Extra-cardiac management of cardiogenic shock in the intensive care unit.

Cardiogenic shock (CS) is a heterogeneous clinical syndrome characterized by low cardiac output leading to end-organ hypoperfusion. Organ dysoxia ranging from transient organ injury to irreversible organ failure and death occurs across all CS etiologies but differing by incidence and type. Herein, we review the recognition and management of respiratory, renal and hepatic failure complicating CS. We also discuss unmet needs in the CS care pathway and future research priorities for generating evidence-based best practices for the management of extra-cardiac sequelae. The complexity of CS admitted to the contemporary cardiac intensive care unit demands a workforce skilled to care for these extra-cardiac critical illness complications with an appreciation for how cardio-systemic interactions influence critical illness outcomes in afflicted patients.

Therapeutic Targeting of LIF Overcomes Macrophage-mediated Immunosuppression of the Local Tumor Microenvironment.

PURPOSE: Leukemia inhibitory factor (LIF) is a multifunctional cytokine with numerous reported roles in cancer and is thought to drive tumor development and progression. Characterization of LIF and clinical-stage LIF inhibitors would increase our understanding of LIF as a therapeutic target. EXPERIMENTAL DESIGN: We first tested the association of LIF expression with transcript signatures representing multiple processes regulating tumor development and progression. Next, we developed MSC-1, a high-affinity therapeutic antibody that potently inhibits LIF signaling and tested it in immune competent animal models of cancer. RESULTS: LIF was associated with signatures of tumor-associated macrophages (TAM) across 7,769 tumor samples spanning 22 solid tumor indications. In human tumors, LIF receptor was highly expressed within the macrophage compartment and LIF treatment drove macrophages to acquire immunosuppressive capacity. MSC-1 potently inhibited LIF signaling by binding an epitope that overlaps with the gp130 receptor binding site on LIF. MSC-1 showed monotherapy efficacy in vivo and drove TAMs to acquire antitumor and proinflammatory function in syngeneic colon cancer mouse models. Combining MSC-1 with anti-PD1 leads to strong antitumor response and a long-term tumor-free survival in a significant proportion of treated mice. CONCLUSIONS: Overall, our findings highlight LIF as a therapeutic target for cancer immunotherapy.

A Novel EGFRvIII T-Cell Bispecific Antibody for the Treatment of Glioblastoma.

T-cell bispecific antibodies (TCB) are engineered molecules that bind both the T-cell receptor and tumor-specific antigens. Epidermal growth factor receptor variant III (EGFRvIII) mutation is a common event in glioblastoma (GBM) and is characterized by the deletion of exons 2-7, resulting in a constitutively active receptor that promotes cell proliferation, angiogenesis, and invasion. EGFRvIII is expressed on the surface of tumor cells and is not expressed in normal tissues, making EGFRvIII an ideal neoantigen target for TCBs. We designed and developed a novel 2+1 EGFRvIII-TCB with optimal pharmacologic characteristics and potent antitumor activity. EGFRvIII-TCB showed specificity for EGFRvIII and promoted tumor cell killing as well as T-cell activation and cytokine secretion only in patient-derived models expressing EGFRvIII. Moreover, EGFRvIII-TCB promoted T-cell recruitment into intracranial tumors. EGFRvIII-TCB induced tumor regression in GBM animal models, including humanized orthotopic GBM patient-derived xenograft models. Our results warrant the clinical testing of EGFRvIII-TCB for the treatment of EGFRvIII-expressing GBMs.

Activity and Resistance of a Brain-Permeable Paradox Breaker BRAF Inhibitor in Melanoma Brain Metastasis.

