Involvement of 5-hydroxytryptamine4 receptor in the exacerbation of neuronal loss by psychological stress in the hippocampus of SHRSP with a transient ischemia

A transient forebrain ischemia produced a delayed neuronal death of the hippocampus pyramidal cells in stroke-prone spontaneously hypertensive rats (SHRSP). Long term exposure of rats to stress has been reported to induce deleterious effects on the brain including morphological neuronal degeneration in the hippocampus. The present study was designed to examine the effects of psychological and physical stress on the ischemia-related neuronal death and the effects of 5-hydroxytryptamine(4) (5-HT(4)) receptor antagonist. SHRSP were exposed to the psychological or physical stress for 60 min in the communication box once or repeatedly for 3 days and occluded. SB204070, a 5-HT(4) receptor antagonist was injected before the occlusion. Seven days after the occlusion, the number of the neurons damaged morphologically was examined. A transient bilateral carotid occlusion produced a neuronal death of the CA1 subfield of the hippocampus in a time-dependent manner between 3 and 10 min. A 4 min occlusion induced very little morphological damage and a 5 min one produced a significant neuronal death. Exposure of rats to the psychological stress during 60 min for 3 days before the ischemic insults damaged the pyramidal cells by 4 min ischemia much more than without stress. Physical stress daily for 3 times also increased the damaged neurons. Pretreatment of SB204070 0.1 mg/kg after the stress exposure for 3 days significantly decreased the neuronal damage exacerbated by the stress exposure; however, it did not alter the damage induced by 4 or 10 min occlusion without stress. These results suggest that the repeated exposure of animals to the stress dramatically exacerbates the neuronal death by a transient ischemia and the 5-HT(4) receptor may be involved in the stress-induced exacerbating mechanism of the neuronal damage.     

Dide-Botcazo syndrome associated with Anton's syndrome after a cardioembolic infarction in the distribution of the bilateral posterior cerebral arteries]

Dide-Botcazo syndrome (Rev Neurol, 1902) is a unique neuropsychological syndrome, characterized by combinations of cortical blindness, amnesia, and topographical disorientation. We report 82-year-old right-handed man manifesting such syndrome associated with Anton's syndrome after a cardioembolic infarction in the distribution of the bilateral posterior cerebral arteries. The MRI study demonstrated recent extensive infarctions bilaterally in the occipital lobes and the medial temporal lobes, and thalamus. Following the resolution of unconsciousness and tetraparesis, the patient persistently presented with denial of cortical blindness (Anton's syndrome), profound anterograde amnesia and retrograde amnesia of about 50 years, severe topographical disorientation, and partial impairment of the tactile and auditory naming for objects. The bilateral extensive damages to the visual area, the memory area, and the connecting areas including the occipital lobe of the non-dominant hemisphere possibly responsible for topographical disorientation, may account for producing Dide-Botcazo syndrome. The syndrome may clinically occur following the "top of the basilar" syndrome.     

NMDA receptor stimulation in the absence of extracellular Ca2+ potentiates Ca2+ influx-dependent cell death system

The meaning of Ca2+ influx in the time course of glutamate stimulation of neuronal cells was addressed. We demonstrated that Ca2+ influx did not work straightforward in the determination of the fate of neuronal cells. There appears to be a critical period for Ca2+ influx to work efficiently in glutamate-induced neuronal cell death. When Ca2+ influx for 5 min from the beginning of glutamate stimulation was allowed in the whole stimulation period for 15 min, potent neuronal cell death could not be attained. On the other hand, when neuronal cells had been pre-treated with glutamate or NMDA for 5-10 min in the absence of extracellular Ca2+ following Ca2+ influx for 5 min fully induced neuronal cell death. APV inhibited this pre-treatment effect. It appears that the pre-treatment of neuronal cells with glutamate or NMDA in the absence of extracellular Ca2+ promotes the Ca2+ influx-dependent process executing cell death. The pre-treatment itself did not change the pattern of intracellular Ca2+ elevation by the activation of NMDA receptors. These results imply that glutamate activation of NMDA receptors consists of two different categories of pathways relating to neuronal cell death, i.e., Ca2+ influx independent and dependent, and that the former facilitates the latter to drive neuronal cells to death. This study clarified a mechanism by which glutamate quickly determines cell fate.     

