CD8+ T cells from feline immunodeficiency virus (FIV) infected cats suppress exogenous FIV replication of their peripheral blood mononuclear cells in vitro

Summary.  Feline immunodeficiency virus (FIV) isolates from domestic cats have been classified into five subtypes, designated A, B, C, D and E. Although many FIV-infected cats may have frequent contact with multiple strains of FIV, they usually become infected with a single FIV subtype. In the present study, we demonstrate that peripheral blood mononuclear cells (PBMC) of FIV infected cats were resistant to exogenous FIV (second virus) replication in vitro and that the resistance of these PBMC was mediated by CD8⁺ T cells. In cats with a low anti-FIV activity of CD8⁺ T cells, the proviral DNA of the second virus inoculated into PBMC was detected intracellularly, and both the second and the originally infecting strain (original virus) were produced in the culture supernatant. In contrast, in cats with a high anti-FIV activity of CD8⁺ T cells, both the proviral DNA of the second virus and the original virus were detected in PBMC intracellularly, but neither virus was produced in the culture supernatant. However, when PBMCs from these cats were depleted of CD8⁺ T cells, the RNA of both viruses was detected in the culture supernatant. These results suggest that CD8⁺ T cells inhibit the late phase of FIV replication after viral integration. Moreover, the inhibition was also effective against FIV strains of different subtypes from that of the original strain. It appears that the CD8⁺ T cell-mediated immune response plays important roles in the maintenance of an asymptomatic state in FIV-infected cats and their resistance to superinfection. Received December 11, 2001; accepted March 22, 2002 Published online June 21, 2002     

Thymic epithelial reticular cell subpopulations in mice defined by monoclonal antibodies

Thymic epithelial reticular cells (TER) are heterogeneous cell populations. Of 14 rat monoclonal antibodies (moAbs) raised against established cell lines of mouse thymic stromal cells (TSC), two were found to recognize TER subpopulations that exhibited distinct intrathymic distributions. MoAb B6TS-1 (IgG2a) recognized the cell-surface determinant mB6TS-1 on TSC in the subcapsular zone, cortico-medullary junction, and medulla. Double staining with antikeratin antiserum showed that, except in the cortex, the distribution of mB6TS-1 bearing cells highly corresponded with that of keratin-positive TER indicating that mB6TS-1 within the thymus was selectively expressed in a particular subpopulation of epithelial cells. Immunoelectronmicroscopy revealed clear polarity in the expression of mB6TS-1 on TER. In the subcapsular zone. TER adherent to fibrous capsule expressed mB6TS-1 on the cell surface that faced the lymphocytes. In the cortico-medullary junction, mB6TS-1 also was found on the side of the TER closely associated with the small blood vessels. The mB6TS-1-bearing cells in the medulla characteristically had cytoplasmic infoldings containing collagen fibrils and amorphous material but did not exhibit the polarity of mB6TS-1-bearing cells. The mB6TS-1-bearing TER were interconnected by desmosomes and tonofilaments, therefore, were easily distinguished from macrophages, dendritic cells, and other components of thymic stroma. In contrast, another moAb AKTS-1 (IgM) stained the keratin-positive TER localized in the subcapsular zone and cortex forming a fine meshwork but did not stain those in the medulla. MoAb B6TS-1 stained thymic nurse cells but not the central cells of thymic rosettes, whereas moAB AKTS-1 did neither. Formation of lymphoid-stromal cell complexes in vitro was not affected by either antibody.     

Acute toxicity,percutaneous absorption and effects on hepatic mixed function oxidase activities of 2,4,4′-trichloro-2′-hydroxydiphenyl ether (Irgasan® DP300) and its chlorinated derivatives

Acute toxicity of 2,4,4-trichloro-2-hydroxydiphenyl ether (Irgasan^® DP300) (I) and its three chlorinated derivatives, 2,3,4,4-tetrachloro-2-hydroxydiphenyl ether (II), 2,4,4,5-tetrachloro-2-hydroxydiphenyl ether (III) and 2,3,4,4,5-pentachloro-2-hydroxydiphenyl ether (IV), in mice were examined by intraperitoneal injection. The LD₅₀ values of Irgasan DP300, II, III and IV were 1,090, 710, 650 and 430 mg/kg, respectively.The percutaneous absorptions of these tritiated compounds were also examined by the application on the backs of mice. The radioactivities in most tissues reached to the maximal levels at 12 h or 18 h after dosing, which corresponded to 11–76% of the maximal levels given by the oral administration (Kanetoshi et al. 1988a). These results show the high percutaneous absorbability of Irgasan DP300 and its chlorinated derivatives.The intraperitoneal administrations of III and IV to rats induced hepatic microsomal aminopyrine N-demethylase and aniline 4-hydroxylase activities similarly to phenobarbital. These chlorinated derivatives also increased cytochrome P-450 content, and the activities of aminopyrine N-demethylase and N-methylaniline N-demethylase in hepatic microsomes from mice. The extents of the increases were similar to those by phenobarbital and 3-methylcholanthrene.     

Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression

Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C–induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with small-molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C–induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.     

Maternal GABAergic and GnRH/corazonin pathway modulates egg diapause phenotype of the silkworm Bombyx mori

Diapause represents a major developmental switch in insects and is a seasonal adaptation that evolved as a specific subtype of dormancy in most insect species to ensure survival under unfavorable environmental conditions and synchronize populations. However, the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration in morphological, physiological, behavioral, and reproductive responses remains unclear. In the bivoltine strain of the silkworm Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother. Here, we show that the hierarchical pathway consists of a γ-aminobutyric acid (GABA)ergic and corazonin signaling system modulating progeny diapause induction via diapause hormone release, which may be finely tuned by the temperature-dependent expression of plasma membrane GABA transporter. Furthermore, this signaling pathway possesses similar features to the gonadotropin-releasing hormone (GnRH) signaling system for seasonal reproductive plasticity in vertebrates.

To ensure survival under unfavorable environmental conditions and synchronize populations, most insect species enter diapause, which is a seasonal adaptation that evolved as a specific subtype of dormancy (1, 2). Diapause is not a passive response to changing conditions but rather an actively induced state that precedes adverse natural situations. Therefore, this diapause phenotype is accompanied by changes in energy metabolism or storage to improve cold/stress tolerance in later life stages, or progeny via reproductive switch (3). Although it has been generally suggested that brain/neuroendocrine systems are associated with this seasonal reproductive plasticity in both vertebrates and invertebrates (3, 4), the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration into morphological, physiological, behavioral, and reproductive responses, known as the diapause syndrome, remains unclear (3).The silkworm Bombyx mori is a typical insect that arrests normal development during early embryogenesis, which is accompanied by metabolic changes in diapause (5, 6). The development of diapause-destined embryos is arrested during the G2 cell cycle stage immediately after the formation of the cephalic lobe and telson and sequential segmentation of the mesoderm (7). The bivoltine strain of B. mori has two generations per year, and progeny diapause is transgenerationally induced as a maternal effect and is determined by the environmental temperature, photoperiod, and nutrient conditions during embryonic and larval development of the mother (5, 6). The temperature signal during the mother’s embryonic development predominantly affects diapause determination, even if silkworms of the bivoltine Kosetsu strain are exposed to all cases of photoperiods during embryonic and larval development. In the Kosetsu strain, when eggs are incubated at 25 °C under continuous darkness, the resultant female moths (25DD) lay diapause eggs in almost all cases. In contrast, incubation of eggs at 15 °C in dark condition results in moths (15DD) that lay nondiapause eggs in almost all cases (6).Embryonic diapause is induced by the diapause hormone (DH) signaling pathway, which consists of highly sensitive and specific interactions between a neuropeptide, DH, and DH receptor (DHR) (6, 8). DH is exclusively synthesized in seven pairs of neurosecretory cells (DH-PBAN–producing neurosecretory cells [DHPCs]) located within the subesophageal ganglion (SG) in the mother’s generation (6). DH is released into the hemolymph during pupal–adult development and acts on the DHR, which belongs to the G protein-coupled receptors (GPCRs) (9). DH levels in the hemolymph are higher in the 25DD than 15DD pupae in the middle of pupal–adult development when the developing ovaries are sensitive to DH (6). Furthermore, the embryonic Bombyx TRPA1 ortholog (BmTRPA1) acts as a thermosensitive channel that is activated at temperatures above ∼21 °C and affects diapause induction through DH release (10). However, there remain questions about the thermal information that is received by BmTRPA1 and linked to DH signaling to induce diapause.From the 1950s, it has been suggested that the DH release was controlled by signals derived from certain region(s) in the brain based on surgical experiments, such as midsagittal bisection or