The effect of instrument type and preflaring on apical file size determination.

AIM: The aim of this study was to investigate the effect of instrument type (K-files and Lightspeed (LS) instruments) and the impact of preflaring on the first file size that binds at working length (WL) in a range of canal types of varying sizes and curvatures. METHODOLOGY: One hundred and twenty-one canals from 60 extracted intact human maxillary and mandibular premolars and molars were utilized. After standard access cavities, a size 6 K-file was inserted into each canal until the tip of the file was visible outside the apical foramen. WL were set 0.5 mm short of these measurements. Each canal was sized using consecutively larger K-files and hand-held LS instruments until one bound at WL. The file sizes were recorded. The same procedures were repeated after coronal and middle third flaring using Profile rotary instruments. RESULTS: Statistical analysis (univariate analysis of variance: ANOVA) showed that overall, the estimate of mean apical diameter with the LS instrument was larger than with the K-file by 9.4 x 10(-2) mm (P < 0.001; 95% confidence interval: 8.7 x 10(-2), 10.2 x 10(-2)), i.e. almost 2 ISO file sizes larger. Flaring of the cervical and middle thirds of the canal had an impact on apical sizing by both types of instruments, with an average increase of 5.3 x 10(-2) mm (P < 0.001; 95% confidence interval: 4.5 x 10(-2), 6.0 x 10(-2)), i.e. approximately 1 ISO file size larger. CONCLUSIONS: Preflaring and use of hand-held LS instruments resulted in an increase in the instrument size that bound at WL. If an operator wishes to determine an accurate master apical file size, canal orifice enlargement should be performed first before the placement of the assessment file.

An adenovirus-Epstein-Barr virus hybrid vector that stably transforms cultured cells with high efficiency.

EBV episomes are nuclear plasmids that are stably maintained through multiple cell divisions in primate and canine cells (J. L. Yates, N. Warren, and B. Sugden, Nature 313:812-815, 1985). In this report, we describe the construction and characterization of an E1-deleted recombinant adenovirus vector system that delivers an EBV episome to infected cells. This adenovirus-EBV hybrid vector system utilizes Cre-mediated, site-specific recombination to excise an EBV episome from a target recombinant adenovirus genome. We demonstrate that this vector system efficiently delivers the EBV episome and stably transforms a large fraction of infected canine D-17 cells. Using a colony-forming assay, we demonstrate stable transformation of 37% of cells that survive the infection. However, maximal transformation efficiency is achieved at doses of the E1-deleted recombinant adenoviruses that are toxic to the infected cells. Consequently, E1-deleted vector toxicity imposes a limitation on our current vector system.

Monitoring of dengue viruses in field-caught Aedes aegypti and Aedes albopictus mosquitoes by a type-specific polymerase chain reaction and cycle sequencing.

Virologic surveillance for dengue through the detection of the prevalent serotype(s) circulating in the human population during inter- and intra-epidemic periods constitutes a reliable sentinel system for dengue outbreaks. We have applied a rapid and sensitive, semi-nested, reverse transcription-polymerase chain reaction (RT-PCR) assay using nonstructural protein 3 gene primers for the type-specific-detection of dengue viruses in artificially infected and in field-caught adult Aedes mosquitoes. In laboratory experiments, the assay was sensitive enough to detect one virus-infected mosquito head in pools of up to 59 uninfected heads. In a prospective field study conducted from April 1995 to July 1996, female adult Ae. aegypti and Ae. albopictus mosquitoes were caught from selected dengue-sensitive areas in Singapore and assayed by RT-PCR. Approximately 20% of 309 mosquito pools were positive for dengue viruses. Of the 23 RT-PCR-positive Ae. aegypti pools (containing 1-17 mosquitoes each), 18 pools (78.3%) were positive for dengue 1 virus. There were 40 RT-PCR-positive Ae. albopictus pools (containing 1-33 mosquitoes each) of which 31 (77.5%) were positive for dengue 1 virus. The predominant virus type responsible for the current dengue epidemic since 1995 was also dengue 1. The geographic locations of the virus-infected mosquitoes correlated with the residences or workplaces of patients within dengue outbreak areas. A total of 43.5% of the positive Ae. aegypti pools and 25.0% of the positive Ae. albopictus pools contained only a single mosquito. Both Aedes species showed similar overall minimum infection rates of 57.6 and 50 per 1,000 mosquitoes. Infected Ae. aegypti were detected as early as six weeks before the start of the dengue outbreaks in 1995 and 1996. However, infected Ae. albopictus appeared later, when the number of cases was increasing. Virologic surveillance by RT-PCR for detecting dengue virus-infected Aedes mosquitoes in the field may serve as an early warning monitoring system for dengue outbreaks.

