In vitro and in vivo acute toxicity of a novel citrate-coated magnetite nanoparticle.

Magnetic nanoparticles (MNps) have become powerful tools for multiple biomedical applications such as hyperthermia drivers, magnetic resonance imaging (MRI) vectors, as well as drug-delivery systems. However, their toxic effects on human health have not yet been fully elucidated, especially in view of their great diversity of surface modifications and functionalizations. Citrate-coating of MNps often results in increased hydrophilicity, which may positively impact their performance as drug-delivery systems. Nonetheless, the consequences on the intrinsic toxicity of such MNps are unpredictable. Herein, novel magnetite (Fe3O4) nanoparticles covered with citrate were synthesized and their potential intrinsic acute toxic effects were investigated using in vitro and in vivo models. The proposed synthetic pathway turned out to be simple, quick, inexpensive, and reproducible. Concerning toxicity risk assessment, these citrate-coated iron oxide nanoparticles (IONps) did not affect the in vitro viability of different cell lines (HaCaT and HepG2). Moreover, the in vivo acute dose assay (OECD test guideline #425) showed no alterations in clinical parameters, relevant biochemical variables, or morphological aspects of vital organs (such as brain, liver, lung and kidney). Iron concentrations were slightly increased in the liver, as shown by Graphite Furnace Atomic Absorption Spectrometry and Perls Prussian Blue Staining assays, but this finding was considered non-adverse, given the absence of accompanying functional/clinical repercussions. In conclusion, this study reports on the development of a simple, fast and reproducible method to obtain citrate-coated IONps with promising safety features, which may be used as a drug nanodelivery system in the short run. (263 words).

Effective combined antiretroviral therapy provides partial immune recovery to mycobacterial antigens in vertically infected, BCG-vaccinated youth living with HIV.

BACKGROUND: We assessed the cytokine response by PBMC of youth living with HIV (YLHIV) under combined antiretroviral therapy (cART) to Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis (BCG) antigens. METHODS: PBMC from 20 Brazilian YLHIV under cART with long-term (≥1 year) virological control, and 20 healthy controls were cultured for 24-96 h under stimulation with BCG, Mtb lysates, ESAT-6 and SEB. We measured TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-10 and IL-17 in culture supernatants using a cytometric bead array. RESULTS: Controls had higher IFN-γ production at 24, 48, 72 and 96 h upon stimulation with BCG lysate, plateauing at 48 h (Median = 1991 vs. 733 pg/mL; p = 0.01), and after 48-72 h of stimulation with Mtb lysate, plateauing at 48 h (3838 vs. 2069 pg/mL; p = 0.049). YLHIV had higher TNF-α production at all time points upon stimulation with ESAT-6, with highest concentration at 36 h (388 vs. 145 pg/mL; p = 0.02). Within the YLHIV group, total CD4 T cell count and CD4/CD8 ratio were associated with IFN-γ response to Mtb lysate and ESAT-6, respectively. CONCLUSIONS: Even under long-term cART, YLHIV seem to have a suboptimal T-helper-1 response to mycobacterial antigens. This can be explained by early immunodeficiency in vertical infection, with lasting damage.

Incidence of tuberculous and non-tuberculous mycobacteria, differentiated by multiplex PCR, in clinical specimens of a large general hospital.

OBJECTIVE: To determine the incidence of Mycobacterium tuberculosis complex and non-tuberculous mycobacterial isolates in the routine setting of a large general hospital using an "in-house" multiplex polymerase chain reaction method and to establish a paradigm for the definitive identification of mycobacteria isolated using semi-automated equipment. METHODS: Established tests, including polymerase chain reaction restriction enzyme analysis, PNB, and NAP inhibition tests as the gold standard, showed 100% agreement with an IS6110/hsp65 multiplex polymerase chain reaction when used to identify stock strains (n = 117). RESULTS: In a subsequent study, 8,790 clinical specimens producing 476 isolates were evaluated with multiplex PCR and also showed 100% agreement in identification using PRA-polymerase chain reaction as the gold standard. The application of this technique to routine analysis was demonstrated in this study. A method was established with the initial application of multiplex PCR for all positive liquid cultures and the subsequent identification of non-tuberculous mycobacteria by polymerase chain reaction restriction enzyme analysis. In total, 77% of isolates belonged to the Mycobacterium tuberculosis complex, and 23% were non-tuberculous mycobacteria. CONCLUSIONS: Several non-tuberculous mycobacterial species were identified, primarily M. avium, but other potentially pathogenic species were also frequently observed, including M. fortuitum, M. abscessus, and M. kansasii. The expeditious communication of these data to the clinical staff was fundamental for the diagnosis of clinical cases. Even in settings where tuberculosis is of major importance, the incidence of non-tuberculous mycobacteria infection is substantial.

