David M. Dorfman

Blockade of Programmed Death-1 Ligands on Dendritic Cells Enhances T Cell Activation and Cytokine Production

Programmed death-1 ligand (PD-L)1 and PD-L2 are ligands for programmed death-1 (PD-1), a member of the CD28/CTLA4 family expressed on activated lymphoid cells. PD-1 contains an immunoreceptor tyrosine-based inhibitory motif and mice deficient in PD-1 develop autoimmune disorders suggesting a defect in peripheral tolerance. Human PD-L1 and PD-L2 are expressed on immature dendritic cells (iDC) and mature dendritic cells (mDC), IFN-γ-treated monocytes, and follicular dendritic cells. Using mAbs, we show that blockade of PD-L2 on dendritic cells results in enhanced T cell proliferation and cytokine production, including that of IFN-γ and IL-10, while blockade of PD-L1 results in similar, more modest, effects. Blockade of both PD-L1 and PD-L2 showed an additive effect. Both whole mAb and Fab enhanced T cell activation, showing that PD-L1 and PD-L2 function to inhibit T cell activation. Enhancement of T cell activation was most pronounced with weak APC, such as iDCs and IL-10-pretreated mDCs, and less pronounced with strong APC such as mDCs. These data are consistent with the hypothesis that iDC have a balance of stimulatory vs inhibitory molecules that favors inhibition, and indicate that PD-L1 and PD-L2 contribute to the poor stimulatory capacity of iDC. PD-L1 expression differs from PD-L2 in that PD-L1 is expressed on activated T cells, placental trophoblasts, myocardial endothelium, and cortical thymic epithelial cells. In contrast, PD-L2 is expressed on placental endothelium and medullary thymic epithelial cells. PD-L1 is also highly expressed on most carcinomas but minimally expressed on adjacent normal tissue suggesting a role in attenuating antitumor immune responses.

Programmed Death-1 (PD-1) is a Marker of Germinal Center-associated T Cells and Angioimmunoblastic T-Cell Lymphoma

Programmed death-1 (PD-1), a member of the CD28 costimulatory receptor family, is expressed by germinal center-associated T cells in reactive lymphoid tissue. In a study of a wide range of lymphoproliferative disorders, neoplastic T cells in 23 cases of angioimmunoblastic lymphoma were immunoreactive for PD-1, but other subtypes of T cell and B cell non-Hodgkin lymphoma, as well as classic Hodgkin lymphoma, did not express PD-1. The pattern of PD-1 immunostaining of neoplastic cells in angioimmunoblastic lymphoma was similar to that reported for CD10, a recently described marker of neoplastic T cells in angioimmunoblastic lymphoma. Tumor-associated follicular dendritic cells in cases of angioimmunoblastic lymphoma were found to express PD-L1, the PD-1 ligand. In addition, PD-1-positive reactive T cells formed rosettes around neoplastic L&H cells in 14 cases of nodular lymphocyte predominant Hodgkin lymphoma studied. These findings, along with data from previous studies, suggest that angioimmunoblastic lymphoma is a neoplasm of germinal center-associated T cells and that there is an association of germinal center-associated T cells and neoplastic cells in nodular lymphocyte predominant Hodgkin lymphoma. PD-1 is a useful new marker for angioimmunoblastic lymphoma and lends further support to a model of T-cell lymphomagenesis in which specific subtypes of T cells may undergo neoplastic transformation and result in specific, distinct histologic, immunophenotypic, and clinical subtypes of T-cell neoplasia.

Impact of Efavirenz on Neuropsychological Performance and Symptoms in HIV-Infected Individuals

