Molecular analysis of bacterial species associated with childhood caries

Although substantial epidemiologic evidence links Streptococcus mutans to caries, the pathobiology of caries may involve more complex communities of bacterial species. Molecular methods for bacterial identification and enumeration now make it possible to more precisely study the microbiota associated with dental caries. The purpose of this study was to compare the bacteria found in early childhood caries (ECC) to those found in caries-free children by using molecular identification methods. Cloning and sequencing of bacterial 16S ribosomal DNAs from a healthy subject and a subject with ECC were used for identification of novel species or uncultivated phylotypes and species not previously associated with dental caries. Ten novel phylotypes were identified. A number of species or phylotypes that may play a role in health or disease were identified and warrant further investigation. In addition, quantitative measurements for 23 previously known bacterial species or species groups were obtained by a reverse capture checkerboard assay for 30 subjects with caries and 30 healthy controls. Significant differences were observed for nine species: S. sanguinis was associated with health and, in order of decreasing cell numbers, Actinomyces gerencseriae, Bifidobacterium, S. mutans, Veillonella, S. salivarius, S. constellatus, S. parasanguinis, and Lactobacillus fermentum were associated with caries. These data suggest that A. gerencseriae and other Actinomyces species may play an important role in caries initiation and that a novel Bifidobacterium may be a major pathogen in deep caries. Further investigation could lead to the identification of targets for biological interventions in the caries process and thereby contribute to improved prevention of and treatment for this significant public health problem.     

Variant lines of mouse kidney cells transformed by an SV40tsA mutant with growth properties of wild-type transformed cells at nonpermissive temperature.

MKSA207 cells, a BALB/c mouse kidney line transformed by a tsA mutant of SV40, are temperature-dependent for the expression of the 'standard transformed phenotype'. At the permissive temperature (33.5 degrees C), the mKSA207 cells resembled wild-type (wt) SV40 transformants; they contained the intranuclear SV40 T antigen, grew to high saturation density in monolayer culture in either 10% or 0.5% serum, and also in methylcellulose suspension culture and became multinucleate in cytochalasin B. At the nonpermissive temperature (39.8 degrees C), the mKSA207 cells lost some of their transformed properties; they grew only to low density in 10% serum, hardly grew at all in 0.5% serum or in methylcellulose suspension culture, and remained mono- or binucleate in cytochalasin B. At 40 degrees C in low serum, mKSA207 cells lost the intranuclear T antigen and when fed 10% serum at 39.8 degrees C, accumulated large amounts of T antigen in the cytoplasm. Derivatives of mKSA207 have been selected at 39.8 degrees C in liquid medium and methylcellulose suspension culture. The heat adapted lines, like wt SV40 transformants, exhibited the standard transformed phenotype at both 33.5 and 39.8 degrees C. It is unlikely that acquisition of temperature-independence for the transformed phenotype was due to reversion of the tsA gene to wild-type because the heat-adapted cell lines displayed the cytoplasmic T antigen at 39.8 degrees C, characteristic of the parental mKSA207 cells and SV40 rescued from one of the heat-adapted lines was temperature sensitive for growth. The T antigen levels (complement fixation units per 10(6) cells) of heat-adapted lines grown at 39.8 degrees C were comparable to those of mKSA207 cells grown at 33.5 or 39.8 degrees C.     

Mapping thymidine kinase-deficient mutants of vaccinia virus by marker rescue with hybrid plasmid DNAs containing portions of the HindIII-J fragment of virus DNA

Five hybrid plasmids were constructed, each containing a portion of the vaccinia virus DNA HindIII-J fragment. These plasmid DNAs were used in marker rescue experiments to map the mutations in the thymidine kinase (TK) gene of three TK- vaccinia virus mutants. The TK gene of each of the three mutants was rescued by DNA from plasmid pPJ701, which contained about one-half of the HindIII-J fragment. Two mutants, 1004B and 1017-1, but not the third, 1016-1, were rescued by DNA of two plasmids, pPJ702 and pPJ703, which contained 16 and 18%, respectively, of one end of the J fragment. Mutant 1016-1 could be rescued by plasmid pPJ705 containing a 1.69-kb fragment of the HindIII-J fragment. The J fragment DNA in plasmid pPJ705 is located adjacent to that and separated by an EcoRI site from pPJ703 in the vaccinia virus genome. These results indicate that the mutation site in the TK gene of 1016-1 differs from that in 1004B or 1017-1 and suggests that the structural gene for the vaccinia virus TK lies near one end of the HindIII-J fragment and spans the EcoRI site.     