Role of endothelin ETA and ETB receptors in the guinea-pig urinary bladder contraction

The distribution and function of endothelin receptors in the guinea-pig urinary bladder were examined. Specific [125I]endothelin-1 binding sites with both the endothelin ET(A) and ET(B) receptor subtypes were distributed in the muscle layer. Endothelin-1 elicited a tonic contraction which was inhibited by cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ123) but not by N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine (BQ788) and which was inhibited more strongly by a combination of BQ123 and BQ788. Sarafotoxin S6c elicited a contraction which was abolished by BQ788. The concentration of endothelin-1 in the muscle layer was 707.0+/-67.5 pg/g wet weight. Thus, endothelin-1 may regulate muscle tone via both subtypes of endothelin receptors in an autocrine manner in the guinea-pig urinary bladder.     

Primary adenovirus-specific cytotoxic T lymphocyte response occurs after viral clearance and liver enzyme elevation

The virus-specific cytotoxic T lymphocyte (CTL) response is a major obstacle to effective delivery of adenovirus gene therapy. However, its relative role in viral clearance, transgene elimination and hepatotoxicity remains unclear. In this paper, we present an analysis of viral clearance and liver toxicity in relation to the induction of the virus-specific CD8 T-cell response revealed by an MHC class I tetramer. A surprisingly high number of tetramer+ CD8 T cells were found in the liver and lung and reached peak values at days 8 and 10, respectively, post-infection. Nearly 100% of these tetramer+ CD8 T cells expressed high levels of granzyme B and IFNgamma. Remarkably, liver viral load and liver enzyme elevation peaked early, at days 2 and 4, respectively, post-infection, before the specific CTL response was detectable. After generation of CTLs, there was only minimal liver damage or further decrease in virus titer. These results indicated that the primary peak response of tetramer+ CTLs does not correlate with the elimination of adenovirus or liver cytotoxic response.     

Mutants in the ADP-ribosyltransferase Cleft of Cholera Toxin Lack Diarrheagenicity but Retain Adjuvanticity