Context Neurologic toxicity is the most commonly reported adverse effect of the antiretroviral drug efavirenz. Contribution In this substudy of a randomized, controlled trial, 12 of 200 (6%) HIV-infected individuals discontinued treatment with efavirenz because of central nervous system symptoms or mood disorders versus 0 of 103 individuals (0%) who were not receiving the drug. Although patients taking efavirenz had more neuropsychological symptoms, such as bad dreams, in the first week of therapy, no statistically significant neuropsychological differences were found at weeks 4, 12, and 24. Implications Some adverse neuropsychological effects associated with efavirenz are probably transient. The Editors Efavirenz is a non-nucleoside reverse transcriptase inhibitor approved for treatment of HIV infection. The drug is potent, is generally well tolerated, and can be administered once daily, making it a preferred treatment option for HIV infection (1, 2). The most commonly reported adverse effect with efavirenz is neurologic toxicity, with more than 50% of patients reporting symptoms in open-label studies (1, 3). Our randomized, controlled study prospectively characterized aspects of the neurologic toxicity of 3 protease inhibitorsparing antiretroviral regimens for the initial treatment of HIV infection. Methods This investigator-initiated trial was a substudy of the AIDS Clinical Trials Group study A5095, a randomized, double-blind trial of 3 antiretroviral regimens: zidovudine and lamivudine in combination with efavirenz; abacavir; or abacavir and efavirenz in combination (4). For simplicity, we will refer to 2 groups: patients who received efavirenz (with or without abacavir) and those who did not. Randomization was performed centrally without reference to center. The study was supported by the National Institutes of Health (NIH) and was approved by the institutional review boards at each of the participating institutions, with each patient providing informed consent to participate in the substudy. All patients at sites taking part in the substudy were invited to participate before randomization for the parent study (Figure 1). Unblinding and within-class substitutions were allowed in cases of treatment-limiting toxicity (we substituted stavudine for zidovudine, didanosine for abacavir, and nevirapine for efavirenz). Participants had not previously received antiretroviral therapy, and their baseline plasma HIV-1 RNA levels were greater than 400 copies/mL. Parent study A5095 enrolled 1147 participants, of whom 303 at 36 clinical trials units volunteered to participate in the additional evaluations for A5097s. Participants were recruited between March 2001 and January 2002. Figure 1. CONSORT diagram of substudy 5097s. The primary measures of neuropsychological performance were the Trail Making Tests (Parts A and B) and the Digit Symbol Substitution Test (part of the Wechsler Adult Intelligence Scale III [5]). A summary neuropsychological Z score (NPZ3) was derived from the sum of the scores from these 3 tests and standardized for age. Positive scores indicated above-normal function, whereas negative scores indicated below-normal function. The entire score was coded as missing if any component of the NPZ3 was not available. The Neurologic AIDS Research Consortium provided administrator training at each site. These tests assessed functioning in the areas of motor persistence, sustained attention, response speed, visuomotor coordination, and conceptual shifting and tracking. Neuropsychometric measures were collected at baseline and at weeks 1, 4, 12, and 24. Testing was performed at each time point to assess symptoms that might be associated with efavirenz use, sleep disorders, anxiety, depression, and history of drug abuse. The instruments are summarized in Table 1. The symptom questionnaire developed for this study is shown in the Appendix Figure. Appendix Figure. Sample participant questionnaire. Table 1. Testing Instruments Whole blood was collected from all participants to determine efavirenz trough concentrations in plasma (13). These data were used to explore relationships between drug exposure and other variables that were evaluated in the study. Statistical Analysis Our substudy was designed to compare neurologic changes from baseline in patients who received efavirenz with changes in those who did not. The study had 90% power to detect a standard deviation of 0.4 for change in the summary neuropsychological performance score from baseline to week 1. We presented descriptive statistics for the study sample and used nonparametric tests to determine treatment differences. Using the nonparametric methods of Hodges and Lehmann (14) and Proc-StatXact software, version 4.0.1 (Cytel Software Corp., Cambridge, Massachusetts), we estimated treatment differences for continuous outcomes with corresponding exact confidence intervals. Generalized estimating equation modeling (a regression method) and the Wei-Johnson