Delivery of Anti-IFNAR1 shRNA to Hepatic Cells Decreases IFNAR1 Gene Expression and Improves Adenoviral Transduction and Transgene Expression.

Chronic liver injury leads to advanced fibrosis, cirrhosis, and hepatocellular carcinoma. Genetical cell treatment related to the use of adenovirus (Ads) has proven to be beneficial and efficient in the recovery of hepatic diseases. Nevertheless, they are highly immunogenic and trigger an immune response where interferons type 1 (IFN-I) play a very important role. Three shRNAs against the Interferon-1 receptor (IFNAR1) were designed and cloned in pENTR/U6 plasmid and amplified in DH5α cells. Huh7 cells were transfected with these plasmids in the presence or absence of 1 × 109 viral particles/ml of adenovirus containing the green fluorescent protein gene used as a reporter. Transfection with the shRNA plasmids partially inhibited the IFNAR1 expression. This inhibition substantially decreased antiviral response, demonstrated by the decrease of IFNAR1, IFN-α, and TNF-α gene expression, and the decrease at protein levels of IFNAR1, Protein kinase RNA-activated (PKR), and phosphorylated STAT1, allowing higher adenoviral transduction and transgene expression. Interestingly it was seen shRNA inhibited macrophage activation. These results suggest that the inhibition of the IFN-I pathway could be a strategy to minimize the immune response against Adenoviral vectors allowing higher Adenovirus transduction extending the transgene expression.

Immune checkpoint expression on peripheral cytotoxic lymphocytes in cervical cancer patients: moving beyond the PD-1/PD-L1 axis.

Immune checkpoint therapy to reverse natural killer (NK) and T cell exhaustion has emerged as a promising treatment in various cancers. While anti-programmed cell death 1 (PD-1) pembrolizumab has recently gained Food and Drug Administration (FDA) approval for use in recurrent or metastatic cervical cancer, other checkpoint molecules, such as T cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) and T cell immunoglobulin and mucin-domain containing-3 (Tim-3), have yet to be fully explored in this disease. We report expression of TIGIT, Tim-3 and PD-1 on subsets of peripheral blood NK (CD56dim/neg CD16bright/dim/neg and CD56bright CD16dim/neg ) and T cells. The percentages of these cells were increased in women with cervical cancer and pre-malignant lesions. PD-1+ NK and T cells were likely to co-express TIGIT and/or Tim-3. These cells, with an apparently 'exhausted' phenotype, were augmented in patients. A subset of cells were also natural killer group 2 member D (NKG2D)- and DNAX accessory molecule 1 (DNAM-1)-positive. PD-1int and PD-1high T cells were notably increased in cervical cancer. Soluble programmed cell death ligand 1 (PD-L1) was higher in cancer patient blood versus healthy donors and we observed a positive correlation between sPD-L1 and PD-1+ T cells in women with low-grade lesions. Within the cancer group, there were no significant correlations between sPD-L1 levels and cervical cancer stage. However, when comparing cancer versus healthy donors, we observed an inverse association between sPD-L1 and total T cells and a correlation between sPD-L1 and CD56dim NK cells. Our results may show an overview of the immune response towards pre-cancerous lesions and cervical cancer, perhaps giving an early clue as to whom to administer blocking therapies. The increase of multiple checkpoint markers may aid in identifying patients uniquely responsive to combined antibody therapies.

CARDIOVASCULAR RISK FACTORS IN ADOLESCENTS: ROLE OF INSULIN RESISTANCE AND OBESITY.

INTRODUCTION: Childhood obesity is a public health problem characterized by early insulin resistance (IR), inflammation, and oxidative stress. The presence of an uninterrupted low-grade inflammatory state impairs metabolic and cardiovascular health. The population is particularly susceptible to develop metabolic disorders related to increased body fat. METHODS: Eighty-three adolescents were recruited and grouped according to HOMA-IR and BMI in either with or without IR and obese or normal-weight respectively. Anthropometric, biochemical, immunological and hormonal variables were determined. Transverse Analytical Study. RESULTS: Obesity, dyslipidemia, IL-6, and C-reactive protein were significantly higher in the IR group than in the non-IR group. Obese adolescents showed increased insulin levels, HOMA-IR, inflammatory markers, and triglycerides; while having lower HDL-C, and adiponectin when compared to normal-weight adolescents. As expected, obesity-related anthropometric markers positively correlated with IR and inflammatory markers while negatively correlated with adiponectin levels. CONCLUSIONS: Early IR, subclinical inflammation, dyslipidemia, and hypoadiponectinemia characterize obesity in adolescents. These factors may increase the risk of future coronary heart disease (CHD) and diabetes mellitus development (DM) in early adulthood.

