Chronic kidney disease in patients with childhood-onset systemic lupus erythematosus.

BACKGROUND: Lupus nephritis (LN) is a frequent manifestation of childhood-onset systemic lupus erythematosus (cSLE) with a potential risk for kidney failure and poor outcomes. This study aimed to evaluate stages III, IV, and V of chronic kidney disease (CKD) and investigate risk factors for CKD in cSLE patients. METHODS: We performed a nationwide observational cohort study in 27 pediatric rheumatology centers, including medical charts of 1528 cSLE patients. Data were collected at cSLE diagnosis, during follow-up, and at last visit or death, between September 2016 and May 2019. RESULTS: Of 1077 patients with LN, 59 (5.4%) presented with CKD, 36/59 (61%) needed dialysis, and 7/59 (11.8%) were submitted for kidney transplantation. After Bonferroni's correction for multiple comparisons (p < 0.0013), determinants associated with CKD were higher age at last visit, urinary biomarker abnormalities, neuropsychiatric involvement, higher scores of disease activity at last visit and damage index, and more frequent use of methylprednisolone, cyclosporine, cyclophosphamide, and rituximab. In the regression model analysis, arterial hypertension (HR = 15.42, 95% CI = 6.12-38.83, p ≤ 0.001) and biopsy-proven proliferative nephritis (HR = 2.83, 95%CI = 1.70-4.72, p ≤ 0.001) increased the risk of CKD, while children using antimalarials had 71.0% lower CKD risk ((1.00-0.29) × 100%) than children not using them. The Kaplan-Meier comparison showed lower survival in cSLE patients with biopsy-proven proliferative nephritis (p = 0.02) and CKD (p ≤ 0.001). CONCLUSIONS: A small number of patients manifested CKD; however, frequencies of dialysis and kidney transplantation were relevant. This study reveals that patients with cSLE with hypertension, proliferative nephritis, and absence of use of antimalarials exhibited higher hazard rates of progression to CKD. A higher resolution version of the Graphical abstract is available as Supplementary information.

Childhood-onset systemic lupus erythematosus-related antiphospholipid syndrome: A multicenter study with 1519 patients.

OBJECTIVE: To assess childhood-onset systemic lupus erythematosus-related antiphospholipid syndrome(cSLE-APS) in a large Brazilian population. METHODS: A retrospective observational cohort study was carried-out in 27 Pediatric Rheumatology university centers, including 1519 cSLE patients. RESULTS: cSLE-APS was observed in 67/1519 (4%) and was diagnosed at disease onset in 39/67 (58%). The median disease duration was 4.9 (0-17) years. Thrombosis recurrences were evidenced in 18/67 (27%) cSLE-APS patients. The most frequent thrombosis sites in cSLE-APS patients were: venous thrombosis in 40/67 (60%), especially deep vein thrombosis in 29/40 (72%); arterial thrombosis in 35/67 (52%), particularly stroke; small vessels thrombosis in 9/67 (13%) and mixed thrombosis in 3/67 (4%). Pregnancy morbidity was observed in 1/67 (1%). Non-thrombotic manifestation associated to cSLE-APS occurred in 21/67 (31%), mainly livedo reticularis in 14/67 (21%), valvar thickening in 4/67 (6%) and valvar vegetations not related to infections in 2/67 (3%). None of them had catastrophic APS. Further analysis demonstrated that the median of SLICC/ACR-DI [1(0-5) vs. 0(0-7),p < 0.0001] was significantly higher in cSLE-APS patients compared to cSLE without APS. The frequencies of cerebrovascular disease (40% vs. 1%,p < 0.0001), polyneuropathy (9% vs. 1%,p < 0.0001), SLICC/ACR-DI ≥1 (57% vs. 27%, p < 0.0001) and intravenous cyclophosphamide use (59% vs. 37%, p < 0.0001) were significantly higher in the former group. CONCLUSIONS: Our large multicenter study demonstrated that cSLE-APS was a rare condition, occurring during disease course with a high accrual damage. Central and peripheral neuropsychiatric involvements were distinctive features of this autoimmune thrombosis.

