Abnormal Uterine Bleeding during Pubertal Induction with Transdermal Estrogen in Individuals with Turner Syndrome.

STUDY OBJECTIVE: Incidence of abnormal uterine bleeding (AUB) during pubertal induction among individuals with Turner syndrome (TS) has not been described previously. We estimated the incidence and characterized factors associated with AUB among individuals with TS. A secondary objective was to evaluate the management of AUB among this patient population. DESIGN, SETTING, PARTICIPANTS, AND INTERVENTION: We conducted a retrospective chart review to evaluate individuals with TS undergoing hormone replacement therapy (HRT) for pubertal induction with transdermal estrogen. A total of 45 participants were identified between January 2007 and June 2019. RESULTS: Of the 45 individuals with TS included, 16 (35%) experienced AUB. Individuals with AUB most commonly experienced prolonged (44%), prolonged and heavy (25%), and intermenstrual (19%) bleeding. Individuals who experienced AUB were more likely to experience spontaneous bleeding (69% vs 28%) and a duration of unopposed estrogen greater than 18 months (63% vs 41%), undergo progestin cycling less often than monthly (69% vs 0%), use a micronized progestin dose of less than 200 mg (25% vs 14%), and be noncompliant with HRT (19% vs 0%) compared with those who did not experience AUB. CONCLUSION: There is a relatively high incidence of AUB among individuals with TS undergoing pubertal induction with transdermal estrogen. Care providers should consider the clinical factors examined to guide monitoring and management of individuals with TS on HRT.

Effect of Subconcussive Head Impact Exposure and Jugular Vein Compression on Behavioral and Cognitive Outcomes after a Single Season of High-School Football: A Prospective Longitudinal Trial.

This prospective longitudinal trial aimed to (1) determine the role of head impact exposure on behavioral/cognitive outcomes, and (2) assess the protective effect(s) of a jugular vein compression (JVC) collar on behavioral/cognitive outcomes after one season of high-school football. Participants included 284 male high-school football players aged 13-18 years enrolled from seven Midwestern high-schools. Schools were allocated to the JVC collar intervention (four teams, 140 players) or no collar/no intervention control (three teams, 144 players) condition. Head impact exposure was measured throughout the season using CSx accelerometers. Outcome measures included post-season parent and adolescent report on Strengths and Weaknesses of ADHD Symptoms and Normal Behavior Scale (SWAN) and Post-Concussion Symptom Inventory (PCSI), as well as adolescent performance on Attention Network Task (ANT), digital Trail Making Task (dTMT), and Cued Switching task. No significant effect of head impact exposure or JVC collar use on post-season SWAN or PCSI scores or performance on dTMT and Cued Switching task were noted. There was no effect of head impact exposure on ANT performance; however, the JVC collar group had greater post-season Alerting network scores than the no collar group (p = 0.026, d = 0.22). Findings provide preliminary evidence that the JVC collar may provide some protection to the alerting attention system. These findings should be interpreted cautiously as a greater understanding of the long-term sequelae of head impact exposure and the role of cumulative head impact exposure behavioral/cognitive outcomes is required.

Age at and indication for diagnosis of Turner syndrome in the pediatric population.

Early diagnosis of Turner syndrome (TS) enables timely intervention and may improve outcomes, but many are still diagnosed late. The objectives of our study were to describe the age and clinical features leading to diagnosis of TS in a large referral center. We hypothesize that newer testing modalities, such as noninvasive prenatal testing (NIPT), may lead to a decline in the age of diagnosis. Medical records of TS patients followed at The Cincinnati Center for Pediatric and Adult TS Care between 1997 and 2016 were reviewed for age at diagnosis, karyotype, and clinical indication(s). Patients (<18 years) were included (n = 239). Thirty-seven percent of patients were diagnosed prenatally or neonatally (≤1 month). The median age of diagnosis was 1.5 (IQR 0.0-10.0) years. If not made during those periods, the median age was 9.3 (IQR 3.2-12.5) years. The most common indications for diagnosis were before birth, unspecified prenatal testing (57%); in neonates/infants, lymphedema (21%); in childhood, short stature (72%); and in adolescence, short stature (45%) followed by pubertal delay with short stature (22%). The age of TS diagnosis in our cohort is young. However, when the diagnosis is not made before 1 year, the median age of diagnosis has not changed in recent years. The age at diagnosis could decrease with prenatal testing, although our study may not have assessed a long enough period of increased use of NIPT. Together with an increase in provider clinical awareness, this may result in more age-appropriate screening of comorbidities and earlier therapeutic intervention.

The effects of internal jugular vein compression for modulating and preserving white matter following a season of American tackle football: A prospective longitudinal evaluation of differential head impact exposure.

