Comparative genomic hybridization: technical development and cytogenetic aspects for routine use in clinical laboratories

PURPOSE: To define, in a phase I study in relapsed non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL), the maximum-tolerated dose (MTD), major toxicities, and possible antitumor activity of bryostatin 1, a macrocyclic lactone. PATIENTS AND METHODS: Bryostatin 1 was delivered by 72-hour continuous infusion every 2 weeks to patients with relapsed NHL or CLL, at doses that ranged from 12 microg/m2 to 180 microg/m2 per course. Correlative investigations included evaluations of total protein kinase C (PKC) in peripheral blood and lymphoid differentiation in patient tumor tissue. RESULTS: Twenty-nine patients were treated, including three patients with CLL and 26 with NHL. Generalized myalgia was the dose-limiting toxicity (DLT) and occurred in two of three patients treated with bryostatin 1 at 180 microg/m2 per course. Myalgias were dose-related and cumulative, and often started in the thighs and calves, improved with activity, were somewhat responsive to analgesics, and often took weeks to resolve once taken off study. Six patients were treated at the MTD of 120 microg/m2 per course. Myalgia, headache, and fatigue were common. Hematologic toxicity was uncommon. Total cumulative doses of bryostatin 1 up to 1,134 microg/m2 have been administered without untoward toxicity. Eleven patients achieved stable disease for 2 to 19 months. An in vitro assay for total PKC evaluation in patient peripheral-blood samples demonstrated activation within the first 2 hours with subsequent downregulation by 24 hours, which was maintained throughout the duration of the 72-hour infusion. CONCLUSION: This phase I study defined the MTD and recommended phase II dose of bryostatin 1, when administered over 72 hours every 2 weeks, to be 120 microg/m2 (40 microg/m2/d for 3 days). Generalized myalgia was the DLT. Future studies will define the precise activity of bryostatin 1 in subsets of patients with lymphoproliferative malignancies and its efficacy in combination with other agents.     

HA-1 and the SMCY-derived peptide FIDSYICQV (H-Y) are immunodominant minor histocompatibility antigens after bone marrow transplantation

BACKGROUND: Allogeneic bone marrow donors can be incompatible at different levels. Even HLA-identical pairs will be still incompatible for numerous minor histocompatibility antigens (mHag). Nevertheless, some incompatibilities are found to be associated with an increased risk of graft-versus-host disease (GVHD), which could be related to the way the immune system recognizes these antigens. METHODS: We determined the specificity of cytotoxic T-cell clones isolated during acute GVHD or during bone marrow graft rejection in patients (n=14) transplanted with marrow from donors who were histoincompatible for different minor and/or major histocompatibility antigens. RESULTS: We found a clear hierarchy among the different types of histoincompatibilities. In three combinations mismatched for a class I allele, all 27 clones isolated during GVHD were specific for the incompatible HLA molecule. In the 11 class I-identical combinations, 14 different mHags were recognized. The mHag HA-1, known to have a significant impact on the development of GVHD, was recognized in the two HA-1-incompatible combinations. In one of these combinations, which was sex mismatched, all 56 clones analyzed were directed against HA-1, demonstrating the dominance of this mHag. In the four HA-1-compatible, sex-mismatched combinations, the anti-H-Y response was directed against one immunodominant epitope rather than against multiple Y-chromosome-encoded epitopes. All male specific cytotoxic T lymphocytes (n=15) recognized the same high-performance liquid chromatography-purified peptide fraction presented by T2 cells. Moreover, all cytotoxic T lymphocytes tested (n=6) were specific for the SMCY-derived peptide FIDSYICQV, originally described as being the H-Y epitope recognized in the context of HLA-A*0201. CONCLUSIONS: Some histocompatibility antigens are recognized in an immunodominant fashion and will therefore be recognized in the majority of mismatched combinations. Only for such antigens, correlations between mismatches and the occurrence of GVHD or graft rejections will be found.     

