Tuft cells, taste-chemosensory cells, orchestrate parasite type 2 immunity in the gut.

The intestinal epithelium forms an essential barrier between a host and its microbiota. Protozoa and helminths are members of the gut microbiota of mammals, including humans, yet the many ways that gut epithelial cells orchestrate responses to these eukaryotes remain unclear. Here we show that tuft cells, which are taste-chemosensory epithelial cells, accumulate during parasite colonization and infection. Disruption of chemosensory signaling through the loss of TRMP5 abrogates the expansion of tuft cells, goblet cells, eosinophils, and type 2 innate lymphoid cells during parasite colonization. Tuft cells are the primary source of the parasite-induced cytokine interleukin-25, which indirectly induces tuft cell expansion by promoting interleukin-13 production by innate lymphoid cells. Our results identify intestinal tuft cells as critical sentinels in the gut epithelium that promote type 2 immunity in response to intestinal parasites.

Activation of Transducin by Bistable Pigment Parapinopsin in the Pineal Organ of Lower Vertebrates.

Pineal organs of lower vertebrates contain several kinds of photosensitive molecules, opsins that are suggested to be involved in different light-regulated physiological functions. We previously reported that parapinopsin is an ultraviolet (UV)-sensitive opsin that underlies hyperpolarization of the pineal photoreceptor cells of lower vertebrates to achieve pineal wavelength discrimination. Although, parapinopsin is phylogenetically close to vertebrate visual opsins, it exhibits a property similar to invertebrate visual opsins and melanopsin: the photoproduct of parapinopsin is stable and reverts to the original dark states, demonstrating the nature of bistable pigments. Therefore, it is of evolutionary interest to identify a phototransduction cascade driven by parapinopsin and to compare it with that in vertebrate visual cells. Here, we showed that parapinopsin is coupled to vertebrate visual G protein transducin in the pufferfish, zebrafish, and lamprey pineal organs. Biochemical analyses demonstrated that parapinopsins activated transducin in vitro in a light-dependent manner, similar to vertebrate visual opsins. Interestingly, transducin activation by parapinopsin was provoked and terminated by UV- and subsequent orange-lights irradiations, respectively, due to the bistable nature of parapinopsin, which could contribute to a wavelength-dependent control of a second messenger level in the cell as a unique optogenetic tool. Immunohistochemical examination revealed that parapinopsin was colocalized with Gt2 in the teleost, which possesses rod and cone types of transducin, Gt1, and Gt2. On the other hand, in the lamprey, which does not possess the Gt2 gene, in situ hybridization suggested that parapinopsin-expressing photoreceptor cells contained Gt1 type transducin GtS, indicating that lamprey parapinopsin may use GtS in place of Gt2. Because it is widely accepted that vertebrate visual opsins having a bleaching nature have evolved from non-bleaching opsins similar to parapinopsin, these results implied that ancestral bistable opsins might acquire coupling to the transducin-mediated cascade and achieve light-dependent hyperpolarizing response of the photoreceptor cells.

Non-specific immunostaining by a rabbit antibody against gustducin α subunit in mouse brain.

Gustducin is a guanosine nucleotide-binding protein functionally coupled with taste receptors and thus originally identified in taste cells of the tongue. Recently, bitter taste receptors and gustducin have been detected in the airways, digestive tracts and brain. The existing studies showing taste receptors and gustducin in the brain were carried out exclusively on frozen sections. In order to avoid the technical shortcomings associated with frozen sectioning, we performed immunofluorescence staining using vibratome-cut sections from mouse brains. Using a rabbit gustducin antibody, we could not detect neurons or astrocytes as reported previously. Rather, we found dense fibers in the nucleus accumbens and periventricular areas. We assumed these staining patterns to be specific after confirmation with conventional negative control staining. For the verification of this finding, we stained gustducin knockout mouse brain and tongue sections with the same rabbit gustducin antibody. Whereas negative staining was confirmed in the tongue, intensive fibers were constantly stained in the brain. Moreover, immunostaining with a goat gustducin antibody could not demonstrate the fibers in the brain tissue. The present study implies a cross immunoreaction that occurs with the rabbit gustducin antibody in mouse brain samples, suggesting that the conventional negative controls may not be sufficient when an immunostaining pattern is to be verified.

Occurrence of gustducin-immunoreactive cells in von Ebner's glands of guinea pigs.