test (a nonparametric method for analyzing incomplete 2-sample data) (15) were used to compare treatment groups longitudinally; both methods assumed that data were missing completely at random. We used the Spearman correlation coefficient, a rank-based method that is robust to extreme observations, to evaluate correlations. All significance testing was performed at an level of 0.05 with no adjustment for multiple testing. All reported P values were 2-sided. To assess the potential effect of any missing data, we performed multiple imputation, analyzed 2 worst-case scenarios (Appendix Table 1 and Appendix Table 2), and conducted an as-treated analysis that excluded patients who discontinued efavirenz therapy. We used SAS software (SAS Institute, Inc., Cary, North Carolina) to perform statistical analyses. Test sources included Elsevier Science (Oxford, United Kingdom) for the Pittsburgh Sleep Quality Index, Mind Garden (Redwood City, California) for the State-Trait Anxiety Inventory for Adults, and the National Institute of Mental Health (Bethesda, Maryland) for the Center for Epidemiologic Studies Depression Scale. Role of the Funding Sources This investigator-initiated protocol was supported by the NIH. Drugs used in the study were donated by pharmaceutical companies whose representatives participated in team discussions. The study was monitored by NIH-contracted monitors and was supervised by a data safety monitoring committee that was appointed by the National Institute of Allergy and Infectious Diseases. The NIH-supported biostatistical team working with the AIDS Clinical Trials Group and the Neurologic AIDS Research Consortium performed the statistical analyses. The protocol team, led by the first author, had final responsibility for the study protocol, case report forms, statistical analysis plan, progress of the study, analysis, and reporting of the data, regardless of outcome. The final version was the sole responsibility of the authors. The team had full access to the data files of the study. Results Baseline Evaluations Recruitment characteristics are displayed in Figure 1; demographic characteristics of the study participants are presented in Table 2. The treatment groups were balanced at baseline with respect to demographic characteristics, neuropsychological measures, and responses to the symptom questionnaire. The sleep disturbance component of the global sleep index demonstrated a baseline difference; the patients who eventually received efavirenz had marginally more sleep disturbances (P= 0.048) (data not shown). Other components of the sleep index, including quality, latency, duration, efficiency, use of sleeping medication, and daytime dysfunction, were similar between groups. Alcohol abuse, drug use, and affective disturbances were infrequent and similar for both groups. Table 2. Baseline Characteristics and Evaluations by Treatment Group Disposition of Study Participants The study allowed for drug substitution from the same class of antiretroviral agents in cases of treatment-limiting toxicity. Table 3 summarizes the modifications that occurred and the respective reasons. Appendix Table 3 gives further details of timing of modifications and the ethnicity of the individuals. Primary end point data (the change in NPZ3 from baseline to week 1) were observed in 283 of the 303 (93.4%) participants. Table 3. Reasons for Modification of Treatment Appendix Table 1. Neuropsychological Performance Appendix Table 2. Results of Sensitivity Analysis by Generalized Estimating Equation Method Appendix Table 3. Status and Ethnicity of Patients Requiring Modification of Treatment Prospective Evaluations Median NPZ3 scores improved in both groups during the study, with the greatest change occurring in the first week of treatment (Figure 2). No statistically significant differences in changes in neuropsychological performance were observed between the groups at any time point. We conducted conventional longitudinal analyses to further investigate differences in neuropsychological scores between the treatment groups. On the basis of these analyses, we had insufficient evidence to conclude that there were treatment differences (generalized estimating equation modeling in which treatment was the only independent variable and an exchangeable correlation structure was assumed, P= 0.176; Wei-Johnson test, P= 0.196). Multiple sensitivity analyses were performed, including single and multiple imputation methods, as-treated analyses, and 2 forms of worst-case scenarios. Details of these analyses are shown in Appendix Table 1. Multiple imputation and as-treated analyses generally provided similar results to observed data at specific weeks; however, the worst-case analyses at weeks 4, 12, and 24 displayed significant differences between groups. These results suggest that differences between groups might exist if the worst-case scenario were true, that is, if patients without data who were receiving efavirenz had wors