Evaluation and Validation of an Enzyme-Linked Immunosorbent Assay and an Immunochromatographic Test for Serological Diagnosis of Severe Acute Respiratory Syndrome

A newly developed severe acute respiratory syndrome (SARS)-specific enzyme-linked immunosorbent assay (ELISA) was further validated to confirm cutoff values and evaluate its diagnostic performance with clinical samples. In parallel, an immunochromatographic test was also evaluated. A total of 227 clinical serum specimens collected from SARS patients were used in the study, together with 385 samples from healthy donors. By use of an immunofluorescent (IF) test as the “gold standard, ” both the ELISA and the immunochromatographic test were able to detect immunoglobulin G antibodies to SARS not only from late-convalescent-stage samples (>21 days from the onset of clinical symptoms), as previously established, but also from early-acute-phase samples (1 to 10 days from onset). The ELISA, using an optical density (OD) of 0.25 as its cutoff value, produced the best sensitivity while maintaining high specificity. It detected SARS-specific antibodies in 58, 70, 75, and 95%, respectively, of the four groups of samples collected from patients 1 to 10 days, 11 to 20 days, 21 to 30 days, and more than 30 days after the onset of clinical symptoms. Similarly, the immunochromatographic test detected SARS-specific antibodies in 55, 68, 81, and 79% of the four groups, respectively. The overall specificities for the ELISA and the rapid test were 99.5 and 97.7%, respectively. Although the positive correlation observed between the ELISA OD values and the IF titers was moderate (r = 0.6915; P < 0.001), the detection rates of both the ELISA and the rapid test were found well in agreement with the IF titers.     

An optimized forward-planning technique for intensity modulated radiation therapy

Intensity modulated radiation therapy (IMRT) has stirred considerable excitement in the radiation oncology community. Its objective is to make the dose conform to the tumor and spare other organs. Instead of resorting to the rather complex inverse-planning, the technique described here is an extension of the conventional treatment planning technique. The beam orientation and wedge angles are chosen in the conventional rule-based manner. However, within each conformal beam's eye view (BEV) field including margin, a number of sub-field openings are added. The smaller field openings are designed to irradiate the tumor, while sparing the normal tissue of the organs at risk (OARs) that intrude into the target region in the BEV. As the number of intrusions into the target BEV increases, the number of sub-fields for each beam increases. The Cimmino simultaneous projection method was employed to obtain the optimized weighting for each field of each beam. In cases where the dose constraints for the tumor and for the OARs are reasonable, it is possible to obtain a plan with a fairly small number of beams that satisfies the specified dose objectives. This is illustrated for the treatment of prostate cancer, where the rectum creates a concavity in the planning target volume. An advantage of this technique is that the quality assurance for the delivery of these plans does not require extensive special efforts.     

Detection of clonal immunoglobulin gene rearrangements in the peripheral blood progenitor cells of patients with multiple myeloma: the potential role of purging with CD34 positive selection

Aims—To determine the extent of clonal cell contamination of peripheral blood progenitor cell (PBPC) collections in patients with multiple myeloma (MM) and to assess the purging efficacy of CD34 positive selection.     

Thymidine kinase-negative bovine herpesvirus type 1 mutant is stable and highly attenuated in calves

