Juan Antonio Gonzalez-Barrios

Improved neurotensin-vector-mediated gene transfer by the coupling of hemagglutinin HA2 fusogenic peptide and Vp1 SV40 nuclear localization signal.

Recently we reported that neurotensin-SPDP-poly-L-lysine (NT-vector) is able to bind plasmid DNA (NT-polyplex) and polyfect cells expressing the high-affinity neurotensin receptor (NTRH) although with low transfecting efficiency: in vitro, 6.5+/-1.5%, and in vivo, 5+/-4%. In this work, we attempted to increase the transfecting efficiency by integrating the hemagglutinin HA2 fusogenic peptide and the Vp1 nuclear localization signal of SV40 to the NT-polyplex (fusogenic-karyophilic-NT-polyplex). Confocal microscopy and flow cytometry analysis showed that the fusogenic-karyophilic-NT-polyplex produced mostly nuclear localization of the plasmid DNA in NTRH-bearing N1E-115 cells. About 50% of N1E-115 cells internalized and expressed the reporter gene when the plasmid DNA was transferred by the fusogenic-karyophilic-NT-polyplex. Although to a less extent, the addition of each viral peptide separately to NT-polyplex (fusogenic-NT-polyplex or karyophilic-NT-polyplex) improved polyfection. Fusogenic-NT-polyplex produced 22.41+/-5.96% of internalization and 20.35+/-0.82% of expression in N1E-115 cells, whereas karyophilic-NT-polyplex yielded 13.75+/-3.88% and 10.94+/-2.04%, respectively. Basal internalization and expression were detected in N1E-115 cells in the presence of 100 nM SR-48692 and in NTRH-lacking cells. The fusogenic-karyophilic-NT-polyplex was microinjected into the substantia nigra to test its ability for gene transfer in vivo. Fusogenic-karyophilic-NT-polyplex internalization was observed within dopamine neurons only. Reporter gene expression was observed in a high proportion of dopamine neurons up to 60 days after NT-polyfection. Both internalization and expression were prevented by SR-48692. Our results show that the fusogenic-karyophilic-NT-polyplex is a highly efficient and specific gene vector and encourage its use to transfer gene of physiological interest to NTRH-bearing neurons.

Nitric oxide and nitric oxide synthases in the fetal cerebral cortex of rats following transient uteroplacental ischemia.

The effect of transient uteroplacental ischemia on nitric oxide (NO) levels, enzymatic activity, and expression of NO synthase (NOS) isoforms was studied in fetal rat brains. Fetuses were subjected to ischemia by clamping the uterine arteries for 5 min on gestational day 17 (GD17). At different times after ischemia, fetuses were delivered by Cesarean section under anesthesia to obtain the brains. Transient uteroplacental ischemia produced a time dependent increase in nitrite levels in the brain, reaching a maximum value (300 +/- 25% of baseline) 24 h after uterine artery occlusion and remaining elevated as long as 48 h. Significantly increased nitrite levels were found in the cerebral cortex but not in the mesencephalon and cerebellum. The ischemia-induced increment in nitrite levels was totally blocked by either L-NAME (10 mg/kg) or AMT (0.65 mg/kg) administered i.p. 1 h before uterine artery occlusion. Both Ca(2+)-dependent and Ca(2+)-independent NOS activities in the cerebral cortex remained significantly increased with respect to controls after 24 h following the ischemia. Reverse transcriptase-polymerase chain reaction showed augmented levels of mRNAs for both nNOS and iNOS when compared with controls at 8 h after ischemia. At 36 h, nNOS mRNA returned to basal levels whereas eNOS mRNA levels increased and iNOS mRNA remained elevated. Our results show that the three NOS isoforms participate in increasing NO levels after transient ischemia and suggest a biphasic and differential regulation of the expression of constitutive NOS isoforms in the rat cerebral cortex.

Transient transfection of human CDNF gene reduces the 6-hydroxydopamine-induced neuroinflammation in the rat substantia nigra

BackgroundThe anti-inflammatory effect of the cerebral dopamine neurotrophic factor (CDNF) was shown recently in primary glial cell cultures, yet such effect remains unknown both in vivo and in 6-hydroxydopamine (6-OHDA) models of Parkinson’s disease (PD). We addressed this issue by performing an intranigral transfection of the human CDNF (hCDNF) gene in the critical period of inflammation after a single intrastriatal 6-OHDA injection in the rat.MethodsAt day 15 after lesion, the plasmids p3xNBRE-hCDNF or p3xNBRE-EGFP, coding for enhanced green florescent protein (EGFP), were transfected into the rat substantia nigra (SN) using neurotensin (NTS)-polyplex. At day 15 post-transfection, we measured nitrite and lipoperoxide levels in the SN. We used ELISA to quantify the levels of TNF-α, IL-1β, IL-6, endogenous rat CDNF (rCDNF) and hCDNF. We also used qRT-PCR to measure rCDNF and hCDNF transcripts, and immunofluorescence assays to evaluate iNOS, CDNF and glial cells (microglia, astrocytes and Neuron/Glial type 2 (NG2) cells). Intact SNs were additional controls.ResultsIn the SN, 6-OHDA triggered nitrosative stress, increased inflammatory cytokines levels, and activated the multipotent progenitor NG2 cells, which convert into astrocytes to produce rCDNF. In comparison with the hemiparkinsonian rats that were transfected with the EGFP gene or without transfection, 6-OHDA treatment and p3xNBRE-hCDNF transfection increased the conversion of NG2 cells into astrocytes resulting in 4-fold increase in the rCDNF protein levels. The overexpressed CDNF reduced nitrosative stress, glial markers and IL-6 levels in the SN, but not TNF-α and IL-1β levels.ConclusionOur results show the anti-inflammatory effect of CDNF in a 6-OHDA rat of Parkinson’s disease. Our results also suggest the possible participation of TNF-α, IL-1β and IL-6 in rCDNF production by astrocytes, supporting their anti-inflammatory role.

