Postnatal maturation of mast cell subpopulations in the rat respiratory tract.

The distribution and enumeration of mast cell subpopulations within the respiratory tract of a high- and low-Ige responder rat strain was determined during postnatal development. Mast cells were identified in adjacent sections by the alcian blue (AB)/safranin (SAF) staining sequence, or using immunoperoxidase to detect the rat mast cell proteinases I (RMCPI) or II (RMCPII). At birth both mucosal mast cells (MMC) and connective tissue mast cells (CTMC) were represented in very low numbers at distinct locations throughout the respiratory tract. The total number of mast cells increased with age. MMC (AB+/RMCPII+ mast cells) were the predominant phenotype in the epithelium and lamina propria of the trachea and the major conducting airways of the lung in all age groups. In contrast, CTMC (AB+/RPMCPI+ and SAF+/RMCPI+ mast cells) predominated in the submucosa of the trachea and major conducting airways as well as in the parenchyma and visceral pleura of the peripheral lung. Both phenotypes co-exist in similar proportions in peribronchial adventitial tissue and adventitia surrounding large blood vessels in neonates as well as adults. In rats the tracheal epithelium is densely populated by MMC from around the time of weaning (3 weeks) and a small but generalized increase in the number of MMC at all sites within the respiratory tract is noted from this time. This increase in MMC frequency in tissue sections with increasing age is mirrored by the levels of circulating serum RMCPII. The number of bone marrow-derived MMC also increased with increasing age prior to weaning, with a significant drop (P less than 0.01) at 4 weeks of age before returning to the peak numbers in 3-week-old rats. The high-IgE responder Brown Norwegian (BN) rat strain constitutively produces significantly more IgE than the low-IgE responder White albino Glaxo (WAG) strain (P less than 0.001) at all ages studied. In contrast, only minor differences between the number and distribution of mast cells in the two strains were observed.     

Computerized follow-up of discrepancies in image interpretation between emergency and radiology departments

Radiographs are ordered and interpreted for immediate clinical decisions 24 hours a day by emergency physicians (EP’s). The Joint Commission for Accreditation of Health Care Organizations requires that all these images be reviewed by radiologists and that there be some mechanism for quality improvement (QI) for discrepant readings. There must be a log of discrepancies and documentation of follow up activities, but this alone does not guarantee effective Q.I. Radiologists reviewing images from the previous day and night often must guess at the preliminary interpretation of the EP and whether follow up action is necessary. EP’s may remain ignorant of the final reading and falsely assume the initial diagnosis and treatment were correct. Some hospitals use a paper system in which the EP writes a preliminary interpretation on the requisition slip, which will be available when the radiologist dictates the final reading. Some hospitals use a classification of discrepancies based on clinical import and urgency, and communicated to the EP on duty at the time of the official reading, but may not communicate discrepancies to the EP’s who initial read the images. Our computerized radiology department and picture archiving and communications system have increased technologist and radiologist productivity, and decreased retakes and lost films. There are fewer face-to-face consultations of radiologists and clinicians, but more communication by telephone and electronic annotation of PACS images. We have integrated the QI process for emergency department (ED) images into the PACS, and gained advantages over the traditional discrepancy log. Requisitions including clinical indications are entered into the Hospital information System and then appear on the PACS along with images and readings. The initial impression, time of review, and the initials of the EP are available to the radiologist dictating the official report. The radiologist decides if there is a discrepancy, and whether it is category I (potentially serious, needs immediate follow-up), category II (moderate risk, follow-up in one day), or category III (low risk, follow-up in several days). During the working day, the radiologist calls immediately for category I discrepancies. Those noted from the evening, night, or weekend before are called to the EP the next morning. All discrepancies with the preliminary interpretation are communicated to the EP and are kept in a computerized log for review by a radiologist at a weekly ED teaching conference. This system has reduced the need for the radiologist to ask or guess what the impression was in the ED the night before. It has reduced the variability in recording of impressions by EP’s, in communication back from radiologists, in the clinical follow-up made, and in the documentation of the whole QI process. This system ensures that EP’s receive notification of their discrepant readings, and provides continuing education to all the EP’s on interpreting images on their patients.     