Phosphatidylinositol 3-kinase is necessary and sufficient for insulin-stimulated stress fiber breakdown

Rat-1 fibroblasts overexpressing the human insulin receptor undergo rapid actin rearrangement in response to insulin. Breakdown of stress fibers present in quiescent cells is followed by transient membrane ruffling and a return of stress fibers. We investigated the signaling pathways that mediate this insulin-stimulated reorganization of the actin cytoskeleton, which was visualized with rhodamine-phalloidin. Treatment of cells with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin prevented insulin action at the preliminary step of stress fiber breakdown. Cellular microinjection of a polyclonal antibody directed against the p85 subunit of PI3-kinase as well as a purified recombinant p85-SH2 domain protein also inhibited actin reorganization. Transient expression of a constitutively active form of PI3-kinase (p110*) was sufficient to cause both stress fiber breakdown and membrane ruffling in the absence of insulin. Microinjection of a polyclonal anti-Shc antibody or dominant negative N17-Ras protein did not affect actin dynamics, and although constitutively active V12-Ras caused modest cytoskeletal reorganization, this effect was blocked by pretreatment with wortmannin. In summary, activation of PI3-kinase is necessary and sufficient to stimulate actin rearrangement, indicating that PI3-kinase may initiate the only signaling cascade required for insulin to induce cytoskeletal restructuring.     

Partial characterization of the cohemolytic factor produced by Streptococcus uberis and comparison with the CAMP-factor

Exosubstances (cohemolysins) produced by Streptococcus agalactiae (CAMP-factor) and Streptococcus uberis (Uberis-factor) showing hemolytic synergism with beta-lysin produced by Staphylococcus aureus were compared. Cohemolytic activity was evaluated in the supernatants of bacterial cultures, before and after ammonium sulfate precipitation. Sheep erythrocytes sensitized with beta-lysin were used as substrate. The assays were performed in microtiter plates and results were expressed as cohemolytic units/ml. Maximum cohemolytic activity was detected, respectively, after 8 h and 14 h of growth in Columbia broth in S. uberis and S. agalactiae cultures. Cohemolytic activities of both microorganisms showed similarities when submitted to various physical and chemical treatments. They were significantly decreased by heating at 60 degrees C and 100 degrees C, or in presence of trypsin, and were abolished in the presence of Tween 20. Activities were found to be stable in crude supernatants and concentrated preparations maintained at -20 degrees C for 3 months. Differences were related to levels of activity and kinetics of detection during the growth cycle. The results indicate the proteic nature, at least in part, of the Uberis factor. Analysis by PAGE in the presence or absence of SDS allowed us to correlate Uberis activity with a protein band with apparent molecular mass of 42 kDa, while CAMP activity was associated with a protein band of 27 kDa.     

Low-density lipoprotein particle size decreases during normal pregnancy in association with triglyceride increases

OBJECTIVE: To characterize the changes in low-density lipoprotein (LDL) peak particle diameter (diameter of the predominant LDL subclass) in relation to changes in serum triglyceride concentration during successive stages of normal gestation and postpartum. METHODS: Nonfasting venous blood was obtained longitudinally during and after uncomplicated primiparous pregnancy from 10 nonsmoking women with no history of metabolic disorders. Plasma LDL diameter was determined by nondenaturing polyacrylamide gel electrophoresis. Serum concentrations of total cholesterol, triglyceride, apolipoprotein B, apolipoprotein A-I, and LDL-cholesterol were measured. Gestational changes were analyzed by one-way repeated-measures analysis of variance and the paired multiple comparison Student-Newman-Keuls test. Pearson coefficients were computed for correlation of serum lipids and LDL diameter. RESULTS: Low-density lipoprotein diameter decreased progressively with advancing gestation, evident by 16-20 weeks relative to 5-12 weeks. Seven of 10 cases were subclass pattern B (diameter less than 255 A) by term, indicating that small, dense particles predominated. The average diameter decrease from early to late gestation was 13 A. All subjects reverted to subclass pattern A (diameter 255 A or more) by 6-12 weeks postpartum, indicating prevalence of large, buoyant LDL. Low-density lipoprotein diameter correlated inversely with concentrations of serum triglyceride (r = -.61, P < .0001), apo B (r = -.66, P < .0001), cholesterol (r = -.53, P < .001), LDL cholesterol (r = -.45, P < .005), and apo A-I (r = -.39, P < .02). CONCLUSION: Gestational triglyceride increases are accompanied by progressive decreases in LDL diameter in a majority of cases. These changes undergo reversal postpartum and therefore are transient. Small, dense LDL particles have a number of properties capable of altering vascular function. However, the consequences of the gestational LDL size decrease for maternal and fetal metabolism remain unknown.     

