Depletion of O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine in three-dimensional collagen cultures of normal human breast epithelial cells.

O6-Alkylguanine--DNA alkyltransferase (AGT) is a protein which removes the promutagenic O6-alkylguanine lesion induced in DNA by alkylating agents. Our results demonstrate that freshly isolated organoids from reduction mammoplasty specimens contain significant levels of AGT activity. AGT activity in breast epithelial cells shows interindividual variation. Constitutive levels of AGT activity remain unchanged during short-term serum-free culture of breast epithelial cells inside three-dimensional rat-tail collagen gel matrix. In the present study, we optimized conditions for depleting AGT activity in human breast epithelial cells cultured in three-dimensional collagen gel matrix using O6-methylguanine and O6-benzylguanine which are substrates for AGT. AGT activity was efficiently inactivated by exposure of cells to O6-methylguanine or O6-benzylguanine. Inactivation with O6-benzylguanine was more rapid, of greater magnitude and consistency and occurred at lower concentrations than with O6-methylguanine. Near-complete inactivation (> 99.5%) of AGT activity was reproducibly achieved with 50 microM O6-benzylguanine. In contrast, 500 microM O6-methylguanine was needed to obtain a maximal effect and this reduced AGT activity by only 53-93% of control. Within 30 min of adding the free base, 50 microM O6-benzylguanine depleted 95% of the levels of AGT compared to 30% inhibition with 500 microM O6-methylguanine. The profile for restoration of AGT activity was different following a 24 h incubation and subsequent removal of each of the guanine derivatives. AGT activity levels remained undetectable for at least 2 days after removal of 50 microM O6-benzylguanine from the medium and recovered to only 53% of control values after an additional 3 days. AGT activity levels remained undetectable for at least 2 days after removal of 50 microM O6-benzylguanine from the medium and recovered to only 53% of control values after an additional 3 days. In contrast, following removal of 500 microM O6-methylguanine, the activity was restored from its nadir of 16% of control values reaching pretreatment levels after 5 days. These results suggest that treatment with O6-benzylguanine may be used to modulate the incidence of transforming mutations in cultured human breast epithelial cells treated with chemical carcinogens which give rise to O6-alkylguanine adducts.     

Case study of a patient with severe nephrotic syndrome

M.T. battled SLE for 9 years before renal failure occurred. She is now free of extrarenal symptoms of lupus, as has been described elsewhere (Ziff & Hilderman, 1983). The patient has regained here appetite, lost 13 pounds, recovered some strength and reestablished her social network. Without the severe proteinuria, M.T. has a chance to improve her nutritional status and increase her albumin. Her pulmonary status may also improve if she can refrain from smoking. Although fatigue is a debilitating feature of lupus, M.T. has increased stamina and is learning energy conservation techniques. Immediately after surgery, she did experience problems with bleeding, pneumonia, and pleural effusions, but the medical and nursing management prevented serious adverse outcomes. Fluid balance is no longer problematic, and M.T. is approaching her ideal body weight as her nutritional status improves. Her serum albumin has increased to about 2.6 gm/dl with some decrease in her proteinuria.     

Glutathione ester protects against hydroxynonenal-induced loss of auditory hair cells.

OBJECTIVE: Test the ability of glutathione monoethyl ester (GSH(e)) to protect auditory hair cells against the ototoxic effects of 4-hydroxy-2,3-nonenal (HNE). STUDY DESIGN AND SETTING: Organ of Corti explants were either untreated or treated with one of a series of four concentrations of GSH(e) for one day, then exposed to HNE. Counts of FITC-phalloidin-labeled hair cells determined both HNE ototoxicity and GSH(e) otoprotection. RESULTS: HNE was toxic to hair cells at physiologically relevant levels, eg, 400 muM, and GSH(e) provided a significant level of protection against HNE ototoxicity (P < 0.05) at all levels tested, ie, 1.16 to 9.3 mM. CONCLUSION: GSH(e) protects auditory hair cells from damage and loss initiated by a naturally occurring ototoxic molecule, ie, HNE (a by-product of oxidative stress). SIGNIFICANCE: Treatment with GSH(e) may be an effective therapy to protect the cochlea against the adverse effects of traumas (eg, electrode insertion) that generate oxidative stress.     

Congenital left atrial wall aneurysm in a patient with neurofibromatosis

A congenital intrapericardial aneurysmal dilatation of the left atrial wall was found in a 28 year old man who presented with atrial fibrillation after a syncopal event. The patient had cutaneous manifestations of neurofibromatosis. The diagnosis was made by cross sectional echocardiography and confirmed by angiocardiography. Surgical excision of the aneurysm resolved the symptoms.     

Evaluation of clinical criteria for the acute respiratory distress syndrome in pediatric patients.

