Olive pollen allergy: searching for immunodominant T-cell epitopes on the Ole e 1 molecule

BACKGROUND: The amino-acid and nucleotide sequence of Ole e 1 (the major antigen of olive pollen) has been described and the IgE antibody response to this major allergen was associated with DR7/DQ2 antigens. With this previous data we try to define the T-cell epitopes implicated in Ole e 1 reactivity. OBJECTIVES: To study the recognition of T cells (derived from allergic and non-allergic Ole e 1 patients) to Ole e 1 synthetic peptides in order to define immunodominant T-cell epitopes. METHODS: We have compared the proliferative response of the peripheral blood mononuclear cells from Ole e 1 sensitized patients vs. non-sensitized controls, induced by 14 Ole e 1 synthetic peptides. Thirty subjects were classified in two groups: group 1 (non-responders against Ole e 1, n=16) and group 2 (Ole e 1 responders, n=14), according to their clinical parameters and the presence or not in their sera of the significant Ole e 1 IgE antibody levels. RESULTS: Our results shown that it is possible to find T cells reactive to Ole e 1 peptides in patients with and without significant levels of Ole e 1 IgE antibodies. However, the percentage of response was higher in patients with IgE antibodies 71.4% vs 25%), and the recognition profile was different: the control group showed a broad reactivity pattern, in contrast, the response by the 'Ole e 1 responders' group was mainly directed against three peptides of the carboxi-terminal region, peptides 10 (91-102), 12 (109-120) and 13 (119-130), with a response frequency of 35.7, 28.5 and 28.5%, respectively. By direct and inhibition test no antibody response was found against the synthetic peptides. CONCLUSIONS: Our data suggest that the regions between 91 and 102 and 109-130 aminoacids on the Ole e 1 molecule are immunodominant T-cell epitopes. These epitopes are not recognized by IgE antibodies.     

Staphylococcal enterotoxin B primes cytokine secretion and lytic activity in response to native bacterial antigens

Superantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured with Streptococcus mutans produced significantly greater amounts of gamma interferon (IFN-gamma) and interleukin-12 than did sham-injected controls. The majority of IFN-gamma production appeared to be CD8(+) T-cell derived since depletion of this cell type dramatically reduced the levels of IFN-gamma. To study host cell damage that may occur following superantigen exposure, we analyzed cytotoxicity to "bystander" fibroblast cells cultured with splenocytes in the presence of bacterial antigens. Prior host exposure to staphylococcal enterotoxin B significantly enhanced fibroblast cytotoxicity in the presence of bacteria. Neutralization of IFN-gamma decreased the amount of cytotoxicity observed. However, a greater reduction was evident when splenocyte-bacterium cocultures were separated from the bystander cell monolayer via a permeable membrane support. Increased cytotoxicity appears to be primarily dependent upon cell-cell contact. Collectively, these data indicate that overproduction of inflammatory cytokines may alter the activity of cytotoxic immune cells. Superantigen exposure exacerbates cytokine production and lytic cell activity when immune cells encounter bacteria in vitro and comparable activities could possibly occur in vivo.     

Expression in human prostate of drug- and carcinogen-metabolizing enzymes: association with prostate cancer risk

The role of two common polymorphisms of enzymes involved in the metabolism of drugs and carcinogens was studied in relation to prostate cancer. The gene encoding one of these enzymes (NAT2) is located in an area where frequent allelic loss occurs in prostate cancer. Mutations at the genes CYP2D6 and NAT2 were analysed by allele-specific polymerase chain reaction and restriction mapping in DNA from 94 subjects with prostate cancer and 160 male healthy control subjects. Eleven prostate specimens were analysed for genotype and enzymatic activities NAT2, CYP2D6 and CYP3A by using the enzyme-specific substrates sulphamethazine and dextromethorphan. Enzyme activities with substrate specificities corresponding to NAT2, CYP2D6 and CYP3A are present in human prostate tissue, with mean +/-s.d. activities of 4.8+/-4.4 pmol min(-1) mg(-1) protein, 156+/-91 and 112+/-72 nmol min(-1) mg(-1) protein respectively. The Km values for the prostate CYP2D6 and CYP3A enzyme activities corresponded to that of liver CYP2D6 and CYP3A activities, and the CYP2D6 enzyme activity is related to the CYP2D6 genotype. The N-acetyltransferase, in contrast, had a higher Km than NAT2 and was independent of the NAT2 genotype. The CYP2D6 and CYP3A enzymes, and an N-acetyltransferase activity that is independent of the regulation of the NAT2 gene, are expressed in human prostate tissue. The presence of carcinogen-metabolizing enzymes in human prostate with a high interindividual variability may be involved in the regulation of local levels of carcinogens and mutagens and may underlie interindividual differences in cancer susceptibility.     