An immunohistochemical examination of guinea-pig taste buds in vallate papillae revealed gustducin-immunoreactive cells in the area of von Ebner's glands, minor salivary glands. Since there have been no reports describing those cells in these locations for other species, we investigated these glands in order both to localize the cells and compare their immunoreactive characteristics with corresponding cells in the vallate taste buds. The gustducin-immunoreactive cells coincided with cells containing no secretory granules in the end portion of the glands, which was supported by the electron-microscopic immunocytochemistry. Double immunofluorescence microscopy confirmed these cells to be entirely immunopositive to type III inositol 1,4,5-triphosphate receptor (IP3R-3), phospholipase Cß2 (PLCß2), and villin and also partly immunopositive to neuron-specific enolase (NSE) and calbindin D-28K. The gustducin-immunoreactive cells in the vallate taste buds exhibited completely the same immunoreactivities for these five molecules. Accordingly, the present results give credence to a consideration that the gustducin-immunnoreactive cells in both locations are identical in function(s) e.g., chemo-reception.

Fixation conditions affect the immunoreactivity of gustducin in rat vallate taste buds.

This study aimed to examine gustducin immunoreactivities when adopting various immunostaining conditions in rat vallate taste buds. The occurrence and intensity of the immunoreactivities exhibited specific patterns in accordance with the fixation time. The immunoreactions were localized to only taste hairs, the upper part of the taste bud, after short fixation periods but then to the cell-body cytoplasm excluding the taste hairs after long fixation periods. These immunohistochemical data suggest that the staining protocols, especially the fixation time, cause discrepancies in gustducin immunoreactivities.

Molecular biomarkers for autoimmune retinopathies: significance of anti-transducin-alpha autoantibodies.

Autoimmune retinopathies (AR) are uncommon retinal degenerations with vision loss associated with unique clinical symptoms and findings and with serum anti-retinal autoantibodies. The experimental and clinical studies corroborate that autoantibodies in high titers can penetrate into the retina affecting function of the target antigens, which leads to retinal dysfunction and degeneration. Anti-recoverin and anti-enolase alpha-enolase autoantibodies were more frequently recognized in AR but autoantibodies with other specificities have also been documented, indicating immunological heterogeneity. Our goal was to examine the associations of anti-retinal autoantibodies with retinopathy in order to identify molecular biomarkers for better diagnosis and prognosis of retinopathies. In these studies we examined 39 patients (10 with cancers) of average age of approximately 57 years old with sudden onset of unexplained progressive vision loss and the presence of circulating serum autoantibodies against 40-kDa retinal protein. The patients presented the retinal phenotype characterized by defects in visual fields and reduced scotopic ERG responses. Anti-40-kDa autoantibodies had specificity to the amino terminus of transducin-alpha. None of the normal subjects' sera had anti-40-kDa autoantibodies. In conclusion, the clinical phenotype of patients with anti-transducin-alpha autoantibodies differed from other phenotypes of AR. These patients, often women at a ratio approximately 2:1, had defects in rod (scotopic) photoreceptor function and typically did not have cancers, whereas the anti-recoverin phenotype is associated with cancer and severe loss of rod and cones function, and anti-enolase retinopathy typically presents with cone dysfunction and is equal in cancer and non-cancer patients. Our studies suggest that anti-transducin autoantibodies can serve as molecular biomarkers for retinal phenotypes and could be used for progression of retinal dysfunction and degeneration.

Proteolytic fragmentation for epitope mapping.

The use of antigen fragments generated by specific proteolytic cleavage is a relatively simple "library" approach for epitope mapping in which possible overlapping fragments are screened with the antibody on Western blots. Proteolytic fragmentation with numerous proteases having different cleavage specificites can be carried out on native and denaturated proteins, generating a small and large number of fragments, respectively. To determine the antigenic site of a monoclonal antibody, we have examined the limited proteolytic digestion of the transducin alpha -subunit with four different proteases and detected antibody binding to fragments by Western blot. Using this approach, the epitope for this antibody was localized within the amino-terminal 17 residues of transducin alpha -subunit.

Autoantibody targets and their cancer relationship in the pathogenicity of paraneoplastic retinopathy.

