microbiome, highlight the importance of strain-level consideration for this species, and explain that while marker gene-based characterization of the composition of the vaginal microbiota does not capture strain-level resolution, whole metagenome sequencing can aid in expanding our understanding of this species in genital health. Recent findings: L. iners exists in the vaginal microbiome as a unique combination of strains. The functional repertoires of these strain combinations are likely wide and contribute to the survival of this species in a variety of vaginal microenvironments. In published studies to date, strain-specific effects are aggregated and may yield imprecise estimates of risk associated with this species. Summary: The worldwide high prevalence of Lactobacillus iners warrants more research into its functional roles in the vaginal microbiome and how it may directly impact susceptibility to infections. By incorporating strain-level resolution into future research endeavors, we may begin to appreciate L. iners more thoroughly and identify novel therapeutic targets for a variety of genital health challenges.

Vaginal Microbiome Metagenome Inference Accuracy: Differential Measurement Error according to Community Composition.

Several studies have compared metagenome inference performance in different human body sites; however, none specifically reported on the vaginal microbiome. Findings from other body sites cannot easily be generalized to the vaginal microbiome due to unique features of vaginal microbial ecology, and investigators seeking to use metagenome inference in vaginal microbiome research are "flying blind" with respect to potential bias these methods may introduce into analyses. We compared the performance of PICRUSt2 and Tax4Fun2 using paired 16S rRNA gene amplicon sequencing and whole-metagenome sequencing data from vaginal samples from 72 pregnant individuals enrolled in the Pregnancy, Infection, and Nutrition (PIN) cohort. Participants were selected from those with known birth outcomes and adequate 16S rRNA gene amplicon sequencing data in a case-control design. Cases experienced early preterm birth (<32 weeks of gestation), and controls experienced term birth (37 to 41 weeks of gestation). PICRUSt2 and Tax4Fun2 performed modestly overall (median Spearman correlation coefficients between observed and predicted KEGG ortholog [KO] relative abundances of 0.20 and 0.22, respectively). Both methods performed best among Lactobacillus crispatus-dominated vaginal microbiotas (median Spearman correlation coefficients of 0.24 and 0.25, respectively) and worst among Lactobacillus iners-dominated microbiotas (median Spearman correlation coefficients of 0.06 and 0.11, respectively). The same pattern was observed when evaluating correlations between univariable hypothesis test P values generated with observed and predicted metagenome data. Differential metagenome inference performance across vaginal microbiota community types can be considered differential measurement error, which often causes differential misclassification. As such, metagenome inference will introduce hard-to-predict bias (toward or away from the null) in vaginal microbiome research. IMPORTANCE Compared to taxonomic composition, the functional potential within a bacterial community is more relevant to establishing mechanistic understandings and causal relationships between the microbiome and health outcomes. Metagenome inference attempts to bridge the gap between 16S rRNA gene amplicon sequencing and whole-metagenome sequencing by predicting a microbiome's gene content based on its taxonomic composition and annotated genome sequences of its members. Metagenome inference methods have been evaluated primarily among gut samples, where they appear to perform fairly well. Here, we show that metagenome inference performance is markedly worse for the vaginal microbiome and that performance varies across common vaginal microbiome community types. Because these community types are associated with sexual and reproductive outcomes, differential metagenome inference performance will bias vaginal microbiome studies, obscuring relationships of interest. Results from such studies should be interpreted with substantial caution and the understanding that they may over- or underestimate associations with metagenome content.

Epidemiologic Evidence on the Role of Lactobacillus iners in Sexually Transmitted Infections and Bacterial Vaginosis: A Series of Systematic Reviews and Meta-Analyses.

ABSTRACT: Although Lactobacillus crispatus -dominated vaginal microbiotas are thought to protect against bacterial vaginosis (BV) and sexually transmitted infections, the role of Lactobacillus iners -dominated microbiotas is less clear. To better understand the impact of L. iners on common cervicovaginal infections, we conducted systematic reviews of the associations between L. iners compared with L. crispatus and 8 outcomes: Chlamydia trachomatis (Ct), BV, human papillomavirus, cervical dysplasia, human immunodeficiency virus, genital herpes, Trichomonas vaginalis , and Neisseria gonorrhoeae . On April 30, 2021, we searched PubMed, Embase, Cochrane Library, and Web of Science for epidemiologic studies of reproductive-age, nonpregnant, cisgender women that used marker gene sequencing to characterize vaginal microbiota composition and presented an effect estimate for the association between L. iners , compared with L. crispatus , and outcomes of interest. For outcomes with ≥3 eligible results presenting the same form of effect estimate, we conducted random-effects meta-analysis. The review protocol was registered prospectively (PROSPERO CRD42020214775). Six Ct studies were included in meta-analysis, which showed L. iners -dominated microbiotas were associated with 3.4-fold higher odds of Ct compared with L. crispatus -dominated microbiotas (95% confidence interval, 2.1-5.4). Three BV studies were included in meta-analysis, which indicated L. iners -dominated microbiotas were associated with 2.1-fold higher prevalence of BV compared with L. crispatus -dominated microbiotas (95% confidence interval, 0.9-4.9). Evidence was too sparse to perform meta-analysis for the remaining outcomes. L. iners -dominated vaginal microbiotas may be suboptimal compared with L. crispatus -dominated microbiotas for BV and Ct. These reviews highlight evidence gaps regarding the remaining outcomes and opportunities to improve epidemiologic rigor in vaginal microbiome science.