transection (11–13). Especially, the operation in nondiapause producers changed them to diapause producers while transection of the protocerebrum had no effect on the diapause producers. These surgical results suggested the involvement of the protocerebrum in the inhibitory control of DH secretion (12, 14). Furthermore, the accumulation of the ovarian 3-hydroxykynurenine (3-OHK) pigment that accompanies the diapause syndrome was affected by injection with γ-aminobutyric acid (GABA) and the plant alkaloid picrotoxin (PTX), which is a widely used ionotropic GABA and glycine receptor antagonist (15, 16), and the selective ionotropic GABA receptor (GABAR) antagonist bicuculline. This suggests that a GABAergic neurotransmission via ionotropic GABAR is involved in DH secretion, which may be active in nondiapause producers but inactive in diapause producers throughout the pupal–adult development (14, 17). In general, ionotropic GABAR is composed of homo- or hetero-pentameric subunits. All known GABAR subunits display a similar structural scheme, with a large N-terminal extracellular domain involved in the formation of a ligand-binding pocket and a pore domain made of four transmembrane alpha-helices (TM1–TM4) (16, 18). Four homologous sequences of the ionotropic GABAR subunit genes were identified as RDL, LCCH3, GRD, and a GRD-like sequence named 8916 in various insects (19). However, the in vivo physiological roles of both signals derived from the brain and the GABAergic pathway in diapause induction have not been previously investigated.Corazonin (Crz) is an undecapeptide neurohormone sharing a highly conserved amino acid (a.a.) sequence across insect lineages and is involved in different physiological functions, such as heart contraction (20), stress response (21, 22), various metabolic activities (23–25), female fecundity (26), melanization of locust cuticles (27), regulation of ecdysis (28, 29), and control of caste identity (30). Moreover, Crz belongs to the gonadotropin-releasing hormone (GnRH) superfamily alongside adipokinetic hormone (AKH) and AKH/Crz-related peptide (ACP). Duplicates of an ancestral GnRH/Crz signaling system occurred in a common ancestor of protostomes and deuterostomes through coevolution of the ligand receptor (31, 32).Herein, we demonstrated that the hierarchical pathway consists of a GABAergic and Crz signaling system modulating progeny diapause induction by acting on DH release. We propose that the PTX-sensitive GABAergic signal may act to chronically suppress Crz release in dorsolateral Crz neurons (under nondiapause conditions) and that diapause conditions (or PTX injection) inhibits GABAergic signaling, resulting in accelerated Crz release, which in turn induces DH release. GABA signaling may be finely tuned by the temperature-dependent expression of the plasma membrane GABA transporter (GAT), which differs between the 25DD and 15DD conditions. Furthermore, this signaling pathway possesses similar features to the GnRH signaling system with respect to seasonal reproductive plasticity in vertebrates.     

Can nurses exclude middle-ear effusion without otoscopy in young asymptomatic children in primary care?

Objective. Scandinavian guidelines recommend controlling middle-ear effusion (MEE) after acute otitis media. The study aim was to determine whether nurses without otoscopic experience can reliably exclude MEE with tympanometry or spectral gradient acoustic reflectometry (SG-AR) at asymptomatic visits. Design. Three nurses were taught to perform examinations with tympanometry and SG-AR. Pneumatic otoscopy by the study physician served as the diagnostic standard. Setting. Study clinic at primary health care level. Patients. A total of 156 children aged 6–35 months. Main outcome measures. Predictive values (with 95% confidence interval) for tympanometry and SG-AR, and the clinical usefulness, i.e. the proportion of visits where nurses obtained the exclusive test result from both ears of the child. Results. At 196 visits, the negative predictive value of type A and C1 tympanograms (tympanometric peak pressure > −200 daPa) was 95% (91–97%). Based on type A and C1 tympanograms, the nurse could exclude MEE at 81/196 (41%) of visits. The negative predictive value of SG-AR level 1 result was 86% (79–91%). Based on SG-AR level 1 results, the nurse could exclude MEE at 29/196 (15%) of visits. Conclusion. Tympanograms with tympanometric peak pressure > −200 daPa (types A and C1) obtained by nurses are reliable test results in excluding MEE. However, these test results were obtained at less than half of the asymptomatic visits and, thus, the usefulness of excluding MEE by nurses depends on the clinical setting.