Summary Calves were vaccinated intranasally (IN) or intravenously (IV) with a thymidine kinase-negative (tk^–) BHV-1 mutant. Vaccinated calves developed neutralizing antibodies but did not show clinical signs of infectious bovine rhinotracheitis (IBR). Vaccination also prevented clinical signs of IBR disease following IN challenge exposure of the calves to parental Los Angeles (LA) and USDA Cooper strains of tk⁺ BHV-1. Nasal swabs were collected for 10 days after the vaccination and the challenge exposures to monitor BHV-1 multiplication. At both 91 and 121 days post vaccination (PV), calves were also stressed for 5 days with dexamethasone (DEX) to induce reactivation of BHV-1 and nasal swabs were obtained. tk^– BHV-1 multiplied in the nasal mucosa of IN vaccinated calves and was also recovered after DEX treatment. Likewise, tk^– BHV-1 was isolated from the buffy coat fraction of IV vaccinated calves, but not from nasal swabs. tk^– BHV-1 vaccination reduced the multiplication of tk⁺ BHV-1 in the nasal mucosa, but did not completely prevent development of a persistent infection by the challenge virus. The phenotypes of viruses isolated from the nasal swabs and buffy coats were analyzed by enzyme assays of extracts from virus-infected cells and by plaque autoradiography. These assays showed that tk^– BHV-1 can persist for at least 3 months in vaccinated calves and may also be transmitted from vaccinated to control calves without revertingin vivo to tk⁺. The results demonstrate that the tk^– BHV mutant is attenuated and efficacious as a vaccine.With 1 Figure     

Acute fibrinous and organizing pneumonia: a histological pattern of lung injury and possible variant of diffuse alveolar damage

CONTEXT: The histologic patterns of diffuse alveolar damage (DAD), bronchiolitis obliterans with organizing pneumonia (BOOP), and eosinophilic pneumonia (EP) are well-recognized histologic patterns of lung injury associated with an acute or subacute clinical presentation. We have recognized acute fibrinous and organizing pneumonia (AFOP) as a histologic pattern, which also occurs in this clinical setting but does not meet the classic histologic criteria for DAD, BOOP, or EP and may represent an underreported variant. OBJECTIVE: To investigate the clinical significance of the AFOP histologic pattern and to explore its possible relationship to other disorders, including DAD and BOOP. DESIGN: Open lung biopsy specimens and autopsy specimens were selected from the consultation files of the Armed Forces Institute of Pathology, which showed a dominant histologic pattern of intra-alveolar fibrin and organizing pneumonia. Varying amounts of organizing pneumonia, type 2 pneumocyte hyperplasia, edema, acute and chronic inflammation, and interstitial widening were seen. Cases with histologic patterns of classic DAD, BOOP, abscess formation, or eosinophilic pneumonia were excluded. To determine the clinical behavior of patients with this histologic finding, clinical and radiographic information and follow-up information were obtained. Statistical analysis was performed using Kaplan-Meier and chi(2) analysis. RESULTS: Seventeen patients (10 men, 7 women) with a mean age of 62 years (range, 33-78 years) had acute-onset symptoms of dyspnea (11), fever (6), cough (3), and hemoptysis (2). Associations believed to be clinically related to the lung disease included definitive or probable collagen vascular disease (3), amiodarone (1), sputum culture positive for Haemophilus influenza (1), lung culture positive for Acinetobacter sp. (1), lymphoma (1), hairspray (1), construction work (1), coal mining (1), and zoological work (1). Six patients had no identifiable origin or association. Follow-up revealed 2 clinical patterns of disease progression: a fulminate illness with rapid progression to death (n = 9; mean survival, 0.1 year) and a more subacute illness, with recovery (n = 8). Histologic analysis and initial symptoms did not correlate with eventual outcome, but 5 of the 5 patients who required mechanical ventilation died (P =.007). CONCLUSIONS: Acute fibrinous and organizing pneumonia is a histologic pattern associated with a clinical picture of acute lung injury that differs from the classic histologic patterns of DAD, BOOP, or EP. Similar to these patterns of acute lung injury, the AFOP pattern can occur in an idiopathic setting or with a spectrum of clinical associations. The overall mortality rate is similar to DAD and therefore may represent a histologic variant; however, AFOP appears to have 2 distinct patterns of disease progression and outcome. The need for mechanical ventilation was the only parameter that correlated with prognosis. None of the patients with a subacute clinical course required mechanical ventilation.     

A flow cytometric method for the measurement of phagocytosis by polymorphonuclear leucocytes

A new method for the measurement of phagocytosis of Candida albicans by human polymorphonuclear leucocytes (PMN) is described using a fluorescence activated cell sorter. We have used acridine orange to discriminate between PMN which have internalised yeast particles and those which have not. This method allows accurate measurement of particle phagocytosis as an event distinct from particle adherence. It also permits detailed examination of the kinetics of phagocytosis, the study of which is likely to be of value in the investigation of diseases where abnormalities of PMN function are suspected.