Associations Between Vaginal Bacteria and Bacterial Vaginosis Signs and Symptoms: A Comparative Study of Kenyan and American Women.

Background: Bacterial colonization and associations with bacterial vaginosis (BV) signs and symptoms (Amsel criteria) may vary between populations. We assessed relationships between vaginal bacteria and Amsel criteria among two populations. Methods: Kenyan participants from the placebo arm of the Preventing Vaginal Infections (PVI) trial and participants from a Seattle-based cross-sectional BV study were included. Amsel criteria were recorded at study visits, and the vaginal microbiota was characterized using 16S rRNA gene sequencing. Logistic regression models, accounting for repeat visits as appropriate, were fit to evaluate associations between bacterial relative abundance and each Amsel criterion. Results: Among 84 PVI participants (496 observations) and 220 Seattle participants, the prevalence of amine odor was 25% and 40%, clue cells 16% and 37%, vaginal discharge 10% and 52%, elevated vaginal pH 69% and 67%, and BV 13% and 44%, respectively. BV-associated bacterium 1 (BVAB1) was positively associated with all Amsel criteria in both populations. Eggerthella type 1, Fannyhessea (Atopobium) vaginae, Gardnerella spp., Sneathia amnii, and Sneathia sanguinegens were positively associated with all Amsel criteria in the Seattle study, and all but discharge in the PVI trial. Conclusions: Core vaginal bacteria are consistently associated with BV signs and symptoms across two distinct populations of women.

Short Report: Using Targeted Urine Metabolomics to Distinguish Between Manganese Exposed and Unexposed Workers in a Small Occupational Cohort.

Objectives: Despite the widespread use of manganese (Mn) in industrial settings and its association with adverse neurological outcomes, a validated and reliable biomarker for Mn exposure is still elusive. Here, we utilize targeted metabolomics to investigate metabolic differences between Mn-exposed and -unexposed workers, which could inform a putative biomarker for Mn and lead to increased understanding of Mn toxicity. Methods: End of shift spot urine samples collected from Mn exposed (n = 17) and unexposed (n = 15) workers underwent a targeted assay of 362 metabolites using LC-MS/MS; 224 were quantified and retained for analysis. Differences in metabolite abundances between exposed and unexposed workers were tested with a Benjamini-Hochberg adjusted Wilcoxon Rank-Sum test. We explored perturbed pathways related to exposure using a pathway analysis. Results: Seven metabolites were significantly differentially abundant between exposed and unexposed workers (FDR ≤ 0.1), including n-isobutyrylglycine, cholic acid, anserine, beta-alanine, methionine, n-isovalerylglycine, and threonine. Three pathways were significantly perturbed in exposed workers and had an impact score >0.5: beta-alanine metabolism, histidine metabolism, and glycine, serine, and threonine metabolism. Conclusion: This is one of few studies utilizing targeted metabolomics to explore differences between Mn-exposed and -unexposed workers. Metabolite and pathway analysis showed amino acid metabolism was perturbed in these Mn-exposed workers. Amino acids have also been shown to be perturbed in other occupational cohorts exposed to Mn. Additional research is needed to characterize the biological importance of amino acids in the Mn exposure-disease continuum, and to determine how to appropriately utilize and interpret metabolomics data collected from occupational cohorts.

Vaginal Bacteria and Risk of Incident and Persistent Infection With High-Risk Subtypes of Human Papillomavirus: A Cohort Study Among Kenyan Women.