The purpose of this clinical trial was to examine whether internal jugular vein compression (JVC)-using an externally worn neck collar-modulated the relationships between differential head impact exposure levels and pre- to postseason changes in diffusion tensor imaging (DTI)-derived diffusivity and anisotropy metrics of white matter following a season of American tackle football. Male high-school athletes (n = 284) were prospectively assigned to a non-collar group or a collar group. Magnetic resonance imaging data were collected from participants pre- and postseason and head impact exposure was monitored by accelerometers during every practice and game throughout the competitive season. Athletes' accumulated head impact exposure was systematically thresholded based on the frequency of impacts of progressively higher magnitudes (10 g intervals between 20 to 150 g) and modeled with pre- to postseason changes in DTI measures of white matter as a function of JVC neck collar wear. The findings revealed that the JVC neck collar modulated the relationships between greater high-magnitude head impact exposure (110 to 140 g) and longitudinal changes to white matter, with each group showing associations that varied in directionality. Results also revealed that the JVC neck collar group partially preserved longitudinal changes in DTI metrics. Collectively, these data indicate that a JVC neck collar can provide a mechanistic response to the diffusion and anisotropic properties of brain white matter following the highly diverse exposure to repetitive head impacts in American tackle football. Clinicaltrials.gov: NCT# 04068883.

Cell type-specific mechanisms coupling protease-activated receptor-1 to infectious colitis pathogenesis.

BACKGROUND: Protease-activated receptor-1 (PAR-1) plays a major role in multiple disease processes, including colitis. Understanding the mechanisms coupling PAR-1 to disease pathogenesis is complicated by the fact that PAR-1 is broadly expressed across multiple cell types. OBJECTIVE: Determine the specific contributions of PAR-1 expressed by macrophages and colonic enterocytes to infectious colitis. METHODS: Mice carrying a conditional PAR-1 allele were generated and bred to mice expressing Cre recombinase in a myeloid- (PAR-1ΔM ) or enterocyte-specific (PAR-1ΔEPI ) fashion. Citrobacter rodentium colitis pathogenesis was analyzed in mice with global PAR-1 deletion (PAR-1-/- ) and cell type-specific deletions. RESULTS: Constitutive deletion of PAR-1 had no significant impact on weight loss, crypt hypertrophy, crypt abscess formation, or leukocyte infiltration in Citrobacter colitis. However, colonic shortening was significantly blunted in infected PAR-1-/- mice, and these animals exhibited decreased local levels of IL-1ß, IL-22, IL-6, and IL-17A. In contrast, infected PAR-1ΔM mice lost less weight and had fewer crypt abscesses relative to controls. PAR-1ΔM mice had diminished CD3+ T cell infiltration into colonic tissue, but macrophage and CD4+ T cell infiltration were similar to controls. Also contrasting results in global knockouts, PAR-1ΔM mice exhibited lower levels of IL-1ß, but not Th17-related cytokines (ie, IL-22, IL-6, IL-17A). Infected PAR-1ΔEPI mice exhibited increased crypt hypertrophy and crypt abscess formation, but local cytokine elaboration was similar to controls. CONCLUSIONS: These studies reveal complex, cell type-specific roles for PAR-1 in modulating the immune response to Citrobacter colitis that are not readily apparent in analyses limited to mice with global PAR-1 deficiency.

Characterizing the effect of exposure to microgravity on anemia: more space is worse.

The effects of space travel have renewed importance with space tourism and plans for long-term missions to the moon and Mars. The study of space anemia is limited by the availability of subjects and extreme conditions. An approach using the accumulated data on human space flight may characterize space anemia. A total of 17 336 hemoglobin (Hb) concentration measures from 721 space missions and controls were used to study acute and long-term effects of duration of exposure to space on Hb decrement. Nearly half of astronauts (48%) landing after long duration missions were anemic. Returning to Earth revealed Hb decrements whose magnitude and time to recover were dependent on exposure to space: -0.61 g/dL (4%), -0.82 g/dL (5%) and -1.66 g/dL (11%) of preflight Hb for mean exposure to space of 5.4, 11.5, and 145 days, respectively. Astronauts returning from a mean 5.4 days in space took 24 days to return to preflight Hb while astronauts 11.5 to 145 days in space took 49 days. Negative effects of microgravity on Hb persisted throughout female and male astronauts' terrestrial lives (-0.001 and -0.002 mg/dL Hb respectively) for every day spent in space (both P < .05). The negative effect of exposure to space was not overcome by a statistically significant effect of being an astronaut compared to controls. Exposure to space showed a dose-response relationship with acute and chronic Hb decrements. Space anemia contributes to the deconditioning of astronauts returning to Earth, and needs to be considered for space travel to other planets, space tourism and for the care of bedridden patients who present similar changes as astronauts.