The therapeutic benefit of approved BRAF and MEK inhibitors (BRAFi/MEKi) in patients with brain metastatic BRAF V600E/K-mutated melanoma is limited and transient. Resistance largely occurs through the restoration of MAPK signaling via paradoxical BRAF activation, highlighting the need for more effective therapeutic options. Aiming to address this clinical challenge, we characterized the activity of a potent, brain-penetrant paradox breaker BRAFi (compound 1a, C1a) as first-line therapy and following progression upon treatment with approved BRAFi and BRAFi/MEKi therapies. C1a activity was evaluated in vitro and in vivo in melanoma cell lines and patient-derived models of BRAF V600E-mutant melanoma brain metastases following relapse after treatment with BRAFi/MEKi. C1a showed superior efficacy compared with approved BRAFi in both subcutaneous and brain metastatic models. Importantly, C1a manifested potent and prolonged antitumor activity even in models that progressed on BRAFi/MEKi treatment. Analysis of mechanisms of resistance to C1a revealed MAPK reactivation under drug treatment as the predominant resistance-driving event in both subcutaneous and intracranial tumors. Specifically, BRAF kinase domain duplication was identified as a frequently occurring driver of resistance to C1a. Combination therapies of C1a and anti-PD-1 antibody proved to significantly reduce disease recurrence. Collectively, these preclinical studies validate the outstanding antitumor activity of C1a in brain metastasis, support clinical investigation of this agent in patients pretreated with BRAFi/MEKi, unveil genetic drivers of tumor escape from C1a, and identify a combinatorial treatment that achieves long-lasting responses. SIGNIFICANCE: A brain-penetrant BRAF inhibitor demonstrates potent activity in brain metastatic melanoma, even upon relapse following standard BRAF inhibitor therapy, supporting further investigation into its clinical utility.

[Hepato-splenic involvement, an unusual finding secondary to cat-scratch disease: a clinical case]. / Compromiso hepatoesplénico, un hallazgo inusual secundario a enfermedad por arañazo de gato: a propósito de un caso.

The infection by Bartonela henselae (BH), the cause of cat scratch disease, it could be asymptomatic or produce local and multisystem illness. The objective of this case report is to document that the hepato-splenic involvement is unusual in BH infection, and the treatment is discussed and individualized in each patient. This case is about an eleven-year girl who presented with findings in abdominal tomography and ultrasound of hepato-splenic abscesses, with later positive serology for BH. In this way, a bibliographic review is carried out to show the low prevalence and incidence of hepato-splenic involvement where the anamnesis and the physical examination are essential to make an early diagnosis and treatment.

La infección por Bartonella henselae (BH) que causa la enfermedad por arañazo de gato puede cursar de manera asintomática, así como presentar manifestaciones locales y sistémicas. El objetivo de este caso es documentar que el compromiso hepatoesplénico es poco habitual en este tipo de infección y el tratamiento, que debe ser personalizado, aún genera controversia. Se presenta un caso de una paciente de 11 años con fiebre de origen de desconocido con hallazgos en tomografía y ecografía abdominal de abscesos hepatoesplénicos, y confirmación de infección por BH a través de métodos serológicos. Se realiza una revisión bibliográfica donde se evidencia la baja frecuencia de compromiso hepatoesplénico. De esta manera, la anamnesis y el examen físico son fundamentales para realizar un rápido diagnóstico y tratamiento.

Compromiso hepatoesplénico, un hallazgo inusual secundario a enfermedad por arañazo de gato: a propósito de un caso / Hepato-splenic involvement, an unusual finding secondary to cat-scratch disease: a clinical case

La infección por Bartonella henselae (BH) que causa la enfermedad por arañazo de gato puede cursar de manera asintomática, así como presentar manifestaciones locales y sistémicas. El objetivo de este caso es documentar que el compromiso hepatoesplénico es poco habitual en este tipo de infección y el tratamiento, que debe ser personalizado, aún genera controversia. Se presenta un caso de una paciente de 11 años con fiebre de origen de desconocido con hallazgos en tomografía y ecografía abdominal de abscesos hepatoesplénicos, y confirmación de infección por BH a través de métodos serológicos. Se realiza una revisión bibliográfica donde se evidencia la baja frecuencia de compromiso hepatoesplénico. De esta manera, la anamnesis y el examen físico son fundamentales para realizar un rápido diagnóstico y tratamiento

The infection by Bartonela henselae (BH), the cause of cat scratch disease, it could be asymptomatic or produce local and multisystem illness. The objective of this case report is to document that the hepato-splenic involvement is unusual in BH infection, and the treatment is discussed and individualized in each patient. This case is about an eleven-year girl who presented with findings in abdominal tomography and ultrasound of hepato-splenic abscesses, with later positive serology for BH. In this way, a bibliographic review is carried out to show the low prevalence and incidence of hepato-splenic involvement where the anamnesis and the physical examination are essential to make an early diagnosis and treatment.

