Pre and postnatal diagnosis of fetus‐in‐fetu

Differentiation of abdominal masses detected on prenatal ultrasound is difficult and requires careful characterization of the mass and precise localization. Differentiation is required in order to distinguish benign from potentially malignant conditions. We describe a case of fetus‐in‐fetu with pre and postnatal imaging.     

Deposition of autofluorescent eosinophil granules in pathologic bone marrow biopsies

Eosinophil granules are intensely autofluorescent when excited by green light. To determine if eosinophils degranulate in the bone marrows of patients with a variety of diseases, we used green light epifluorescence microscopy to examine deparaffinized and dezenkerized sections of 49 bone marrow core biopsies. In 14 of the biopsies, there was striking extracellular deposition of intensely autofluorescent eosinophil granules in addition to numerous intact eosinophils. Among the 14 specimens with extracellular autofluorescence were seven cases of leukemia, four cases of non-Hodgkin's lymphoma, two cases of myelofibrosis, and one case of pancytopenia with eosinophilia. In the remaining 35 specimens, only intact eosinophils were identifiable. There was no extracellular autofluorescence in three normal marrows, four marrows from AIDS patients, or three biopsies from patients with idiopathic thrombocytopenic purpura (ITP). We conclude that green light epifluorescence microscopy identifies extracellular deposits of eosinophil granules in bone marrow biopsies of some neoplastic disorders and in diseases associated with reticulin fibrosis.     

X-linked chronic granulomatous disease: correction of NADPH oxidase defect by retrovirus-mediated expression of gp91-phox

Chronic granulomatous disease (CGD) is an inherited immunodeficiency resulting from the inability of an individual's phagocytes to produce superoxide anions because of defective NADPH oxidase. The disease may be treated by bone marrow transplantation and as such is a candidate for somatic gene therapy. Two thirds of patients have defects in an X- linked gene (X-CGD) encoding gp91-phox, the large subunit of the membrane cytochrome b-245 component of NADPH oxidase. Epstein-Barr virus-transformed B-cell lines from patients with CGD provide a model system for the disease. We have used retrovirus-mediated expression of gp91-phox to reconstitute functionally NADPH oxidase activity in B-cell lines from three unrelated patients with X-CGD. The protein is glycosylated and membrane associated, and the reconstituted oxidase is appropriately activated via protein kinase C. The kinetics of superoxide production by such reconstituted cells is similar to that of normal B-cell lines. These data show the potential of gene therapy for this disease.     

Enterohemorrhagic Escherichia coli O157:H7 gal mutants are sensitive to bacteriophage P1 and defective in intestinal colonization

Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal(+) strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal(+) parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal(+) revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.     

Alternative Mechanism of Cholera Toxin Acquisition by Vibrio cholerae: Generalized Transduction of CTXΦ by Bacteriophage CP-T1

Horizontal transfer of genes encoding virulence factors has played a central role in the evolution of many pathogenic bacteria. The unexpected discovery that the genes encoding cholera toxin (ctxAB), the main cause of the profuse secretory diarrhea characteristic of cholera, are encoded on a novel filamentous phage named CTXPhi, has resulted in a renewed interest in the potential mechanisms of transfer of virulence genes among Vibrio cholerae. We describe here an alternative mechanism of cholera toxin gene transfer into nontoxigenic V. cholerae isolates, including strains that lack both the CTXPhi receptor, the toxin coregulated pilus (TCP), and attRS, the chromosomal attachment site for CTXPhi integration. A temperature-sensitive mutant of the V. cholerae generalized transducing bacteriophage CP-T1 (CP-T1ts) was used to transfer a genetically marked derivative of the CTX prophage into four nontoxigenic V. cholerae strains, including two V. cholerae vaccine strains. We demonstrate that CTXPhi transduced by CP-T1ts can replicate and integrate into these nontoxigenic V. cholerae strains with high efficiency. In fact, CP-T1ts transduces the CTX prophage preferentially when compared with other chromosomal markers. These results reveal a potential mechanism by which CTXPhi(+) V. cholerae strains that lack the TCP receptor may have arisen. Finally, these findings indicate an additional pathway for reversion of live-attenuated V. cholerae vaccine strains.