The effect of the interferon-gamma-inducible processing machinery on the generation of a naturally tumor-associated human cytotoxic T lymphocyte epitope within a wild-type and mutant p53 sequence context

The human wild-type (wt) p53.264-272 peptide is a universal tumor antigen and recognized by HLA-A*0201 (A2.1)-restricted CTL. Generation of this epitope by constitutive 20S proteasomes is prevented by a p53 R to H hotspot mutation at the C-terminal flanking residue 273. We report on the impact of the interferon-gamma (IFN-gamma)-inducible proteasomal activator PA28 (11S regulator) and the immunoproteasome on the in vitro and cellular processing of wt and mutant (mut) p53 substrates. We found that production of the antigenic 264-272 peptide from wt p53 by constitutive as well as immunoproteasomes is accelerated and amplified by the PA28 activator. PA28 and (immuno)proteasomes were not capable to reconvert the resistance of epitope release from mut p53. Maximum and accelerated antigen production in vitro and on the cellular level required the IFN-gamma-inducible interaction of immunoproteasomes and PA28. We conclude that efficient processing of p53.264272 from wt p53 is governed by the proteasome/PA28 complex. These studies have important implications for p53-specific cancer immunotherapy and demonstrate that the effects of the immunoproteasome and PA28 are influenced by the individual epitope and its flanking sequence context.     

Detection of expansion regions in Portuguese bipolar families

We have studied 24 families with multiple affected members with bipolar disorder to test the hypothesis that in those families clinically showing genetic anticipation [Macedo et al., 1999] we would find large repeat expansions. The families meeting inclusion criteria had a minimum of two affected members over two generations and showed marked anticipation both in terms of age of onset and disease severity. We used the repeat expansion detection (RED) method to test patients (n = 24) and controls from these families and unrelated controls (n = 53). We also genotyped patients and family members from two families with large expansions at the known expansion loci on chromosomes 13, 17, and 18. The RED method revealed a higher number of large expansions in patients compared with controls (t-test; P < 0.0055: Mann-Whitney U; P = 0.02). The patients with the largest expansions were typed at the specific loci on chromosomes 13, 17, and 18 and the chromosome 18 expansion locus segregated with disease in one family, and a second family showed segregation with the expansion located at the SCA8 locus on chromosome 13. Genetic anticipation had been analyzed in this cohort of families, with correction for potential ascertainment bias, possible proband effects, cohort effects, regression to the mean, gender effects, and maternal vs. paternal transmission. None of these potential confounds appeared to account for the observed anticipation. We also identified that the presence of large expansions in affected family members derives primarily from two families from the genetically isolated Azores population. One family shows segregation with the chromosome 18 locus, whereas the other family segregates with expansions at the SCA8 locus. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:854-857, 2000.     

Rheumatic heart disease: proinflammatory cytokines play a role in the progression and maintenance of valvular lesions

Heart lesions of rheumatic heart disease (RHD) patients contain T-cell clones that recognize heart proteins and streptococcal M peptides. To functionally characterize heart-infiltrating T lymphocytes, we evaluated their cytokine profile, both directly in situ and in T-cell lines derived from the heart (HIL). Interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, and IL-10 expressions were characterized in 20 heart tissue infiltrates from 14 RHD patients by immunohistochemistry. IFN-gamma-, TNF-alpha-, and IL-10-positive cells were consistently predominant, whereas IL-4 was scarce in the valves. In agreement with these data, the in vitro experiments, in which 13 HILs derived from heart samples of eight patients were stimulated with M5 protein and the immunodominant M5 (81-96) peptide, IL-4 was detected in HIL derived from the atrium (three of six) but not from the valve (zero of seven). IFN-gamma and IL-10 production were detected in culture supernatants in 11 of 13 and 6 of 12 HILs, respectively. The predominant IFN-gamma and TNF-alpha expression in the heart suggests that Th1-type cytokines could mediate RHD. Unlike in reversible myocardium inflammation, the significantly lower IL-4 expression in the valvular tissue (P = 0.02) may contribute to the progression of the RHD leading to permanent valvular damage (relative risk, 4.3; odds ratio, 15.8). The lack of IL-4 in vitro production by valve-derived HIL also emphasizes the more severe tissue destruction in valves observed in RHD.     

Outbreak of carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme in Curitiba,Brazil

Carbapenem-resistant Acinetobacter baumannii isolates were obtained from eight patients in two hospitals in Curitiba, Brazil. The isolates were multiresistant, belonged to a single strain, and produced the OXA-23 carbapenemase. Treatment options were limited, although the isolates were susceptible to polymyxin B in vitro. The strain contributed to the deaths of five patients.     

Immunoglobulins and other serological parameters in Chagas'' disease: evidence for increased IgA levels in the chronic digestive form.

Immunoglobulin levels were measured in serum samples from 36 patients with different clinical forms of chronic Chagas' disease. Increased IgA levels were observed in 50% of the patients in the chronic digestive group and there was a significant correlation with the severity of the disease. IgG and IgM levels were within the normal range. Anti-ssDNA antibodies and EVI (endothelium, vessels and interstitium) antibodies were found in some patients with different clinical forms of the disease.     