Polymorphism of the beta3-adrenergic receptor and lipid profile in patients with rheumatoid arthritis and systemic lupus erythematosus treated with chloroquine.

We investigated the effect of beta 3-adrenergic receptor (beta(3)AR) polymorphism on lipid profiles in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) treated with chloroquine. One hundred sixty-eight subjects were classified into three groups: 61 RA patients, 57 SLE patients, and 50 healthy subjects. All patients fulfilled the 1987 and 1982 classification criteria for RA and SLE, respectively, of the American College of Rheumatology. Demographic data and clinical characteristics of the patients were registered. Fasting lipid profile determination and leukocyte genomic DNA isolation from peripheral blood was performed in all the participants. Screening of the beta(3)-AR gene polymorphic region (exon 1) was done by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Quantitative and qualitative variables were analyzed using analysis of variance (ANOVA) with the LSD and chi(2) tests, respectively. An association between the arg64/arg64 beta(3)-AR genotype and high levels of triglycerides (TG) and very low-density lipoprotein cholesterol (VLDL-c) was found in three RA patients ( P=0.01), two of them taking chloroquine. Arg64/arg64 beta(3)-AR polymorphism may contribute to increased TG and VLDL-c in RA patients, independently of chloroquine treatment.

T(H)1/T(H)2 cytokine profile, metalloprotease-9 activity and hormonal status in pregnant rheumatoid arthritis and systemic lupus erythematosus patients.

During the course of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), several immune and neuroendocrine changes associated with pregnancy may exert positive (amelioration) or negative (exacerbation) effects on the clinical outcome. In order to shed light on the mechanisms underlying these responses, we performed a prospective longitudinal study in RA and SLE pregnant women, including healthy pregnant women as a control group. Cytokine messenger RNA (mRNA) expression assessed by quantitative competitive polymerase chain reaction (PCR) in peripheral blood mononuclear cells (PBMC), cytokine levels and lymphocyte proliferation responses (LPR) following phytohaemagglutinin (PHA) stimulation of PBMC, plasma metalloprotease-9 activity (MMP-9) and hormonal status during pregnancy were determined. TNFa was the most abundant cytokine mRNA expressed in PBMC in all groups studied (healthy pregnant women, RA and SLE pregnant patients). However, a general TH2 response reflected by high IL-10 levels was found in RA, as well as SLE, patients. A significant change in IFN-gamma was observed in RA patients but only during the first trimester of pregnancy. This compared with a major TH1 response in healthy pregnant women. Interestingly, our study showed a homogeneous hormonal pattern in RA and SLE patients. Although decreased cortisol levels were observed in all patients studied, this is possibly related to the remission of disease activity status brought about by steroid treatment before and during pregnancy. In summary, we suggest that complex immune and hormonal networks are involved in pregnancy and that rheumatic diseases are very dynamic immune processes that cannot be described with a clear-cut cytokine profile. Furthermore, the observations in this study may reflect treatment-related immune effects more than those associated with disease.

Genotypes and S-gene variability of Mexican hepatitis B virus strains.

The genotypes and subtypes of 15 Mexican hepatitis B virus strains were determined by sequencing and phylogenetic analysis of the small S-gene. The most predominant strains were found to be divergent genotype/subtype F/adw4 strains (66.6%), followed by A/adw2 (20.0%), D/ayw3 (6.7%), and G/adw2 (6.7%). The S-genes of the Mexican genotype F strains and two Nicaraguan strains described previously formed a subcluster with more than 4% divergence from the other strains within this genotype. The Mexican strains within genotypes A and D showed the highest homology with strains from Europe and the United States. Ten amino acid substitutions not described previously were found in the S-genes of strains from nine chronic carriers, whereas the S gene in strains from six acute hepatitis B patients were highly conserved as compared to their respective genotypes. One genotype F strain from an HBsAg positive chronic carrier had a T to A mutation at position 647, forming a translational stop at codon 216. Two genotype F strains from HBsAg negative chronic carriers had a Val180 instead of an Ala found in the other genotype F strains. This study shows that a divergent genotype F predominates in Mexican strains analyzed, which presented amino acid substitutions not reported previously outside the a determinant.

Anticonvulsant effects of Magnolia grandiflora L. in the rat.