Cholera toxin (CT), the most commonly used mucosal adjuvant in experimental animals, is unsuitable for humans because of potent diarrhea-inducing properties. We have constructed two CT-A subunit mutants, e.g., serine→ phenylalanine at position 61 (S61F), and glutamic acid→ lysine at 112 (E112K) by site-directed mutagenesis. Neither mutant CT (mCT), in contrast to native CT (nCT), induced adenosine diphosphate-ribosylation, cyclic adenosine monophosphate formation, or fluid accumulation in ligated mouse ileal loops. Both mCTs retained adjuvant properties, since mice given ovalbumin (OVA) subcutaneously with mCTs or nCT, but not OVA alone developed high-titered serum anti-OVA immunoglobulin G (IgG) antibodies (Abs) which were largely of IgG1 and IgG2b subclasses. Although nCT induced brisk IgE Ab responses, both mCTs elicited lower anti-OVA IgE Abs. OVA-specific CD4⁺ T cells were induced by nCT and by mCTs, and quantitative analysis of secreted cytokines and mRNA revealed a T helper cell 2 (Th2)-type response. These results now show that the toxic properties of CT can be separated from adjuvanticity, and the mCTs induce Ab responses via a Th2 cell pathway.The major enterotoxins produced by Vibrio cholerae and Escherichia coli, termed cholera toxin (CT)¹ and heatlabile toxin (LT), respectively, are multisubunit macromolecules composed of two structurally, functionally, and immunologically separate A and B subunits (1–3). The B subunit of each toxin consists of five identical 11.6-kD peptides, but differ from each other in that the B subunit of CT (CT-B) only binds to GM1 ganglioside (4), whereas the B subunit of LT (LT-B) binds GM1 as well as asialo GM1 and GM2 (5). After the B subunit binds to epithelial cell GM1 or GM2 receptors, the A subunit reaches the cytosol, and after activation, binds to nicotinamide adenosine diphosphate and catalyzes ADP ribosylation of Gs-α (6). This GTP-binding protein activates adenyl cyclase with subsequent elevation of cAMP, which in epithelial cells results in secretion of water and chloride ions into the small intestine (7).Both CT and LT are immunogenic and mucosal exposure results in secretory IgA (S-IgA) and serum Abs, which are almost entirely restricted to CT-B or LT-B (8, 9). Further, both toxins are also strong mucosal adjuvants for coadministered, unrelated protein Ags when given by oral, intranasal, or parenteral routes (8–10). We have shown that induction of maximal mucosal S-IgA and serum IgG Ab responses correlated directly with Ag-specific CD4⁺ Th cells secreting IL-4 and IL-5 in mice orally immunized with protein Ag and CT as adjuvant (10). Further, detailed analysis showed that CT elicits adjuvant responses by inducing Ag-specific CD4⁺ Th2-type cells which produce high levels of IL-4 and IL-5, responsible for supporting subsequent development of systemic IgG1 and IgG2b subclass, IgE and S-IgA Ab responses (11). On the other hand, oral immunization with LT promotes IgM, IgG1, IgG2a, IgG2b, and S-IgA Ab responses, which are supported by a mixed CD4⁺ Th1- and Th2-type response associated with IFN-γ, IL-4, IL-5, IL-6, and IL-10 production (12). Furthermore, when IL-4 levels produced by CD4⁺ T cells were compared when CT or LT were used as adjuvants, Ag-specific IL-4 production was lower when LT was used (11, 12).Earlier studies have attempted to dissociate toxicity from adjuvanticity of CT and LT; however, it was shown that a mutant LT toxin, E112K, which had single amino acid substitution in the ADP-ribosyltransferase active center, was nontoxic and also lacked adjuvanticity (13). Recently, two groups have reported that single amino acid substitution mutants of LT, R7K, and R192G, which are outside of the ADP-ribosyltransferase cleft, were nontoxic, but retained adjuvant properties (14, 15). However, one of these LT mutants retained some ADP-ribosyltransferase activity and could potentially cause diarrhea in humans.To develop an ideal nontoxic mucosal adjuvant, we constructed two CT mutants, S61F and E112K, by site-directed mutagenesis, which have single amino acid substitutions in the ADP-ribosyltransferase active center (16). Neither mutant showed toxic activity, and in the present study these mutants were used to assess adjuvant properties as well as signaling pathways involved in adjuvanticity.     

Orally Administered Prion Protein Is Incorporated by M Cells and Spreads into Lymphoid Tissues with Macrophages in Prion Protein Knockout Mice