BACKGROUND: Bacterial vaginosis (BV) is associated with an increased risk of high-risk human papillomavirus (hrHPV), whereas Lactobacillus-dominated vaginal microbiotas are associated with reduced burden of hrHPV. Few epidemiologic studies have prospectively investigated the relationships between vaginal bacteria and hrHPV, particularly among women from countries in Africa. METHODS: We conducted a prospective cohort study nested within the Preventing Vaginal Infections trial to evaluate associations between vaginal bacteria and hrHPV incidence and persistence. Sexually active, HIV-seronegative women aged 18 to 45 years who had a vaginal infection at screening were eligible to enroll. Analyses were restricted to participants enrolled in Kenya and randomized to placebo. At enrollment and months 2, 4, 6, 8, 10, and 12, hrHPV testing, quantitative polymerase chain reaction (measuring taxon quantity per swab), and 16S rRNA gene amplicon sequencing of the vaginal microbiota were performed. Generalized estimating equations multinomial logistic regression models were fit to evaluate associations between vaginal bacteria and incident and persistent hrHPV. RESULTS: Eighty-four participants were included in this analysis. Higher concentrations of Lactobacillus crispatus were inversely associated with persistent hrHPV detection. Specifically, 1 tertile higher L. crispatus concentration was associated with 50% reduced odds of persistent hrHPV detection (odds ratio, 0.50; 95% confidence interval, 0.29-0.85). CONCLUSIONS: This study is consistent with reports that vaginal L. crispatus is associated with reduced susceptibility to hrHPV persistence. Evidence from in vitro studies provides insight into potential mechanisms by which L. crispatus may mediate hrHPV risk. Future studies should further explore in vivo mechanisms that may drive this relationship and opportunities for intervention.

Sexual behaviour change following HIV testing services: a systematic review and meta-analysis.

INTRODUCTION: Learning one's HIV status through HIV testing services (HTS) is an essential step toward accessing treatment and linking to preventive services for those at high HIV risk. HTS may impact subsequent sexual behaviour, but the degree to which this varies by population or is true in the setting of contemporary HIV prevention activities is largely unknown. As part of the 2019 World Health Organization Consolidated Guidelines on HTS, we undertook a systematic review and meta-analysis to determine the effect of HTS on sexual behaviour. METHODS: We searched nine electronic databases for studies published between July 2010 and December 2019. We included studies that reported on at least one outcome (condom use [defined as the frequency of condom use or condom-protected sex], number of sex partners, HIV incidence, STI incidence/prevalence). We included studies that prospectively assessed outcomes and that fit into one of three categories: (1) those evaluating more versus less-intensive HTS, (2) those of populations receiving HTS versus not and (3) those evaluating outcomes after versus before HTS. We conducted meta-analyses using random-effects models. RESULTS AND DISCUSSION: Of 29 980 studies screened, 76 studies were included. Thirty-eight studies were randomized controlled trials, 36 were cohort studies, one was quasi-experimental and one was a serial cross-sectional study. There was no significant difference in condom use among individuals receiving more-intensive HTS compared to less-intensive HTS (relative risk [RR]=1.03; 95% CI: 0.99 to 1.07). Condom use was significantly higher after receiving HTS compared to before HTS for individuals newly diagnosed with HIV (RR = 1.65; 95% CI: 1.36 to 1.99) and marginally significantly higher for individuals receiving an HIV-negative diagnosis (RR = 1.63; 95% CI: 1.01 to 2.62). Individuals receiving more-intensive HTS reported fewer sex partners at follow-up than those receiving less-intensive HTS, but the finding was not statistically significant (mean difference = -0.28; 95% CI: -3.66, 3.10). CONCLUSIONS: Our findings highlight the importance of using limited resources towards HTS strategies that focus on early HIV diagnosis, treatment and prevention services rather than resources dedicated to supplementing or enhancing HTS with additional counselling or other interventions.

The vaginal microbiota, high-risk human papillomavirus infection, and cervical cytology: results from a population-based study.