Disease presentation of 1312 childhood-onset systemic lupus erythematosus: influence of ethnicity.

OBJECTIVE: To evaluate the influence of ethnicity in presentation of childhood-onset systemic lupus erythematosus (cSLE) patients. METHODS: This multicenter study included cSLE patients (American College of Rheumatology criteria) followed in 27 Pediatric Rheumatology services of Brazil. Ethnicities were classified in four groups according to the parents' and all four grandparents' self-reported ethnicity. The statistical analysis was performed using the Bonferroni's correction (p < 0.0027). RESULTS: According to ethnic groups, 1537 cSLE patients were classified in Caucasian (n = 786), African-Latin American (n = 526), Asian (n = 8), and others/unknown (n = 217). Comparisons between 1312 African-Latin American and Caucasian revealed similar median age at cSLE diagnosis [12.2(2.6-18) vs. 12.1(0.3-18) years, p = 0.234], time interval to diagnosis [0.25(0-12) vs. 0.3(0-10) years, p = 0.034], and SLEDAI-2K score [14(0-55) vs. 14(0-63), p = 0.781] in both groups. The mean number of diagnostic criteria according to SLICC (6.47 ± 1.911 vs. 5.81 ± 1.631, p < 0.0001) and frequencies of maculopapular lupus rash (8% vs. 3%, p < 0.0001), palate oral ulcers (17% vs. 11%, p = 0.001), tongue oral ulcers (4% vs. 1%, p = 0.001), and nonscarring alopecia (29% vs. 16%, p < 0.0001) were significantly higher in African-Latin American, whereas malar rash (45% vs. 58%, p < 0.0001) was more frequent in Caucasian. The presence of anti-phospholipid antibody (23% vs. 12%, p < 0.0001), low complement levels (58% vs. 41%, p < 0.0001), and isolated direct Coombs test (10% vs. 5%, p = 0.001) was also significantly higher in the former group. CONCLUSIONS: Our study demonstrated that disease presentation severity of African-Latin American cSLE patients is comparable with Caucasian. Mucocutaneous manifestations and autoantibodies profile were the only distinctive features of the former group. The unique mixed background of Brazilian patients probably minimized race diversity spectrum of these patients. Key Points • Our study demonstrated that disease presentation severity of African-Latin American cSLE patients is comparable with Caucasian. • Mucocutaneous manifestations and autoantibodies profile were the only distinctive features of African-Latin American cSLE patients. • African-Latin American cSLE patients had more often anti-phospholipid antibodies and hypocomplementemia. • The unique mixed background of Brazilian patients probably minimized race diversity spectrum of these patients.

Evaluation of metabolic reactivity in macrophages from mice with chronic sodium arsenite intake and experimental carcinogenesis.

 Arsenic is carcinogenic to human beings, and environmental exposure to arsenic is a public health issue that affects large populations around the world. Thus, studies are needed to determine the mode of action of arsenic and to prevent harmful effects that arise from arsenic intake. In particular, knowledge of the effects of arsenic exposure in individuals who are undergoing a carcinogenesis process is lacking. The present study was performed in mice to evaluate the effect of chronic As3+ administration on peritoneal and alveolar macrophages; the As3+ was administered in drinking water over 9 months and there was a two-stage carcinogenesis process. At the end of the experiment, the number of tumors stabilized to below the control values, but the tumors showed increased malignancy. Our objective was to evaluate the systemic effects of chronic As3+ingestion in a population of macrophages that was derived from the peritoneal cavity and the broncho-alveolar trunk of cancerized mice since they are the first line of defense in the immune system. The results showed that the macrophages under all conditions retained their ability to self-regulate their metabolic reactivity. This feature was more evident in peritoneal macrophages than in alveolar macrophages. Furthermore, an increase in the number of macrophages from animals receiving higher doses of As3+ compared to untreated animals was observed. These findings indicate that certain parameters associated with two-stage skin carcinogenesis are modified by the presence of As3+ in drinking water.

