Utility of quantitative flow cytometry immunophenotypic analysis of CD5 expression in small B-cell neoplasms.

CONTEXT: The value of assessing CD5 expression in the differential diagnosis of small B-cell neoplasms is well established. Assessment is usually done qualitatively. OBJECTIVES: To assess CD5 expression levels by quantitative flow cytometry immunophenotyping and to determine possible differences among various small B-cell neoplasms. DESIGN: We performed 4-color flow cytometry analysis on specimens of peripheral blood and bone marrow aspirate and quantified CD5 expression in various small B-cell lymphomas and leukemias. We also assessed CD5 levels in peripheral blood samples of healthy blood donors. RESULTS: Cases of chronic lymphocytic leukemia and mantle cell lymphoma had higher levels of CD5 compared with control B cells (P < .001). Cases of marginal zone lymphoma and hairy cell leukemia had CD5 levels similar to control B cells (P = .35 and P = .14, respectively), whereas cases of follicular lymphoma and lymphoplasmacytic lymphoma had significantly lower CD5 levels than control B cells (P < .001 and P = .04, respectively). In B-cell neoplasms, a high level of CD5 expression was correlated with a homogeneous pattern of positive events, whereas lower CD5 levels were correlated with heterogeneous patterns of positive events. CONCLUSIONS: Using flow cytometric immunophenotypic analysis to quantify CD5 levels can aid in diagnosis. CD5 expression levels are higher in patients with chronic lymphocytic leukemia and mantle cell lymphoma, and expression is observed in a homogeneous pattern, as compared with other B-cell neoplasms that are either negative for CD5 or express CD5 at lower levels with a heterogeneous pattern. However, there is some overlap in CD5 expression levels between a subset of atypical chronic lymphocytic leukemia and marginal zone lymphoma cases.

Immunostaining for rapid diagnosis of acute promyelocytic leukemia with the tetramethylrhodamine-5-isothiocyanate-conjugated anti-promyelocytic leukemia monoclonal antibody PG-M3.

CONTEXT: Anti-promyelocytic leukemia (PML) immunofluorescence staining is a known diagnostic tool for rapid diagnosis of acute promyelocytic leukemia (APL). OBJECTIVE: We describe our methods using the recently developed, commercially available, tetramethylrhodamine-5-isothiocyanate-labeled PG-M3 anti-PML antibody for APL testing. DESIGN: Immunofluorescence staining with the tetramethylrhodamine-5-isothiocyanate-labeled PG-M3 antibody was used to detect PML-RARA in bone marrow aspirate and/or peripheral blood smears from 30 patients with acute leukemia. The results were compared with those of concurrent testing with our in-house polyclonal anti-PML antibody and with established tests. RESULTS: All APL cases showed a positive (fine/microgranular) immunofluorescence staining pattern, whereas non-APL cases showed a negative (chunky/macrogranular) pattern. These results, which were available within 2 hours, were validated by testing with the polyclonal anti-PML antibody and with established cytogenetic and molecular testing methods. CONCLUSIONS: We validated the utility of the tetramethylrhodamine-5-isothiocyanate-labeled anti-PML antibody PG-M3 for the diagnosis of APL. Our results indicate that immunofluorescence staining with this antibody is a rapid and reliable method for the diagnosis of APL.

The mojastin mutant Moj-DM induces apoptosis of the human melanoma SK-Mel-28, but not the mutant Moj-NN nor the non-mutated recombinant Moj-WN.

In this study, three recombinant mojastin peptides (Moj-WN, Moj-NN, and Moj-DM) were produced and compared functionally. Recombinant Moj peptides were purified as GST-fusions. GST-Moj-WN and GST-Moj-NN inhibited ADP-induced platelet aggregation in platelet rich plasma. The GST-Moj-WN had an IC(50) of 160nM, while the GST-Moj-NN had an IC(50) of 493nM. The GST-Moj-DM did not inhibit platelet aggregation. All three GST-Moj peptides inhibited SK-Mel-28 cell adhesion to fibronectin. The GST-Moj-WN inhibited the binding of SK-Mel-28 cells to fibronectin with an IC(50) of 11nM, followed by the GST-Moj-NN (IC(50) of 28nM), and the GST-Moj-DM (IC(50) of 46nM). The GST-Moj peptides' ability to induce apoptosis on SK-Mel-28 cells was determined using Annexin-V-FITC and nuclear fragmentation assays. Cells were incubated with 5muM GST-Moj peptides for 24h. At 5microM GST-Moj-DM peptide, 13.56%+/-2.08 of treated SK-Mel-28 cells were in early apoptosis. The GST-Moj-DM peptide also caused nuclear fragmentation as determined by fluorescent microscopy and Hoechst staining. The GST-Moj-WN and GST-Moj-NN peptides failed to induce apoptosis. We characterized the SK-Mel-28 integrin expression, as the first step in determining r-Moj binding specificity. Our results indicate that SK-Mel-28 cells express alphavbeta3, alphav, alpha6, beta1, and beta3 integrin receptors.

Molecular evolution of PIII-SVMP and RGD disintegrin genes from the genus Crotalus.