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases. Infection by the oral route is assumed to be important, although its pathogenesis is not understood. Using prion protein (PrP) knockout mice, we investigated the sequence of events during the invasion of orally administered PrPs through the intestinal mucosa and the spread into lymphoid tissues and the peripheral nervous system. Orally administered PrPs were incorporated by intestinal epitheliocytes in the follicle-associated epithelium and villi within 1 hour. PrP-positive cells accumulated in the subfollicle region of Peyer''s patches a few hours thereafter. PrP-positive cells spread toward the mesenteric lymph nodes and spleen after the accumulation of PrPs in the Peyer''s patches. The number of PrP molecules in the mesenteric lymph nodes and spleen peaked at 2 days and 6 days after inoculation, respectively. The epitheliocytes in the follicle-associated epithelium incorporating PrPs were annexin V-positive microfold cells and PrP-positive cells in Peyer''s patches and spleen were CD11b-positive and CD14-positive macrophages. Additionally, PrP-positive cells in Peyer''s patches and spleen were detected in the vicinity of peripheral nerve fibers in the early stages of infection. These results indicate that orally delivered PrPs were incorporated by microfold cells promptly after challenge and that macrophages might act as a transporter of incorporated PrPs from the Peyer''s patches to other lymphoid tissues and the peripheral nervous system.Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative diseases that infect humans and both wild and domestic animals. They include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle.¹ The common neuropathological features within the central nervous system (CNS) of TSEs are seen as a spongiform pathology, neuronal loss,² glial activation,³ and the accumulation of an abnormal and protease-resistant conformer of the scrapie-associated prion proteins (PrP-res or PrP^Sc),⁴ which are closely associated with the infection.⁵It has been reported that variant CJD in humans is most likely to have occurred because of the transmission of BSE after the consumption of beef contaminated with the BSE agent.⁶ Therefore, the oral route of TSE infection is widely assumed to be important under natural conditions. Many of the infectious agents accumulate in the gut-associated lymphoid tissues (GALT) after oral infection, such as the Peyer''s patches and mesenteric lymph nodes (MLN) before spreading to the CNS.⁷ It is necessary for the infectious agents to cross the intestinal epithelium before they can accumulate in the GALT. In addition, there are microfold cells (M cells) within the follicle-associated epithelium (FAE) that are specialized for the transepithelial transport of macromolecules and particles.⁸ One in vitro study has demonstrated that M cells actively transcytose the scrapie agents into the basolateral side of the epithelium.⁹ However, it is still a matter of controversy as to whether M cells may be involved in the in vivo transport of the infectious agents across the intestinal epithelium. After alimentary uptake of the infectious agents, they accumulate in the GALT and the lymphoreticular systems (eg, the spleen and other peripheral lymph nodes) long before they are detected in the CNS.¹⁰ As the GALT and the lymphoreticular systems are highly innervated, they are believed to be important sites for the infectious agents to gain contact with the nervous system (ie, neuroinvasion).¹¹ Once neuroinvasion occurs, the infectious agents reach their initial CNS target sites by spreading in a retrograde direction along efferent nerve fibers.¹²In the lymphoid tissues, it is believed that the macrophages, dendritic cells (DCs), and follicular dendritic cells (FDCs) are involved in the transportation and replication of the infectious agents. Macrophages are prevalent candidates for both spread¹³ and clearance¹⁴ of the infectious agents. DCs can capture and retain protein antigens in a nondegraded state.^15,16 These characteristics suggest that the macrophages and DCs may act as a transporter of the infectious agents from the gut to lymphoid tissues. FDCs express high levels of cellular PrPs (PrP^c), and therefore an early accumulation of PrP^Sc is seen in them.^17,18 Many studies of the alimentary pathogenesis of TSEs have been conducted to elucidate how infectious agents spread from the GALT to the CNS, although this has not been clearly determined yet. Therefore, the aim of the present study was to reveal the cells involved in the early stages of the pathogenesis of oral TSE infection, such as the sites of entry, spread, and neuroinvasion.     

The nasal dendritic cell-targeting Flt3 ligand as a safe adjuvant elicits effective protection against fatal pneumococcal pneumonia