Symptomatic polyautoimmunity at diagnosis of 1463 childhood-onset lupus: A Brazilian multicenter study.

OBJECTIVE: To evaluate symptomatic polyautoimmunity (PA) at childhood-onset systemic lupus erythematosus(cSLE) diagnosis, and its association with demographic data, disease activity, clinical manifestations and laboratorial abnormalities in a large Brazilian cSLE population. METHODS: A multicenter retrospective study was performed in 1463 cSLE(ACR criteria) patients from 27 Pediatric Rheumatology services. Symptomatic PA was defined according to the presence of more than one concomitant autoimmune disease(AD) and symptomatic multiple autoimmune syndrome(MAS) was defined as three or more AD. An investigator meeting was held to define the protocol. Demographic data, SLICC classification criteria and SLEDAI-2K were evaluated. RESULTS: At cSLE diagnosis symptomatic PA was observed in 144/1463(9.8%) and symptomatic MAS occurred in solely 10/1463(0.7%). In the former group the more frequently observed associated AD were Hashimoto thyroiditis n = 42/144(29%), antiphospholipid syndrome n = 42/144(29%), autoimmune hepatitis n = 26/144(18%) and type 1 diabetes mellitus n = 23/144(15.9%). Further comparisons between cSLE patients with and without PA showed a higher median age(p = 0.016) and lower mean SLICC criteria (p = 0.039) in those with PA. Additionally, these cSLE patients had less renal involvement(35% vs. 44%, p = 0.038) and red blood cell cast(6% vs. 12%, p = 0.042) and more antiphospholipid antibodies(29% vs. 15%, p < 0.0001). CONCLUSIONS: Approximately 10% of cSLE had symptomatic PA at diagnosis, particularly endocrine autoimmune disorders and antiphospholipid syndrome. Lupus was characterized by a mild disease onset and MAS was infrequently evidenced. Further studies are necessary to determine if this subgroup of cSLE patients have a distinct genetic background with a less severe disease and a better long-term outcome.

Diffuse alveolar hemorrhage in childhood-onset systemic lupus erythematosus: a severe disease flare with serious outcome.

OBJECTIVE: To evaluate prevalence, clinical manifestations, laboratory abnormalities and treatment in a multicenter cohort study including 847 childhood-onset systemic lupus erythematosus (cSLE) patients with and without diffuse alveolar hemorrhage (DAH), as well as concomitant parameters of severity. METHODS: DAH was defined as the presence of at least three respiratory symptoms/signs associated with diffuse interstitial/alveolar infiltrates on chest x-ray or high-resolution computer tomography and sudden drop in hemoglobin levels. Statistical analysis was performed using Bonferroni correction (p < 0.0022). RESULTS: DAH was observed in 19/847 (2.2%) cSLE patients. Cough/dyspnea/tachycardia/hypoxemia occurred in all cSLE patients with DAH. Concomitant parameters of severity observed were: mechanical ventilation in 14/19 (74%), hemoptysis 12/19 (63%), macrophage activation syndrome 2/19 (10%) and death 9/19 (47%). Further analysis of cSLE patients at DAH diagnosis compared to 76 cSLE control patients without DAH with same disease duration [3 (1-151) vs. 4 (1-151) months, p = 0.335], showed higher frequencies of constitutional involvement (74% vs. 10%, p < 0.0001), serositis (63% vs. 6%, p < 0.0001) and sepsis (53% vs. 9%, p < 0.0001) in the DAH group. The median of disease activity score(SLEDAI-2 K) was significantly higher in cSLE patients with DAH [18 (5-40) vs. 6 (0-44), p < 0.0001]. The frequencies of thrombocytopenia (53% vs. 12%, p < 0.0001), intravenous methylprednisolone (95% vs. 16%, p < 0.0001) and intravenous cyclophosphamide (47% vs. 8%, p < 0.0001) were also significantly higher in DAH patients. CONCLUSIONS: This was the first study to demonstrate that DAH, although not a disease activity score descriptor, occurred in the context of significant moderate/severe cSLE flare. Importantly, we identified that this condition was associated with serious disease flare complicated by sepsis with high mortality rate.

