A novel antithrombin gene mutation: slippage and mispairing as a mechanism of genetic disease

Treatment options for acute leukemia relapsing after allogeneic BMT include conventional chemotherapy or a second transplant; however, results are rather discouraging, the first option being associated with poor survival and the second with a high mortality rate. More recently, donor leukocyte infusion (DLI) from the original donor has been used for relapsed patients in an attempt to induce a graft-versus-leukemia (GVL) effect. This procedure is partially devoid of the toxicity inherent to a second BMT. At our Institution, a 36-year-old patient with biphenotypic AML in second complete remission after relapse following allogeneic BMT was treated with peripheral blood stem cell (PBSC)-enriched donor leukocytes, obtained after in vivo priming with rhG-CSF. The patient experienced extensive cGVHD but developed a testicular relapse while in full hematologic remission. After irradiation of the sanctuary site he remains free of disease, still with chronic GVHD, 21 months after bone marrow relapse. This case suggests that immunologically privileged sites are inaccessible to GVHD/GVL effect. This should be considered when planning salvage transplants procedures in patients at risk for extramedullary involvement.     

Kinetics of adhesion molecule expression and spatial organization using targeted sampling fluorometry

Cellular interactions with the vascular wall under flow conditions are controlled, in part, by the density of adhesion molecules on endothelial cells. The spatial arrangement and absolute levels of these molecules over the endothelium are therefore important determinants of cellular localization. Many biochemical and functional studies have characterized the interactions between leukocytes and endothelial monolayers, but no reliable method has been reported for quantifying the spatial expression of adhesion molecules on intact endothelial cell monolayers. We report the development of targeted sampling fluorometry (TSF), which uses standard immunostaining, fluorescence microscopy and digital image analysis techniques to analyze cell surface molecule expression on a cell-by-cell basis. This technique is performed on an intact monolayer and results in cellular intensity distributions that reflect spatial heterogeneity in adhesion molecule expression. We demonstrate the use of targeted sampling fluorometry in a study of the kinetics of tumor necrosis factor alpha-induced activation of human umbilical vein endothelial cell monolayers and show that the spatial patterns of adhesion molecule expression correlate with the locations of bound lymphocytes.     

Involvement of calcium in interactions between gingival epithelial cells and Porphyromonas gingivalis

Porphyromonas gingivalis, a periodontal pathogen can invade primary cultures of gingival epithelial cells. This invasion was significantly inhibited (74-81%) by thapsigargin and 1,2-bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid, acetoxymethyl ester (BAPTA/AM), but not by EDTA or amiloride. Release of Ca2+ from an intracellular store and the subsequent increase in cytosolic [Ca2+] may, therefore, be involved in the invasion process, while Ca2+ influx is not. Moreover, cytosolic [Ca2+] was found to increase transiently in about 30% of gingival epithelial cells acutely exposed to P. gingivalis, but not in unexposed cells, or in cells exposed to noninvasive Escherichia coli. These findings indicate that P. gingivalis invasion of epithelial cells is correlated with activation of [Ca2+]-dependent host cell signaling systems.     

Characterization of a novel DNA binding domain within the amino-terminal region of the RAG-1 protein

Rag-1 and Rag-2 are the critical components of the V-(D)-J recombinase required for site-specific recombination of the antigen receptor genes. In this study, we have examined the ability of recombinant (r) Rag-1 and Rag-2 to bind the recombination signal sequences (RSS) and have determined that rRag-1, but not rRag-2, is able to directly bind DNA. rRAG-1 DNA binding activity was found to reside within a novel amino-terminal arginine-rich (RR) domain with partial homology to a variety of nucleic acid binding domains. Although the RR-domain did not demonstrate RSS-specificity, this DNA binding domain may stabilize the interaction of RAG-1 with, or increase the affinity for, the V-(D)-J recombination signals.     

Effect of an enzymatic rinse on salivary levels of Streptococcus mutans and lactobacilli in periodontally treated patients

