Imaging African trypanosomes.

Trypanosoma brucei are extracellular kinetoplastid parasites transmitted by the blood-sucking tsetse fly. They are responsible for the fatal disease human African trypanosomiasis (HAT), also known as sleeping sickness. In late-stage infection, trypanosomes cross the blood-brain barrier (BBB) and invade the central nervous system (CNS) invariably leading to coma and death if untreated. There is no available vaccine and current late-stage HAT chemotherapy consists of either melarsoprol, which is highly toxic causing up to 8% of deaths, or nifurtimox-eflornithine combination therapy (NECT), which is costly and difficult to administer. There is therefore an urgent need to identify new late-stage HAT drug candidates. Here, we review how current imaging tools, ranging from fluorescent confocal microscopy of live immobilized cells in culture to whole-animal imaging, are providing insight into T. brucei biology, parasite-host interplay, trypanosome CNS invasion and disease progression. We also consider how imaging tools can be used for candidate drug screening purposes that could lead to new chemotherapies.

Detection of translocations affecting the BCL6 locus in B cell non-Hodgkin's lymphoma by interphase fluorescence in situ hybridization.

Structural alterations in 3q27 affecting the BCL6 locus are among the most frequent changes in B-NHL. The aim of the present study was to establish an interphase-FISH assay for the detection of all diverse BCL6 translocations in B-NHL. Two different approaches were tested, one using a PAC-clone spanning the major breakpoint region (MBR) of BCL6 (span-assay), and another using two BAC clones flanking the MBR (flank-assay). Interphase FISH with the span-assay detected the various BCL6 translocations in seven B-NHL cell lines. The dual-color flank-assay was evaluated in two laboratories independently: in normal controls, the cutoff level for false-positive signals was 2.6%, whereas the cutoff level for false-negatives in the seven cell lines was 7.5%. To test the feasibility of the FISH strategies, 30 samples from patients with B-NHL with cytogenetic abnormalities of 3q27 were evaluated with both assays. In 21 cases, the span-assay indicated a BCL6 rearrangement. In 18 of the 21 cases, the dual-color flank-assay confirmed the translocation including 12 different partner chromosomal loci. The three false-positive cases detected with the span-assay showed trisomy of chromosome 3 by cytogenetic analyses, and they were correctly classified as non-rearranged with the flank-assay. In summary, our FISH strategy using two differently labeled flanking BCL6 BAC probes provides a robust, sensitive, and reproducible method for the detection of common and uncommon abnormalities of BCL6 gene in interphase nuclei. The routine application of this assay to patients with B-NHL will allow the assessment of the diagnostic and prognostic significance of BCL6 rearrangements.

ARL1 has an essential role in Trypanosoma brucei.

Myristoyl-CoA protein:NMT (N-myristoyl transferase) catalyses the N-myristoylation of cellular proteins with a range of functions and is essential for viability in the protozoan parasites, Leishmania major and Trypanosoma brucei. In our investigations to define the essential downstream targets of NMT, we have focused on the ARF (ADP-ribosylation factor) family of proteins, as growth arrest in Saccharomyces cerevisiae mutants with reduced NMT activity correlates with decreased modification of members of this group of proteins. We have identified nine ARF/ARLs (where ARL stands for ARF-like) encoded in the T. brucei and T. cruzi genomes and ten in L. major. The T. brucei ARL1 protein is expressed only in the mammalian bloodstream form of the parasite, in which it is localized to the Golgi apparatus. RNAi (RNA interference) has been used to demonstrate that ARL1 is essential for viability in these infective cells. Before cell death, depletion of ARL1 protein results in disintegration of the Golgi structure and a delay in exocytosis of the abundant GPI (glycosylphosphatidylinositol)-anchored VSG (variant surface glycoprotein) to the parasite surface.

Cloning, heterologous expression and antigenicity of a schistosome cercarial protease.

A gene coding for the 30 kDa Schistosoma mansoni cercarial protease was amplified using the polymerase chain reaction (PCR) from genomic DNA templates. Cloning and sequencing of several independent PCR clones revealed the presence of an intron additional to the one described in the original cloning of the gene. The 3 exons were cloned into expression vectors so that they could be expressed as separate glutathione-S-transferase (GST) translational fusions. Recombinant bacteria carrying these expression plasmids expressed the fusion proteins at high levels. Western blotting of bacterial lysates with sera raised against the native S. mansoni cercarial protease showed that all 3 exons were recognized. Thus we have produced recombinant bacteria capable of providing large amounts of an S. mansoni antigen for immunological studies and evaluation as a candidate vaccine.

Zonal distribution of alveolar macrophages, type II pneumonocytes, and alveolar septal connective tissue gaps in adult human lungs.

