Single-center randomized trial of T-reg graft alone vs T-reg graft plus tacrolimus for the prevention of acute GVHD.

ABSTRACT: Allogeneic hematopoietic cell transplantation (HCT) is a curative therapy for hematological malignancies for which graft-versus-host disease (GVHD) remains a major complication. The use of donor T-regulatory cells (Tregs) to prevent GVHD appears promising, including in our previous evaluation of an engineered graft product (T-reg graft) consisting of the timed, sequential infusion of CD34+ hematopoietic stem cells and high-purity Tregs followed by conventional T cells. However, whether immunosuppressive prophylaxis can be removed from this protocol remains unclear. We report the results of the first stage of an open-label single-center phase 2 study (NCT01660607) investigating T-reg graft in myeloablative HCT of HLA-matched and 9/10-matched recipients. Twenty-four patients were randomized to receive T-reg graft alone (n = 12) or T-reg graft plus single-agent GVHD prophylaxis (n = 12) to determine whether T-reg graft alone was noninferior in preventing acute GVHD. All patients developed full-donor myeloid chimerism. Patients with T-reg graft alone vs with prophylaxis had incidences of grade 3 to 4 acute GVHD of 58% vs 8% (P = .005) and grade 3 to 4 of 17% vs 0% (P = .149), respectively. The incidence of moderate-to-severe chronic GVHD was 28% in the T-reg graft alone arm vs 0% with prophylaxis (P = .056). Among patients with T-reg graft and prophylaxis, CD4+ T-cell-to-Treg ratios were reduced after transplantation, gene expression profiles showed reduced CD4+ proliferation, and the achievement of full-donor T-cell chimerism was delayed. This study indicates that T-reg graft with single-agent tacrolimus is preferred over T-reg graft alone for the prevention of acute GVHD. This trial was registered at www.clinicaltrials.gov as #NCT01660607.

Improved outcomes for relapsed/refractory Hodgkin lymphoma after autologous transplantation in the era of novel agents.

The treatment landscape of relapsed/refractory (R/R) classic Hodgkin lymphoma (cHL) has evolved significantly over the past decade after the approval of brentuximab vedotin (BV) and the programmed death-1 (PD-1) inhibitors. We evaluated how outcomes and practice patterns have changed for patients with R/R cHL who underwent autologous hematopoietic cell transplantation (AHCT) at our institution from 2011 to 2020 (N = 183) compared with those from 2001 to 2010 (N = 159) and evaluated prognostic factors for progression-free survival (PFS) and overall survival (OS) in both eras. OS was superior in the modern era with a trend toward lower nonrelapse mortality beyond 2 years after transplant. Among patients who progressed after AHCT, 4-year postprogression survival increased from 43.3% to 71.4% in the modern era, reflecting increasing use of BV and the PD-1 inhibitors. In multivariable analysis for patients that underwent transplant in the modern era, age ≥45 years, primary refractory disease, and lack of complete remission pre-AHCT were associated with inferior PFS, whereas receipt of a PD-1 inhibitor-based regimen pre-AHCT was associated with superior PFS. Extranodal disease at relapse was associated with inferior OS. Our study demonstrates improved survival for R/R cHL after AHCT in the modern era attributed to more effective salvage regimens allowing for better disease control pre-AHCT and improved outcomes for patients who progressed after AHCT. Excellent outcomes were observed with PD-1 inhibitor-based salvage regimens pre-AHCT and support a randomized trial evaluating immunotherapy in the second line setting.

Post-infusion CAR TReg cells identify patients resistant to CD19-CAR therapy.

