Simplified and accurate nonradioactive polynucleotide gene probe assay for identification of enterotoxigenic Escherichia coli.

The present study describes a colony hybridization setup for identification of enterotoxigenic Escherichia coli obviating the need for advanced equipment and radioactive isotopes. With a modest laboratory arrangement, polynucleotide gene probes were produced in large quantities. The probes were labeled with digoxigenin and, after hybridization, detected with an antidigoxigenin alkaline phosphatase conjugate. With an established isotope-based oligonucleotide hybridization assay as reference, a blinded study on a large battery of enterotoxigenic and nonenterotoxigenic bacteria revealed a satisfactory sensitivity and specificity of the nonradioactive assay.     

Immunohistochemical study of nasopharyngeal carcinoma using monoclonal keratin antibodies

Nasopharyngeal carcinoma (NPC) provides a unique opportunity to evaluate distinctive epidemiologic features and a possible etiologic relationship with Epstein-Barr virus (EBV) in human malignancy. The lack of a uniformly accepted pathologic classification for NPC has limited the application of this data, although the World Health Organization (WHO) developed a classification that may solve this problem. Monoclonal keratin antibodies were used for staining of NPC for evaluation of its assistance in diagnosis and classification. In the present immunohistochemical study, monoclonal keratin antibodies, designated AE1, AE2, and AE3, and a polyclonal keratin antibody (RAK) were used for study of the presence of keratin in 121 cases of NPC obtained from China and the United States. AE1 monoclonal antibody, which recognizes keratin protein classes 56.5K, 50K, and 40K, was shown to be the most sensitive and specific for NPC tumor cells among the keratin antibodies studied. In addition, some different keratin expression patterns could be identified between different kinds of epithelium and different tumor groups, with possible relevance to the histogenesis of the histologic subtypes of NPC.     

Adrenoreceptor blockade in angiotensin-induced hypertension: effect on rat coronary arteries and myocardium

Adrenoreceptor blockade has been used to separate the actions of elevated blood pressure, angiotensin II, and catecholamines on the coronary vasculature and myocardium of rats. Twenty-two male Wistar-Kyoto rats received phentolamine (an alpha-receptor blocker, 10 mg/kg body weight) and/or propranolol (a beta-receptor blocker, 1 mg/kg body weight) followed by an infusion for 2 hours of angiotensin amide (1.7 micrograms/min/kg) or saline. Sections of left ventricle were examined by light and electron microscopy. Blood pressure was elevated only in animals receiving angiotensin II with or without propranolol. Epicardial arteries were devoid of lesions in all animals. Small intramural arteries and arterioles in the hypertensive animals exhibited vasoconstriction, endothelial cell vacuolization with bleb formation, and medial smooth muscle cell fragmentation and necrosis. Foci of irreversible ischemic or anoxic myocardial injury consisting of contraction zones and bands and translocated mitochondria with granular matrix densities were seen in angiotensin-infused animals. Similar but less severe myocardial changes were found in the animals pretreated with propranolol. Vascular lesions were also seen in animals receiving phentolamine, propranolol, and angiotensin II; but myocardial alterations consisted solely of areas with contraction zones. Vascular but not myocardial lesions were observed in animals that received angiotensin II and phentolamine. It is concluded that angiotensin II can produce vascular injury in the absence of elevated systemic blood pressure or catecholamine effects. In contrast, irreversible myocardial injury seems to depend upon the increased pressure and/or coronary artery vasoconstriction associated with angiotensin administration.     

Genetic relationship of putative colonization factor O166 to colonization factor antigen I and coli surface antigen 4 of enterotoxigenic Escherichia coli.

