Mycobacterial disease associated with aplastic anaemia

Haematological changes associated with infectious disease are relatively common but true aplastic anaemia secondary to infection is rare. We describe a patient wit disseminated Mycobacterium avium-intracellulare infection and who had a histologically proven remission of his aplastic anemia accompanying antimycobacterial therapy. We also review the literature on the haematological changes associated with mycobacterial infections and other infectious causes of aplastic anaemia.     

Increased fatty acid synthase is a therapeutic target in mesothelioma.

Many common human cancer tissues express high levels of fatty acid synthase (FAS), the primary enzyme for the synthesis of fatty acids, and the differential expression of FAS between normal and neoplastic tissues has led to the consideration of FAS as a target for anticancer therapy. To investigate the potential of targeting FAS for the treatment of pleural mesothelioma, we first determined whether FAS is overexpressed in human mesothelioma. By immunohistochemistry, we found 22 of 30 human mesothelioma tissue samples tested to express significantly increased levels of FAS compared with normal tissues, including mesothelium. To further explore FAS as a therapeutic target in mesothelioma, we established a nude mouse xenograft model for human mesothelioma using the H-Meso cell line. The i.p. xenografts of this cell line have high levels of FAS expression and fatty acid synthesis pathway activity and grow along mesothelial surfaces in a manner similar to the growth pattern of human mesothelioma. Growth of these tumor xenografts was essentially abolished in mice treated with weekly i.p. injections of C75, a synthetic, small molecule inhibitor of FAS, at levels that resulted in no significant systemic toxicity except for reversible weight loss. These results suggest that FAS may be an effective target for pharmacological therapy in a high proportion of human mesotheliomas.     

Functional interleukin-2 receptors are expressed on natural killer-like leukemic cells from a dog with cutaneous lymphoma

We identified a dog with large granular lymphocytic leukemia and cutaneous lymphoma that exhibited constitutive expression of interleukin-2 (IL-2) receptors by the leukemic peripheral blood lymphocytes. The leukemic cells phenotypically resembled natural killer (NK) cells, and their surface IL-2 receptors were functional, as determined by the capacity to bind human recombinant IL-2 with high- affinity resulting in the transduction of proliferation signals and in the development of lymphokine-activated killer cell activity. These cells produced IL-2 spontaneously, and they may have maintained their proliferative state through an IL-2-dependent autocrine growth pathway. Our results indicate that neoplastic lymphocytes of syndromes that involve circulating leukemic cells with dermotropism can originate from NK-like cells. Additionally, the data also suggest that proliferative conditions such as these may be the result of the aberrant production of IL-2. Further, this case illustrates the potential for the use of hematopoietic malignancies in the dog as a suitable animal model for immune targeting of IL-2 receptors as a novel treatment approach for similar malignancies of human beings.     

Two acquired immunodeficiency syndrome-associated Burkitt's lymphomas produce specific anti-i IgM cold agglutinins using somatically mutated VH4-21 segments

We analyzed the reactivity and the structure of the VH and VL segments of two IgM monoclonal antibodies (MoAbs) produced by spontaneously in vitro outgrowing cell lines, HBL-2 and HBL-3, established from two acquired immunodeficiency syndrome (AIDS) patients with Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL). These B-cell clones were representative of the respective neoplastic parental clones, as determined by immunophenotypic and molecular genetic analysis. The IgM MoAbs were highly specific for the i determinant on red blood cells (cold agglutinins), but bound none of the other eight self and nine foreign antigens (Ags) tested, including those most commonly recognized by natural antibodies or autoantibodies. Structural analysis showed that the IgM MoAb VH segment sequences were 93.5% and 84.2% identical with that of the germline VH4-21 gene, which encodes the vast majority of cold agglutinins that are specific for the i/l carbohydrate Ag and are produced under chronic lymphoproliferative conditions. The HBL-2 MoAb VH4-21 gene segment was juxtaposed with 20P3 and JH6 genes and paired with a V lambda 1 segment, the sequence of which was 95.5% identical to that of the germline Humlv117 gene; the HBL-3 MoAb VH4-21 gene segment was juxtaposed with DXP'1 and JH5 genes and paired with a V lambda 1 segment, the sequence of which was 86.7% identical to that of the germline Humlv1L1 gene. The high degree of conservation of the VH4-21 gene in the human population, the nature of the nucleotide differences in the expressed VH4-21 segments, and the presence of nucleotide substitutions in the HBL-2 and HBL-3 IgM MoAb JH and/or J lambda segments suggested that the MoAb V segments underwent a process of somatic hypermutation. This was formally shown in the HBL-3 MoAb VH segment, by differentially targeted polymerase chain reaction amplification of the HBL-3 MoAb-producing cell genomic DNA. In addition, cloning and sequencing of the genomic DNA from fibroblasts of the same patient whose neoplastic B cells gave rise to the HBL-3 cell line yielded a germline copy of the VH4-21 gene. Thus, the expression of VH4-21 gene products may be involved in a self Ag-driven process of clonal B-cell expansion and selection associated with BL in these AIDS patients.     