Approximately 60% of patients with large B cell lymphoma treated with chimeric antigen receptor (CAR) T cell therapies targeting CD19 experience disease progression, and neurotoxicity remains a challenge. Biomarkers associated with resistance and toxicity are limited. In this study, single-cell proteomic profiling of circulating CAR T cells in 32 patients treated with CD19-CAR identified that CD4+Helios+ CAR T cells on day 7 after infusion are associated with progressive disease and less severe neurotoxicity. Deep profiling demonstrated that this population is non-clonal and manifests hallmark features of T regulatory (TReg) cells. Validation cohort analysis upheld the link between higher CAR TReg cells with clinical progression and less severe neurotoxicity. A model combining expansion of this subset with lactate dehydrogenase levels, as a surrogate for tumor burden, was superior for predicting durable clinical response compared to models relying on each feature alone. These data credential CAR TReg cell expansion as a novel biomarker of response and toxicity after CAR T cell therapy and raise the prospect that this subset may regulate CAR T cell responses in humans.

A Composite Endpoint for Treatment Benefit According to Patient Preference.

Complex disorders usually affect multiple symptom domains measured by several outcomes. The importance of these outcomes is often different among patients. Current approaches integrate multiple outcomes without considering patient preferences at the individual level. In this paper, we propose a new composite Desirability of Outcome Ranking (DOOR) that integrates individual level ranking of outcome importance and define a winning probability measuring the overall treatment effect. Stratified randomization can be performed based on the participants' baseline outcome rankings. A Wilcoxon-Mann-Whitney U-statistic is used to average the pairwise DOOR between one treated and one control patient, considering the difference in these patients' ranking of outcome importance. We use both theoretical and empirical methods to examine the statistical properties of our method and to compare with conventional approaches. We conclude that the proposed composite DOOR properly reflects patient-level preferences and can be used in pivotal trials or comparative effectiveness trials for a patient-centered evaluation of overall treatment benefits.

Development of immunosuppressive myeloid cells to induce tolerance in solid organ and hematopoietic cell transplant recipients.

Replacement of failed organs followed by safe withdrawal of immunosuppressive drugs has long been the goal of organ transplantation. We studied changes in the balance of T cells and myeloid cells in the blood of HLA-matched and -mismatched patients given living donor kidney transplants followed by total lymphoid irradiation, anti-thymocyte globulin conditioning, and donor hematopoietic cell transplant to induce mixed chimerism and immune tolerance. The clinical trials were based on a conditioning regimen used to establish mixed chimerism and tolerance in mice. In preclinical murine studies, there was a profound depletion of T cells and an increase in immunosuppressive polymorphonuclear (pmn) myeloid-derived suppressor cells (MDSCs) in the spleen and blood following transplant. Selective depletion of pmn MDSCs in mice abrogated mixed chimerism and tolerance. In our clinical trials, patients given an analogous tolerance conditioning regimen developed similar changes, including profound depletion of T cells and a marked increase in MDSCs in blood posttransplant. Posttransplant pmn MDSCs transiently increased expression of lectin-type oxidized LDL receptor-1, a marker of immunosuppression, and production of the T-cell inhibitor arginase-1. These posttransplant pmn MDSCs suppressed the activation, proliferation, and inflammatory cytokine secretion of autologous T-cell receptor microbead-stimulated pretransplant T cells when cocultured in vitro. In conclusion, we elucidated changes in receptors and function of immunosuppressive myeloid cells in patients enrolled in the tolerance protocol that were nearly identical to those of MDSCs required for tolerance in mice. These trials were registered at www.clinicaltrials.gov as #NCT00319657 and #NCT01165762.

Immune reconstitution and infectious complications following axicabtagene ciloleucel therapy for large B-cell lymphoma.

Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has significantly improved outcomes in the treatment of refractory or relapsed large B-cell lymphoma (LBCL). We evaluated the long-term course of hematologic recovery, immune reconstitution, and infectious complications in 41 patients with LBCL treated with axicabtagene ciloleucel (axi-cel) at a single center. Grade 3+ cytopenias occurred in 97.6% of patients within the first 28 days postinfusion, with most resolved by 6 months. Overall, 63.4% of patients received a red blood cell transfusion, 34.1% of patients received a platelet transfusion, 36.6% of patients received IV immunoglobulin, and 51.2% of patients received growth factor (granulocyte colony-stimulating factor) injections beyond the first 28 days postinfusion. Only 40% of patients had recovered detectable CD19+ B cells by 1 year, and 50% of patients had a CD4+ T-cell count <200 cells per µL by 18 months postinfusion. Patients with durable responses to axi-cel had significantly longer durations of B-cell aplasia, and this duration correlated strongly with the recovery of CD4+ T-cell counts. There were significantly more infections within the first 28 days compared with any other period of follow-up, with the majority being mild-moderate in severity. Receipt of corticosteroids was the only factor that predicted risk of infection in a multivariate analysis (hazard ratio, 3.69; 95% confidence interval, 1.18-16.5). Opportunistic infections due to Pneumocystis jirovecii and varicella-zoster virus occurred up to 18 months postinfusion in patients who prematurely discontinued prophylaxis. These results support the use of comprehensive supportive care, including long-term monitoring and antimicrobial prophylaxis, beyond 12 months after axi-cel treatment.

Image-based scaling laws for somatic growth and pulmonary artery morphometry from infancy to adulthood.

Pulmonary artery (PA) morphometry has been extensively explored in adults, with particular focus on intra-acinar arteries. However, scaling law relationships for length and diameter of extensive preacinar PAs by age have not been previously reported for in vivo human data. To understand preacinar PA growth spanning children to adults, we performed morphometric analyses of all PAs visible in the computed tomography (CT) and magnetic resonance (MR) images from a healthy subject cohort [n = 16; age: 1-51 yr; body surface area (BSA): 0.49-2.01 m2]. Subject-specific anatomic PA models were constructed from CT and MR images, and morphometric information-diameter, length, tortuosity, bifurcation angle, and connectivity-was extracted and sorted into diameter-defined Strahler orders. Validation of Murray's law, describing optimal scaling exponents of radii for branching vessels, was performed to determine how closely PAs conform to this classical relationship. Using regression analyses of vessel diameters and lengths against orders and patient metrics (BSA, age, height), we found that diameters increased exponentially with order and allometrically with patient metrics. Length increased allometrically with patient metrics, albeit weakly. The average tortuosity index of all vessels was 0.026 ± 0.024, average bifurcation angle was 28.2 ± 15.1°, and average Murray's law exponent was 2.92 ± 1.07. We report a set of scaling laws for vessel diameter and length, along with other morphometric information. These provide an initial understanding of healthy structural preacinar PA development with age, which can be used for computational modeling studies and comparison with diseased PA anatomy.NEW & NOTEWORTHY Pulmonary artery (PA) morphometry studies to date have focused primarily on large arteries and intra-acinar arteries in either adults or children, neglecting preacinar arteries in both populations. Our study is the first to quantify in vivo preacinar PA morphometry changes spanning infants to adults. For preacinar arteries > 1 mm in diameter, we identify scaling laws for vessel diameters and lengths with patient metrics of growth and establish a healthy PA morphometry baseline for most preacinar PAs.

Analysis of the Whole CDR3 T Cell Receptor Repertoire after Hematopoietic Stem Cell Transplantation in 2 Clinical Cohorts.

