Regulation of gene expression by retinoids.

Vitamin A serves as substrate for the biosynthesis of several derivates (retinoids) which are important for cell growth and cell differentiation as well as for vision. Retinoic acid is the major physiologically active form of vitamin A regulating the expression of different genes. At present, hundreds of genes are known to be regulated by retinoic acid. This regulation is very complex and is, in turn, regulated on many levels. To date, two families of retinoid nuclear receptors have been identified: retinoic acid receptors and retinoid X receptors, which are members of the steroid hormone receptor superfamily of ligand-activated transcription factors. In order to regulate gene expression, all-trans retinal needs to be oxidized to retinoic acid. All-trans retinal, in turn, can be produced during oxidation of all-trans retinol or in a retinol-independent metabolic pathway through cleavage of ß-carotene with all-trans retinal as an intermediate metabolite. Recently it has been shown that not only retinoic acid is an active form of vitamin A, but also that all-trans retinal can play an important role in gene regulation. In this review we comprehensively summarize recent literature on regulation of gene expression by retinoids, biochemistry of retinoid receptors, and molecular mechanisms of retinoid-mediated effects on gene regulation.

[Adoptive T-cell therapy of rhabdomyosarcoma]. / Adoptive T-Zell-Therapie des Rhabdomyosarkoms.

AIMS: To improve survival of patients with advanced rhabdomyosarcomas (RMS), we aimed to adoptively transfer T-cells with redirected specificity for the fetal acetylcholine receptor (AChR), an RMS-specific cell surface antigen. METHODS: A "second generation" chimeric antigen receptor (CAR) with a combined CD28-CD3ζ signaling domain was derived from our previously described chimeric antigen receptor composed of an extracellular human anti-fAChR antibody fragment, an Fc hinge region, and the intracellular T-cell receptor zeta chain. Lymphocytes from the peripheral blood were modified by retroviral transduction and monitored by FACS analysis. Cytotoxicity of modified T-cells towards RMS cells was recorded by MTT-based viability tests; expression of co-stimulatory molecules and anti-apoptotic genes was studied by FACS and qRT-PCR analysis. RESULTS: Co-stimulatory molecules were expressed in low levels on RMS cells giving the rationale to generate a CD28-CD3ζ signalling CAR (chimeric antigen receptor) for redirecting T-cells. T-cells were successfully engineered with the "second generation" AChR-specific chimeric antigen receptor. Despite of high CAR expression engineered T-cells showed low killing efficiency towards RMS compared to redirected killing of CD20+ lymphoma or CEA-expressing adenocarcinoma cell lines when redirected by CD20- and/or CEA-specific CAR. CONCLUSIONS: Data suggest that RMS cells exhibit resistance to a T-cell attack redirected by a fAChR-specific CAR. Inhibition of anti-apoptotic pathways in those cells may improve sensitivity to conventional as well as T-cell-based therapeutics.

Onconeural versus paraneoplastic antigens?

A hallmark of naturally occurring tumor immunity is the aberrant expression of so called "onconeural antigens" or "paraneoplastic antigens". At present, these two terms are used as synonyms for proteins which are normally expressed only in neuronal tissues, but in the process of carcinogenesis, they can be detected in tumors located outside the nervous system. As neuronal tissues are immunopriveleged zones, expression of these proteins in tumor cells can induce an autoimmune response, which manifests in the generation of autoantibodies and/or specific cytotoxic T-cells. Whether or not such immune responses necessarily lead to paraneoplastic syndromes or to a beneficial antitumor response or both is not fully understood. In this review we comprehensively summarize recent literature on paraneoplastic antigens including the corresponding neurological syndromes. A unified classification is proposed with "onconeural antigens" as collective term and a number of subgroups including the recently discovered cancer-retina antigens. Certain onconeural antigens can serve as paraneoplastic antigens under conditions which have yet to be defined, implying that the paraneoplastic function is not inherent to the antigen. The potential of onconeural antigens in cancer diagnostics and treatment strategies is discussed.

SEREX identification of new tumour-associated antigens in cutaneous T-cell lymphoma.