Effect of Sodium Arsenite on Mouse Skin Carcinogenesis.

Arsenic is carcinogenic in human beings, and environmental exposure to arsenic is a public health issue that affects large populations worldwide. Thus, studies are needed to determine the mode of action of arsenic and prevent harmful effects arising from arsenic intake. The present study assessed the influence of sodium arsenite (As(3+)) on potentially carcinogenic processes that are either pre-existing or concomitant with chronic intake of water containing As(3+). Experiments using SenCar mice were designed to evaluate the effect of chronic administration of As(3+) (2, 20, or 200 mg of As(3+)/L) in drinking water that overlapped to varying degrees with a 2-stage carcinogenesis protocol carried out over 9 months. The results showed a time-dependent pattern. During early stages of carcinogenesis (6-12 weeks), animals exposed to As(3+) and the carcinogenesis protocol showed increased numbers of tumors compared to control animals. During late carcinogenesis (16-30 weeks), the number of tumors stabilized to below control values, but the tumors showed increased malignancy. These findings indicate that the outcomes of the 2-stage skin carcinogenesis protocol are modified by the presence of arsenite in drinking water, which increases the rate of carcinoma development.

Induction and persistence of large γH2AX foci by high linear energy transfer radiation in DNA-dependent protein kinase-deficient cells.

PURPOSE: To evaluate the cell response to DNA double-strand breaks induced by low and high linear energy transfer (LET) radiations when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an essential protein of the nonhomologous end-joining repair pathway, lacks kinase activity. METHODS AND MATERIALS: CHO10B2, a Chinese hamster ovary cell line, and its derived radiosensitive mutant cell line, irs-20, lacking DNA-PKcs activity, were evaluated after 0 to 3 Gy of γ-rays, plateau and Bragg peak protons, and lithium beams by clonogenic assay, and as a measurement of double-strand breaks, phosphorylated H2AX (γH2AX) foci number and size were quantified by immunocytofluorescence. RESULTS: Irs-20 exhibited greater radiosensitivity and a higher amount of γH2AX foci than CHO10B2 at 6 hours after irradiation for all types of radiations. Remarkably, CHO10B2 and irs-20 maintained their difference in radiosensitivity after high-LET radiation. Six hours after low-LET radiations, irs-20 did not reach basal levels of γH2AX at high doses, whereas CHO10B2 recovered basal levels for all doses. After high-LET radiation, only CHO10B2 exhibited a reduction in γH2AX foci, but it never reached basal levels. Persistent foci in irs-20 confirmed a repair deficiency. Interestingly, after 30 minutes of high-LET radiation both cell lines exhibited large foci (size>0.9 µm2) related to the damage nature, whereas at 6 hours irs-20 showed a higher amount of large foci than CHO10B2, with a 7-fold increase at 3 Gy, that could also be associated to radiosensitivity. CONCLUSIONS: We demonstrated, for the first time, an association between deficient DNA-PKcs activity and not only high levels of H2AX phosphorylation but also persistence and size increase of γH2AX foci after high-LET irradiation.

Phosphorylation and subcellular localization of p27Kip1 regulated by hydrogen peroxide modulation in cancer cells.

The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H(2)O(2)) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H(2)O(2) removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H(2)O(2) (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H(2)O(2) scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1.

H2O2 scavenging inhibits G1/S transition by increasing nuclear levels of p27KIP1.