Activity involvement, risk-taking, demographic variables, and other drug use: prediction of trying smokeless tobacco

Four activity participation variables (clubs, sports, church, and parties); two indices of "risk-taking" (preference for risk-taking, getting into trouble at school); three demographic variables (sex, ethnic group, socioeconomic status); and two drug use variables (trial of cigarettes and alcohol) were examined as correlates and prospective predictors of trial of smokeless tobacco in two cohorts of seventh graders in urban Los Angeles. The data were analyzed separately for males and females. Cross-sectional logistic regression analyses indicated that correlates of trying smokeless tobacco among the seventh-grade cohorts or among these same cohorts in the eighth grade (considering those persons who had not tried smokeless tobacco in seventh grade) generally included being white, trying cigarettes, risk-taking, and attending parties. Prospective logistic regression analyses with data from subjects who had not tried smokeless tobacco in the seventh grade indicated that predictors of subsequent trial of it generally included only being white and having tried cigarettes. Sports participation predicted onset only in one cohort of female subjects but not in males. Some activities that have been proposed as being predictive of smokeless tobacco use (e.g., sports participation) are generally irrelevant for a large sample of young adolescents in urban Los Angeles. White male cigarette smokers, regardless of the activities they have engaged in, are most likely to try smokeless tobacco.     

Single step enrichment of blood dendritic cells by positive immunoselection

Dendritic cells (DC) for cancer immunotherapy protocols are generated most commonly by in vitro differentiation of monocytes with exogenous cytokines (Mo-DC). However, Mo-DC differ in their molecular phenotype and function from blood DC (BDC). Clinical isolation of BDC has been limited to the use of density gradients, which result in low yields of variable purity. We have developed a DC enrichment platform, which uses the CMRF-44 (IgM) or CMRF-56 (IgG) monoclonal antibodies (mAb) to select BDC that express these antigens after a short overnight incubation. After culture of peripheral blood mononuclear cells (PBMC) in autologous/AB serum, biotinylated CMRF-44 was used to select DC in a single step immuno-magnetic bead procedure; this produced populations containing up to 99% CMRF-44(+) cells, including up to 67% CMRF-44(+) CD14(-) CD19(-) DC, from an initial starting population of approximately 0.5%. We observed consistent differences in the purities obtained from individual donors with a mean of 54% CMRF-44(+) cells (range 19-99%). Similar results were obtained using biotinylated CMRF-56 mAb, an antibody identifying a comparable population in cultured PBMC. We recovered an average of 54% and 66% of the available BDC in separations performed with the CMRF-44 and CMRF-56 mAb, respectively. The reproducibility of the procedure and the ability to perform it in a closed sterile system makes it suitable for clinical use. Larger scale preparations starting from apheresis derived PBMC will produce sufficient BDC for immunotherapy protocols. The purified BDC elicited strong allogeneic mixed leukocyte reactions and HLA classes II- and I-restricted antigen-specific primary immune responses.     

Characterization of a P1-deficient strain of Streptococcus mutans that expresses the SpaA protein of Streptococcus sobrinus.

The Streptococcus sobrinus SpaA protein and the Streptococcus mutans P1 protein share 66% sequence homology at the amino acid level. To determine if the SpaA protein can be expressed in S. mutans and functionally replace the P1 protein, the spaA gene of S. sobrinus 6715 was isolated from plasmid pX1303 and inserted into the Escherichia coli-Streptococcus shuttle vector pVA838. The resulting plasmid pX1600 was transformed into the P1-deficient strain S. mutans 834 that has defects in saliva-mediated aggregation and in the ability to adhere to saliva-coated hydroxyapatite surfaces. Western blot (immunoblot) analysis of cellular protein fractions of S. mutans 834 (pX1600) detected in mutanolysin-solubilized cell walls a major protein of 210 kDa with an electrophoretic mobility similar to that of S. sobrinus SpaA protein and a minor 210-kDa protein and a major 64-kDa protein in the extracellular protein fraction. Analysis of virulence traits showed that expression of SpaA protein by S. mutans 834(pX1600) cells had restored the ability of the S. mutans 834 cells to aggregate in the presence of saliva or salivary agglutinin but not to adhere to saliva-coated hydroxyapatite. This cell aggregation was inhibited specifically by antisera to S. sobrinus SpaA protein. These results indicate that SpaA plays a role in the virulence of S. sobrinus by specifically interacting with fluid-phase salivary agglutinin to mediate cell aggregation.     