Plasmid DNA from two strains of enterotoxigenic Escherichia coli harboring genes encoding coli surface antigen 4 (CS4) and from seven Indian enterotoxigenic E. coli isolates cross-hybridized at low stringency but not at high stringency with two polynucleotide probes derived from the colonization factor antigen I (CFA/I) operon. Low-stringency Southern blot hybridization of PstI-digested plasmid DNA from the seven Indian isolates yielded characteristic restriction fragment patterns, distinct from those of CS4- and CFA/I-associated plasmid DNA. Two of the Indian strains were transformed with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator of CFA/I and CS4 genes. The cfaD transformants produced large amounts of putative colonization factor O166 (PCFO166) irrespective of whether the nutrient agar contained bile salts, a growth factor otherwise required for adequate PCFO166 expression. A considerable interstrain variation in the level of PCFO166 production could be explained by differences in the proportion of bacteria that were fimbriated, as visualized by electron microscopy. The N-terminal amino acid sequence of PCFO166 fimbrial protein showed a high degree of homology with the corresponding sequences of CFA/I and CS4.     

Juvenile xanthogranuloma. Clinical and pathologic characterization

The changing name of the juvenile xanthogranuloma bears witness to the evolution of knowledge and experience of its varied clinical and histologic presentations. This study characterizes the clinical, microscopic, and some immunohistochemical features of 34 cases. The salient clinical findings include a bimodal age distribution inclusive of adults, a male:female ratio of 4:1, occasional multiplicity of lesions (20%), and common presentation in the cephalad area. Histologic findings include varied architectural patterns, cellular participation in various proportions by foamy histiocytes, epithelioid monocytes, lymphocytes, plasma cells, eosinophils, Touton giant cells, and spindle cells of two forms (dendritic and fusiform). S100-positive dendritic cells comprised a minor, but important, component at expansion zones. The significance of these findings is described.     

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP)

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.      

Distribution of cell surface antigens in histiocytosis X cells. Quantitative immunoelectron microscopy using monoclonal antibodies

We have shown previously that cells comprising the cutaneous infiltrates of histiocytes X (HXCs), like Langerhans cells (LCs), react with monoclonal anti-T6 antibody. HXCs also react with anti-T4/4b and anti-Ia-like antibodies. To further define the patterns of antibody reactivity in HXCs, we performed immunoelectron microscopy on two clear-cut cases of histiocytosis X using the immunoperoxidase technique. The reaction product of diaminobenzidine, indicating anti-T6 antibody reactivity, was easily detected along cell membranes of HXCs and at sites of possible endocytosis. Anti-T4/4b and anti-Ia antibodies had less cell membrane reactivity. Computerized image analysis aided in discriminating the patterns of anti-T6 antibody in HXCs and from LCs and confirmed that anti-T6 antibody staining was the most intense of the antibodies evaluated. These findings (1) document antigenic similarities and differences between HXCs and LCs on an ultrastructural level, (2) add support to the concept that HXCs are abnormal proliferations of LCs, and (3) demonstrate the association of the cell surface antigen, T6, with apparent endocytotic activity.     

Immunohistochemical characterization of seminoma and its inflammatory cell infiltrate

Monoclonal antibodies and the avidin-biotin immunoperoxidase technique were used to study the expression of major histocompatibility complex antigens and the nature of the inflammatory cell infiltrate in 10 testicular seminomas. Tumor cells did not react with anti-HLA-A,B,C, anti-HLA-DR, anti-HLA-DQ, and anti-beta 2 microglobulin antibodies to major histocompatibility antigens. All of the 10 tumors contained a slight to marked inflammatory cell infiltrate at the periphery of the tumor, in the connective tissue septa, and in the tumor lobules. The lymphocytes were predominantly T cells; B lymphocytes were rare. The tissue available for study from seven tumors showed tumor lobules separated by delicate fibrovascular septa; T lymphocytes with a cytotoxic-suppressor phenotype predominated in this area in six tumors. In the four tumors in which peripheral tissue was available for study, cells with a helper-inducer phenotype predominated at the tumor margin. Tissue from three tumors showed stromal sclerosis and a dense lymphohistiocytic infiltrate separating individual and small nests of tumor cells; T cells with a helper-inducer phenotype predominated in these cases. Aggregates of macrophages that expressed OKM-1 and Leu-M3 were present in eight of 10 tumors. These findings indicate that two types of immune reactions may be operating: a delayed-type hypersensitivity response at the periphery and a cytotoxic-suppressor effector mechanism in the tumor lobules. Furthermore, major histocompatibility complex antigens are not involved in eliciting the inflammatory response.