A major cause of morbidity and mortality for patients who undergo hematologic stem cell transplantation (HSCT) is acute graft-versus-host disease (aGVHD), a mostly T cell-mediated disease. Examination of the T cell receptor (TCR) repertoire of HSCT recipients and the use of next-generation nucleotide sequencing have raised the question of whether features of TCR repertoire reconstitution might reproducibly associate with aGVHD. We hypothesized that the peripheral blood TCR repertoire of patients with steroid-nonresponsive aGVHD would be less diverse. We also hypothesized that patients with GVHD who shared HLA might also share common clones at the time of GVHD diagnosis, thereby potentially providing potential clinical indicators for treatment stratification. We further hypothesized that HSCT recipients with the same HLA mismatch might share a more similar TCR repertoire based on a potentially shared focus of alloreactive responses. We studied 2 separate patient cohorts and 2 separate platforms for measuring TCR repertoire. The first cohort of patients was from a multicenter Phase III randomized double-blinded clinical trial of patients who developed aGVHD (NCT01002742). The second cohort comprised samples from biobanks from 2 transplantation centers and the Center for International Blood and Marrow Transplant Research of patients who underwent mismatched HSCT. There were no statistically significant differences in the TCR diversity of steroid responders and nonresponders among patients with aGVHD on the day of diagnosis. Most clones in the repertoire were unique to each patient, but a small number of clones were found to be both exclusive to and shared among aGVHD nonresponders. We were also able to show a strong correlation between the presence of Vß20 and Vß29 and steroid responsiveness. Using the Bhattacharya coefficient, those patients who shared the same HLA mismatch were shown to be no more similar to one another than to those who had a completely different mismatch. Using 2 separate clinical cohorts and 2 separate platforms for analyzing the TCR repertoire, we have shown that the sampled human TCR repertoire is largely unique to each patient but contains glimmers of common clones of subsets of clones based on responsiveness to steroids in aGVHD on the day of diagnosis. These studies are informative for future strategies to assess for reproducible TCR responses in human alloreactivity and possible markers of GVHD responsiveness to therapy.

Nonmyeloablative TLI-ATG conditioning for allogeneic transplantation: mature follow-up from a large single-center cohort.

Nonmyeloablative total lymphoid irradiation and antithymocyte globulin (TLI-ATG) conditioning is protective against graft-versus-host disease (GVHD), while retaining graft-versus-tumor activity across various hematologic malignancies. We report our comprehensive experience using TLI-ATG conditioning in 612 patients with hematologic malignancies who underwent allogeneic transplantation at Stanford University from 2001 to 2016. All patients received granulocyte colony-stimulating factor-mobilized peripheral blood grafts and cyclosporine and mycophenolate mofetil for GVHD prophylaxis. The median age was 60 years (range, 21-78), with a median follow-up of 6.0 years (range, 1.0-16.4). Common diagnoses included acute myeloid leukemia (AML; n = 193), myelodysplastic syndrome (MDS; n = 94), chronic lymphocytic leukemia (CLL; n = 80), non-Hodgkin lymphoma (NHL; n = 175), and Hodgkin lymphoma (HL; n = 35). Thirty-four percent of patients had a comorbidity index ≥3, 30% had a high to very high disease risk index, and 56% received unrelated donor grafts, including 15% with HLA-mismatched donors. Ninety-eight percent underwent transplant in the outpatient setting, and 57% were never hospitalized from days 0 through 100. The 1-year rates of nonrelapse mortality (NRM), grade II-IV acute GVHD, and extensive chronic GVHD were 9%, 14%, and 22%, respectively. The 4-year estimates for overall and progression-free survival were 42% and 32% for AML, 30% and 21% for MDS, 67% and 43% for CLL, 68% and 45% for NHL, and 78% and 49% for HL. Mixed chimerism correlated with the risk of relapse. TLI-ATG conditioning was well tolerated, with low rates of GVHD and NRM. Durable remissions were observed across hematologic malignancies, with particularly favorable outcomes for heavily pretreated lymphomas. Several efforts are underway to augment donor chimerism and reduce relapse rates while maintaining the favorable safety and tolerability profile of this regimen.

High human herpesvirus 6 viral load in pediatric allogeneic hematopoietic stem cell transplant patients is associated with detection in end organs and high mortality.

Human Herpes Virus 6 (HHV-6) reactivation occurs in approximately half of patients following allogeneic hematopoietic stem cell transplant (HSCT). While encephalitis and delayed engraftment are well-documented complications of HHV-6 following HSCT, the extent to which HHV-6 viremia causes disease in children is controversial. We performed a retrospective review of HHV-6 reactivation and possible manifestations in pediatric allogeneic HSCT patients at a single institution. Of 89 children and young adults who underwent allogeneic HSCT over a three-and-a-half-year period, 34 patients reactivated HHV-6 early post-transplant. Unrelated donor stem cell source and lack of antiviral prophylaxis were risk factors for the development of HHV-6 viremia. Viremia correlated with the presence of acute graft-versus-host disease, but not chronic graft-versus-host disease. We identified two subgroups within the viremic patients-a high-risk viremic and tissue-positive group that reactivated HHV-6 and had suspected end-organ disease and a low-risk viremic but asymptomatic group that reactivated HHV-6 but did not exhibit symptoms or signs of end-organ disease. Peak viral load was found to be strongly associated with mortality. Prospective studies in larger numbers of patients are needed to further investigate the role of HHV-6 in causing symptomatic end-organ disease as well as the association of viral load with mortality.