Immune cell profiling of the cerebrospinal fluid enables the characterization of the brain metastasis microenvironment.

Brain metastases are the most common tumor of the brain with a dismal prognosis. A fraction of patients with brain metastasis benefit from treatment with immune checkpoint inhibitors (ICI) and the degree and phenotype of the immune cell infiltration has been used to predict response to ICI. However, the anatomical location of brain lesions limits access to tumor material to characterize the immune phenotype. Here, we characterize immune cells present in brain lesions and matched cerebrospinal fluid (CSF) using single-cell RNA sequencing combined with T cell receptor genotyping. Tumor immune infiltration and specifically CD8+ T cell infiltration can be discerned through the analysis of the CSF. Consistently, identical T cell receptor clonotypes are detected in brain lesions and CSF, confirming cell exchange between these compartments. The analysis of immune cells of the CSF can provide a non-invasive alternative to predict the response to ICI, as well as identify the T cell receptor clonotypes present in brain metastasis.

Circulating tumour DNA from the cerebrospinal fluid allows the characterisation and monitoring of medulloblastoma.

The molecular characterisation of medulloblastoma, the most common paediatric brain tumour, is crucial for the correct management and treatment of this heterogenous disease. However, insufficient tissue sample, the presence of tumour heterogeneity, or disseminated disease can challenge its diagnosis and monitoring. Here, we report that the cerebrospinal fluid (CSF) circulating tumour DNA (ctDNA) recapitulates the genomic alterations of the tumour and facilitates subgrouping and risk stratification, providing valuable information about diagnosis and prognosis. CSF ctDNA also characterises the intra-tumour genomic heterogeneity identifying small subclones. ctDNA is abundant in the CSF but barely present in plasma and longitudinal analysis of CSF ctDNA allows the study of minimal residual disease, genomic evolution and the characterisation of tumours at recurrence. Ultimately, CSF ctDNA analysis could facilitate the clinical management of medulloblastoma patients and help the design of tailored therapeutic strategies, increasing treatment efficacy while reducing excessive treatment to prevent long-term secondary effects.

Game-Based Learning Interventions to Foster Cross-Cultural Care Training: A Scoping Review.

Objective: Differences in cultural background between health providers and patients can reduce effective access to health services in multicultural settings. Health sciences educators have recently suggested that game-based learning may be effective for cross-cultural care training. This scoping review maps published knowledge on educational games intended to foster cross-cultural care training and highlights the research gaps for future research. Materials and Methods: A scoping review searched PubMed, Eric, Embase, Lilacs, PsycINFO, and Google Scholar for theoretical and empirical research, using terms relevant to cross-cultural care and game-based learning. A participatory research framework engaged senior medical students and participatory research experts in conducting and evaluating the review. Results: Forty-one documents met the inclusion criteria, all from developed countries. The most common source of publication was nursing and medicine (39%; 16/41) and used the cultural competence approach (44%; 18/41). Around one-half of the publications (51%; 21/41) were theoretical and 39% (16/41) were empirical. Empirical studies most commonly used mixed methods (44%; 7/16), followed by strictly quantitative (31%; 5/16) or qualitative (25%; 4/16) approaches. There were no randomized controlled trials and only one study engaged end-users in the design. Empirical studies most frequently assessed role-play-related games (44%; 7/16) and used game evaluation-related outcomes or learning-related outcomes. None used patient-oriented outcomes. Findings suggest that educational games are an effective and engaging educational intervention for cross-cultural care training. Conclusions: The paucity of studies on educational games and cross-cultural care training precludes a systematic review. Future empirical studies should focus on randomized counterfactual designs and patient-related outcomes. We encourage involving end-users in developing content for educational games.

Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program.

Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.

LIF regulates CXCL9 in tumor-associated macrophages and prevents CD8+ T cell tumor-infiltration impairing anti-PD1 therapy.