Salivary immunoglobulins in a patient with IgA deficiency

The saliva of an individual with selective IgA deficiency was found to contain IgG and IgM, with some of the IgM linked to secretory component. Some specimens showed evidence of low molecular weight immunoglobulin fragments, presumed to be the result of proteolysis.     

Kinetics of estimated human muscle capillary blood flow during recovery from exercise

The kinetic characteristics of muscle capillary blood flow (Qcap) during recovery from exercise are controversial (e.g. one versus two phases). Furthermore, it is not clear how the overall Qcap kinetics are temporally associated with muscle oxygen uptake (VO2m) kinetics. To address these issues, we examined the kinetics of Qcap estimated from the rearrangement of the Fick equation (Qcap=VO2m/C(a-v)O2) using the kinetics of pulmonary VO2 (VO2p, primary component) and deoxy-haemoglobin concentration ([HHb]) as indices of VO2m and C(a - v)O2 (arterio-venous oxygen difference) kinetics, respectively. VO2p (l min-1) was measured breath by breath and [HHb] (microm) was measured by near infrared spectroscopy during moderate (M; below lactate threshold, LT) and heavy exercise (H, above LT) in nine subjects. The kinetics of Qcap were biphasic, with an initial fast phase (tauI; M=9.3+/-4.9 s and H=6.0+/-3.8 s) followed by a slower phase 2 (tauP; M=29.9+/-8.6 s and H=47.7+/-26.0 s). For moderate exercise, the overall kinetics of Qcap (mean response time [MRT], 36.1+/-8.6 s) were significantly slower than the kinetics of VO2p (tauP; 27.8+/-5.3 s) and [HHb] (MRT for [HHb]; 16.2+/-6.3 s). However, for heavy exercise, there was no significant difference between MRT-[HHb] (34.7+/-10.4 s) and tauP for VO2p (32.3+/-6.7 s), while MRT for Qcap (48.7+/-21.8 s) was significantly slower than MRT for [HHb] and tauP for VO2p. In conclusion, during recovery from exercise the estimated Qcap kinetics were biphasic, showing an early rapid decrease in blood flow. In addition, the overall kinetics of Qcap were slower than the estimated VO2m kinetics.     

Human bone cell cultures in biocompatibility testing. Part I: osteoblastic differentiation of serially passaged human bone marrow cells cultured in alpha-MEM and in DMEM

Well-characterised human osteoblastic bone marrow cell cultures are a useful in vitro tool to analyse bone tissue/biomaterials interactions. In this work, human bone marrow was cultured in experimental conditions described to favour osteoblastic differentiation and, serially passaged cells were cultured in two widely used culture media, minimum essential medium Eagle, alpha modification (alpha-MEM) and Dulbecco's modified Eagle's medium (DMEM). Cultures were grown for 35 d and compared concerning morphologic appearance on scanning electron microscopy (SEM), cell viability/proliferation, total protein content, activity of alkaline phosphatase (ALP) and ability to form calcium phosphate deposits. Results showed that cell proliferation was similar in cultures grown in the two media but ALP activity and ability to form mineralised deposits were lower in DMEM cultures. In both experimental situations, osteoblastic parameters were strongly reduced on cell passage, particularly from the first to the second subculture. In the experimental conditions used (presence of ascorbic acid, sodium beta-glycerophosphate and dexamethasone in the primary and secondary cultures), osteoblastic differentiation was observed in the first and second subcultures grown in alpha-MEM and in the first subculture grown in DMEM. These results underline the importance of the definition of the experimental conditions in studies involving bone cell cultures.     

Determination of Blomia tropicalis-specific IgE and IgG subclasses in atopic dermatitis patients

BACKGROUND: To verify the importance of Blomia tropicalis in atopic dermatitis (AD), we determined the cutaneous reactivity and the serum level of B. tropicalis-specific IgE and IgG subclasses in AD patients. METHODS: B. tropicalis-specific IgE and IgG subclasses were determined in AD patients and compared with bronchial asthma (BA) patients and a control group (CG) of nonatopic subjects. Specific IgE was obtained by skin prick test and RAST. B. tropicalis-specific IgG subclasses were determined by ELISA. The data were statistically analyzed by chi-square test (Mantel-Haenszel) and odds ratio (OR). RESULTS: We detected positive skin prick tests in 61.76% of AD and 83.33% of BA patients, and in 12.5% of the CG. RAST was positive in 44.12% of AD and in 61.90% of BA patients, but not in the CG. B. tropicalis-specific IgG1 and IgG2 subclasses showed no significant differences between the three groups. IgG3 subclass positivity was statistically significant in AD patients (41.17%) when compared to BA patients (14.29%) and the CG (16.67%). The determination of B. tropicalis-specific IgG4 was positive in 32.35% of AD patients, 21.43% of BA patients, and 8.33% of the CG. CONCLUSIONS: These results confirm that the storage mite B. tropicalis is an important allergen in AD. It is possible that IgG3 activates the complement in AD patients, releasing vasoactive amines that further amplify the allergic reaction. The positive results of the B. tropicalis-specific IgG4 found in AD and BA were probably due to chronic exposure to this storage mite in the home environment.