Paraneoplastic retinopathies (PR), including cancer-associated retinopathy (CAR) or the closely related melanoma-associated retinopathy (MAR) occur in a small subset of patients with retinal degeneration and systemic cancer. This autoimmune syndrome is characterized by sudden, progressive loss of vision in association with circulating anti-retinal autoantibodies. The PR syndromes are heterogeneous, may produce a number of ocular symptoms, and may be associated with several different neoplasms, including lung, breast, prostate, gynecological, and colon cancer, melanoma, and hematologic malignancies. We examined the onset of retinopathy in correlation to the diagnosis of cancer and the presence of specific anti-retinal autoantibodies in PR patients. In some patients without diagnosed malignant tumors, the onset of ocular symptoms and the presence of autoantibodies preceded the diagnosis of cancer by months to years, including anti-recoverin, anti-transducin-alpha, and anti-carbonic anhydrase II antibodies. Although anti-retinal autoantibodies may not be a good predictor of a specific neoplasm, they can be used as biomarkers for different subtypes of retinopathy. Identification of autoantibodies involved in autoimmune-mediated PR will help elucidate the mechanisms underlying the PR syndromes and develop targeted therapies for these sight-threatening disorders.

Existence of subtypes of gustducin-immunoreactive cells in the vallate taste bud of guinea pigs.

Vallate taste buds in the guinea-pig tongue were immunohistochemically investigated with regard to the colocalization of gustducin with calbindin-D28K (=spot 35 protein) and type III inositol triphosphate receptor (IP(3)R-3) in order to characterize gustducin-immunoreactive cells. Individual taste bud cells ranged from totally immunopositive to totally immunonegative for these three molecules. Among the immunoreactive cells, gustducin-immunoreactive cells were divided into two cell populations: one immunopositive and the other immunonegative for calbindin-D28K. Applying our previous data to the present results, the former cells should belong to Type III cells designated by electron microscopy. This finding provides new evidence regarding the taste bud types of cells expressing gustducin in the guinea pig.

Structure of bovine fungiform taste buds and their immunoreactivity for gustducin.

The taste buds of bovine fungiform papillae were studied by light and electron microscopy using both histological and immunohistochemical methods. The taste buds existed in the epithelium of the apical region of the papillae. By electron microscopy, two types of taste cells, namely type I and type II cells, could be classified according to the presence of dense-cored vesicles, the cytoplasmic density and the cell shape. Type I cells were thin, had an electron-dense cytoplasm containing dense-cored vesicles, and possessed long thick apical processes in the taste pore. Type II cells were thick, had an electron-lucent cytoplasm containing many electron-lucent vesicles, rather than dense-cored vesicles, and possessed microvilli in the taste pore. Immunohistochemical staining with an antiserum against gustducin was investigated by both light and electron microscopy using the avidin-biotin complex (ABC) method. Some, but not all, of the type II cells exhibited gustducin immunoreactivity, whereas none of the type I cells showed any immunoreactivity.

Autoantibodies to transducin in a patient with melanoma-associated retinopathy.

PURPOSE: To report novel immunoreactivity in a patient with melanoma-associated retinopathy. DESIGN: Retrospective case report and experimental study. METHODS: A 32-year-old woman with a history of metastatic melanoma presented with bilateral decreased visual acuity. Electroretinography, Goldmann perimetry, immunohistochemistry, and Western blotting of her serum were performed. RESULTS: Electroretinography showed a "negative" B-wave. Paracentral and central scotomas were observed on Goldmann perimetry. Antibodies to a retinal transducin were demonstrated by Western blotting. No immunoreactivity to retinal bipolar cells was detected by immunohistochemistry. CONCLUSION: Melanoma-associated retinopathy can be related to a variety of antiretinal antibodies. Recognition of transducin, a novel melanoma-associated retinopathy antigen, may be important for identifying and treating patients with night blindness and melanoma.

Bitter taste transduced by PLC-beta(2)-dependent rise in IP(3) and alpha-gustducin-dependent fall in cyclic nucleotides.

Current evidence points to the existence of multiple processes for bitter taste transduction. Previous work demonstrated involvement of the polyphosphoinositide system and an alpha-gustducin (Galpha(gust))-mediated stimulation of phosphodiesterase in bitter taste transduction. Additionally, a taste-enriched G protein gamma-subunit, Ggamma(13), colocalizes with Galpha(gust) and mediates the denatonium-stimulated production of inositol 1,4,5-trisphosphate (IP(3)). Using quench-flow techniques, we show here that the bitter stimuli, denatonium and strychnine, induce rapid (50-100 ms) and transient reductions in cAMP and cGMP and increases in IP(3) in murine taste tissue. This decrease of cyclic nucleotides is inhibited by Galpha(gust) antibodies, whereas the increase in IP(3) is not affected by antibodies to Galpha(gust). IP(3) production is inhibited by antibodies specific to phospholipase C-beta(2) (PLC-beta(2)), a PLC isoform known to be activated by Gbetagamma-subunits. Antibodies to PLC-beta(3) or to PLC-beta(4) were without effect. These data suggest a transduction mechanism for bitter taste involving the rapid and transient metabolism of dual second messenger systems, both mediated through a taste cell G protein, likely composed of Galpha(gust)/beta/gamma(13), with both systems being simultaneously activated in the same bitter-sensitive taste receptor cell.