The ethyl ether (EE) and hydroalcoholic extract (HE) of Magnolia grandiflora L. (Magnoliaceae) seeds, a popular plant utilized in the Mexican traditional medicine because of its antispasmodic as well as other reported pharmacological effects, were studied in adult male Wistar rats. EE and HE orally administered in a single dose of 250 mg/kg (calculated on lipidic base) and 200 mg/kg, exhibited abolition of the extensor reflex of maximal electric induced-seizure test in 50 and 40% of the experimental animals, respectively. They significantly prolonged the sleeping time induced by pentobarbital and only the ethanol extract induced hypothermia. No neurological deficit was exhibited by either extract according to the gait, stance and righting test. Although ulterior toxicological and pharmacological insight is necessary, these results suggest that the chemical constituents of this plant could have utility in the control of epileptic patients presenting convulsive seizures.

Anticonvulsant activity of Casimiroa edulis in comparison to phenytoin and phenobarbital.

An aqueous extract of Casimiroa edulis leaves was tested in adult male Wistar rats for anticonvulsant activity utilizing two models of experimental epilepsy: maximal electroshock seizure (MES) and subcutaneously injected metrazole (METsc). Single dose of 100 mg/kg C. edulis vacuum dried aqueous extracts (VDA) orally administered to experimental animals elicited 50% and 70% abolition of MES and METsc-induced seizures, respectively. Two firmly established antiepileptic drugs in human therapy, phenytoin (PHT) and phenobarbital (PB), abolished 90% of MES-induced seizures, whereas an 80% and 100% absence of clonic seizures was attained in METsc test, correspondingly. The seizure abolition observed in C. edulis VDA treated rats was comparable with the anticonvulsive pattern exhibited by PHT and PB. These results suggest that potencially antiepileptic compounds are present in C. edulis extracts that deserve the study of their identity and mechanism of action.

[Oral administration of diphenylhydantoin sodium (DFH-Na or phenytoin) predictably affects the liver and kidney of Sprague Dawley rats]. / La administración oral de difenilhidantoinato sódico (DFH-Na ó Fenitoina) afecta previsiblemente hígado y riñón de ratas Sprague-Dawley.

Phenytoin and its vehicle were orally administered to adult Sprague-Dawley rats during 7, 14 and 30 days at doses of 300 and 450 mg/kg/24 hr., respectively. We found: 1) Increased liver DNA concentration in subgroups of animals treated with 450 mg at 7 (P < 0.02) and 15 days (P < 0.001) Phenytoin serum levels were 19 ug/ml. 2) Increased protein concentration with 300 mg at 7 (P < 0.01) and 15 days (P < 0.001), respectively. 3) Cloudy swelling, vacuolar degeneration, liver sinusoids disappearance and lymphocytic cells infiltrate in subgroups of rats receiving vehicle throughout 6, 14 and 15 days correspondingly. The former lesion was found in all subgroups, except that 450 mg treated animals liver more severely affected. 4) Increased DNA concentration in kidney of subgroups receiving 450 mg/kg throughout 7 (P < 0.05), 15 (P < 0.001) and 30 days (P < 0.001), correspondingly. 5) Increased protein concentration in rats receiving 450 mg during 15 days (P < 0.001) and severely decreased at 30 days period. 6) Cloudy swelling was found in all treated animals subgroups. Seven cellular and tissue lesions were caused by vehicle at 15 and 30 days periods. 450 mg of phenytoin predominantly caused tissue condensation and vacuolar degeneration in kidney cortex. 7) propylene glycol do affect liver and kidney at doses below TD-50. Phenytoin stimulate kidney and liver cell proliferation. Caution should be observed when using parenteral phenytoin.

[Modified procedure for screening anticonvulsants. Re-evaluation of sodium diphenylhydantoin and phenobarbital]. / Procedimiento modificado para tamizaje anticonvulsionante. Reevalucación del difenilhidantoinato sódico y el fenobarbital.