Abnormalities of cytoplasmic Ca2+ in platelets from patients with uremia

Uremic patients have a hemorrhagic tendency, often associated with prolonged bleeding times and decreased platelet function in vitro. Whether these defects result from abnormalities in plasma factors affecting platelet activity, platelet surface receptors, intracellular platelet mediators, or other aspects of platelet behavior is unknown. To examine the possibility that the abnormality in platelet function may result from aberrations in Ca2+ homeostasis, blood was obtained from 29 patients with severe uremia. The platelets were washed, loaded with the Ca2+ -sensitive probes indo-1 and aequorin, gel-filtered, and resuspended in either plasma or buffer. Of the 29 patients, seven had template bleeding times prolonged to 11 minutes or more, but platelet aggregation in plasma was not consistently impaired in these patients. However, in aequorin-loaded platelets from the patients with long bleeding times, the highest elevation of cytoplasmic calcium [( Ca2+]i) in response to the Ca2+ ionophore A23187, arachidonate, adenosine diphosphate (ADP), or epinephrine was lower than that seen in platelets from both uremic patients with less prolonged bleeding times and normal volunteers. The reduced [Ca2+]i response was associated with decreased aggregation of gel-filtered platelets suspended in buffer. Suspending washed aequorin-loaded uremic platelets in normal plasma for 20 minutes did not reverse the decreased agonist-induced rise in [Ca2+]i; platelets from a normal donor resuspended in uremic plasma aggregated and produced a normal increase in [Ca2+]i in response to agonists. We conclude that the platelet defect seen in some patients with uremia is associated with a decreased rise in platelet [Ca2+]i after stimulation and that this is a manifestation of an intrinsic platelet defect.     

Tumor necrosis factor-alpha downregulates protein S secretion in human microvascular and umbilical vein endothelial cells but not in the HepG- 2 hepatoma cell line

Protein S deficiency, which is associated with thrombosis, can either be inherited or acquired. Recently, we reported that a decrease in free protein S was observed in 19 of 25 persons with HIV/AIDS. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), has been reported to be elevated in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients and has been shown to induce a procoagulant state on the surface of endothelial cells. We report here that recombinant TNF-alpha (rTNF-alpha) downregulated protein S synthesis in the SV-40T transfected human microvascular endothelial cell line (HMEC-1) model system by approximately 70% and in primary human umbilical vein and dermal microvascular endothelial cell cultures by approximately 50%. Using the HMEC-1 model, Northern blot analysis showed a decrease in protein S RNA at 24 hours that was corroborated by Western blot analysis and enzyme- linked immunosorbent assay (ELISA) quantification. Evidence supporting the specificity of the TNF-alpha effect included the following: (1) TNF- alpha down-regulation of protein S was completely blocked by TNF neutralizing antibody; (2) the effect was transient, and protein S was restored to near normal levels after TNF was removed from cell cultures; (3) an antibody directed to the TNF RI (55-kD receptor) was shown to mimic the action of TNF-alpha on HMEC-1 cells; and (4) other proinflammatory cytokines, interleukin (IL)-1, IL-6, and TGF-beta, had no effect on protein S secretion. However, TNF-alpha showed no regulatory control over protein S synthesis in the human hepatocellular carcinoma cell line HepG-2. We suggest that TNF-alpha downregulation of protein S may be a mechanism for localized procoagulant activity and thrombosis recently reported in some AIDS patients with associated protein S deficiency.     

FDG-PET for Pharmacodynamic Assessment of the Fatty Acid Synthase Inhibitor C75 in an Experimental Model of Lung Cancer

Purpose Fatty acid synthase (FAS) is an emerging target for anticancer therapy with a variety of new FAS inhibitors being explored in preclinical models. The aim of this study was to use positron emission tomography with [¹⁸F]fluorodeoxyglucose (FDG-PET) to monitor the effects of the FAS inhibitor C75 on tumor glucose metabolism in a rodent model of human A549 lung cancer. Materials and Methods After a baseline FDG-PET scan, C75 was administered and post-treatment scans were performed serially. FAS activity was measured in treated animals ex vivo by [¹⁴C]acetate incorporation in animals euthanized in parallel to those imaged. Results Longitudinally measured metabolic volumes of interest and tumor/background ratios demonstrated a transient, reversible decrease in glucose metabolism and tumor metabolic volume after treatment, with the peak effect seen at 4 h. FDG-PET measurements correlated with changes in tumor FAS activity measured ex vivo. Conclusions Because C75 causes an effect that is shorter in duration than expected, modification of the current weekly dosing regimen should be considered. These results demonstrate the utility of small animal FDG-PET in assessing the pharmacodynamics of new anticancer agents in preclinical models. Jae Sung Lee and Hajime Orita contributed equally to this work     

The effect of chitosan on the migration of neutrophil-like HL60 cells, mediated by IL-8

Research interest in chitosan stems in part from the demonstrated wound healing properties. The benefits of chitosan as a therapeutic agent appear to be paradoxical because chitosan also elicits neutrophil infiltration indicative of an inflammatory response. While the affinity between chitosan and neutrophils has been well documented, the underlying mechanism is unclear. To our knowledge, no studies have investigated the consequences of chitosan-neutrophil interaction to explain neutrophil migration. To that end, transwell migration assays to chitosan of varying extent of acetylation were conducted using a differentiated model cell line (HL60-PMN) in order to assess the effect of chitosan chemistry and the resultant physical properties such as charge and hydrophobicity on neutrophil migration. As chitosan N-acetylation increased, neutrophil migration increased and chitosan became less positively charged and more hydrophobic. Moreover, HL60-PMN cells secreted the potent neutrophil chemokine IL-8, also known as CXCL8, when exposed to chitosan and IL-8 levels increased with N-acetylation, and migration was inhibited by anti-IL-8 antibodies. Collectively these results suggest that chitosan-neutrophil interaction is encouraged by material properties, results in IL-8 secretion, and causes migration of neutrophils to chitosan. The implication is that the wound healing properties of chitosan may be enhanced through the attenuation of overabundant neutrophils, and thus the inflammatory response, simply by changing chitosan N-acetylation.