Differentiation of the lingual and palatal gustatory epithelium of the rat as revealed by immunohistochemistry of alpha-gustducin.

We used alpha-gustducin, a taste-cell-specific G protein to investigate the onset of taste transduction and its relation to the development of the palatal and lingual taste buds. Frozen cryostat and paraffin sections were prepared from the palatal and lingual gustatory epithelium of the rat from birth till postnatal day 21 (PN 21d). At PN 1-7d, alpha-gustducin-immunoreactive solitary ovoid or bipolar cells were scattered among the oral epithelium either horizontally along the oral surface or vertically oriented between the basal lamina and oral surface. In the circumvallate and foliate papillae, these cells became wrapped in alpha-gustducin-immunonegative cells surrounded by an extracellular space forming a bud-like structure. Simultaneously, different stages of typical taste buds were recognized, but alpha-gustducin was only expressed in some neonatally developed pored buds. At PN 1d, alpha-gustducin was expressed in pored taste buds with a relatively higher frequency recorded in the soft palate as compared with the nasoincisor, circumvallate, and foliate papillae. The immunoreactive cells were spindle shaped with elongated processes extending from the base to the pore of the taste buds. During the second week, the solitary cells could no longer be recognized while the total counts of immunoreactive cells within the taste buds gradually increased. We argue that taste transduction is essentially required from the time of birth and can be fulfilled by both of the solitary chemosensory cells, which are immunoreactive for alpha-gustducin and scattered in the oral epithelium, and the taste cells within the mature taste buds. Moreover, the onset of taste transduction accomplished by the palatal taste buds developed earlier than that achieved by taste buds in the circumvallate and foliate papillae.

Contribution of phosphoinositide-dependent signalling to photomotility of Blepharisma ciliate.

The effect of experimental procedures designed to modify an intracellular phosphoinositide signalling pathway, which may be instrumental in the photophobic response of the protozoan ciliate Blepharisma japonicum, has been investigated. To assess this issue, the latency time of the photophobic response and the cell photoresponsiveness have been assayed employing newly developed computerized videorecording and standard macro-photographic methods. Cell incubation with neomycin, heparin and Li+, drugs known to greatly impede phosphoinositide turnover, causes evident dose-dependent changes in cell photomotile behaviour. The strongest effect on photoresponses is exerted by neomycin, a potent inhibitor of polyphosphoinositide hydrolysis. The presence of micromolar concentrations of neomycin in the cell medium causes both prolongation of response latency and decrease of cell photoresponsiveness. Neomycin at higher concentrations (> 10 microM) abolishes the cell response to light at the highest applied intensity. A slightly lower inhibition of cell responsiveness to light stimulation and prolongation of response latency are observed in cells incubated in the presence of heparin, an inositol trisphosphate receptor antagonist. Lithium ions, widely known to deplete the intracellular phosphoinositide pathway intermediate, inositol trisphosphate, added to the cell medium at millimolar level, also cause a slowly developing inhibitory effect on cell photoresponses. Mastoparan, a specific G-protein activator, efficiently mimics the effect of light stimulation. In dark-adapted ciliates, it elicits ciliary reversal with the response latency typical for ciliary reversal during the photophobic response. Sustained treatment of Blepharisma cells with mastoparan also suppresses the photoresponsiveness, as in the case of cell adaptation to light during prolonged illumination. The mastoparan-induced responses can be eliminated by pretreatment of the cells with neomycin. Moreover, using antibodies raised against bovine transducin, a cross-reacting protein with an apparent molecular mass of about 55 kDa in the Blepharisma cortex fraction is detected on immunoblots. The obtained results indirectly suggest that the changes in internal inositol trisphosphate level, possibly elicited by G-protein-coupled phospholipase C, might play a role in the photophobic response of Blepharisma. However, further experiments are necessary to clarify the possible coupling between the G-protein and the putative phospholipase C.

Ultrastructural localization of gustducin immunoreactivity in microvilli of type II taste cells in the rat.