Cancer response to immunotherapy depends on the infiltration of CD8+ T cells and the presence of tumor-associated macrophages within tumors. Still, little is known about the determinants of these factors. We show that LIF assumes a crucial role in the regulation of CD8+ T cell tumor infiltration, while promoting the presence of protumoral tumor-associated macrophages. We observe that the blockade of LIF in tumors expressing high levels of LIF decreases CD206, CD163 and CCL2 and induces CXCL9 expression in tumor-associated macrophages. The blockade of LIF releases the epigenetic silencing of CXCL9 triggering CD8+ T cell tumor infiltration. The combination of LIF neutralizing antibodies with the inhibition of the PD1 immune checkpoint promotes tumor regression, immunological memory and an increase in overall survival.

The Genomic and Immune Landscapes of Lethal Metastatic Breast Cancer.

The detailed molecular characterization of lethal cancers is a prerequisite to understanding resistance to therapy and escape from cancer immunoediting. We performed extensive multi-platform profiling of multi-regional metastases in autopsies from 10 patients with therapy-resistant breast cancer. The integrated genomic and immune landscapes show that metastases propagate and evolve as communities of clones, reveal their predicted neo-antigen landscapes, and show that they can accumulate HLA loss of heterozygosity (LOH). The data further identify variable tumor microenvironments and reveal, through analyses of T cell receptor repertoires, that adaptive immune responses appear to co-evolve with the metastatic genomes. These findings reveal in fine detail the landscapes of lethal metastatic breast cancer.

Genetic heterogeneity and actionable mutations in HER2-positive primary breast cancers and their brain metastases.

Brain metastases constitute a challenge in the management of patients with HER2-positive breast cancer treated with anti-HER2 systemic therapies. Here we sought to define the repertoire of mutations private to or enriched for in HER2-positive brain metastases. Massively parallel sequencing targeting all exons of 254 genes frequently mutated in breast cancers and/or related to DNA repair was used to characterize the spatial and temporal heterogeneity of HER2-positive breast cancers and their brain metastases in six patients. Data were analyzed with state-of-the-art bioinformatics algorithms and selected mutations were validated with orthogonal methods. Spatial and temporal inter-lesion genetic heterogeneity was observed in the HER2-positive brain metastases from an index patient subjected to a rapid autopsy. Genetic alterations restricted to the brain metastases included mutations in cancer genes FGFR2, PIK3CA and ATR, homozygous deletion in CDKN2A and amplification in KRAS. Shifts in clonal composition and the acquisition of additional mutations in the progression from primary HER2-positive breast cancer to brain metastases following anti-HER2 therapy were investigated in additional five patients. Likely pathogenic mutations private to or enriched in the brain lesions affected cancer and clinically actionable genes, including ATR, BRAF, FGFR2, MAP2K4, PIK3CA, RAF1 and TP53. Changes in clonal composition and the acquisition of additional mutations in brain metastases may affect potentially actionable genes in HER2-positive breast cancers. Our observations have potential clinical implications, given that treatment decisions for patients with brain metastatic disease are still mainly based on biomarkers assessed in the primary tumor.

Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma.

Cell-free circulating tumour DNA (ctDNA) in plasma has been shown to be informative of the genomic alterations present in tumours and has been used to monitor tumour progression and response to treatments. However, patients with brain tumours do not present with or present with low amounts of ctDNA in plasma precluding the genomic characterization of brain cancer through plasma ctDNA. Here we show that ctDNA derived from central nervous system tumours is more abundantly present in the cerebrospinal fluid (CSF) than in plasma. Massively parallel sequencing of CSF ctDNA more comprehensively characterizes the genomic alterations of brain tumours than plasma, allowing the identification of actionable brain tumour somatic mutations. We show that CSF ctDNA levels longitudinally fluctuate in time and follow the changes in brain tumour burden providing biomarkers to monitor brain malignancies. Moreover, CSF ctDNA is shown to facilitate and complement the diagnosis of leptomeningeal carcinomatosis.

GATA-3 dose-dependent checkpoints in early T cell commitment.