We have previously shown that a pneumococcal surface protein A (PspA)-based vaccine containing DNA plasmid encoding the Flt3 ligand (FL) gene (pFL) as a nasal adjuvant prevented nasal carriage of Streptococcus pneumoniae. In this study, we further investigated the safety and efficacy of this nasal vaccine for the induction of PspA-specific antibody (Ab) responses against lung infection with S. pneumoniae. C57BL/6 mice were nasally immunized with recombinant PspA/Rx1 (rPspA) plus pFL three times at weekly intervals. When dynamic translocation of pFL was initially examined, nasal pFL was taken up by nasal dendritic cells (DCs) and epithelial cells (nECs) but not in the central nervous systems, including olfactory nerve and epithelium. Of importance, nasal pFL induced FL protein synthesis with minimum levels of inflammatory cytokines in the nasal washes (NWs) and bronchoalveolar lavage fluid (BALF). NWs and BALF as well as plasma of mice given nasal rPspA plus pFL contained increased levels of rPspA-specific secretory IgA and IgG Ab responses that were correlated with elevated numbers of CD8(+) and CD11b(+) DCs and interleukin 2 (IL-2)- and IL-4-producing CD4(+) T cells in the nasal mucosa-associated lymphoid tissues (NALT) and cervical lymph nodes (CLNs). The in vivo protection by rPspA-specific Abs was evident in markedly reduced numbers of CFU in the lungs, airway secretions, and blood when mice were nasally challenged with Streptococcus pneumoniae WU2. Our findings show that nasal pFL is a safe and effective mucosal adjuvant for the enhancement of bacterial antigen (Ag) (rPspA)-specific protective immunity through DC-induced Th2-type and IL-2 cytokine responses.     

Six-month aerobic exercise training ameliorates central sleep apnea in patients with chronic heart failure

BackgroundSleep-disordered breathing (SDB) is common in patients with heart failure and carries an independent risk for poor long-term prognosis. We aimed to study the effects of supervised, aerobic exercise training for 6 months on SDB in patients with chronic heart failure.Methods and ResultsWe enrolled 18 patients having both systolic dysfunction (left ventricular ejection fraction <45%) and SDB (apnea-hypopnea index [AHI] >10). The exercise group comprised 10 patients who participated in our cardiac rehabilitation program for 6 months, and the remaining 8 patients served as control. AHI (median [interquartile range]) was unchanged in the control group patients at 6-month intervals (30.4 [19.9–36.3] versus 36.6 [8.6–39.4], NS). In contrast, AHI was significantly decreased in the exercise group from 24.9 [19.2–37.1] to 8.8 [5.3–10.1] (P < .01). In the exercise group, the numbers of central sleep apnea per night was significantly decreased (152 [124–244] versus 50 [24–67], P < .01) after exercise training, but those of obstructive apnea/hypopnea were unchanged (42 [7–94] versus 18 [7–54], NS). In addition, exercise training significantly increased peak oxygen consumption and decreased minute ventilation to carbon dioxide production slope (both P < .01).ConclusionsSix-month, aerobic exercise training increased exercise capacity and improved central sleep apnea in patients with chronic heart failure from systolic dysfunction.     

Oral immunization with Porphyromonas gingivalis outer membrane protein and CpG oligodeoxynucleotides elicits T helper 1 and 2 cytokines for enhanced protective immunity

The aim of this study was to evaluate the efficacy of an oral vaccine containing the 40‐kDa outer membrane protein of Porphyromonas gingivalis (40K‐OMP) and synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) to control oral infection by P. gingivalis. Oral immunization with 40K‐OMP plus CpG ODN induced significant 40K‐OMP‐specific serum immunoglobulin G (IgG), IgA, and saliva IgA antibody responses. The 40K‐OMP‐specific CD4⁺ T cells induced by oral 40K‐OMP plus CpG ODN produced both T helper type 1 (Th1; interferon‐γ) and Th2 (interleukin‐4) cytokines. Furthermore, increased frequencies of CD11c⁺ B220⁺ dendritic cells (DCs) and CD11c⁺ CD11b⁺ DCs with upregulated expression of CD80, CD86, CD40, and major histocompatibility complex class II molecules were noted in spleen, Peyer’s patches, and cervical lymph nodes. Immunized mice were then infected orally with P. gingivalis to determine whether the immune responses induced by oral 40K‐OMP plus CpG ODN were capable of suppressing the bone resorption caused by P. gingivalis infection. Mice given 40K‐OMP plus CpG ODN showed significantly reduced bone loss associated with oral infection by P. gingivalis. Oral administration of 40K‐OMP together with CpG ODN induces Th1‐type and Th2‐type cells, which provide help for protective immunity against P. gingivalis infection. This may be an important tool for the prevention of chronic periodontitis.