Alveolar macrophages. VI. Regulation of alveolar macrophage-mediated suppression of lymphocyte proliferation by a putative T cell.

Alveolar macrophages (AM) from normal rats suppressed antigen- or mitogen-stimulated blastogenic responses in cultures of splenic or lymph node lymphocytes, high levels of suppression often being observed when added AM comprised as few 0.6% of the total cells in culture. The efficiency of AM-mediated suppression of spleen cell blastogenesis declined with the age of the spleen cell donors, was severely curtailed by pretreatment of donors with low levels of cyclophosphamide, and was depleted by adult thymectomy coupled with thoracic duct drainage. The suppressive activity of AM was most obvious at high cell density, was unaffected by the presence of indomethacin in the cultures, or by prior X-irradiation of the spleen cells. Fractionation of spleen cells by velocity sedimentation yielded cell populations of greatly varying sensitivities to AM-mediated suppression, from small splenocytes (sedimentation velocity 1.1-2.8 mm/h) which were almost totally refractory to AM-suppression when assayed in isolation from the remainder of the spleen cell population, to larger cells (sedimentation velocity greater than 3.,5 mm/h) exhibiting high levels of sensitivity. Fractionation of spleen cells by glass wool adherence indicated decreased sensitivity to AM-suppression in the effluent population. Examination of the suppressive activity of individual subpopulations of AM separated by velocity sedimentation indicated that the larger macrophages were the most active in vitro. Suppressive activity of this nature was not seen with unstimulated peritoneal macrophages, but was observed when 'activated' peritoneal exudate cells were tested. These data are discussed in terms of a two-cell model for suppression of blastogenesis, the ultimate effector cell being a macrophage, the activity of which is controlled by a long-lived, recirculating lymphocyte, which we have provisionally designated as a T lymphocyte.     

Cellular fatty acid composition of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

Strains of Actinobacillus actinomycetemcomitans isolated from deep pockets of patients with juvenile periodontitis were analyzed for their content of cellular fatty acids. Oral Haemophilus strains, morphologically and biochemically similar to Haemophilus aphrophilus, were also examined for their content of cellular fatty acids. The extractable lipids of the actinobacilli represented approximately 10% of the cell dry weight, with the bound lipids representing 2 to 5%. The major fatty acids consisted of myristic (C14:0) and palmitic (C16:0) acids and a C16:1 acid, possibly palmitoleic acid, accounting for 21, 35, and 31% of the total extractable fatty acids, respectively. Haemophilus strains had a similar cellular fatty acid content.     

Streptococcus mutans Genes That Code for Extracellular Proteins in Escherichia coli K-12

Chromosomal DNA from Streptococcus mutans 6715 (serotype g) was cloned into Escherichia coli K-12 by using the cosmid pJC74 cloning vector and a bacteriophage λ in vitro packaging system. Rabbit antiserum against S. mutans extracellular proteins was used for immunological screening of the clone bank. Twenty-one clones produced weak to strong precipitin bands around the colonies, but only after the λ c1857 prophage was induced by being heated to lyse the E. coli cells. None of the clones expressed enzyme activity for several known S. mutans extracellular enzymes. One of these clones contained a 45-kilobase recombinant plasmid designated pYA721. An 8.5-kilobase fragment of S. mutans DNA from pYA721 was isolated and recloned into the BamHI restriction site of the plasmid vector pACYC184 to construct pYA726. pYA726 contained all, or nearly all, of the gene for a surface protein antigen (the spaA protein) of S. mutans 6715. This was deduced from immunological studies in which extracts of cells harboring pYA726 reacted with antisera against both purified 6715 spaA protein (about 210,000 daltons) and the immunologically similar antigen I/II of serotype c strains of S. mutans. In addition, the S. mutans spaA protein was found to possess at least one antigenic determinant not present on the protein specified by pYA726. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of E. coli clone extracts revealed that pYA726 produced a polypeptide with a molecular mass of about 180,000 daltons which was predominantly found in the periplasmic space of E. coli cells. Antisera to the spaA protein of S. mutans reacted with extracellular protein from representative strains of S. mutans serotypes a, c, d, e, f, and g, but not b.