Improving T-cell therapy for relapsed EBV-negative Hodgkin lymphoma by targeting upregulated MAGE-A4.

PURPOSE: Patients with Hodgkin lymphoma (HL) relapsing after hematopoietic stem cell transplant have limited options for long-term cure. We have shown that infused cytotoxic T cells (CTL) targeting Epstein Barr virus (EBV)-derived proteins induced complete remissions in EBV(+) HL patients. A limitation of this approach is that up to 70% of relapsed HL tumors are EBV-negative. For these patients, an alternative is to target the cancer/testis antigen MAGE-A4 present in EBV antigen-negative HL tumors. Furthermore, epigenetic modification by clinically available demethylating agents can enhance MAGE-A4 expression in previously MAGE-negative tumors. EXPERIMENTAL DESIGN: We explored the feasibility of combining adoptive T cell therapy with epigenetic modification of tumor antigen expression. We further characterized MAGE-A4-specific T-cell phenotype and function, and examined the effects of the epigenetic modifying drug decitabine on these T cells. RESULTS: Cytotoxic T cells were generated specifically recognizing MAGE-A4 expressed by autologous HL targets and tumor cell lines. Decitabine-previously shown to increase tumor antigen expression in HL-did not compromise MAGE-A4-specific T-cell phenotype and function. In patients treated with decitabine, expanded MAGE-A4-specific T cells had a broader antitumor T cell repertoire, consistent with increased antigen stimulation in vivo. CONCLUSIONS: Adoptive transfer of MAGE-A4-specific T cells, combined with epigenetic modifying drugs to increase expression of the protein, may improve treatment of relapsed HL.

The histone deacetylase inhibitor entinostat (SNDX-275) induces apoptosis in Hodgkin lymphoma cells and synergizes with Bcl-2 family inhibitors.

OBJECTIVE: Based on promising in vitro and in vivo activity of several histone deacetylase inhibitors in Hodgkin lymphoma (HL), we investigated SNDX-275, an oral class 1 isoform-selective histone deacetylase inhibitors in HL-derived cell lines. MATERIALS AND METHODS: Proliferation and cell death were examined by MTS assay, Annexin V/propidium iodide, and fluorescence-activated cell sorting analysis. Gene and protein expression were measured by reverse transcriptase polymerase chain reaction, Western blotting, and immunohistochemical analysis. A multiplex assay was used to determine cytokines and chemokines. RESULTS: SNDX-275 induced cell death in a dose- and time-dependent manner with an IC(50) at the sub- and lower micromolar range at 72 hours. At the molecular level, SNDX-275 increased histone H3 acetylation, upregulated p21 expression, and activated the intrinsic apoptosis pathway by downregulating the X-linked inhibitor of apoptosis protein. SNDX-275 downregulated expression of antiapoptotic Bcl-2 and Bcl-xL proteins without altering Mcl-1 or Bax levels. Combination studies demonstrated that two Bcl-2 inhibitors (ABT-737 and obatoclax) significantly enhanced the effect of SNDX-275. SNDX-275 modulated the level of several cytokines and chemokines, including interleukin-12 p40-70, interferon-inducible protein-10, RANTES (regulated on activation, normal T expressed and secreted), interleukin-13, interleukin-4, and thymus and activation-regulated chemokine and variably induced the cancer/testis antigen expression of MAGE-A4 and survivin in HL cell lines. CONCLUSIONS: SNDX-275 has antiproliferative activity in HL cell lines, involving several mechanisms: induction of apoptosis, regulation of cytokines and chemokines, and alteration of cancer/testis antigens. Clinical investigation of SNDX-275 alone or in combination with Bcl-2 inhibitors is warranted in patients with HL. Phase 2 studies with SNDX-275 in HL are ongoing, and future clinical studies should investigate combinations with SNDX-275.

High-avidity cytotoxic T lymphocytes specific for a new PRAME-derived peptide can target leukemic and leukemic-precursor cells.

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies, but is absent on normal tissues, including hematopoietic progenitor cells, and may therefore be an appropriate candidate for T cell-mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity, PRAME-specific cytotoxic T lymphocytes (CTLs), we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal, PRAME-specific CTL lines and elicited high-avidity CTLs, with a high proportion of cells recognizing a previously uninvestigated HLA-A*02-restricted epitope, P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME(+) hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts, but also against leukemic progenitor cells as assessed by colony-forming-inhibition assays, which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors, indicating that this approach may be of value for immunotherapy of PRAME(+) hematologic malignancies.