Several types of disintegrins have been isolated from Crotalus spp rattlesnakes, including RGD disintegrins, and PIII-SVMPs. We isolated six cDNAs from snake venom glands using RT-PCR. Three RGD disintegrins (atroxatin, mojastin, and viridistatin) and three PIII-SVMPs (catroriarin, scutiarin, and viristiarin) cDNAs were isolated from the rattlesnakes Crotalus atrox, Crotalus scutulatus scutulatus, and Crotalus viridis viridis, respectively. Atroxatin and Viridistatin shared 90% amino acid identity to each other, and 87% identity to Mojastin. Scutiarin and Viristiarin were identical. All PIII-SVMPs isolated in this study shared the highest amino acid identity with Catrocollastatin. cDNA and protein sequences for RGD disintegrins, one MVD disintegrin, and PIII-SVMPs of the genus Crotalus (present in the NCBI database), were used in phylogenetic analysis. Neighbor-joining analysis of PIII-SVMP and RGD/MVD disintegrin-coding DNA sequences showed that these groups of genes separate into separate clades. A Phi(ST) pairwise comparison and Analysis of Molecular Variance (AMOVA) between PIII-SVMPs and RGD/MVD disintegrins showed significant genetic differences. Mutations observed in ten of the cDNAs analyzed did not affect Cys-coding sequences. Our K(A)/K(S) data suggest that rapid evolution occurred between the genes coding for PIII-SVMPs resulting, in the production of RGD disintegrin-coding genes. However, once these genes diverged, mutations in the PIII-SVMP-coding genes were accumulated less frequently.

Molecular barcoding, DNA from snake venom, and toxinological research: Considerations and concerns.

The problem of species identification in toxinological research and solutions such as molecular barcoding and DNA extraction from venom samples are addressed. Molecular barcoding is controversial with both perceived advantages and inherent problems. A method of species identification utilizing mitochondrial DNA from venom has been identified. This method could result in deemphasizing the importance of obtaining detailed information on the venom source prior to analysis. Additional concerns include; a cost prohibitive factor, intraspecific venom variation, and venom processing issues. As researchers demand more stringent records and verification, venom suppliers may be prompted to implement improved methods and controls.

Genetic variation of a disintegrin gene found in the American copperhead snake (Agkistrodon contortrix).

Disintegrins are small, non-enzymatic proteins produced in snake venom. PCR and DNA sequencing analysis of genomic DNA for all subspecies of the copperhead snake (Agkistrodon contortrix) were analyzed for the presence of a disintegrin gene. Four samples each of the subspecies: A. c. contortrix, A. c. laticinctus, A. c. mokasen, A. c. phaeogaster, and A. c. pictigaster were collected from different locations across their geographic range and analyzed. A single PCR fragment from each sample was obtained, containing exon and intron sequences. The disintegrins identified in this study shared the highest amino acid identity to contortrostatin and acostatin b chain. Neighbor joining analysis of the disintegrin haplotypes and bootstrap tests of significance grouped the A. contortrix subspecies into two clades. The A. c. mokasen samples collected in Kentucky were grouped in one clade, while the A. c. contortrix, A. c. laticinctus, A. c. phaeogaster, and A. c. pictigaster samples collected in Texas, Louisiana, and Missouri were grouped in a different clade. Analysis of molecular variance (AMOVA) and PhiST pairwise comparisons showed significant genetic variation between subspecies. Nucleotide substitution analysis suggests the rapid evolution of disintegrin genes in A. contortrix subspecies.

Disintegrin, hemorrhagic, and proteolytic activities of Mohave rattlesnake, Crotalus scutulatus scutulatus venoms lacking Mojave toxin.

Venom from the Mohave rattlesnake, Crotalus scutulatus scutulatus, has been reported to be either: (1) neurotoxic; (2) hemorrhagic, or both (3) neurotoxic and hemorrhagic. In this study, 14 Mohave rattlesnakes from Arizona and Texas (USA) were analyzed for the presence of disintegrins and Mojave toxin. All venom samples were analyzed for the presence of hemorrhagic, proteolytic and disintegrin activities. The venoms were each chromatographed by reverse phase and their fractions tested for disintegrin activity. All specimens containing Mojave toxin were the most toxic and lacked proteolytic, hemorrhagic and disintegrin activities. In contrast, the venoms containing these activities lacked Mojave toxin. Two disintegrin genes, scutustatin and mojavestatin, were identified by PCR of genomic sequences. Scutustatin is a highly conserved disintegrin, while mojavestatin shows low conservation to other known disintegrins. Venoms with the highest LD50 measurements lacked both disintegrin genes, while the specimens with intermediate and low LD50 contained both genes. The intermediate LD50 group contained Mojave toxin and both disintegrin genes, but lacked hemorrhagic and disintegrin activity. Our results raise the possibility that scutustatin and mojavestatin are not expressed in the intermediate LD50 group, or that they may not be the same disintegrins responsible for the disintegrin activity found in the venom. Therefore, it is possible that Mohave rattlesnakes may produce more than two disintegrins.