The distribution of alveolar macrophages, Type II cells, and alveolar septal connective tissue discontinuities or gaps in two neighboring alveoli from a human lung obtained at surgery, and preserved by vascular perfusion-fixation, was studied by electron microscopy. Discontinuities or gaps are defined as complete interruptions of all the connective tissue elements of the alveolar septum, including the basement membranes. Serial-sectioning of the alveoli, and the creation of montages of the entire circumference of each alveolus at intervals of every twentieth section (approximately 1.6 micron) at a magnification of X 2,160 permitted precise identification of cells and connective tissue gaps and allowed the reconstruction, by computer techniques, of the alveolar walls in 3 dimensions. These studies showed that all of the 48 alveolar macrophages identified, and over two thirds of all Type II cells and alveolar septal gaps, were located or bordered on alveolar septal junctional zones (within 10 microns of septal junctions). The profiles of 739 alveoli examined by light microscopy from 6 lungs similarly preserved by vascular perfusion-fixation, in which the alveolar surface lining was well fixed, also showed alveolar macrophages preferentially distributed in alveolar junctional zones. These were compared with 242 alveolar profiles from 3 other vascularly-fixed lungs and 971 alveolar profiles from 9 lungs fixed by way of the airways, in which the alveolar surface lining was lost. In these lungs, most of the alveolar macrophages were in the alveolar air spaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Schistosoma mansoni: anomalous immunogenic properties of a 27 kDa larval serine protease associated with protective immunity.

A cationic Schistosoma mansoni cercarial antigen was shown to be a serine protease as it was capable of hydrolysing N-acetyl-DL-phenylalanine beta-naphthyl ester (NAPBNE) after precipitation by immunoelectrophoresis, and this reaction was modulated by the serine protease inhibitors phenylmethanesulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DEP). The antigen in the immunoprecipitin arcs could also be radio-isotope labelled with tritiated DFP. The peptidolytic enzyme identified in immunoelectrophoresis with polyspecific sera and radio-isotope labelled with tritiated DFP had a relative molecular size of approximately 27 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and evidence obtained after partial purification, SDS-PAGE and immunoblotting supported this size estimate for the enzyme. A rabbit antiserum raised against the peptidolytic antigen reacted against a doublet of antigens at 27/28 kDa in immunoelectrophoresis arcs and against an antigen of 60 kDa in Western immunoblots of crude cercarial homogenate. However, the latter serum precipitated the cationic antigen in immunoelectrophoresed cercarial homogenates only after pre-incubation of the homogenates with PMSF. Fractions containing the partially purified protease also degraded radio-isotope labelled human IgG. The reactivity of a range of polyspecific and monospecific rabbit antisera in Western blots with larval extracts indicated that antibody responses against the 27/28 kDa doublet may be modulated. When immunized with material which contained the 27 kDa enzyme as a major constituent, and which was secreted by S. mansoni cercariae during transformation, only 5 of 16 mice produced antibody to this antigen that was detectable in Western blots. The 5 antibody 'responder' mice were significantly (P < 0.001) protected against challenge with a percutaneous infection of S. mansoni cercariae compared with a group of a mice also immunized with CTF, but which had not produced antibodies against the 27/28 kDa doublet. The results indicate that the 27 kDa serine protease of S. mansoni larvae is a target that is sensitive to immunological attack.

The BCL11 gene family: involvement of BCL11A in lymphoid malignancies.

Many malignancies of mature B cells are characterized by chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32.3 and result in deregulated expression of the translocated oncogene. t(2;14)(p13;q32.3) is a rare event in B-cell malignancies. In contrast, gains and amplifications of the same region of chromosome 2p13 have been reported in 20% of extranodal B-cell non-Hodgkin lymphomas (B-NHL), in follicular and mediastinal B-NHL, and in Hodgkin disease (HD). It has been suggested that REL, an NF-kappaB gene family member, mapping within the amplified region, is the pathologic target. However, by molecular cloning of t(2;14)(p13;q32.3) from 3 cases of aggressive B-cell chronic lymphocytic leukemia (CLL)/immunocytoma, this study has shown clustered breakpoints on chromosome 2p13 immediately upstream of a CpG island located about 300 kb telomeric of REL. This CpG island was associated with a Krüppel zinc finger gene (BCL11A), which is normally expressed at high levels only in fetal brain and in germinal center B-cells. There were 3 major RNA isoforms of BCL11A, differing in the number of carboxy-terminal zinc fingers. All 3 RNA isoforms were deregulated as a consequence of t(2;14)(p13;q32.3). BCL11A was highly conserved, being 95% identical to mouse, chicken, and Xenopus homologues. BCL11A was also highly homologous to another gene (BCL11B) on chromosome 14q32.1. BCL11A coamplified with REL in B-NHL cases and HD lymphoma cell lines with gains and amplifications of 2p13, suggesting that BCL11A may be involved in lymphoid malignancies through either chromosomal translocation or amplification.