Antigen-specific cytotoxic T lymphocytes can target chemoresistant side-population tumor cells in Hodgkin lymphoma.

Side-population (SP) analysis has been used to identify progenitor cells from normal and malignant tissues as well as revealing tumor cells with increased resistance to radiation and chemotherapy. Despite enhanced chemoresistance, tumor SP cells may still express tumor-associated antigens (TAAs), which may render them susceptible to elimination by the immune system. In this study, we show that both Hodgkin lymphoma (HL) cell lines and primary HL tumor samples contain a distinct SP phenotype. Importantly, while these cells showed increased resistance to gemcitabine, a commonly used drug for the treatment of refractory HL, HL SP cells also expressed higher levels of the TAAs MAGEA4, SSX2, survivin, and NY-ESO-1, which allowed them to be specifically recognized and killed by TAA-specific cytotoxic T lymphocytes. This study suggests that chemoresistant HL SP cells can be targeted by the immune system, providing a rationale for combined chemotherapy and immunotherapy for the treatment of HL.

Efficient and rapid osteoinduction in an immune-competent host.

Osteoinductive systems to induce targeted rapid bone formation hold clinical promise, but development of technologies for clinical use that must be tested in animal models is often a difficult challenge. We previously demonstrated that implantation of human cells transduced with Ad5F35BMP2 to express high levels of bone morphogenetic protein-2 (BMP2) resulted in rapid bone formation at targeted sites. Inclusion of human cells in this model precluded us from testing this system in an immune-competent animal model, thus limiting information about the efficacy of this approach. Here, for the first time we demonstrate the similarity between BMP2-induced endochondral bone formation in a system using human cells in an immune-incompetent mouse and a murine cell-based BMP2 gene therapy system in immune-competent animals. In both cases the delivery cells are rapidly cleared, within 5 days, and in neither case do they appear to contribute to any of the structures forming in the tissues. Endochondral bone formation progressed through a highly ordered series of stages that were both morphologically and temporally indistinguishable between the two models. Even longterm analysis of the heterotopic bone demonstrated similar bone volumes and the eventual remodeling to form similar structures. The results suggest that the ability of BMP2 to rapidly induce bone formation overrides contributions from either immune status or the nature of delivery cells.

Oxygen tension directs chondrogenic differentiation of myelo-monocytic progenitors during endochondral bone formation.

Synthesis of bone requires both essential progenitors to form the various structures and the correct microenvironment for their differentiation. To identify these factors, we have used a system that exploits bone morphogenetic protein's ability to induce endochondral bone formation rapidly. One of the earliest events observed was the influx and proliferation of fibroblastic cells that express both vascular smooth muscle cell markers, alpha smooth muscle actin (alpha SMA), smooth muscle myosin heavy chain, and the monocytic marker CD68. The expression of these factors was lost by days 4 to 5, coincident with the up-regulation of Sox9 and the appearance of chondrocytes. Studies with a cyclization recombination (Cre)/lox system, in which a myeloid-specific promoter driving Cre recombinase can irreversibly unblock expression of beta-galactosidase only in cells of myeloid origin, showed specific activity in the newly formed chondrocytes. These results suggest that early chondrocyte progenitors are of myeloid origin. Simultaneous with this recruitment, we determined that a numbers of these cells were in a hypoxic state, indicative of a low-oxygen environment. The cells in the hypoxic regions were undergoing chondrogenesis, whereas cells in adjacent normoxic regions appeared to be assembling into new vessels, suggesting that the oxygen microenvironment is critical for establishment of the cartilage.

Novel compound enables high-level adenovirus transduction in the absence of an adenovirus-specific receptor.

Viral vectors are extensively used to deliver foreign DNA to cells for applications ranging from basic research to potential clinical therapies. A limiting step in this process is virus uptake and internalization into the target cells, which is mediated by membrane receptors. Although it is possible to modify viral capsid proteins to target the viruses, such procedures are complex and often unsuccessful. Here we present a rapid, inexpensive system for improving transduction of cells, including those that lack receptors for adenovirus fiber proteins. Addition of GeneJammer (Stratagene, La Jolla, CA) during the adenovirus transduction led to a significant increase in both the total number of transduced cells and the level of transgene expression per cell. Studies using cell lines deficient in adenovirus receptors demonstrated that addition of GeneJammer provided a novel cellular entry mechanism for the virus. These findings were tested in a cell-based gene therapy system for the induction of bone, which is contingent on high-level expression of the transgene. Inclusion of GeneJammer in either Ad5BMP2 or Ad5F35BMP2 transduction of a variety of cells demonstrated a correlating increase in bone formation. The results suggest a novel and versatile method for achieving high-level transduction using adenovirus.