Human Pumilio-2 is expressed in embryonic stem cells and germ cells and interacts with DAZ (Deleted in AZoospermia) and DAZ-Like proteins

Early in development, a part of the embryo is set aside to become the germ cell lineage that will ultimately differentiate to form sperm and eggs and transmit genetic information to the next generation. Men with deletions encompassing the Y-chromosome DAZ genes have few or no germ cells but are otherwise healthy, indicating they harbor specific defects in formation or maintenance of germ cells. A DAZ homolog, DAZL (DAZ-Like), is found in diverse organisms, including humans and is required for germ cell development in males and/or females. We identified proteins that interact with DAZ proteins to better understand their function in human germ cells. Here, we show that PUM2, a human homolog of Pumilio, a protein required to maintain germ line stem cells in Drosophila and Caenorhabditis elegans, forms a stable complex with DAZ through the same functional domain required for RNA binding, protein–protein interactions and rescue of Pumilio mutations in flies. We also show that PUM2 is expressed predominantly in human embryonic stem cells and germ cells and colocalizes with DAZ and DAZL in germ cells. These data implicate PUM2 as a component of conserved cellular machinery that may be required for germ cell development.

Solitary fibrous tumor of the orbit

We report three cases of an orbital soft tissue lesion that fulfills the histologic, immunohistochemical, and electron microscopic criteria for solitary fibrous tumor, an entity previously described as a pleural tumor, but recently reported to occur in other locations. All three patients presented with proptosis. Two of the patients were cured by simple excision, and one patient had two recurrences, the last recurrence incompletely excised. The findings indicate that solitary fibrous tumor can occur in the orbit and, like solitary fibrous tumors of other anatomic sites, may behave in a nonaggressive or occasionally, locally aggressive fashion, with as yet no metastatic potential demonstrated in orbital lesions.

DAZ Family Proteins Exist Throughout Male Germ Cell Development and Transit from Nucleus to Cytoplasm at Meiosis in Humans and Mice

Abstract The human DAZ gene family is expressed in germ cells and consists of a cluster of nearly identical DAZ (deleted in azoospermia) genes on the Y chromosome and an autosomal homolog, DAZL (DAZ-like). Only the autosomal gene is found in mice. Y-chromosome deletions that encompass the DAZ genes are a common cause of spermatogenic failure in men, and autosomal homologs of DAZ are essential for testicular germ cell development in mice and Drosophila. Previous studies have reported that mouse DAZL protein is strictly cytoplasmic and that human DAZ protein is restricted to postmeiotic cells. By contrast, we report here that human DAZ and human and mouse DAZL proteins are present in both the nuclei and cytoplasm of fetal gonocytes and in spermatogonial nuclei. The proteins relocate to the cytoplasm during male meiosis. Further observations using human tissues indicate that, unlike DAZ, human DAZL protein persists in spermatids and even spermatozoa. These results, combined with findings in diverse species, suggest that DAZ family proteins function in multiple cellular compartments at multiple points in male germ cell development. They may act during meiosis and much earlier, when spermatogonial stem cell populations are established.

Precursor B-cell lymphoblastic lymphoma: a predominantly extranodal tumor with low propensity for leukemic involvement.

Precursor B-cell lymphoblastic lymphoma (B-LBL) is uncommon and accounts for less than 10% of cases of lymphoblastic lymphoma. We collected 25 cases of B-LBL, occurring in children and adults, and report the clinical and histologic features. Patients with concurrent precursor B-cell acute lymphoblastic leukemia (B-ALL) or a history thereof were excluded. There was no evidence of bone marrow disease at the time of diagnosis in 23 patients; two patients had focal (<5%) involvement. Immunophenotypic analysis was performed in all cases using flow cytometry or immunohistochemical methods. The treatment and survival data available for a subset of patients with B-LBL were compared with those from a series of patients with B-ALL at our institution. The median age was 20 years (range, 5–68 yrs); 22 (88%) patients were younger than 35 years of age. There were 17 males and 8 females. The primary sites of disease were skin (nine cases), bones (five cases), soft tissue (four cases), lymph nodes, (three cases), breast (two cases), stomach and colon (one case), and mediastinum (one case). Clinical stage was stage I in 13 cases, stage II in seven cases, stage III in three cases, and stage IV in two cases. Histologically, each neoplasm was diffuse and composed of small to medium-sized lymphoid cells with blastic nuclear chromatin and a high mitotic rate. All cases were positive for B-cell antigens and terminal deoxynucleotidyl transferase. Thirteen (76.4%) of 17 cases analyzed were positive for CD10 and 13 (54.1%) of 24 cases assessed were positive for CD20. Of 14 patients with available survival data, all achieved complete clinical response after combination chemotherapy (13 patients) or surgical excision followed by local irradiation (one patient). Five (35.7%) patients subsequently relapsed, including the patient who had received only irradiation, and four of these patients died after a median survival time of 60 months. None of the patients had leukemia, although one patient developed extensive bone marrow involvement. Nine patients remained in complete remission and were alive at the last follow up (range, 6–144 months). Unlike precursor T-cell lymphoblastic lymphoma, which commonly involves lymph nodes and the mediastinum, B-LBL usually involves extranodal sites, most often the skin, and rarely presents as a mediastinal mass. With aggressive chemotherapy, patients with precursor B-LBL rarely develop leukemia and appear to have a better prognosis than do patients with B-ALL.