State-of-the-art principles and practices of medical economics

Genomic DNA from 23 patients with isolated growth hormone (GH) deficiency (12 males and 11 females: heights -4.9 +/- 1.4 SDS) was screened for GH gene deletions by restriction endonuclease analysis of polymerase chain reaction amplification products. Three unrelated patients had typical features of severe GH deficiency and deletions (6.7 kb in two and 7.6 kb in one) of the GH gene. The two patients with 6.7-kb deletions developed growth-attenuating anti-GH antibodies whereas the patient with the 7.6-kb deletion continued to grow with GH replacement therapy. Our finding that 3/23 (approximately 13%) Brazilian subjects had GH gene deletions agrees with previous studies of severe isolated GH deficiency subjects in other populations. Two of three subjects (67%) with deletions developed blocking antibodies despite administration of exogenous GH at low doses. Interestingly, only 1/10 of cases with affected relatives or parental consanguinity had GH-1 gene deletions.     

The effect of changing excitation frequency on parallel conductance in different sized hearts

OBJECTIVE: An important component of the ventricular volume measured using the conductance catheter technique is due to parallel conductance (Vc), which results from the extension of the electric field beyond the ventricular blood pool. Parallel conductance volume is normally estimated using the saline dilution method (Vc(saline dilution)), in which the conductivity of blood in the ventricle is transiently increased by injection of hypertonic saline. A simpler alternative has been reported by Gawne et al. [12]. Vc(dual frequency) is estimated from the difference in total conductance measured at two exciting frequencies and the method is based on the assumption that parallel conductance is mainly capacitive and hence is negligible at low frequency. The objective of this study was to determine whether the dual frequency technique could be used to substitute the saline dilution method to estimate Vc in different sized hearts. METHODS: The accuracy and linearity of a custom-built conductance catheter (CC) system was initially assessed in vitro. Subsequently, a CC and micromanometer were inserted into the left ventricle of seven 5 kg pigs (group 1) and six 50 kg pigs (group 2). Cardiac output was determined using thermodilution (group 1) and an ultrasonic flow probe (group 2) from which the slope coefficient (alpha) was determined. Steady state measurements and Vc estimated using saline dilution were performed at frequencies in the range of 5-40 kHz. All measurements were made at end-expiration. Finally, Vc was estimated from the change in end-systolic conductance between 5 kHz and 40 kHz using the dual frequency technique of Gawne et al. [12]. RESULTS: There was no change in measured volume of a simple insulated cylindrical model when the stimulating frequency was varied from 5-40 kHz. Vc(saline dilution) varied significantly with frequency in group 1 (8.63 +/- 2.74 ml at 5 kHz; 11.51 +/- 2.65 ml at 40 kHz) (p = 0.01). Similar results were obtained in group 2 (69.43 +/- 27.76 ml at 5 kHz; 101.24 +/- 15.21 ml at 40 kHz) (p < 0.001). However, the data indicate that the resistive component of the parallel conductance is substantial (Vc at 0 Hz estimated as 8.01 ml in group 1 and 62.3 ml in group 2). There was an increase in alpha with frequency in both groups but this did not reach significance. The correspondence between Vc(dual frequency) and Vc(saline dilution) methods was poor (group 1 R2 = 0.69; group 2 R2 = 0.22). CONCLUSION: At a lower excitation frequency of 5 kHz a smaller percentage of the electric current extends beyond the blood pool so parallel conductance is reduced. While parallel conductance is frequency dependent, it has a substantial resistive component. The dual frequency method is based on the assumption that parallel conductance is negligible at low frequencies and this is clearly not the case. The results of this study confirm that the dual frequency technique cannot be used to substitute the saline dilution technique.     

