Molecular typing of Haemophilus influenzae using a DNA probe and multiplex PCR.

The use of new molecular typing methods for the characterization of Haemophilus influenzae strains is reported. Sixty-four isolates of H. influenzae originating from different types of infection and obtained from eight hospitals across Canada were first analysed for restriction polymorphism. Chromosomal DNA fragments generated by two different combinations of restriction endonucleases were electrophoresed and transferred to nylon membranes before hybridization with a species specific 32P-labelled DNA fragment (5 kb) used as a probe. The combinations Bg/II/PstI led to 11 typing groups (A-K) and BamHI/Bg/II/PstI to 14 sub-groups, respectively. Most of the isolates retrieved from cerebrospinal fluids (10/13; 76.9%) were classified in two groups (A and B) and two sub-groups. Isolates from respiratory tract infections were mostly found in groups C and E (24/32; 75.0%), and divided into seven sub-groups. Selected ampicillin-resistant, beta-lactamase-negative strains were also found in groups C and E (11/14; 78.6%). Isolates from conjunctivitis and acute otitis media were classified in various groups. All biotypes (I-VIII) and serotypes (none, a-f) were spread among the typing groups although biotype I prevailed in groups A, B, and G; II in group E (sub-group 6); and III in group C. A PCR approach derived from the typing system was also tested. A set of 25-mer primers was selected from the 5-kb DNA probe for the amplification of a 317-bp region. This set of primers was used concomitantly in a PCR multiplex assay with a set of primers selected from the nucleotide sequence of the gene encoding the H. influenzae P1 protein. This multiplex assay was also able to discriminate the clonal origin of some H. influenzae strains because size polymorphism was observed in PCR products. The PCR approach was then used to determine the genetic relatedness of H. influenzae strains found persistently in sputa of some patients with cystic fibrosis. Genetically related strains could be isolated from some patients even after antibiotherapy and months between visits, whereas other patients showed distinct strains. In summary, our typing system is able to provide new characteristics for strains having identical biotype or serotype. The rapid PCR alternative may prove useful for specific epidemiological and strain-tracking studies.

Isolation of IFAPa-400 cDNAs: evidence for a transient cytostructural gene activity common to the precursor cells of the myogenic and the neurogenic cell lineages.

Differentiation of neural and muscle cells is characterized by a switch in the expression of the type of intermediate filament protein subunit. In these lineages, vimentin is transiently expressed in the initial stages of development and is gradually replaced by a tissue specific protein. We have identified a giant developmentally regulated antigen (IFAPa-400) which colocalizes with vimentin in the precursor cells of the neurogenic and myogenic lineages of the chick embryo [Chabot and Vincent (1990) Dev. Brain Res. 54, 195-204; Cossette and Vincent (1991) J. Cell Sci. 98, 251-260]. Based on the expression of this protein during neurogenesis and myogenesis, we hypothesize that IFAPa-400 and vimentin define a special intermediate filament network, common to the non-differentiated cells derived from the neuroectoderm and those of the myogenic tissues. We report here the isolation and sequence of partial cDNAs encoding more than 400 amino acids of the carboxy-terminus of this protein. RNA blot analysis and in situ hybridization indicate that IFAPa-400 represents a bona fide developmentally regulated gene product. These results further confirm that IFAPa-400 mRNA transcripts are limited to the early precursor cells of both neurogenic and myogenic lineages.

Localization of molecules with restricted patterns of expression in morphogenesis: an immunohistochemical approach.

In a search for molecules with restricted patterns of expression during development, monoclonal antibodies were raised against different transitory structures of the chick embryo. Mice were immunized with cell suspensions from lightly homogenized embryonic tissues explanted from morphogenetically active regions. A convenient immunohistochemical assay was used to screen the hybridoma supernatants on a large scale. It relied on the use of poly(ethylene glycol) as embedding medium. Its water miscibility allowed, in a one-step incubation with antibody-containing supernatants, the dewaxing and rehydration of the tissue sections as well as antibody binding. We report here the usefulness of this approach in selecting monoclonals with unique patterns of immunoreactivity. In this study, cephalic neural crest cells in early or late phase of migration, together with their surrounding tissues, were used as immunogens. The monoclonal antibodies obtained have been classified into regional, cell-lineage, cell-cycle or extracellular material-associated markers. The information provided by the direct visualization of the immunoreactivity of the various monoclonal antibodies on tissue sections, as early as the first round of screening, allows rapid determination of the subsequent strategy to be followed for further characterization of the individual markers.