Bioinspired multivalent DNA network for capture and release of cells

Capture and isolation of flowing cells and particulates from body fluids has enormous implications in diagnosis, monitoring, and drug testing, yet monovalent adhesion molecules used for this purpose result in inefficient cell capture and difficulty in retrieving the captured cells. Inspired by marine creatures that present long tentacles containing multiple adhesive domains to effectively capture flowing food particulates, we developed a platform approach to capture and isolate cells using a 3D DNA network comprising repeating adhesive aptamer domains that extend over tens of micrometers into the solution. The DNA network was synthesized from a microfluidic surface by rolling circle amplification where critical parameters, including DNA graft density, length, and sequence, could readily be tailored. Using an aptamer that binds to protein tyrosine kinase-7 (PTK7) that is overexpressed on many human cancer cells, we demonstrate that the 3D DNA network significantly enhances the capture efficiency of lymphoblast CCRF-CEM cells over monovalent aptamers and antibodies, yet maintains a high purity of the captured cells. When incorporated in a herringbone microfluidic device, the 3D DNA network not only possessed significantly higher capture efficiency than monovalent aptamers and antibodies, but also outperformed previously reported cell-capture microfluidic devices at high flow rates. This work suggests that 3D DNA networks may have broad implications for detection and isolation of cells and other bioparticles.

A targeted mutational landscape of angioimmunoblastic T-cell lymphoma

The genetics of angioimmunoblastic T-cell lymphoma (AITL) are very poorly understood. We defined the mutational landscape of AITL across 219 genes in 85 cases from the United States and Europe. We identified ≥2 mutations in 34 genes, nearly all of which were not previously implicated in AITL. These included loss-of-function mutations in TP53 (n = 4), ETV6 (n = 3), CCND3 (n = 2), and EP300 (n = 5), as well as gain-of-function mutations in JAK2 (n = 2) and STAT3 (n = 4). TET2 was mutated in 65 (76%) AITLs, including 43 that harbored 2 or 3 TET2 mutations. DNMT3A mutations occurred in 28 (33%) AITLs; 100% of these also harbored TET2 mutations (P < .0001). Seventeen AITLs harbored IDH2 R172 substitutions, including 15 with TET2 mutations. In summary, AITL is characterized by high frequencies of overlapping mutations in epigenetic modifiers and targetable mutations in a subset of cases.

Normal d-dimer levels in emergency department patients suspected of acute pulmonary embolism

OBJECTIVES We sought to determine:1) whether normal D-dimer enzyme-linked immunosorbent assay (ELISA) assays predicted the absence of pulmonary embolism (PE) in the high-volume emergency department (ED) of the Brigham and Womens Hospital, and 2) whether ED physicians accepted normal D-dimer levels as confirmation of no PE without further diagnostic testing such as lung scanning, chest computed tomography (CT) scanning, or pulmonary angiography. BACKGROUND Although the plasma D-dimer ELISA is a sensitive screening test for excluding acute PE, this laboratory marker has not been widely integrated into clinical algorithms such as creatine kinase-MB fraction or troponin testing for acute myocardial infarction. METHODS We mandated that ED physicians order D-dimer ELISA tests on all patients suspected of acute PE. We reviewed the clinical record of each ED patient initially evaluated for suspected PE during the year 2000. We determined whether additional imaging tests for PE were obtained and whether the final diagnosis was PE. RESULTS Of 1,106 D-dimer assays, 559 were elevated and 547 were normal. Only 2 of 547 had PE despite a normal D-dimer. The sensitivity of the D-dimer ELISA for acute PE was 96.4% (95% confidence interval [CI]: 87.5% to 99.6%), and the negative predictive value was 99.6% (95% CI: 98.7% to >99.9%). Nevertheless, 24% of patients with normal D-dimers had additional imaging tests for PE. CONCLUSIONS The D-dimer ELISA has a high negative predictive value for excluding PE. By paying more attention to normal D-dimer results, fewer chest CT scans and lung scans will be required, and improvements may be realized in diagnostic efficiency and cost reduction.