BACKGROUND: The relationship between the vaginal microbiota, high-risk human papillomavirus infection, and abnormal cervical cytology has not been well characterized. Our objective was to characterize the vaginal microbiota in a stratified random sample of women from a population-based study in Appalachia. METHODS: We analyzed a random sample of 308 women in the Community Access, Resources and Education: Project 3 study across 16 clinics in Ohio and West Virginia. Using Illumina MiSeq sequencing of 16S rRNA gene amplicons, we characterized the vaginal microbiota among (I) 109 women randomly chosen with abnormal cervical cytology (i.e., the majority were atypical squamous cells of undetermined significance (n=55) and low-grade squamous intraepithelial lesions (n=45) while n=6 were high-grade squamous intraepithelial lesions and n=3 were atypical glandular cells); (II) 110 high-risk human papillomavirus infection only without cytologic abnormality; and (III) 89 women from a stratified random sample without cytologic abnormalities (negative for intraepithelial lesion or malignancy or any human papillomavirus infection). Among the women with abnormal cervical cytology (n=109), 80 had human papillomavirus infection, the majority of which were positive for a high-risk type (n=61). RESULTS: Nearly all of the women were non-Hispanic White (94.5%), and the mean age was 26 (IQR=21-39) years. Women with abnormal cervical cytology or who were HPV+ were more likely to have a diverse vaginal microbiota characterized by higher Gardnerella vaginalis relative abundance, compared to women without cytologic abnormalities whose communities were more likely to be Lactobacillus spp. dominant (P<0.04). Women without cytologic abnormalities had a higher prevalence of L. iners dominated communities than women with abnormal cervical cytology and HR HPV+ respectively (P<0.04), and L. gasseri relative abundance was differentially greater among these women compared to women with abnormal cervical cytology or who were high-risk HPV+ (Linear discriminant analysis effect size =4.17; P=0.0009). After adjustment for age, white race, current smoking, and ≥2 male partners in the last year, however, we did not detect differences in the vaginal microbiota community states across the three outcome groups. CONCLUSIONS: Compared to women without cytologic abnormalities, the vaginal microbiota of women with abnormal cervical cytology or who were high-risk HPV+ were characterized by a diverse community with increased relative abundance of G. vaginalis and reduced relative abundance of L. gasseri. However, these differences were attenuated after adjustment for other factors. Further study and validation of these differences for prognostic use is warranted.

Lessons from Suppressive Therapy and Periodic Presumptive Treatment for Bacterial Vaginosis.

PURPOSE OF REVIEW: Suppressive therapy and periodic presumptive treatment (PPT) are distinct but related strategies that have been used to reduce the incidence of bacterial vaginosis (BV). Here, we review clinical trial evidence of the effectiveness of suppressive therapy and PPT to reduce BV, and discuss their roles for women who frequently experience symptomatic or asymptomatic BV. RECENT FINDINGS: Among women who were recently and successfully treated for symptomatic BV, suppressive therapy with twice-weekly metronidazole gel for 16 weeks reduces the likelihood of recurrent symptomatic BV and is currently recommended by the Centers for Disease Control and Prevention for prevention of recurrent BV. The premise of PPT is to provide regimens used to treat BV at regular intervals to reduce the overall frequency of BV, regardless of symptoms. Three PPT trials were conducted using different routes (oral or intravaginal), doses, and frequencies of administration. Each trial demonstrated a significant reduction in BV over the course 12 months, ranging from a 10 to 45% decrease. PPT regimens that substantially reduce the frequency of BV over time could be evaluated in clinical trials to assess whether a reduced frequency of BV leads to subsequent reductions in BV-associated sequelae. While both suppressive therapy and PPT reduce BV, their impact wanes following cessation of the regimen. Given the high prevalence of BV globally and burden of adverse reproductive health outcomes among women with BV, there is a critical need for more effective treatments that produce durable shifts in the microbiota towards vaginal health.

Development and application of a high throughput one-pot extraction protocol for quantitative LC-MS/MS analysis of phospholipids in serum and lipoprotein fractions in normolipidemic and dyslipidemic subjects.

Progress toward better diagnosis and treatment of lipid metabolism-related diseases requires high throughput approaches for multiplexed quantitative analysis of structurally diverse lipids, including phospholipids (PLs). This work demonstrates a simplified "one-pot" phospholipid extraction protocol, as an alternative to conventional liquid-liquid extraction. Performed in a 96-well format, the extraction was coupled with high throughput UPLC and multiplexed tandem mass spectrometry (MS/MS) detection, allowing non-targeted quantification of phosphatidylcholines (PC), sphingomyelins (SM), lysophosphatidylcholines (LPC), phosphatidylethanolamines (PE), and phosphatidylinositols (PI). Using 50 µL aliquots of serum samples from 110 individuals, lipoproteins were fractionated by size, and analyzed for phospholipids and non-polar lipids including free cholesterol (FC), cholesteryl esters (CEs) and triglycerides (TGs). Analysis of serum samples with wide range of Total-TG levels showed significant differences in PL composition. The correlations of molar ratios in lipoprotein size fractions, SM/PL with FC/PL, PE/PL with TG/CE, and PE/PL with PI/PL, demonstrate the applicability of the method for quantitative composition analysis of high, low and very-low density lipoproteins (HDL, LDL and VLDL), and characterization of lipid metabolism related disease states.