Root surface caries is prevalent in patients with both treated and untreated periodontal disease. The major etiologic factor has been identified as microbial plaque. In periodontally treated patients, significantly higher root caries prevalence and incidence have been found in patients with high levels of Streptococcus mutans and Lactobacilli in saliva. Reducing the levels of S. mutans and Lactobacilli in saliva may lower the risk of root caries development. The purpose of this investigation was to determine the effect of an oral enzymatic rinse on the salivary counts of S. mutans and Lactobacilli in periodontally treated patients. Fifteen adult subjects participated in a double-blind, cross-over designed clinical trial. Each subject had previously undergone comprehensive periodontal therapy and had been maintained on a regular program of supportive periodontal therapy. Paraffin-stimulated whole saliva was collected from each participant. Each subject was then randomly given either the enzymatic rinse product or a control rinse and instructed to rinse with one tablespoonful twice a day for 2 weeks, after which saliva samples were taken. After a washout period, salivary samples were again taken, and the subjects received the alternate rinse product. Two weeks later, final salivary samples were taken. The salivary samples were serially diluted and incubated aerobically on selective culture media. S. mutans and Lactobacilli were counted on the basis of colonial morphology. Pretreatment and posttreatment salivary counts of S. mutans and Lactobacilli were analyzed using the Wilcoxon matched-pairs signed-rank test at the 5% level of significance. Analysis of data revealed that neither the test nor the control rinse significantly lowered salivary counts of either species in the sample population.     

Isolation of bioactive compounds from Helix aspersa nerve tissue and the effect of pQPPLPRYamide on heart, esophagus and central neurons of H. aspersa and rectum of Anodonta woodiana

1. Tumour necrosis factor-alpha (TNF-alpha) is implicated in the pathogenesis of many pulmonary and airway diseases. TNF-alpha stimulation may release interleukin-8 (IL-8) in airways mediated via an increase in intracellular oxidant stress. In the present study, we have assessed leukosequestration and IL-8 release in the airways in response to intratracheal administration of human recombinant TNF-alpha, and examined the modulatory role of endogenous NO by pretreatment with a NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). 2. TNF-alpha (10(2)-10(-4) u) was administered intratracheally in male guinea-pigs which were anaesthetized with urethane and were ventilated artificially. TNF-alpha induced a time- and dose-related increase in neutrophil numbers and a concomitant increase in human IL-8 equivalent level retrieved from bronchoalveolar lavage (BAL) with the peak effect at 10(3) u at 6 h of TNF-alpha injection (late phase). Intratracheal administration of recombinant human (rh)IL-8 (0.025, 0.25, 2.5 ng) producing a similar range of human IL-8 equivalent levels in BAL as measured in our results induced neutrophil recovery in BAL fluid to a similar extent. Administration of anti-IL-8 antibody prevented the late phase of neutrophil recruitment induced by TNF-alpha or rhIL-8. 3. Pretreatment with L-NAME significantly enhanced the TNF-alpha (10(3) u)-induced neutrophil recruitment and human IL-8 equivalents production at 6 h, but not at 1 h of TNF-alpha administration (early phase). L-Arginine reversed the responses to L-NAME. Pretreatment with 0.2% DMSO (i.v.) significantly inhibited TNF-alpha-induced neutrophil recruitment and human IL-8 equivalents release both in the early and late phase of the responses. Pretreatment with DMSO also inhibited the enhancement effect of L-NAME on the late phase of TNF-alpha-induced responses. DMSO failed to modify exogenous rhIL-8-induced neutrophil recruitment. Neither L-NAME nor DMSO alone induced any significant change in neutrophil numbers or human IL-8 equivalent level in BAL fluid. 4. Neutrophil depletion by cyclophosphamide pretreatment failed to modify TNF-alpha-induced human IL-8 equivalent release. 5. The expression of beta 2-integrin, CD11b/CD18 on neutrophils was increased only in the late but not early phase of TNF-alpha stimulation. L-NAME failed to modify these responses. 6. In conclusion, we demonstrated that NO may be an important endogenous inhibitor of TNF-alpha-induced leukocyte chemotaxis via inhibition of IL-8 production. Thus, the production of NO in airway inflammatory diseases may play a negative feedback role in self-limiting the magnitude of inflammatory responses.     

Effect of rendering procedures on the scrapie agent

A pool of scrapie-infected sheep brains was used to spike mixtures of porcine bone and intestine. These were processed in pilot-scale facsimiles of 12 rendering procedures that were in use within the European Union in 1991, and three that were not. Meat and bone meal, and tallow, were produced from the rendered tissues. Suspensions of all the meat and bone meal samples, and two of the tallow samples were assayed in mice for scrapie infectivity. Neither of the tallow samples had any detectable infectivity but the meat and bone meal samples were positive, except for those produced by processes involving exposure to hyperbaric steam. In addition, greaves were produced from the scrapie-spiked raw materials by an atypical low-temperature process and subjected to solvent extraction with hot heptane. The treated greaves were then exposed to steam to drive off residual solvent. Although the starting titre of infectivity in these greaves was low, there appeared to be no reduction in infectivity as a result of the treatments with hot heptane and steam. However, there was no detectable infectivity in the meat and bone meal prepared from the greaves produced by the atypical low-temperature process after it had been exposed to hyperbaric steam.