Baculovirus-mediated expression of a Helicobacter pylori protein-based multiepitope hybrid gene induces a potent B cell response in mice.

Helicobacter pylori is a gram-negative bacterium that is present in over half of the world's population. The colonization of the stomach́s gastric mucosa by H. pylori is related to the onset of chronic gastritis, peptic ulcer, and cancer. The estimated deaths from gastric cancer caused by this bacterial infection are in the 15,000-150,000 range. Current treatment for controlling the colonization of H. pylori includes the administration of two to four antibiotics and a gastric ATPase proton pump inhibitor. Nevertheless, the bacterium has shown increased resistance to antibiotics. Despite an extensive list of attempts to develop a vaccine, no approved vaccine against H. pylori is available. Recombinant viruses are a novel alternative for the control of primary pathogenic agents. In this work, we employed a baculovirus that carries a Thp1 transgene coding for nine H. pylori epitopes, some from the literature, and others were selected in silico from the sequence of H. pylori proteins (carbonic anhydrase, urease B subunit, gamma-glutamyl transpeptidase, Lpp20, Cag7, and CagL). We verified the expression of this hybrid multiepitopic protein in HeLa cells. Mice were inoculated with the recombinant baculovirus Bac-Thp1 using various administration routes: intranasal, intragastric, intramuscular, and a combination of intranasal and intragastric. We identified a strong adjuvant-independent IgG-antibody response in the serum of recombinant baculovirus-Thp1 inoculated mice, which was specific for a strain of H. pylori isolated from a human patient. The bacterium-specific IgG-antibodies were present in sera 125 days after the first vaccine administration. Also, H. pylori-specific IgA-antibodies were found in feces at 82 days after the first inoculation. A baculovirus-based vaccine for H. pylori is promising for controlling this pathogen in humans.

Gene Therapy for Treatment of Chronic Hyperammonemia in a Rat Model of Hepatic Encephalopathy.

INTRODUCTION AND AIM: Hepatic encephalopathy (HE), caused by hyperammonemia resulting from liver disease, is a spectrum of neuropsychiatric and motor disorders that can lead to death. Existing therapies are deficient and alternative treatments are needed. We have shown that gene therapy with a baculovirus vector containing the glutamine synthetase (Bac-GS) gene is efficient for reducing ammonia levels in an acute hyperammonemia rat model. However, the most common condition resulting from liver disease is chronic hyperammonemia. In this work, Bac-GS was evaluated in bile-duct ligated rats, a chronic liver disease model with hyperammonemia and some characteristics of Type C HE. MATERIAL AND METHODS: Bac-GS was tested for mediating GS overexpression in HeLa cells and H9C2 myotubes. For determining the utility of Bac-GS for the reduction of ammonia levels in a chronic hyperammonemia animal model, four groups of rats were treated: control, sham, ligated with Bac-GS and ligated with Bac-GFP. Baculoviruses were injected i.m. 18 days post-surgery. Blood was drawn 2, 3 and 4 weeks post-surgery and plasma ammonia concentrations were quantified. RESULTS: In protein lysates of cells and myotubes transduced with Bac-GS, a 44 kDa band corresponding to GS was detected. Significant results were obtained in the hyperammonemic bile-duct ligated rat model, as plasma ammonia was reduced to normal levels 3 days after treatment with Bac-GS. Furthermore, a transitory effect of Bac-GS was observed. CONCLUSION: Our results show that gene therapy by delivering GS is a promising alternative for treatment of hyperammonemia in acute-on-chronic liver failure patients with HE.

Ablation of Kv3.1 and Kv3.3 potassium channels disrupts thalamocortical oscillations in vitro and in vivo.

The genes Kcnc1 and Kcnc3 encode the subunits for the fast-activating/fast-deactivating, voltage-gated potassium channels Kv3.1 and Kv3.3, which are expressed in several brain regions known to be involved in the regulation of the sleep-wake cycle. When these genes are genetically eliminated, Kv3.1/Kv3.3-deficient mice display severe sleep loss as a result of unstable slow-wave sleep. Within the thalamocortical circuitry, Kv3.1 and Kv3.3 subunits are highly expressed in the thalamic reticular nucleus (TRN), which is thought to act as a pacemaker at sleep onset and to be involved in slow oscillatory activity (spindle waves) during slow-wave sleep. We showed that in cortical electroencephalographic recordings of freely moving Kv3.1/Kv3.3-deficient mice, spectral power is reduced up to 70% at frequencies <15 Hz. In addition, the number of sleep spindles in vivo as well as rhythmic rebound firing of TRN neurons in vitro is diminished in mutant mice. Kv3.1/Kv3.3-deficient TRN neurons studied in vitro show approximately 60% increase in action potential duration and a reduction in high-frequency firing after depolarizing current injections and during rebound burst firing. The results support the hypothesis that altered electrophysiological properties of TRN neurons contribute to the reduced EEG power at slow frequencies in the thalamocortical network of Kv3-deficient mice.