A system for non-invasively measuring blood pressure on a treadmill.

Measurement of signal to artifact ratio yielded optimal frequencies for a Korotkoff-based automatic system for measuring blood pressure on a treadmill. Maximal Korotkoff sounds occurred just above the crease of the elbow over the brachial artery. Output of a piezoelectric microphone and charge amplifier was measured during treadmill exercise for frequency bands from 8 to 57 Hz. An automatic system filtered output from 40 to 45 Hz, rectified it, then compared it to a threshold that had fixed and exponentially decaying components. For five subjects, the system decisions of systolic and diastolic pressures compared well with those of two observers using stethoscopes. The system shows promise for improved measurements of blood pressure during treadmill exercise.

Tumor regression and induction of anti-tumor immunity by local chemotherapy of guinea-pigs bearing a line-10 hepatocarcinoma.

Local administration of a low dosage of the active cyclophosphamide derivative 4-hydroperoxy-cyclophosphamide (4-HPCY) at the site of antigenic stimulation strongly enhances T-cell-mediated immune responses in both mice and guinea-pigs. Such immunopotentiation is related to functional elimination of suppressor cells from the draining lymph nodes. In the present study we examined the potential immunotherapeutic effects of local cytostatic drug administration in strain-2 guinea-pigs bearing a line-10 hepatocarcinoma. This tumor, when injected intradermally (10(6) cells) metastasizes within 7 days into the draining lymph node and untreated animals die within 60-80 days from metastatic growth. In sensitization experiments, using irradiated line-10 tumor cells, potentiation of delayed-type hypersensitivity reactivity was observed with local administration of low dosages of 4-HPCY. Intralesional treatment with increasing dosages of 4-HPCY, when started 7 days after tumor-cell inoculation and continued for 3 weeks, resulted in a dose-dependent regression of the primary tumor. Cure rates of up to 75% were achieved. All cured animals showed strong delayed-type hypersensitivity reactivity towards line-10 cells and were resistant to a rechallenge with 10(6) line-10 tumor cells. When treatment was started at a very late stage of the disease (day 14) only a small number of animals were cured. However, when local chemotherapy was preceded by one (non-curative) systemic dose of cyclophosphamide, a 57% cure rate was obtained. Again, all cured animals showed strong delayed-type hypersensitivity reactivity and protective immunity to line-10 tumor cells. Tumor immunity was transferable to naive recipients with immune spleen cells and was T-cell-dependent. Other cytostatic drugs, selected for local immunopotentiating capacity, notably etoposide (VP16) and cis-platinum (cis-Pt) were similarly effective in the local chemotherapy protocol.

Immunotherapeutic effects of local chemotherapy with an active metabolite of cyclophosphamide.

A new approach is described for the use of cytostatic drugs as biological response modifiers (BRM's). Under restricted conditions, strong potentiation of T-effector function can be obtained through preferential elimination of suppressor cells. Recent studies from our group have demonstrated that such conditions are fulfilled when low dosages of certain drugs are injected at a site of low antigenic stimulation. Furthermore, local injection of cytostatic drugs not only enhances the development of cell-mediated immunity in non-sensitized animals, but also facilitates the reversal of existing immunological tolerance. These data shed new light on the tumor regression and induction of tumor immunity observed upon intralesional chemotherapy in experimental tumor models.

Identification and quantitation of the expression of T cell surface markers during the development of chronic relapsing experimental allergic encephalomyelitis (CREAE) in the guinea pig.

As there were discrepancies in previous data on the T cell nature of cells infiltrating the meninges at all stages of chronic relapsing experimental allergic encephalomyelitis (CREAE), experiments have been performed using a further monoclonal antibody (Mab) recognizing total T cell populations and the classic E rosetting technique. Cytospins were prepared of the meningeal inflammatory cells obtained by washing the brains of these animals, and stained by indirect immunoperoxidase. It was found that the T cell, as defined by both E rosetting and staining with the Mab CT5, is the major cell type found in the meninges during the development of CREAE. However, the staining with the Mab CT7, which recognizes a functionally relevant antigen, showed that there is a discrepancy between the numbers of lymphocytes stained compared to the results with CT5 and E rosettes. Furthermore, the antigen recognized by CT7 appeared to be modulated during the disease. The possible functional relevance and its relation to clinical remission and relapse are discussed.

Changes in lymphocyte subsets after treatment with cyclophosphamide and during the development of contact sensitivity in the guinea pig.

