Molecular characterization of Waldenstrom's macroglobulinemia reveals frequent occurrence of two B-cell clones having distinct IgH VDJ sequences.

PURPOSE: Malignant B lineage cells in Waldenstrom's macroglobulinemia (WM) express a unique clonotypic IgM VDJ. The occurrence of biclonal B cells and their clonal relationships were characterized. EXPERIMENTAL DESIGN: Bone marrow and blood from 20 WM patients were analyzed for clonotypic VDJ sequences, clonal B-cell frequencies, and the complementary determining region 3 profile. RESULTS: Two different clonotypic VDJ sequences were identified in 4 of 20 WM. In two cases, partner clones had different VDJ rearrangements, with one clonotypic signature in bone marrow and a second in blood. For both cases, the bone marrow clone was hypermutated, whereas the blood clone was germ line or minimally mutated. In two other cases, partner clones shared a common VDJ rearrangement but had different patterns of somatic mutations. They lacked intraclonal diversity and were more abundant in bone marrow than in blood. VDJ mutation profiles suggested they arose from a common IgM progenitor. Single-cell analysis in one case indicated the partner clones were reciprocally expressed, following rules of allelic exclusion. CONCLUSIONS: The existence of two B-cell clones having distinct VDJ sequences is common in WM, suggesting that frequent transformation events may occur. In two cases, the partner clones had distinct tissue distributions in either blood or bone marrow, were of different immunoglobulin isotypes, and in one case exhibited differential response to therapy. The contributions of each clone are unknown. Their presence suggests that WM may involve a background of molecular and cellular events leading to emergence of one or more malignant clones.     

Dichapetalin-type triterpenoids and lignans from the aerial parts of Phyllanthus acutissima

Chemical investigation of the aerial parts of Phyllanthus acutissima resulted in the isolation of five new dichapetalin-type triterpenoids, acutissimatriterpenes A-E ( 1- 5), and two new lignans, acutissimalignans A ( 6) and B ( 7), along with two known lignans and three known ellagic acid derivatives. The structures of 1- 7 were determined mainly on the basis of spectroscopic methods. The compounds obtained were evaluated for cytotoxic and anti-HIV-1 activities.     

Small Intestinal Manometric Studies in Patients with Familial Visceral Myopathies

We studied jejunal manometry on 10 patients with type 1 familial visceral myopathy (FVM), and one patient each with types II and III. Two patients of type I and both patients of types II and III had intestinal pseudo-obstruction syndrome. The record was obtained in each patient for 4 to 5 hours during fasting, and 1 hour after feeding. In type I FVM, migrating motor complexes were present in six and absent in four patients. In these four patients (two with intestinal pseudo-obstruction syndrome) with absent migrating motor complexes, there was infrequent low-amplitude contractions during fasting, and after feeding. In six patients with migrating motor complexes, the motility indices of phases 2, 3, and fed period were 59%, 49%, and 24% of those of control subjects, respectively, and the frequency of contractions of phases 2, 3 and the fed period were 70%, 79%, and 32% of those of control subjects, respectively. In both patients with types II and III FVM, only infrequent low-amplitude contractions were recorded during fasting and after feeding. We concluded that intestinal contractions in patients with familial visceral myopathies were weak, and the weakness was more severe in patients with intestinal pseudo-obstruction syndrome.     

Iso-electricfocusing of Bithynia snail antigens for IgG- and IgG(1-4)-ELISA detection of human opisthorchiasis

Diagnosis of opisthorchiasis is confirmed by the presence of characteristic eggs and worms. However, misdiagnosis may occur in light infections, and also due to the morphological similarity of opisthorchid eggs to other species. A finding of specific immune mediators can help confirm infection. This study used indirect ELISA to detect total IgG and IgG(1-4) with selected antigens of Bithynia siamensis goniomphalos extract, which were derived by liquid-phase iso-electricfocusing (IFE). Antigens (Iso-AgF) from 20 IEF fractionated fractions were selected based on a high ELISA-OD ratio between pooled-positive and pooled-negative sera. Iso-AgF 7, 7, 6, 2, and 10 resulted in high OD-ratio to total IgG, IgG1, 2, 3, and 4, respectively. A full-scale ELISA was conducted with sera from 50 opisthorchiasis cases, 196 from other parasitic-disease cases, and 35 healthy controls. Iso-AgF7 to IgG1 showed the best result, with sensitivity, specificity, positive and negative predictive value of 100, 96, 86, and 100%, respectively, at a cut-off 0.221. Low cross-reactivity to IgG1 was found in one case each of gnathostomiasis, trichinellosis, toxocariasis, angiostrongyliasis, bancroftian filariasis, enterobiasis, neurocysticercosis, and taeniasis. Thus, Iso-AgF7 to IgG1 was a good candidate antigen to be developed for detection of antibodies against Opisthorchis viverrini.     

Discrimination of O. viverrini, C. sinensis, H. pumilio and H. taichui using nuclear DNA-based PCR targeting ribosomal DNA ITS regions

Small liver and minute intestinal flukes are highly prevalent in Southeast Asia, and in mixed infections, their eggs are difficult to differentiate morphologically in fecal samples. PCR assays targeting the ITS regions in ribosomal DNA were designed to identify and differentiate species. The PCR amplicons of Opisthorchis viverrini, Clonorchis sinensis, Haplorchis pumilio, and Haplorchis taichui were 800, 820, 1250, and 930 bp for the ITS1 region, and 380, 390, 380, and 530 bp for ITS2, respectively. The ITS1-region amplicon sizes successfully differentiated 4 species, while only H. taichui were significantly different from the other 3 species in the ITS2 region. PCR assays were employed for preliminary analysis using fecal samples diagnosed as having “small trematode eggs” by modified thick smear, showing 76.2% sensitivity for ITS1 and 95.2% for ITS2.     

A single chain Fv specific against Western equine encephalitis virus

A recombinant single chain Fv (scFv) specific against Western equine encephalitis virus (WEE) was developed and characterized. The scFv was generated from 11D2 hybridoma producing anti-WEE antibody reactive to E1 component of viral envelope glycoprotein. V(L) and V(H) gene segments of 11D2 scFv were generated and joined together with a (gly4ser)3 linker by polymerase chain reaction (PCR). The resulting scFv was successfully expressed in P. pastoris expression system. Fifteen individual plasmids were tested and six of them were shown to drive scFv expression. DNA sequence analysis from three productive plasmids showed that they all carried the same VL and V(H) gene segments with a few base differences. Comparison of 11D2 scFv DNA sequence to the Kabat database showed that VH of 11D2 antibody belonged to subgroup IIID and subfamily XIV, while VL domain did not belong to any known subgroup or subfamily. Western blot analysis of 11D2 scFv using anti-c-myc antibody for detection showed different band pattern among clones derived from different plasmids. This was thought to be due to the different glycosylation where amino acid substitution occurred. Successful purification of 11D2 scFv could be done by immobilized metal affinity chromatography with an unoptimized yield of 700 microg/L. Functional studies showed that 11D2 scFv could bind to its respective WEE antigen as demonstrated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). The binding affinity of 11D2 scFv is reasonably good compared to the parental 11D2 bivalent monoclonal antibody (MAb). Thus, 11D2 scFv and its derivatives have a potential use as immunotherapeutic and immunodiagnostic agents of WEE infections.