GATA-3 expression is crucial for T cell development and peaks during commitment to the T cell lineage, midway through the CD4(-)CD8(-) (double-negative [DN]) stages 1-3. We used RNA interference and conditional deletion to reduce GATA-3 protein acutely at specific points during T cell differentiation in vitro. Even moderate GATA-3 reduction killed DN1 cells, delayed progression to the DN2 stage, skewed DN2 gene regulation, and blocked appearance of the DN3 phenotype. Although a Bcl-2 transgene rescued DN1 survival and improved DN2 cell generation, it did not restore DN3 differentiation. Gene expression analyses (quantitative PCR, RNA sequencing) showed that GATA-3-deficient DN2 cells quickly upregulated genes, including Spi1 (PU.1) and Bcl11a, and downregulated genes, including Cpa3, Ets1, Zfpm1, Bcl11b, Il9r, and Il17rb with gene-specific kinetics and dose dependencies. These targets could mediate two distinct roles played by GATA-3 in lineage commitment, as revealed by removing wild-type or GATA-3-deficient early T lineage cells from environmental Notch signals. GATA-3 worked as a potent repressor of B cell potential even at low expression levels, so that only full deletion of GATA-3 enabled pro-T cells to reveal B cell potential. The ability of GATA-3 to block B cell development did not require T lineage commitment factor Bcl11b. In prethymic multipotent precursors, however, titration of GATA-3 activity using tamoxifen-inducible GATA-3 showed that GATA-3 inhibits B and myeloid developmental alternatives at different threshold doses. Furthermore, differential impacts of a GATA-3 obligate repressor construct imply that B and myeloid development are inhibited through distinct transcriptional mechanisms. Thus, the pattern of GATA-3 expression sequentially produces B lineage exclusion, T lineage progression, and myeloid-lineage exclusion for commitment.

Hallazgos electrocardiográficos en pacientes hipertensos del programa de vigilancia y control de la hipertensión arterial de ASSBASALUD ESE, Manizales, Colombia / Electrocardiographic findings in hypertensive patients of the hypertension surveillance and control program at ASSBASALUD ESE, Manizales, Colombia

Antecedentes. La hipertensión arterial sistémica es una enfermedad multifactorial que incluye la elevación de las cifras de tensión arterial, factores de riesgo cardiovascular e hipertrofia ventricular izquierda. Objetivo. Analizar los cambios electrocardiográficos en pacientes hipertensos inscritos en el programa de hipertensión arterial en ASSBASALUD Empresa Social del Estado, Manizales, Colombia, 2012. Materiales y métodos. Estudio poblacional de corte transversal. 102 pacientes. Variables electrocardiográficas analizadas: índices de Cornell, Sokolow Lyon y Macruz; cambios en la onda P y cambios en el eje. Se tomó electrocardiograma. Resultados. Edad promedio de 62 años. El 58,5% de los pacientes estudiados se clasificaron como pre-hipertensos, el 18,6% tenían frecuencia cardíaca superior a 100 latidos por minuto. Se encontraron cambios en la onda P en el 26,3%. En el 34% el eje estaba desviado. 64,6% tenían índice de Macruz anormal. 10,8% mostraron aumento en el voltaje del índice Sokolow-Lyon. En un 10,8% el voltaje de Cornell estaba elevado. En un 5,1% la fuerza terminal de onda P fue mayor a 40. Un 7,8% tenían bloqueos de rama derecha. En los pacientes con niveles altos de la presión arterial se encontró relación significativa con la elevación en los índices de Cornell (p=0,000) y de Sokolow-Lyon (p=0,016). Se encontró relación significativa entre los valores elevados de la presión arterial sistólica y los cambios en los índices de Cornell (p=0,010) y Sokolow-Lyon (p=0.001), los valores de la presión arterial diastólica se asociaron más a cambios en el Sokolow-Lyon (p=0,001). Conclusiones. Se confirma en esta población de hipertensos la asociación entre hipertensión arterial y cambios electrocardiográficos, especialmente en los índices de Cornell y de Sokolow-Lyon.