Human apolipoprotein A-I gene promoter mutation influences plasma low density lipoprotein cholesterol response to dietary fat saturation

Numerous studies have demonstrated an association between polycyclic aromatic hydrocarbons (PAHs) and lymphocyte toxicity. The present study shows that, consistent with its effects on Ca2+ homeostasis, benzo[a]pyrene (BaP) induces apoptosis in Daudi cells. Terminal deoxynucleotidal transferase-mediated dUTP-biotin nick end labeling (TUNEL) analysis at 18 h revealed a significant increase in the number of cells undergoing apoptosis in response to BaP (75%), BaP-7, 8-dihydrodiol (110%), and BaP-7,8-9,10-diol epoxide (BPDE) (215%) over DMSO vehicle control cultures. By 36 h, the trend toward increasing numbers of apoptotic cells continued with the parent compound producing a 125% increase over control values and the 7, 8-dihydrodiol and BPDE metabolites producing 195% and 370% increases over controls, respectively. DNA fragmentation assays demonstrated the presence of internucleosomal cleavage products consistent with the increasing numbers of TUNEL-positive cells responding to PAHs at 18 and 36 h. Analysis of poly(ADP-ribose) polymerase (PARP) protein in BaP- and BaP-7,8-dihydrodiol-treated cells strongly suggested the involvement of cysteine proteases by the appearance of an 85-kD fragment derived from hydrolytic cleavage of PARP, a phenomenon that has been associated with apoptosis in many systems. Immunoblot analysis demonstrated that both BaP and its 7,8-dihydrodiol metabolite affected a pathway involving Bcl-2 and Bax cytosolic proteins. Daudi cells undergoing apoptosis at 36 h in response to 10 microM BaP, the parent compound, expressed moderately reduced amounts of Bcl-2 (78% of vehicle controls). At the same time point, the 7,8-dihydrodiol and BDPE metabolites at 3 microM resulted in Bcl-2 protein expression that was 52% of that seen in vehicle controls. Parallel samples analyzed for expression of Bax protein displayed a 130% increase over vehicle control in Bax expression in response to the parent compound, while the 7,8-dihydrodiol metabolite produced a 257% increase in Bax. Furthermore, the effects on increased Bax expression were observed as early as 3 h after PAH exposure. The apoptotic response to PAHs in Daudi cells was sensitive to 4-h pretreatment with 0.3 microM alpha-naphthoflavone (ANF), a known inhibitor of cytochrome P450. In TUNEL assays of cells exposed to PAHs following pretreatment with ANF, at 18 h there was a significant reduction in the number of cells undergoing apoptosis in response to ANF compared to cells that were not pretreated with the compound. The effect of the parent compound at 18 h was completely blocked with ANF pretreatment, while ANF exerted a relatively weaker, but significant, effect on BaP-7, 8-dihydrodiol-induced apoptosis. With regard to modulation of expression of apoptosis-related proteins, Bax expression was restored to that observed in vehicle-control cultures at all time points tested (3, 18, and 36 h). Bcl-2 expression was most responsive to ANF at later time points following PAH exposure (18 and 36 h); however, Bcl-2 appeared to be more sensitive to the effects of ANF alone. Taken together, these data suggest that modulation of Bcl-2 family proteins, perhaps secondary to altered Ca2+ homeostasis, plays an important role in human B cell apoptosis induced by BaP.