Evidence in vitro of glial cell priming in the taiep rat.

Cultured glial cells from the cerebellum of 15-day-old taiep rats produced NO, increased iNOS levels, up-regulated iNOS expression and promoted TNF release when stimulated with LPS and IFNgamma. These responses were much greater than in control cells. In taiep glial cells, NO production and iNOS levels and expression induced by the co-stimulatory signal were resistant to the inhibitory effect of TGFbeta1. The glial cell priming might have been generated by oligodendrocyte alteration in taiep rats.

Increased nitric oxide levels and nitric oxide synthase isoform expression in the cerebellum of the taiep rat during its severe demyelination stage

We have previously reported progressive reactive astrocytes in the cerebellum of taiep rats, one of the most regions affected by demyelination, and activation of cerebellar glial cells in vitro. Based on the hypothesis that activated glial cells produce high levels of reactive nitrogen intermediates, we assessed the production of nitric oxide (NO) and the expression of the three NO synthases (NOS) in the cerebellum of 6-month-old taiep rats. A significant 40% increase of NO levels was measured in taiep rats when compared with controls. The protein and mRNA levels of the three NOS isoforms were also significantly increased. In contrast to controls, immunostaining assays against nNOS or iNOS showed an increased number of immunoreactive glial cells in the granular layer (nNOS) and Purkinje layer (iNOS) of cerebellum of taiep rats. Microglia-macrophages and both CD4- and CD8-immunoreactive cells were observed in cerebellar white matter of taiep rats only, thus suggesting other possible cell sources of those NOSs. Differences in the cellular location for eNOS immunoreactivity were not observed. The enhanced levels of NO, NOS proteins, mRNAs, and NOS immunoreactivities in glial cells and microglia strongly suggest glial activation together with the professional immune cells can aggravate the demyelination of aged taiep rats.

Subacute Zinc Administration and L-NAME Caused an Increase of NO, Zinc, Lipoperoxidation, and Caspase-3 during a Cerebral Hypoxia-Ischemia Process in the Rat

Zinc or L-NAME administration has been shown to be protector agents, decreasing oxidative stress and cell death. However, the treatment with zinc and L-NAME by intraperitoneal injection has not been studied. The aim of our work was to study the effect of zinc and L-NAME administration on nitrosative stress and cell death. Male Wistar rats were treated with ZnCl2 (2.5 mg/kg each 24 h, for 4 days) and N-ω-nitro-L-arginine-methyl ester (L-NAME, 10 mg/kg) on the day 5 (1 hour before a common carotid-artery occlusion (CCAO)). The temporoparietal cortex and hippocampus were dissected, and zinc, nitrites, and lipoperoxidation were assayed at different times. Cell death was assayed by histopathology using hematoxylin-eosin staining and caspase-3 active by immunostaining. The subacute administration of zinc before CCAO decreases the levels of zinc, nitrites, lipoperoxidation, and cell death in the late phase of the ischemia. L-NAME administration in the rats treated with zinc showed an increase of zinc levels in the early phase and increase of zinc, nitrites, and lipoperoxidation levels, cell death by necrosis, and the apoptosis in the late phase. These results suggest that the use of these two therapeutic strategies increased the injury caused by the CCAO, unlike the alone administration of zinc.

Prophylactic Subacute Administration of Zinc Increases CCL2, CCR2, FGF2, and IGF-1 Expression and Prevents the Long-Term Memory Loss in a Rat Model of Cerebral Hypoxia-Ischemia

Prophylactic subacute administration of zinc decreases lipoperoxidation and cell death following a transient cerebral hypoxia-ischemia, thus suggesting neuroprotective and preconditioning effects. Chemokines and growth factors are also involved in the neuroprotective effect in hypoxia-ischemia. We explored whether zinc prevents the cerebral cortex-hippocampus injury through regulation of CCL2, CCR2, FGF2, and IGF-1 expression following a 10 min of common carotid artery occlusion (CCAO). Male rats were grouped as follows: (1) Zn96h, rats injected with ZnCl2 (one dose every 24 h during four days); (2) Zn96h + CCAO, rats treated with ZnCl2 before CCAO; (3) CCAO, rats with CCAO only; (4) Sham group, rats with mock CCAO; and (5) untreated rats. The cerebral cortex-hippocampus was dissected at different times before and after CCAO. CCL2/CCR2, FGF2, and IGF-1 expression was assessed by RT-PCR and ELISA. Learning in Morris Water Maze was achieved by daily training during 5 days. Long-term memory was evaluated on day 7 after learning. Subacute administration of zinc increased expression of CCL2, CCR2, FGF2, and IGF-1 in the early and late phases of postreperfusion and prevented the CCAO-induced memory loss in the rat. These results might be explained by the induction of neural plasticity because of the expression of CCL2 and growth factors.