Changes in lymphocyte subpopulations were investigated in guinea pig lymph nodes during the development of contact sensitivity to 2,4-dinitrofluorobenzene (DNFB) and following the injection of cyclophosphamide (CY; 300 mg/kg) using a panel of monoclonal antibodies against guinea pig lymphocyte surface markers. Application of a sensitizing dose of DNFB to the ear resulted in a significant increase in the number of cells recovered from the draining auricular and cervical lymph nodes, 4 and 6 days post sensitization. A significant increase in the number of cells in the contralateral cervical node was found at day 6 but not at day 4. At 4 days postsensitization the proportions of Ia positive lymphocytes were higher than those of immunoglobulin positive B cells in the draining auricular and cervical node and the contralateral cervical lymph nodes suggesting T-cell "activation". Four and six days after sensitization there were no significant changes in the proportions of Pan T and T suppressor/cytotoxic (Ts/c) positive lymphocytes in the draining auricular and cervical lymph nodes. Although contact sensitivity is "classically" a T-cell mediated phenomenon there was a significant increase in the proportion of B cells found in the draining auricular node 4 days after sensitization as compared to the "normal" auricular node. 1, 2, 3 and 7 days after injection of CY there was a significant depletion in the proportion of B-lymphocytes in the cervical lymph node. This effect was maximal between 2 and 3 days after CY and was paralleled by an increase in the proportion of Pan T positive cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Local administration of various cytostatic drugs after subcutaneous immunization enhances delayed-type hypersensitivity reaction to sheep red blood cells in mice.

Delayed hypersensitivity reaction in mice was enhanced with various anti-cancer drugs administered at the site of antigenic stimulation during 4 days following sensitization. The immunopotentiating effect of the presented local chemotherapy protocol is thought to result from impairment of a regulatory circuit, with activated suppressor T cells (Ts) as target rather than Ts precursors or Ts-inducing antigen-presenting cells.

Immunocytochemical identification and quantitation of mononuclear cells in the meninges during the development of chronic relapsing experimental allergic encephalomyelitis (CREAE) in the guinea pig.

To investigate the role of mononuclear cells in the meninges at all stages of chronic relapsing experimental allergic encephalomyelitis, juvenile guinea pigs were inoculated with isogeneic spinal cord in Freund's complete adjuvant (FCA) in parallel with animals inoculated with FCA alone as age-matched controls. Cytospins were prepared of the meningeal inflammatory cells obtained by washing the brains of these animals. These cytospins were stained by indirect immunoperoxidase, using a panel of monoclonal antibodies (Mabs) recognizing "activated" macrophages (M phi s), Ia antigen, total T cells and a putatively T-cell-suppressor subset, and an antiserum against immunoglobulins. The inflammatory response was quantitated and the proportions of the different cell types were determined. It was found that the total number of infiltrating cells correlated with the neurological symptoms of the disease. "Activated" M phi s increased significantly during the disease, in line with clinical signs. The expression of the Ia antigen, found on both lymphocytes and M phi s, also appeared to correlate with the disease. There was no increase in putative T-suppressor-cells during remission but there was a significant rise in the proportion of both cells staining with anti-immunoglobulins and plasma cells during relapse.

Production of monoclonal antibodies defining guinea pig T-cell surface markers and a strain 13 Ia-like antigen: the value of immunohistological screening.

The production and characterization of eight monoclonal antibodies (MAbs) against surface markers of guinea pig T-cells is reported. MAbs CT5 and CT7 define putative pan-T-cell markers. CT5, however, also reacts with the B-cell leukemic line L2C. MAb CT6 is reactive with less than 30% of peripheral T-cells. MAbs CT1, 2, 3, 4, and 8 are reactive with lymphocytes, but not with germinal center B-cells. In addition to the CT's, a MAb (CI-13.1) has been prepared that reacts with an Ia-like antigen on cells of strain 13 and outbred guinea pigs, but not with cells of strain 2 animals. CI-13.1 cross-reacts with human tissue sections: About 30% of the OKIa-positive dendritic cells in the human dermis are recognized by CI-13.1. In the course of production and characterization, various binding assays and an immunohistological method were used for determining the antibody specificity. Immunohistological screening was found to be the most informative method.

Studies of the characteristics, distribution and physiological role of a large cardioactive peptide in Lymnaea stagnalis.

Large cardioactive peptide (LCP, heat-stable, mol. wt greater than or equal to 1500) occurs in the ventricle and CNS, and in high concentrations in the auricle. The LCP haemolymph concentrations in fed animals are ca. 35 times higher than those in starved snails. The excitatory effects of LCP on auricle, ventricle and oesophagus are similar to those of some (putative) neurotransmitters. LCP has no effects on the penis retractor muscle. Its effects on the auricle are much more prolonged than those of the transmitters. It is suggested that LCP is a neurohormone, which is transported to the auricle via the nervus intestinalis, and released in response to feeding stimuli.