Laxative use and testing for Clostridium difficile in hospitalized adults: An opportunity to improve diagnostic stewardship.

BACKGROUND: It is recommended that that only unformed stool from patients with diarrhea be tested for Clostridium difficile infection. We determined the prevalence of and patient characteristics associated with antecedent laxative receipt among hospitalized adults undergoing C difficile testing. METHODS: In a case-control study of 5,452 C difficile tests from 5 hospitals in Southeast Michigan, patients who received laxatives (docusate, senna, polyethylene glycol 3350, bisacodyl, and magnesium hydroxide) in the 24 or 48 hours before testing were identified. Logistic regression was performed to identify patient characteristics associated with laxative receipt before testing. RESULTS: In 535 (9.8%) and 707 (13%) tests, patients received laxatives in the 24 and 48 hours before testing, respectively. The odds of antecedent laxative receipt were significantly greater for patients residing on a surgical service than a medical service (24 hours odds ratio [OR], 2.5; 95% confidence interval [CI], 2.1-3.1; 48 hours OR, 2.7; 95% CI, 2.3-3.2), patients residing in an intensive care unit (ICU) than a non-ICU (24 hours OR, 1.3; 95% CI, 1.0-1.6; 48 hours OR, 1.3; 95% CI, 1.1-1.6), and patients whose Elixhauser Comorbidity Score was 4 or higher (24 hours OR, 1.4; 95% CI, 1.1-1.7; 48 hours OR, 1.4; 95% CI, 1.2-1.7). CONCLUSIONS: Among patients tested for C difficile, antecedent laxative use was common. Improving diagnostic stewardship around C difficile testing, particularly in surgical and ICU patients, is a significant opportunity and priority for quality improvement.

Myeloid-Derived Suppressor Cell Survival and Function Are Regulated by the Transcription Factor Nrf2.

Tumor-induced myeloid-derived suppressor cells (MDSC) contribute to immune suppression in tumor-bearing individuals and are a major obstacle to effective immunotherapy. Reactive oxygen species (ROS) are one of the mechanisms used by MDSC to suppress T cell activation. Although ROS are toxic to most cells, MDSC survive despite their elevated content and release of ROS. NF erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates a battery of genes that attenuate oxidative stress. Therefore, we hypothesized that MDSC resistance to ROS may be regulated by Nrf2. To test this hypothesis, we used Nrf2(+/+)and Nrf2(-/-)BALB/c and C57BL/6 mice bearing 4T1 mammary carcinoma and MC38 colon carcinoma, respectively. Nrf2 enhanced MDSC suppressive activity by increasing MDSC production of H2O2, and it increased the quantity of tumor-infiltrating MDSC by reducing their oxidative stress and rate of apoptosis. Nrf2 did not affect circulating levels of MDSC in tumor-bearing mice because the decreased apoptotic rate of tumor-infiltrating MDSC was balanced by a decreased rate of differentiation from bone marrow progenitor cells. These results demonstrate that Nrf2 regulates the generation, survival, and suppressive potency of MDSC, and that a feedback homeostatic mechanism maintains a steady-state level of circulating MDSC in tumor-bearing individuals.

Cross-talk among myeloid-derived suppressor cells, macrophages, and tumor cells impacts the inflammatory milieu of solid tumors.

MDSC and macrophages are present in most solid tumors and are important drivers of immune suppression and inflammation. It is established that cross-talk between MDSC and macrophages impacts anti-tumor immunity; however, interactions between tumor cells and MDSC or macrophages are less well studied. To examine potential interactions between these cells, we studied the impact of MDSC, macrophages, and four murine tumor cell lines on each other, both in vitro and in vivo. We focused on IL-6, IL-10, IL-12, TNF-α, and NO, as these molecules are produced by macrophages, MDSC, and many tumor cells; are present in most solid tumors; and regulate inflammation. In vitro studies demonstrated that MDSC-produced IL-10 decreased macrophage IL-6 and TNF-α and increased NO. IL-6 indirectly regulated MDSC IL-10. Tumor cells increased MDSC IL-6 and vice versa. Tumor cells also increased macrophage IL-6 and NO and decreased macrophage TNF-α. Tumor cell-driven macrophage IL-6 was reduced by MDSC, and tumor cells and MDSC enhanced macrophage NO. In vivo analysis of solid tumors identified IL-6 and IL-10 as the dominant cytokines and demonstrated that these molecules were produced predominantly by stromal cells. These results suggest that inflammation within solid tumors is regulated by the ratio of tumor cells to MDSC and macrophages and that interactions of these cells have the potential to alter significantly the inflammatory milieu within the tumor microenvironment.