Local estrogen axis in the human bone microenvironment regulates estrogen receptor-positive breast cancer cells.

BACKGROUND: Approximately 70% of all breast cancers express the estrogen receptor, and are regulated by estrogen. While the ovaries are the primary source of estrogen in premenopausal women, most breast cancer is diagnosed following menopause, when systemic levels of this hormone decline. Estrogen production from androgen precursors is catalyzed by the aromatase enzyme. Although aromatase expression and local estrogen production in breast adipose tissue have been implicated in the development of primary breast cancer, the source of estrogen involved in the regulation of estrogen receptor-positive (ER+) metastatic breast cancer progression is less clear. METHODS: Bone is the most common distant site of breast cancer metastasis, particularly for ER+ breast cancers. We employed a co-culture model using trabecular  bone tissues obtained from total hip replacement (THR) surgery specimens to study ER+ and estrogen receptor-negative (ER-) breast cancer cells within the human bone microenvironment. Luciferase-expressing ER+ (MCF-7, T-47D, ZR-75) and ER- (SK-BR-3, MDA-MB-231, MCF-10A) breast cancer cells were cultured directly on bone tissue fragments or in bone tissue-conditioned media, and monitored over time with bioluminescence imaging (BLI). Bone tissue-conditioned media were generated in the presence vs. absence of aromatase inhibitors, and testosterone. Bone tissue fragments were analyzed for aromatase expression by immunohistochemistry. RESULTS: ER+ breast cancer cells were preferentially sustained in co-cultures with bone tissues and bone tissue-conditioned media relative to ER- cells. Bone fragments analyzed by immunohistochemistry revealed expression of the aromatase enzyme. Bone tissue-conditioned media generated in the presence of testosterone had increased estrogen levels and heightened capacity to stimulate ER+ breast cancer cell proliferation. Pretreatment of cultured bone tissues with aromatase inhibitors, which inhibited estrogen production, reduced the capacity of conditioned media to stimulate ER+ cell proliferation. CONCLUSIONS: These results suggest that a local estrogen signaling axis regulates ER+ breast cancer cell viability and proliferation within the bone metastatic niche, and that aromatase inhibitors modulate this axis. Although endocrine therapies are highly effective in the treatment of ER+ breast cancer, resistance to these treatments reduces their efficacy. Characterization of estrogen signaling networks within the bone microenvironment will identify new strategies for combating metastatic progression and endocrine resistance.

Breast Cancer Cell Colonization of the Human Bone Marrow Adipose Tissue Niche.

BACKGROUND/OBJECTIVES: Bone is a preferred site of breast cancer metastasis, suggesting the presence of tissue-specific features that attract and promote the outgrowth of breast cancer cells. We sought to identify parameters of human bone tissue associated with breast cancer cell osteotropism and colonization in the metastatic niche. METHODS: Migration and colonization patterns of MDA-MB-231-fLuc-EGFP (luciferase-enhanced green fluorescence protein) and MCF-7-fLuc-EGFP breast cancer cells were studied in co-culture with cancellous bone tissue fragments isolated from 14 hip arthroplasties. Breast cancer cell migration into tissues and toward tissue-conditioned medium was measured in Transwell migration chambers using bioluminescence imaging and analyzed as a function of secreted factors measured by multiplex immunoassay. Patterns of breast cancer cell colonization were evaluated with fluorescence microscopy and immunohistochemistry. RESULTS: Enhanced MDA-MB-231-fLuc-EGFP breast cancer cell migration to bone-conditioned versus control medium was observed in 12/14 specimens (P = .0014) and correlated significantly with increasing levels of the adipokines/cytokines leptin (P = .006) and IL-1ß (P = .001) in univariate and multivariate regression analyses. Fluorescence microscopy and immunohistochemistry of fragments underscored the extreme adiposity of adult human bone tissues and revealed extensive breast cancer cell colonization within the marrow adipose tissue compartment. CONCLUSIONS: Our results show that breast cancer cells migrate to human bone tissue-conditioned medium in association with increasing levels of leptin and IL-1ß, and colonize the bone marrow adipose tissue compartment of cultured fragments. Bone marrow adipose tissue and its molecular signals may be important but understudied components of the breast cancer metastatic niche.