A modified antiepileptic screening procedure to test anticonvulsant drugs is shown. Diphenylhydantoin sodium salt (DFH-Na) and phenobarbital (Phb) were tested throughout 8 h, at hourly intervals after a single oral drug intake in rats. Another group was tested at steady stage of DFH-Na during 7 days period. A single dose of DFH-Na was orally administrated to male and female rats (30 mg/kg) and after testing throughout 8 h: 0, 5, 28, 38, 52, 70 and 75% and 10, 18, 50, 35, 62, 50 and 70% protection against MES, was found. Only 20% protection was found in females to METsc test on the 6th and 7th h. However, 80, 60, 60 and 20% males were found protected against METsc test from the 4th to the 8th hour. Maximum blood serum levels were 2 micrograms/ml. Phenobarbital at doses 12 mg/kg in males and females as well showed: 30, 64, 66, 74, 84, 90, 40, 34% and 14, 36, 53, 41, 55, 70, 82 and 82% protection against MES, respectively. On the other hand, 60, 60, 46, 47, 94, 100, 80 and 20% and 80, 80, 46, 70, 60, 40, 80 and 20% of males and females were protected against METsc test, respectively. An average of 8 micrograms/ml and 12 micrograms/ml of Phb serum levels were found since the 1 to the 8th and 5th hours, correspondingly. A 28% protection of both male and female rats to MES test was found following 7 days of treatment with DFH-Na (30 mg/kg) treatment. Also an average of 10% female and males were found protected against METsc test.

Progesterone fate in rabbit cornea.

Excised cornea from adult New Zealand rabbits were incubated with progesterone-4-14C in Eagle's media for 96 hr. Samples were inactivated at intervals of 24 hr incubation periods. The following metabolites of progesterone were isolated: 20 alpha-Hydroxy-4-pregnen-3-one, 20-hydroxy-4-pregnen-3-one, 5 alpha-pregnane-3,20-dione; 5 beta-pregnane-3,20-dione and 6 beta-hydroxy-4-pregnen-3,20-dione. 20 alpha-Hydroxy-pregnen-3-one was the predominant metabolite of progesterone-4-14C. A linear increase was observed throughout 96 hr. The opposite was found for 5 alpha and 5 beta pregnane-3,20-dione. Compounds remaining at the origin of the paper chromatograms contained 6 beta-hydroxy-4-pregnen-3,20-dione and other still unidentified metabolites of progesterone-4-14C. Presence of 20 alpha and 20 beta-reductase; 5 alpha and 5 beta-reductase and 6 beta-hydroxylase enzyme systems are involved in corneal progesterone metabolism. No fungal neither bacterial enzymatic biotransformation occurred in the culture media.

Valproic acid effects on leukocytes and platelets of Sprague-Dawley rats.

A study with valproic acid (VPA) was performed in Sprague-Dawley rats with the aim of relating drug serum levels to variations of leukocytes, platelets, coagulation time and other haematological parameters. VPA, 20 and 240 mg/kg, were administered orally, three times a day in groups of rats of both sexes throughout 7, 14, 30, 60 and 90 day periods. Total blood leukocytes averaged 7296 +/- 443 in control as well as in treated groups. Lymphocytes and monocytes decreased in all five groups when receiving the TD50 of 240 mg/kg (P less than 0.05). The VPA fasting serum levels were detectable if administered at 8 hr intervals. Nevertheless, VPA caused modification of the chosen parameters. VPA t1/2 averaged 3 hr in rats. Thus, even if the VPA effects were observed in our experiments, we suggest dividing the whole VPA dose into eight portions to be administered at 3 hr intervals in rats to attain constant serum VPA levels as well as constant drug effects.

Phenytoin effects on tissues of Mongolian gerbils (Meriones unguiculatus).

1. Phenytoin was intraperitoneally injected in six different experimental groups of adult Mongolian gerbils of both sexes at doses of 5, 100 and 150 mg/kg of body wt during 7 and 14 days. 2. Their brain, cerebellum, liver and kidney DNA, RNA and protein content were measured by conventional procedures. 3. The reference values of these rodents' cellular DNA were calculated through hepatocytes isolated following an intracardiac perfusion with collagenase in Hanks solution. 4. A concentration of 7.2 micrograms of DNA per 10(6) cells was found. 5. Phenytoin did not affect tissues of groups of animals receiving daily doses of 5 mg/kg of weight during 7 and 14 days of treatment; although a slight increase of protein concentration was observed in liver (P less than 0.01) and kidney (P less than 0.05). 6. RNA values were found to be significantly decreased in cerebellum (P less than 0.001), brain (P less than 0.05) and kidney (P less than 0.05) of those animals receiving the drug throughout 14 days. 7. DNA concentration was found to be decreased in all four tissues obtained from animals treated with daily doses of phenytoin of 100 (P less than 0.001) and 150 (P less than 0.001) mg/kg of body wt throughout 14 days. 8. These results indicate that phenytoin affected tissue cellularity only when administered at high doses and 14 days of interval of drug administration (P less than 0.001).