Prophylactic Chronic Zinc Administration Increases Neuroinflammation in a Hypoxia-Ischemia Model

Acute and subacute administration of zinc exert neuroprotective effects in hypoxia-ischemia animal models; yet the effect of chronic administration of zinc still remains unknown. We addressed this issue by injecting zinc at a tolerable dose (0.5 mg/kg weight, i.p.) for 14 days before common carotid artery occlusion (CCAO) in a rat. After CCAO, the level of zinc was measured by atomic absorption spectrophotometry, nitrites were determined by Griess method, lipoperoxidation was measured by Gerard-Monnier assay, and mRNA expression of 84 genes coding for cytokines, chemokines, and their receptors was measured by qRT-PCR, whereas nitrotyrosine, chemokines, and their receptors were assessed by ELISA and histopathological changes in the temporoparietal cortex-hippocampus at different time points. Long-term memory was evaluated using Morris water maze. Following CCAO, a significant increase in nitrosative stress, inflammatory chemokines/receptors, and cell death was observed after 8 h, and a 2.5-fold increase in zinc levels was detected after 7 days. Although CXCL12 and FGF2 protein levels were significantly increased, the long-term memory was impaired 12 days after reperfusion in the Zn+CCAO group. Our data suggest that the chronic administration of zinc at tolerable doses causes nitrosative stress, toxic zinc accumulation, and neuroinflammation, which might account for the neuronal death and cerebral dysfunction after CCAO.

Genetic diversity of Mycobacterium tuberculosis and transmission associated with first-line drug resistance: A first analysis in Jalisco, Mexico

OBJECTIVES The objectives of this study were to analyse the frequency of gene mutations associated with antitubercular drug resistance in clinical samples from the population of Jalisco State (Mexico) and to evaluate the genetic variability of Mycobacterium tuberculosis and multidrug-resistant (MDR) M. tuberculosis strains to describe the frequency of various families. METHODS Clinical isolates of M. tuberculosis obtained from Jalisco State were analysed. Isolates were subjected to drug susceptibility testing, and mutations were characterised by sequencing, followed by genotyping using spoligotyping and mycobacterial interspersed repetitive units-variable-number of tandem repeats (MIRU-VNTR). Moreover, the prevalence of mutations was analysed by phylogenetic lineages. RESULTS Resistant strains were analysed by sequencing of katG, inhA and rpoB genes to determine the presence of mutations associated with isoniazid and rifampicin resistance. In MDR, monoresistant and polyresistant isolates, mutations were found in 17 (54.84%) of 31 strains. Spoligotyping identified six different strain lineages [T1 (25.40%), H3 (7.94%), MANU (4.76%), X1 (3.17%), EAI5 (1.59%) and LAM1 (1.59%)], with the remaining strains identified as orphans. In additional tree-based identification, a dendrogram of spoligotype patterns generated five different similarity clusters. When combining 24-loci MIRU-VNTR and spoligotyping approaches, the results shows that there is no cluster formation, indicating low transmission of the samples. CONCLUSIONS This study using spoligotyping and MIRU-VNTR showed that the analysed strains were not related to each other since no two identical strains were found. Families with the highest prevalence in the study were orphans followed by T family.

Growth factor-enriched autologous plasma improves wound healing after surgical debridement in odontogenic necrotizing fasciitis: a case report

BackgroundOdontogenic necrotizing fasciitis of the neck is a fulminant infection of odontogenic origin that quickly spreads along the fascial planes and results in necrosis of the affected tissues. It is usually polymicrobial, occurs frequently in immunocompromised patients, and has a high mortality rate.Case presentationA 69-year old Mexican male had a pain in the maxillar right-canine region and a swelling of the submental and submandibular regions. Our examination revealed local pain, tachycardia, hyperthermia (39°C), and the swelling of bilateral submental and submandibular regions, which also were erythematous, hyperthermic, crepitant, and with a positive Godet sign. Mobility and third-degree caries were seen in the right mandibular canine. Bacteriological cultures isolated streptococcus pyogenes and staphylococcus aureus. The histopathological diagnosis was odontogenic necrotizing fasciitis of the submental and submandibular regions. The initial treatment was surgical debridement and the administration of antibiotics. After cultures were negative, the surgical wound was treated with a growth factor-enriched autologous plasma eight times every third day until complete healing occurred.ConclusionsThe treatment with a growth factor-enriched autologous plasma caused a rapid healing of an extensive surgical wound in a patient with odontogenic necrotizing fasciitis. The benefits were rapid tissue regeneration, an aesthetic and a functional scar, and the avoidance of further surgery and possible complications.