The aim of the present study was to evaluate cell cycle regulation by scavenging H(2)O(2) in tumor cells. A significant arrest in the G1 phase of the cell cycle was demonstrated in CH72-T4 carcinoma cells exposed to catalase, associated with a decrease in cyclin D1 and an increase in the CDK inhibitory protein p27(KIP1). Moreover, we found a differential intracellular distribution of p27(KIP1), which remained in the nucleus after catalase treatment. In vivo experiments showed an increase in nuclear levels of p27(KIP1) associated with the inhibition of tumor growth by H(2)O(2) scavenging, confirming in vitro results. To conclude, H(2)O(2) scavenging may induce cell cycle arrest through the modulation of cyclin D1 and p27(KIP1) levels and nuclear localization of p27(KIP1). To our knowledge, this is the first report that demonstrates that the modulation of ROS alters the intracellular localization of a key regulatory protein of G1/S transition.

Induction and rejoining of DNA double strand breaks assessed by H2AX phosphorylation in melanoma cells irradiated with proton and lithium beams.

PURPOSE: The aim of this study was to evaluate the induction and rejoining of DNA double strand breaks (DSBs) in melanoma cells exposed to low and high linear energy transfer (LET) radiation. METHODS AND MATERIALS: DSBs and survival were determined as a function of dose in melanoma cells (B16-F0) irradiated with monoenergetic proton and lithium beams and with a gamma source. Survival curves were obtained by clonogenic assay and fitted to the linear-quadratic model. DSBs were evaluated by the detection of phosphorylated histone H2AX (gammaH2AX) foci at 30 min and 6 h post-irradiation. RESULTS: Survival curves showed the increasing effectiveness of radiation as a function of LET. gammaH2AX labeling showed an increase in the number of foci vs. dose for all the radiations evaluated. A decrease in the number of foci was found at 6 h post-irradiation for low LET radiation, revealing the repair capacity of DSBs. An increase in the size of gammaH2AX foci in cells irradiated with lithium beams was found, as compared with gamma and proton irradiations, which could be attributed to the clusters of DSBs induced by high LET radiation. Foci size increased at 6 h post-irradiation for lithium and proton irradiations in relation with persistent DSBs, showing a correlation with surviving fraction. CONCLUSIONS: Our results showed the response of B16-F0 cells to charged particle beams evaluated by the detection of gammaH2AX foci. We conclude that gammaH2AX foci size is an accurate parameter to correlate the rejoining of DSBs induced by different LET radiations and radiosensitivity.

Oxidative metabolism of lung macrophages exposed to sodium arsenite.

Arsenic pollution has become increasingly severe. It occurs as the result of geological processes and different human activities. Arsenic toxicity at the respiratory level occurs mainly by inhalation of products of coal combustion. The aim of this study was to evaluate sodium arsenite (As(3+)) toxicity in murine alveolar macrophages (AMs) in vitro and its association with the alterations in cell metabolism. No changes in viability, apoptosis or cell area were detected in AMs treated with As(3+) concentrations up to 2 microM for 24-96 h. A marked decrease in these end-points was observed for As(3+) concentrations ranging from 2.5 microM to 10 microM. Regarding the dynamics of the endo-exocytic process triggered by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell incorporation, no variations were detected for As(3+) concentrations lower than 2 microM while higher concentrations markedly modified this response. MTT specific activity, as a measure of cell metabolic activity, was not modified irrespective of the As(3+) concentration assayed. However, nitroblue tetrazolium (NBT) specific activity, as a measure of superoxide anion generation, is responsive but only to low As(3+) doses. Although this study focuses on lung macrophages, the effects of As(3+) described herein may also apply to the response of macrophages residing in other organs. Arsenite modifies the metabolic and the oxidative status of AMs in vitro. When macrophages are in an As(3+) rich medium, they exhibit a reduction in respiratory burst levels and lose their intrinsic capacity to respond to toxicants.

Selective radiosensitization by nitric oxide in tumor cell lines.