Background. Systemic blood pressure (HBP) is a multifactorial disease that includes elevated blood pressure levels, cardiovascular risk factors and left ventricular hypertrophy. Objective. To analyze the electrocardiographic changes in hypertensive patients enrolled in the hypertension surveillance and control program at ASSBASALUD ESE, Manizales, Colombia in 2012. Materials and methods. Cross-sectional study that involved 102 patients. The variables used were electrocardiogram indexes as Cornell, Sokolow Lyon, Macruz and P wave duration. It took a 12-lead electrocardiogram for each patient executed at ASBASALUD ESE, La Asunción. Results. The average age was 62 years, 58.5% were in the prehypertension range, also 18.6% had abnormal heart rate, 26.3% abnormal P wave duration, 34% abnormal cardiac axis, 64.6% abnormal Macruz index, 10.8% abnormal Sokolow-Lyon index, 10.8% abnormal Cornell index, 5.1% positive P-wave terminal force, 7.8% had right bundle branch block. The rank of hypertension showed significant relationship with the level of Cornell (p=0.000) and Sokolow-Lyon (p=0.016) indexes. A significant relationship between systolic blood pressure value and Cornell (p=0.010) and Sokolow-Lyon indexes were found (p=0.001), likewise diastolic blood pressure value and Sokolow-Lyon index value showed relationship (p=0.001). Conclusions. It is confirmed, in this population of hypertensive patients, that the indexes show variation according to level of hypertension, as well with the values of systolic and diastolic blood pressure are Cornell and Sokolow-Lyon indexes.

The reality of heart failure in Latin America.

Heart failure (HF) data in Latin America (LA) were reviewed to guide health service planning in the prevention and treatment of HF. The HF epidemiology and the adequacy of relevant health service provision related to HF in LA are not well delineated. A systematic search of the electronic databases and the World Health Organization website was undertaken for HF in LA. LA countries have reduced gross income and lower total expenditure on health per capita. LA is a heterogeneous region with HF risk factors of developed and nondeveloped countries, including lower risk of raised blood glucose levels, obesity, tobacco, and aging, whereas systemic hypertension (SH), rheumatic fever, and Chagas' disease (C'D) are higher in LA. Main etiologies of HF in LA are idiopathic dilated cardiomyopathy (from 1.3% to 37%), C'D (from 1.3% to 21%), ischemic (from 68% to 17%), SH (from 14% to 76%), valvular (from 3% to 22%), and alcohol related (from 1.1% to 8%). The prognosis of C'D HF is worse than for other etiologies. Chronic HF is the cause of death in 6.3% of cases. Decompensated HF is the main cause of cardiovascular hospitalization. The prevalence of systolic HF varies from 64% to 69%. LA is under the awful paradox of having the HF risk factors and HF epidemiology of developed countries with the added factors of SH, C'D, and rheumatic fever. Overall, in the scenario of lower total expenditure on health per capita and lower gross national income per capita, new strategies are essential for prevention and treatment of HF in LA.

High TGFbeta-Smad activity confers poor prognosis in glioma patients and promotes cell proliferation depending on the methylation of the PDGF-B gene.

TGFbeta acts as a tumor suppressor in normal epithelial cells and early-stage tumors and becomes an oncogenic factor in advanced tumors. The molecular mechanisms involved in the malignant function of TGFbeta are not fully elucidated. We demonstrate that high TGFbeta-Smad activity is present in aggressive, highly proliferative gliomas and confers poor prognosis in patients with glioma. We discern the mechanisms and molecular determinants of the TGFbeta oncogenic response with a transcriptomic approach and by analyzing primary cultured patient-derived gliomas and human glioma biopsies. The TGFbeta-Smad pathway promotes proliferation through the induction of PDGF-B in gliomas with an unmethylated PDGF-B gene. The epigenetic regulation of the PDGF-B gene dictates whether TGFbeta acts as an oncogenic factor inducing PDGF-B and proliferation in human glioma.

Definition of regulatory network elements for T cell development by perturbation analysis with PU.1 and GATA-3.

PU.1 and GATA-3 are transcription factors that are required for development of T cell progenitors from the earliest stages. Neither one is a simple positive regulator for T lineage specification, however. When expressed at elevated levels at early stages of T cell development, each of these transcription factors blocks T cell development within a different, characteristic time window, with GATA-3 overexpression initially inhibiting at an earlier stage than PU.1. These perturbations are each associated with a distinct spectrum of changes in the regulation of genes needed for T cell development. Both transcription factors can interfere with expression of the Rag-1 and Rag-2 recombinases, while GATA-3 notably blocks PU.1 and IL-7Ralpha expression, and PU.1 reduces expression of HES-1 and c-Myb. A first-draft assembly of the regulatory targets of these two factors is presented as a provisional gene network. The target genes identified here provide insight into the basis of the effects of GATA-3 or PU.1 overexpression and into the regulatory changes that distinguish the developmental time windows for these effects.