Gustducin is a transducin-like G protein (guanine nucleotide-binding protein) that is expressed in taste bud cells. Gustducin is believed to be involved in bitter and possibly sweet taste transduction. In the present study, we demonstrate that a subset of type II cells displays immunoreactivity to antisera directed against gustducin in taste buds of rat circumvallate papilla. Immunogold particles are present both in the microvilli and cytoplasm of the immunoreactive cells. Quantitative analysis of the data suggests that the number of colloidal gold particles (P<0.001) and nanogold particles (P<0.01) in the immunoreactive type II cells are much greater than in type I cells. There are also approximately 2.5 times (P<0.05) as many colloidal gold particles associated with the microvilli versus the cytoplasm in the immunoreactive type II cells. The ultrastructural distribution of gustducin immunoreactivity is consistent with its proposed role in the initial events of sensory transduction by gustatory receptor cells.

The timing of alpha-gustducin expression during cell renewal in rat vallate taste buds.

The G protein subunit alpha-gustducin is expressed in a subset of light (Type II) but not in dark (Type I) cells in rat vallate taste buds. The thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) is incorporated into DNA during the S-phase of the cell cycle and can be used to determine the time of origin of a cell. In this study, 31 rats were injected with BrdU (50 mg/kg i.p.) and perfused at various times, from 2.5 to 10.5 days, following BrdU administration. Vallate papillae were embedded in polyester wax, cut into 4 microm transverse sections, and characterized with antibodies to BrdU and alpha-gustducin. Sections were processed for indirect immunofluorescence or with an immunoperoxidase procedure. From immunoperoxidase material on 21 rats, counts of alpha-gustducin- and BrdU-labeled cells were obtained from 300-800 taste bud profiles at each survival time; a total of 4122 taste bud profiles were examined. Cells with nuclei immunoreactive for BrdU occurred within the taste buds at 2.5 days and double-labeled cells were clearly evident at 3.5 days; a small number of double-labeled cells were seen as early as 2.5 days. Double-labeled cells reached a peak at 6.5 days and did not decline significantly by 10.5 days. Cells labeled for BrdU but not alpha-gustducin peaked at 5.5 days and showed a significant decline by 8.5 days. These latter cells included light cells not expressing alpha-gustducin and dark cells, which have previously been shown to have a shorter life span than light cells. These data suggest that expression of alpha-gustducin appears very early in a cell's life span and that these cells are longer lived than many of the cells that do not express this G protein.

Preparation and characterization of monoclonal antibodies specific for lauroylated isoform of bovine transducin alpha-subunit: immunohistochemical analysis of bovine retinas.

The photoreceptor G protein transducin [alpha- and beta gamma-subunits (T alpha/T beta gamma)] plays a central role in the visual transduction process. The amino-terminus of bovine T alpha is modified by one of four distinct fatty acids-laurate (C12:0), myristate (C14:0), C14:1 (5-cis), and C14:2 (5-cis, 8-cis)-but the biological significance and the localization of the four isoforms of T alpha are poorly understood. To investigate the cellular distribution of each isoform, we prepared monoclonal antibodies against a synthetic C12:0-, C14:0-, C14:1-, or C14:2-nonapeptide corresponding to the N-terminal region of T alpha. Among several types of antibodies isolated, only one type, represented by LA4, reacted specifically with the C12:0-peptide as well as purified T alpha but not with the other proteins in bovine retinal homogenate, including recoverin, indicating that the epitope comprises both C12:0 and the N-terminal amino acids of T alpha. Immunohistochemical analyses of bovine retinal sections by LA4 showed the uniform distribution of C12:0-T alpha in almost all the rod outer segments. Hence, it seemed unlikely that each isoform of T alpha was localized in specific cells. This observation, together with evidence for a possible functional diversity among the isoforms, suggests that the four isoforms of T alpha in a single rod cell may contribute simultaneously to a fine tuning of the photon-signal transduction process.

Immunochemical detection of GTP-binding protein in cephalopod photoreceptors by anti-peptide antibodies.

We prepared polyclonal antibodies (Pab) against the following peptides: partial sequences of bovine transducin alpha subunit including the ADP-ribosylation sites sensitive to cholera toxin (CTX) and pertussis toxin (PTX) and the N-terminus of Drosophila GTP-binding protein q alpha (DGqN); Pab CTX, Pab PTX and Pab DGqN, respectively. These antibodies were specific to the peptides used as antigen and no crossreactivity was observed. Pab CTX and Pab PTX reacted with bovine transducin alpha subunit and the reactivity was lost by preincubation with the specific antigen peptide. Proteins of 41-42 kDa in octopus and squid photoreceptor membranes were recognized by Pab DGqN but not by Pab CTX or Pab PTX. Anti-alpha antibody (GA/1) reacted with the same bands as Pab DGqN recognized. These results suggest that the major GTP-binding protein in cephalopod photoreceptors is a Gq-type, similar to Drosophila Gq.