High-throughput analysis of T cell–monocyte interaction in human tuberculosis

The lack of efficient tools for identifying immunological correlates of tuberculosis (TB) protection or risk of disease progression impedes the development of improved control strategies. To more clearly understand the host response in TB, we recently established an imaging flow cytometer-based in-vitro assay, which assesses multiple aspects of T cell–monocyte interaction. Here, we extended our previous work and characterized communication between T cells and monocytes using clinical samples from individuals with different TB infection status and healthy controls from a TB endemic setting. To identify T cell–monocyte conjugates, peripheral blood mononuclear cells (PBMC) were stimulated with ds-Red-expressing Mycobacterium bovis bacille Calmette–Guérin or 6-kDa early secreted antigenic target (ESAT 6) peptides for 6 h, and analyzed by imaging flow cytometer (IFC). We then enumerated T cell–monocyte conjugates using polarization of T cell receptor (TCR) and F-actin as markers for synapse formation, and nuclear factor kappa B (NF-κB) nuclear translocation in the T cells. We observed a reduced frequency of T cell–monocyte conjugates in cells from patients with active pulmonary tuberculosis (pTB) compared to latent TB-infected (LTBI) and healthy controls. When we monitored NF-κB nuclear translocation in T cells interacting with monocytes, the proportion of responding cells was significantly higher in active pTB compared with LTBI and controls. Overall, these data underscore the need to consider multiple immunological parameters against TB, where IFC could be a valuable tool.     

Antithymocyte globulin has limited efficacy and substantial toxicity in unselected anemic patients with myelodysplastic syndrome

Antithymocyte globulin (ATG) has recently been popularized as an effective treatment in myelodysplastic syndrome (MDS). We treated 8 anemic MDS patients (refractory anemia [RA] and refractory anemia with excess blasts [RAEB-1]) with ATG (40 mg/kg/d for 4 days) and prednisone in a phase 2 trial. The study was stopped early according to a preset termination rule because of lack of efficacy. There were no salutary responses. Toxicities included serum sickness (in all patients), transient neutropenia and thrombocytopenia, diarrhea, vomiting, and syncope with a generalized seizure. At least 3 patients had the HLA-DR15 (DR2) allele. We conclude that the risk-benefit ratio of ATG in an unselected population of MDS patients may be unfavorable, and more work is needed to define the subset of patients who will respond to ATG before its widespread use can be recommended.     

Both B and T lymphocytes may be clonally involved in myelofibrosis with myeloid metaplasia

A combination of magnetic cell sorting (MACS) and fluorescent in situ hybridization (FISH) techniques was used to detect clonal cytogenetic markers in different myeloid and lymphoid cell types of the peripheral blood from 4 patients with myelofibrosis with myeloid metaplasia (MMM) that was associated with either a 13q- or a 20q- karyotypic abnormality. Interphase cytogenetics studies demonstrated abnormal clonal FISH signal patterns in neutrophil, myeloid, erythroid, megakaryocyte, and B- and T-cell preparations in 3 of the 4 patients. In one patient, FISH results were within normal limits in T cells and slightly abnormal in B cells. In general, the percentage of abnormal nuclei was variable in both lymphocyte populations but always higher in B lymphocytes compared with T lymphocytes. The current study provides direct evidence for the clonal involvement of both B and T lymphocytes in MMM. A larger study is needed to clarify the relevance of the observed interpatient heterogeneity in clonal constitution.     

Methotrexate induced seizures associated with acute reversible magnetic resonance imaging (MRI) changes in a patient with acute lymphoblastic leukemia

A 27-year-old woman with acute lymphoblastic leukemia (ALL) had an episode of generalized tonic-clonic seizure after therapy with intrathecal and intravenous methotrexate (MTX). Magnetic resonance imaging (MRI) of her head showed meningeal, cortical and subcortical enhancement that was new when compared to a study done prior to therapy. Subsequent imaging 10 days later showed partial resolution of these findings. The association of seizures and MTX in ALL and the corresponding MRI changes are discussed.     

The effect of absolute lymphocyte count recovery kinetics on survival after autologous stem cell transplantation for non-Hodgkin's lymphoma

Absolute lymphocyte count (ALC) >or=500 cells/microl at day 15 after autologous stem cell transplantation (ASCT) is a powerful independent, prognostic indicator for survival in multiple hematological malignancies. A limitation in these studies was the selection of a single time point (day 15 post-ASCT) as the only discriminator of clinical outcome in relation to ALC recovery. We hypothesized there is a continuous and not discrete relationship between ALC recovery and clinical outcome post-ASCT in NHL. Therefore, we analyzed 274 consecutive patients who underwent ASCT for NHL between 1987 and 2001. The primary end point was to assess the impact of the kinetics of post-ASCT lymphocyte recovery>or=500 cells/microl (K-ALC) on overall survival (OS) and progression-free survival (PFS). K-ALC was a predictor of OS and PFS when the Cox proportional hazards model was used with K-ALC entered as a continuous variable (p<0.0001). Multivariate analysis demonstrated K-ALC recovery post-ASCT to be an independent prognostic indicator for OS and PFS. These data support our hypothesis that the K-ALC post-ASCT is associated with clinical outcome in NHL.