The radiosensitizing effect of nitric oxide (NO) on mouse and human tumor cells with different degrees of malignancy was evaluated. Cells pre-treated with the NO donor diethylenetriamine-NO (DETA-NO), were irradiated with gamma rays. Survival curves were obtained by clonogenicity and fitted to the linear-quadratic model. Results demonstrated an association between radiosensitization and degree of malignancy. The more malignant the cell line, the higher the degree of radiosensitization by DETA-NO. In conclusion, the differential radiosensitizing effect of DETA-NO shown here is of great interest for the potential use of NO in radiotherapy, due to an enhanced radiation effect on tumor vs. normal tissue.

Kinetics of MTT-formazan exocytosis in phagocytic and non-phagocytic cells.

MTT is taken up by cells by endocytosis and reduced to formazan in the endosomal/lysosomal compartment. Formazan is deposited intracellularly as blue granules and is later exocytosed as needle-like formazan crystals. The present study involves an analysis of the pattern of exocytosis of MTT in different cell types showing clearcut differences in the response that can be associated to their ability to phagocytose. To further assess the characteristics of the exocytic mechanism of MTT/formazan, different experimental conditions were assayed. When culture medium with decreasing serum concentration was used as a metabolic modulator no variations were observed in the proportion of cells with formazan crystals. Conversely, the markedly sensitivity of phagocytic cells to increasing concentrations of genistein constituted a remarkable difference with non-phagocytic cells. These results must be considered when the modulation of MTT exocytosis is used as a signal of the progress of human diseases.

Imbalance of antioxidant enzymes in tumor cells and inhibition of proliferation and malignant features by scavenging hydrogen peroxide.

The aim of this study was to evaluate the endogenous alterations of the antioxidant enzymes in tumor cells and to specifically compensate the resulting changes in the levels of reactive oxygen species (ROS) to control the malignant growth. We determined and compared the activities of antioxidant enzymes and the levels of superoxide anion (O2*-) and hydrogen peroxide (H2O2) in tumor cell lines with different degrees of malignancy, paired with regard to their origin (PB/CH72T4, PDV/PDVC57, and HBL-100/MCF-7). An increase in superoxide dismutase activity and a decrease in the activities of H2O2-detoxifying enzymes, as a function of malignancy, coupled with a rise in H2O2 and a decrease in O2*- were demonstrated. Treatment of cells with exogenous catalase showed a dose-dependent inhibition of proliferation. This inhibition was also demonstrated in several cell lines of different tissue origin and species, suggesting a general role of H2O2 in cell proliferation. Moreover, stable expression of human catalase in MCF-7 cells inhibited proliferation and also reverted malignant features. We conclude that H2O2 played a crucial and general role in the regulation of proliferation and that an endogenous imbalance in antioxidant enzymes could be a relevant event in the carcinogenesis process.

Cell-based quantitative evaluation of the MTT assay.

OBJECTIVE: To analyze the bioreduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) on a per cell basis and evaluate its modulation as a function of different stages of cell metabolism. STUDY DESIGN: Following MTT bioreduction, total optical density (TOD), cell area and specific activity (TOD/area) of V79 cells and cultured macrophages were recorded for individual cells by means of digital image analysis. The effect of different serum (0-10% vol/vol) or genistein (0-100 microM) concentrations was used to modulate the MTT-specific activity response. RESULTS: As cells in culture are heterogeneous in cell size, the contribution of each cell to the total amount of formazan formed per dish is variable. The production of formazan per cell as a result of MTT bioreduction was found to be proportional to cell size. CONCLUSION: Specific MTT-reducing activity was analyzed in phagocytes and nonphagocyte cells, revealing the utility of this variable in evaluating the MTT assay at the single-cell level.

Age-dependent change in reactive oxygen species and nitric oxide generation by rat alveolar macrophages.