A randomized clinical trial of therapeutic hypothermia mode during transport for neonatal encephalopathy.

OBJECTIVE: To determine if temperature regulation is improved during neonatal transport using a servo-regulated cooling device when compared with standard practice. STUDY DESIGN: We performed a multicenter, randomized, nonmasked clinical trial in newborns with neonatal encephalopathy cooled during transport to 9 neonatal intensive care units in California. Newborns who met institutional criteria for therapeutic hypothermia were randomly assigned to receive cooling according to usual center practices vs device servo-regulated cooling. The primary outcome was the percentage of temperatures in target range (33°-34°C) during transport. Secondary outcomes included percentage of newborns reaching target temperature any time during transport, time to target temperature, and percentage of newborns in target range 1 hour after cooling initiation. RESULTS: One hundred newborns were enrolled: 49 to control arm and 51 to device arm. Baseline demographics did not differ with the exception of cord pH. For each subject, the percentage of temperatures in the target range was calculated. Infants cooled using the device had a higher percentage of temperatures in target range compared with control infants (median 73% [IQR 17-88] vs 0% [IQR 0-52], P < .001). More subjects reached target temperature during transport using the servo-regulated device (80% vs 49%, P <.001), and in a shorter time period (44 ± 31 minutes vs 63 ± 37 minutes, P = .04). Device-cooled infants reached target temperature by 1 hour with greater frequency than control infants (71% vs 20%, P < .001). CONCLUSIONS: Cooling using a servo-regulated device provides more predictable temperature management during neonatal transport than does usual care for outborn newborns with neonatal encephalopathy.

Reading abilities in school-aged preterm children: a review and meta-analysis.

AIM: Children born preterm (at ≤32wks) are at risk of developing deficits in reading ability. This meta-analysis aims to determine whether or not school-aged preterm children perform worse than those born at term in single-word reading (decoding) and reading comprehension. METHOD: Electronic databases were searched for studies published between 2000 and 2013, which assessed decoding or reading comprehension performance in English-speaking preterm and term-born children aged between 6 years and 13 years, and born after 1990. Standardized mean differences in decoding and reading comprehension scores were calculated. RESULTS: Nine studies were suitable for analysis of decoding, and five for analysis of reading comprehension. Random-effects meta-analyses showed that children born preterm had significantly lower scores (reported as Cohen's d values [d] with 95% confidence intervals [CIs]) than those born at term for decoding (d=-0.42, 95% CI -0.57 to -0.27, p<0.001) and reading comprehension (d=-0.57, 95% CI -0.68 to -0.46, p<0.001). Meta-regressions showed that lower gestational age was associated with larger differences in decoding (Q[1]=5.92, p=0.02) and reading comprehension (Q[1]=4.69, p=0.03) between preterm and term groups. Differences between groups increased with age for reading comprehension (Q[1]=5.10, p=0.02) and, although not significant, there was also a trend for increased group differences for decoding (Q[1]=3.44, p=0.06). INTERPRETATION: Preterm children perform worse than peers born at term on decoding and reading comprehension. These findings suggest that preterm children should receive more ongoing monitoring for reading difficulties throughout their education.

Molecular classification of endometriosis and disease stage using high-dimensional genomic data.