OBJECTIVE: The primary goal of this study was to evaluate the validity of the North American-European Consensus Committee (NAECC) definition for acute respiratory distress syndrome (ARDS) in pediatric patients. A secondary aim was to evaluate the threshold value for the PaO2/FiO2 ratio, used to determine which pediatric patients have ARDS. DESIGN: Retrospective cohort study. SETTING: Pediatric intensive care unit. PATIENTS: Pediatric intensive care unit patients who required mechanical ventilation, died, and underwent autopsy between January 1, 1996, and December 31, 2002 (n = 34). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Clinical and chest radiograph information was collected retrospectively through chart review using a standardized data collection tool. Data included the criteria specified in the NAECC definition of ARDS and demographic information. We calculated the sensitivity, specificity, positive predictive value, negative predictive value, and likelihood ratio of clinical diagnosis of ARDS compared with a pathologic diagnosis. The threshold value of PaO2/FiO2 was identified by plotting receiver operating characteristics curves and comparing the areas under the curves. The NAECC definition yielded a sensitivity of 80.7% (95% confidence interval 60-92%), specificity of 71.4% (95% confidence interval 30-95), positive predictive value of 91.3% (95% confidence interval 70-98), negative predictive value of 50.0% (95% confidence interval 20-78), and likelihood ratio of 2.82. A PaO2/FiO2 <150 had a slightly higher (but not significantly different) specificity for ARDS than a value >200 (71% vs. 86%, p = .15) without changing sensitivity. CONCLUSIONS: Our study suggests the need for further research with larger number of children to identify an optimal Pao2/Fio2 threshold for identifying ARDS in this population.     

Mechanisms involved in uptake of Bordetella bronchiseptica by mouse dendritic cells.

The invasion and intracellular survival of Bordetella bronchiseptica in mouse dendritic cells were investigated. The results obtained suggest that B. bronchiseptica binds specifically to glycosylated receptors present on the plasma membrane of dendritic cells, thereby inducing a signal that triggers an actin polymerization-dependent phagocytic process, probably via a protein kinase-dependent transducing phosphorylation signal. The energy required for the uptake process by host cells is provided mainly by the glycolytic pathway. An intact microtubule system and de novo protein synthesis in eukaryotic and prokaryotic cells are essential for efficient uptake and intracellular survival. The interaction of B. bronchiseptica with dendritic cells may be pertinent to natural infections that follow a chronic clinical course and predispose to secondary infections, and to the T-cell response involved in protective immunity following infections caused by Bordetella spp.     

Antibody responses in the serum and respiratory tract of mice following oral vaccination with liposomes coated with filamentous hemagglutinin and pertussis toxoid.

Mice were orally vaccinated with liposomes coated with filamentous hemagglutinin (FHA) and detoxified pertussis toxin (PT) of Bordetella pertussis. FHA- and PT-specific immunoglobulin G (IgG) was detected in serum, and both IgG and IgA were detected in lung washes following the immunization. Antibody responses in mice immunized with liposomes coated with FHA and PT were significantly higher than those in mice immunized with free FHA and PT, which demonstrated the adjuvanticity of the liposome carrier. The results indicate the potential usefulness of this approach for eliciting immune responses against FHA and PT (and perhaps other pertussis antigens) in humans and its possible utility in large-scale vaccination to protect against both B. pertussis infection and disease.     

During active viscerocutaneous leishmaniasis the anti-P2 humoral response is specifically triggered by the parasite P proteins.

In this work we show that in the sera from dogs naturally infected with the protozoan parasite Leishmania infantum there are antibodies that react specifically against the parasite acidic ribosomal proteins LiP2a and LiP2b, and that each one of the Leishmania P proteins elicits a specific humoral immune response. Using synthetic peptides, the antigenic epitope of these proteins has been mapped in a single region located adjacent to the C-terminal domain highly conserved among the eukaryotic P proteins. The anti-P antibodies elicited during the Leishmania infection do not recognize the conserved C-terminal domain of the parasite P proteins, in contrast with the findings reported in Chagas' disease or systemic lupus erythematosus. The antigenic epitopes of the LiP2a and LiP2b are almost identical in amino acid sequence. No reactivity against Trypanosoma cruzi and human P proteins was found in sera from L. infantum-infected dogs.     

Quantitative Analysis of Mycobacterial and Propionibacterial DNA in Lymph Nodes of Japanese and European Patients with Sarcoidosis

The cause(s) of sarcoidosis is unknown. Mycobacterium spp. are suspected in Europe and Propionibacterium spp. are suspected in Japan. The present international collaboration evaluated the possible etiological links between sarcoidosis and the suspected bacterial species. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, one from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from two institutes in Japan and three institutes in Italy, Germany, and England. Genomes of Propionibacterium acnes, Propionibacterium granulosum, Mycobacterium tuberculosis, Mycobacterium avium subsp. paratuberculosis, and Escherichia coli (as the control) were counted by quantitative real-time PCR. Either P. acnes or P. granulosum was found in all but two of the sarcoid samples. M. avium subsp. paratuberculosis was found in no sarcoid sample. M. tuberculosis was found in 0 to 9% of the sarcoid samples but in 65 to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of P. acnes or P. granulosum were far more than those of M. tuberculosis. P. acnes or P. granulosum was found in 0 to 60% of the tuberculosis and control samples, but the total numbers of genomes of P. acnes or P. granulosum in such samples were less than those in sarcoid samples. Propionibacterium spp. are more likely than Mycobacteria spp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.