Incidence of tuberculous and non-tuberculous mycobacteria, differentiated by multiplex PCR, in clinical specimens of a large general hospital

OBJECTIVE: To determine the incidence of Mycobacterium tuberculosis complex and non-tuberculous mycobacterial isolates in the routine setting of a large general hospital using an "in-house" multiplex polymerase chain reaction method and to establish a paradigm for the definitive identification of mycobacteria isolated using semi-automated equipment. METHODS: Established tests, including polymerase chain reaction restriction enzyme analysis, PNB, and NAP inhibition tests as the gold standard, showed 100% agreement with an IS6110/hsp65 multiplex polymerase chain reaction when used to identify stock strains (n = 117). RESULTS: In a subsequent study, 8,790 clinical specimens producing 476 isolates were evaluated with multiplex PCR and also showed 100% agreement in identification using PRA-polymerase chain reaction as the gold standard. The application of this technique to routine analysis was demonstrated in this study. A method was established with the initial application of multiplex PCR for all positive liquid cultures and the subsequent identification of non-tuberculous mycobacteria by polymerase chain reaction restriction enzyme analysis. In total, 77% of isolates belonged to the Mycobacterium tuberculosis complex, and 23% were non-tuberculous mycobacteria. CONCLUSIONS: Several non-tuberculous mycobacterial species were identified, primarily M. avium, but other potentially pathogenic species were also frequently observed, including M. fortuitum, M. abscessus, and M. kansasii. The expeditious communication of these data to the clinical staff was fundamental for the diagnosis of clinical cases. Even in settings where tuberculosis is of major importance, the incidence of non-tuberculous mycobacteria infection is substantial.

Peptidase activities in rats treated chronically with N(omega)-nitro-L-arginine methyl ester (L-NAME).

The chronic treatment of rats with N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) biosynthesis, results in hypertension. This inhibition of NO production results in activation of the renin-angiotensin system, with increased activity of the carboxypeptidase angiotensin I-converting enzyme (ACE). Since chronic NO inhibition increases ACE activity, we hypothesized that this inhibition could also affect the activities of other peptidases involved in cardiovascular functions. To test this possibility, we examined the activities of aminopeptidase M (APM), dipeptidyl peptidase IV (DPP IV), metalloendopeptidase 24.15 (MEP 24.15) and neutral endopeptidase 24.11 (NEP 24.11) in rat brain, heart, kidney, liver, lung and thoracic aorta. Male Wistar rats were treated chronically with L-NAME (80mgkg(-1) per day) administered in the drinking water for 4 weeks and their organs then removed and processed for the determination of peptidase activities. Treatment with L-NAME did not significantly alter the activities of the four peptidases in brain, heart, kidney, liver and lung. In contrast, in aorta, the activity of APM was slightly but significantly reduced whereas those of DPP IV and MEP 24.15 were markedly enhanced; NEP 24.11 was not detected in this tissue. Immunoblotting for DPP IV and MEP 24.15 showed increased expression in aortic tissue. Neither L-NAME (1-100microM) nor the NO donors sodium nitroprusside and 3-morpholinosydnonimine (SIN-1; 1-100microM) had any consistent effect on the activity of recombinant MEP 24.15 or renal DPP IV. The importance of MEP 24.15 in peptide metabolism was confirmed in pentobartibal-anesthetized rats pretreated with the MEP 24.15 inhibitor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate (JA2), which significantly potentiated the hypotensive response to bradykinin. The altered peptidase activities seen in aorta may contribute to modulating vascular responses in this model of hypertension.

Immunochemical detection of Lonomia obliqua caterpillar venom in rats.

Severe cases of human envenoming by caterpillars of the saturniid moth Lonomia obliqua in Brazil can result in renal damage, leading to renal failure, and intracerebral hemorrhaging. In this work, we used immunohistochemical staining with rabbit antiserum raised against L. obliqua venom to examine venom distribution in selected tissues of the brain (cerebellum and hippocampus), kidneys, and liver of male Wistar rats injected with a single dose of venom (200 microg/kg, i.v.) and sacrificed 6, 18, 24, and 72 hours later. The immunolabeling of GFAP was also examined to assess the venom effects on perivascular astrocytic end-feet in the microvasculature of the hippocampus and cerebellum. Venom was detected in the kidneys (6 and 18 hours) and in the liver (6 hours) but not in the brain at any of the time intervals examined. In contrast, immunolabeling for GFAP revealed astrogliosis in the cerebellum and enhanced expression of this protein in the glial processes of the cerebellum and hippocampus, with a maximum response from 24 hours onwards. The high immunoreactivity seen in the kidneys agreed with the renal damage and dysfunction reported for some patients. The lack of venom detection in the brain, despite the altered expression of GFAP in astrocytes, suggested either that the venom does not enter this organ or that its entrance is transient and fast. Alternatively, the circulating venom may induce the release of mediators that could serve as second messengers to provoke the late astrocytic reactivity and astrogliosis. It is possible that both of these mechanisms may contribute to the effects observed.