Endometriosis (E), an estrogen-dependent, progesterone-resistant, inflammatory disorder, affects 10% of reproductive-age women. It is diagnosed and staged at surgery, resulting in an 11-year latency from symptom onset to diagnosis, underscoring the need for less invasive, less expensive approaches. Because the uterine lining (endometrium) in women with E has altered molecular profiles, we tested whether molecular classification of this tissue can distinguish and stage disease. We developed classifiers using genomic data from n = 148 archived endometrial samples from women with E or without E (normal controls or with other common uterine/pelvic pathologies) across the menstrual cycle and evaluated their performance on independent sample sets. Classifiers were trained separately on samples in specific hormonal milieu, using margin tree classification, and accuracies were scored on independent validation samples. Classification of samples from women with E or no E involved 2 binary decisions, each based on expression of specific genes. These first distinguished presence or absence of uterine/pelvic pathology and then no E from E, with the latter further classified according to severity (minimal/mild or moderate/severe). Best performing classifiers identified E with 90%-100% accuracy, were cycle phase-specific or independent, and used relatively few genes to determine disease and severity. Differential gene expression and pathway analyses revealed immune activation, altered steroid and thyroid hormone signaling/metabolism, and growth factor signaling in endometrium of women with E. Similar findings were observed with other disorders vs controls. Thus, classifier analysis of genomic data from endometrium can detect and stage pelvic E with high accuracy, dependent or independent of hormonal milieu. We propose that limited classifier candidate genes are of high value in developing diagnostics and identifying therapeutic targets. Discovery of endometrial molecular differences in the presence of E and other uterine/pelvic pathologies raises the broader biological question of their impact on the steroid hormone response and normal functions of this tissue.

Coculturing human endometrial epithelial cells and stromal fibroblasts alters cell-specific gene expression and cytokine production.

OBJECTIVE: To determine the effects of coculturing endometrial epithelial cells (eEC) with paired endometrial stromal fibroblasts (eSF) on cell-specific gene expression and cytokine secretion patterns. DESIGN: In vitro study. SETTING: University research laboratory. PATIENT(S): Endometrial biopsies were obtained from premenopausal women. INTERVENTION(S): Polarized eEC and subject-paired eSF were cultured for 12.5 hours alone (monoculture) or combined in a two-chamber coculture system without cell-cell contact. Cells and conditioned media were analyzed for global gene expression and cytokine secretion, respectively. Purified, endometrial tissue-derived eEC and eSF isolated by fluorescent activated cell sorting (FACS) were used as noncultured controls. MAIN OUTCOME MEASURE(S): Cell-specific global gene expression profiling and analysis of secreted cytokines in eEC/eSF cocultures and respective monocultures. RESULT(S): Transepithelial resistance, diffusible tracer exclusion, expression of tight junction proteins, and apical/basolateral vectorial secretion confirmed eEC structural and functional polarization. Distinct transcriptomes of eEC and eSF were consistent with their respective lineages and their endometrial origin. Coculture of eEC with eSF resulted in altered cell-specific gene expression and cytokine secretion. CONCLUSION(S): This coculture model provides evidence that interactions between endometrial functionally polarized epithelium and stromal fibroblasts affect cell-specific gene expression and cytokine secretion underscoring their relevance when modeling endometrium in vitro.

Molecular probe technology detects bacteria without culture.

BACKGROUND: Our ultimate goal is to detect the entire human microbiome, in health and in disease, in a single reaction tube, and employing only commercially available reagents. To that end, we adapted molecular inversion probes to detect bacteria using solely a massively multiplex molecular technology. This molecular probe technology does not require growth of the bacteria in culture. Rather, the molecular probe technology requires only a sequence of forty sequential bases unique to the genome of the bacterium of interest. In this communication, we report the first results of employing our molecular probes to detect bacteria in clinical samples. RESULTS: While the assay on Affymetrix GenFlex Tag16K arrays allows the multiplexing of the detection of the bacteria in each clinical sample, one Affymetrix GenFlex Tag16K array must be used for each clinical sample. To multiplex the clinical samples, we introduce a second, independent assay for the molecular probes employing Sequencing by Oligonucleotide Ligation and Detection. By adding one unique oligonucleotide barcode for each clinical sample, we combine the samples after processing, but before sequencing, and sequence them together. CONCLUSIONS: Overall, we have employed 192 molecular probes representing 40 bacteria to detect the bacteria in twenty-one vaginal swabs as assessed by the Affymetrix GenFlex Tag16K assay and fourteen of those by the Sequencing by Oligonucleotide Ligation and Detection assay. The correlations among the assays were excellent.