Immunosenescence is an age-associated dysregulation of the immune function, which contributes to increased susceptibility to disease in the elderly. Alveolar macrophages (AM) are known phagocytes that generate reactive oxygen species (ROS) and nitric oxide (NO), essential mediators for host defence. We studied phagocytosis, ROS and NO production in AM obtained from young, adult and senescent rats (1-2, 9-12 and 18-24 months old, respectively) after exposure to lipopolysaccharide (LPS, 0.1-10 microg mL(-1)), 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.1 microg mL(-1)) or LPS + TPA in culture. Phagocytosis was significantly lower in control AM from adult rats than in AM from young animals. Nevertheless, AM from adult animals pretreated with LPS exhibited higher phagocytic capacity than AM from younger animals. ROS was identified by the NBT test at single cell level and quantified by automated image analysis. When TPA was added to all three populations, AM from adult and senescent animals responded more than AM from young animals. All LPS-stimulated AM produce more NO than controls. However, NO production increased three-, four- and two-fold in young, adult and senescent animals, respectively. Our results demonstrate that AM from young, adult and senescent animals display differential responsiveness to inflammatory mediators. Therefore, aging processes markedly affect AM metabolic functions and may further compromise the lung immune defence response, increasing adverse long-term health effects.

Radiation effects on oxidative metabolism in young and aged rat alveolar macrophages.

The effect of ionizing radiation on metabolic functions of alveolar macrophages (AM) have been well studied. However, variations associated to age have not been established yet. The aim of this work was to perform a comparative study on irradiated alveolar macrophages from young and aged rat lungs. Cell viability and occurrence of apoptosis as well as production of nitric oxide (NO), generation of superoxide anion (O2*-) and total antioxidant capacity were analyzed in vitro after exposure to gamma-irradiation with 10, 25, 50 and 75 Gy. Cell viability decreased only in the aged population at the higher doses. Morphological features of apoptosis were clearly evidenced in irradiated alveolar macrophages from aged animals although the DNA fragmentation assay for apoptosis showed no differences for either of the populations studied. NO production and total reactive antioxidant potential (TRAP) levels showed a dose-dependent modulation. Low radiation doses inhibited the production of NO and decreased TRAP levels whereas higher doses enhanced the NO production and increased the TRAP levels in both macrophage populations. Generation of O2*- was always higher in the aged population for all the doses assayed. We conclude that in vitro young alveolar macrophages exhibited higher radioresistance over the whole range of doses as compared to the aged macrophage population. Our results show that the aging process markedly affects the radioresistance of phagocytic cells. Therefore, immune defense and inflammatory response of lungs from aged patients should be considered when planning radiotherapy protocols.

Regulation of phospholipase C activation by the number of H(2) receptors during Ca(2+)-induced differentiation of mouse keratinocytes.

We have reported previously that the histamine H(2) receptor (H(2)R) can stimulate the phospholipase C (PLC) signaling pathway in mouse keratinocytes. In the present work, we examined the physiological mechanisms involved in this activation by studying histamine metabolism and H(2)R expression and coupling during mouse keratinocyte differentiation. Ca(2+)-induced differentiation decreased histidine decarboxylase (HDC) mRNA, the enzyme responsible for histamine synthesis, by 68.9+/-5.0%. Concomitantly, intracellular histamine content and its release into the extracellular medium were reduced significantly by 68.2+/-2.0 and 74.1+/-1.7%, respectively. Binding of [3H]tiotidine to H(2)Rs present on the surface of whole cells was also decreased by cellular differentiation [(18.17+/-2.1)x10(4) vs. (6.27+/-0.87)x10(4) sites/cell, undifferentiated and differentiated cells, respectively], without affecting H(2)R affinity. Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the H(2)R mRNA showed that the expression was also down-regulated at the transcriptional level. Moreover, the inhibition of H(2)R expression strongly affected the ability of the receptor to induce PLC activation. Our findings suggest that H(2)R signaling through the PLC second messenger system is inhibited during keratinocyte differentiation by an autocrine loop involving down-regulation of H(2)R expression and inhibition of histamine metabolism.