Pyogenic arthritis of the sacro-iliac joint. Long-term follow-up

Nine cases of sacro-iliac pyarthrosis are presented. The difficulty in localizing the infection is attributable mostly to failure to appreciate the posteriorly situated physical findings. This, and the difficulty with early roentgenographic demonstration of the lesion, may lead to unnecessary abdominal exploration (as in two of our patients) or to prolonged delay in diagnosis and hence spread of the infection. Awareness of the usual physical findings and prompt use of radioisotope scanning to localize the infection led to earlier diagnosis and avoidance of surgery in three patients seen recently. Antibiotic therapy, with or without surgery, led to cure in all patients, with minimum sequelae.     

Purification and characterization of Clostridium difficile glutamate dehydrogenase

Recombinant Clostridium difficile glutamate dehydrogenase (L-glutamate:NAD oxidoreductase, EC 1.4.1.2) was purified 177-fold to electrophoretic homogeneity with a 62% recovery through a four-step procedure involving gel filtration and ion-exchange and dye affinity chromatography. The approximate molecular weights of the native enzyme by gel filtration and subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were consistent with a hexameric structure for the purified enzyme. The enzyme-catalyzed glutamate oxidation was an NAD-dependent sequential process in which NADP could not be substituted as coenzyme. Several dinucleotide analogs of NAD structurally altered in either the pyridine or the purine moiety were observed to function as coenzymes when substituted for NAD. Nicotinamide mononucleotide did not serve as a coenzyme for glutamate oxidation. Product inhibition by NADH was competitive with respect to NAD. In deadend inhibition studies, adenosine diphosphoribose was shown to be an effective coenzyme-competitive inhibitor.     

Mechanical consequences of core drilling and bone-grafting on osteonecrosis of the femoral head

We employed an anatomically realistic three-dimensional finite-element model to explore several biomechanical variables involved in coring or bone-grafting of a segmentally necrotic femoral head. The mechanical efficacy of several variants of these procedures was indexed in terms of their alteration of the stress:strength ratio in at-risk necrotic cancellous bone. For coring alone, the associated structural compromise was generally modest, provided that the tract did not extend near the subchondral plate. Cortical bone-grafting was potentially of great structural benefit for femoral heads in which the graft penetrated deeply into the superocentral or lateral aspect of the lesion, ideally with abutment against the subchondral plate. By contrast, central or lateral grafts that stopped well short of the subchondral plate were contraindicated biomechanically because they caused marked elevations in stress on the necrotic cancellous bone. Calculated levels of stress were relatively insensitive to variations in the diameter of the graft.     

Restoration of squamous mucosa after ablation of Barrett's esophageal epithelium

BACKGROUND: Antireflux therapy has generally failed to induce regression of Barrett's epithelium. It was hypothesized that squamous epithelium could be restored if the columnar tissue was ablated while gastric acid secretion was suppressed. METHODS: Ten white men with Barrett's esophagus received 40 mg of omeprazole daily. Thereafter, every 2-5 weeks they underwent videotaped endoscopies to argon laser photoablate columnar tissue, obtain biopsy specimens, and assess results. Squamous re-epithelialization was assessed by correlation of videotapes and directed biopsies. RESULTS: Patients had one to eight areas ablated, totaling 0.5-12.0 cm2. Videotape assessments were corroborated by biopsy in all but one instance. Thirty-eight of 40 treatment locations partially or completely re-epithelialized with squamous tissue. Squamous regrowth appeared to occur by spread from contiguous squamous borders and de novo from glandular tissue. Regrowth was influenced by the extent of squamous borders and completeness of ablations. Nonablated glandular tissue persisted beneath squamous epithelium. CONCLUSIONS: Ablation of Barrett's epithelium and suppression of acid secretion facilitated squamous re-epithelialization. A progenitor cell within the metaplastic tissue has the potential to differentiate normally.