Perivascular human endometrial mesenchymal stem cells express pathways relevant to self-renewal, lineage specification, and functional phenotype.

Human endometrium regenerates on a cyclic basis from candidate stem/progenitors whose genetic programs are yet to be determined. A subpopulation of endometrial stromal cells, displaying key properties of mesenchymal stem cells (MSCs), has been characterized. The endometrial MSC (eMSC) is likely the precursor of the endometrial stromal fibroblast. The goal of this study was to determine the transcriptome and signaling pathways in the eMSC to understand its functional phenotype. Endometrial stromal cells from oocyte donors (n = 20) and patients undergoing benign gynecologic surgery (n = 7) were fluorescence-activated cell sorted into MCAM (CD146)(+)/PDGFRB(+) (eMSC), MCAM (CD146)(-)/PDGFRB(+) (fibroblast), and MCAM (CD146)(+)/PDGFRB(-) (endothelial) populations. The eMSC population contained clonogenic cells with a mesenchymal phenotype differentiating into adipocytes when cultured in adipogenic medium. Gene expression profiling using Affymetrix Human Gene 1.0 ST arrays revealed 762 and 1518 significantly differentially expressed genes in eMSCs vs. stromal fibroblasts and eMSCs vs. endothelial cells, respectively. By principal component and hierarchical clustering analyses, eMSCs clustered with fibroblasts and distinctly from endothelial cells. Endometrial MSCs expressed pericyte markers and were localized by immunofluorescence to the perivascular space of endometrial small vessels. Endometrial MSCs also expressed genes involved in angiogenesis/vasculogenesis, steroid hormone/hypoxia responses, inflammation, immunomodulation, cell communication, and proteolysis/inhibition, and exhibited increased Notch, TGFB, IGF, Hedgehog, and G-protein-coupled receptor signaling pathways, characteristic of adult tissue MSC self-renewal and multipotency. Overall, the data support the eMSC as a clonogenic, multipotent pericyte that displays pathways of self-renewal and lineage specification, the potential to respond to conditions during endometrial desquamation and regeneration, and a genetic program predictive of its differentiated lineage, the stromal fibroblast.

A model for shear stress sensing and transmission in vascular endothelial cells.

Arterial endothelial cell (EC) responsiveness to flow is essential for normal vascular function and plays a role in the development of atherosclerosis. EC flow responses may involve sensing of the mechanical stimulus at the cell surface with subsequent transmission via cytoskeleton to intracellular transduction sites. We had previously modeled flow-induced deformation of EC-surface flow sensors represented as viscoelastic materials with standard linear solid behavior (Kelvin bodies). In the present article, we extend the analysis to arbitrary networks of viscoelastic structures connected in series and/or parallel. Application of the model to a system of two Kelvin bodies in parallel reveals that flow induces an instantaneous deformation followed by creeping to the asymptotic response. The force divides equally between the two bodies when they have identical viscoelastic properties. When one body is stiffer than the other, a larger fraction of the applied force is directed to the stiffer body. We have also probed the impact of steady and oscillatory flow on simple sensor-cytoskeleton-nucleus networks. The results demonstrated that, consistent with the experimentally observed temporal chronology of EC flow responses, the flow sensor attains its peak deformation faster than intracellular structures and the nucleus deforms more rapidly than cytoskeletal elements. The results have also revealed that a 1-Hz oscillatory flow induces significantly smaller deformations than steady flow. These results may provide insight into the mechanisms behind the experimental observations that a number of EC responses induced by steady flow are not induced by oscillatory flow.