BACKGROUND: Cutaneous T-cell lymphoma (CTCL) is a clonal lymphoproliferative disorder of mainly CD4+ T cells, with primary manifestation in the skin. OBJECTIVES: To detect new CTCL-associated antigens for immunological therapies and to define their specificity in terms of RNA expression and seroreactivity. METHODS: A newly constructed CTCL cDNA phage library was screened and cross-reactivities against the detected clones were tested using 15 mycosis fungoides and six Sézary syndrome sera. The mRNA expression of the identified genes was analysed by reverse transcription-polymerase chain reaction (RT-PCR) using 22 tumour tissues, nine cell lines and up to 29 different types of normal tissue. RESULTS: We identified nine different tumour antigens (HD-CL-01 to HD-CL-09) of which seven clones had high homology to genes with known functions. Several of these genes had previously been associated with cancer, namely inositol 1,4,5-triphosphate 5-phosphatase, vimentin, aldose reductase and elongation factor-1alpha. Variations in the deduced protein sequences were observed in three cases, mostly due to variations in protein length. The individual clones were recognized by up to 56% of patients' sera, while control sera were negative except in one case. Using RT-PCR, we found a frequent expression of these new tumour antigens in tumour specimens (26-100%). In contrast to humoral specificity, specific mRNA was also detected in selected normal tissues (29-89%). CONCLUSIONS: SEREX (serological identification of antigens by recombinant expression cloning) identified multiple tumour-associated antigens in CTCL. The serological specificity and the high percentage of reactive sera of CTCL patients against several clones suggest these genes as potential targets for diagnostic and prognostic purposes.

Seroreactivity against MAGE-A and LAGE-1 proteins in melanoma patients.

BACKGROUND: Cancer-testis antigens exemplify a growing number of tumour antigens which are expressed in a variety of malignancies, but not in normal tissues other than germ cells, primarily those of the testis. OBJECTIVES: To investigate the humoral response to known cancer-testis antigens in melanoma patients. METHODS: We used phage clones coding for seven different melanoma antigens MAGE-A or LAGE-1A proteins. These clones were isolated using the newly developed DNA hybridization analysis of recombinantly expressed cDNA libraries (HYREX) approach. HYREX combines the advantage of a nonradioactive library screening method with the possibility of subsequently analysing the serological response to the recombinant proteins. We isolated clones coding for MAGE-A1, -A3, -A4b, -A6, -A9 and -A12, as well as LAGE-1A. Additionally, we correlated gene expression and seroreactivity. RESULTS: Between 13% and 27% of sera (n = 15) were reactive against individual tumour antigens. We found the presence of specific antibodies was, with only two exceptions, generally correlated with mRNA expression of the antigen within cell lines derived from the same patient. While cross-reactivity of patients' IgG might play a role in these cases, antibodies from patients' sera were able to distinguish even the closely related MAGE-A3 and -A6. In general, the mRNA expression frequency was higher than the detected IgG responses. CONCLUSIONS: Antibody recognition of specific tumour antigens by patients' sera may be used for evaluating the possible immunogenicity of new antigens; serological tests could be used for tumour monitoring purposes.

Towards defining specific antigens for cutaneous lymphomas.

Cutaneous lymphomas (CLs) are a heterogeneous group of malignancies of T-cell (cutaneous T-cell lymphoma, CTCL) or B-cell (cutaneous B-cell lymphoma, CBCL) origin with primary manifestation in the skin. CLs are difficult to treat in their advanced stages, especially as there is no curative treatment available. Immunological therapies might be a promising alternative, but the prerequisite for such strategies is the knowledge of tumor-specific antigens. This paper is reviewing the methods used today for identifying such antigens with special respect to CLs. The most successful strategies for the discovery of new tumor antigens include the cytotoxic T-cell approach using either genetic or biochemical tools, or synthetic peptide libraries leading to so-called mimotopes. A second strategy utilizes antibodies for screening recombinant libraries: either monoclonal antibodies generated against tumor cells or the so-called SEREX approach using antibodies of the patient's serum. Especially the antibody-based strategies led to several new antigens expressed in CTCL. Finally, already known tumor antigens have been evaluated as possible targets for CLs. A growing list of tumor antigens can be summarized for CLs, especially CTCL, which include cTAGE-1, SCP-1, GBP-TA, several mimotopes, SC5, LAGE-1, and NY-ESO-1, as well as the GAGE and MAGE-A groups. Perspectives on basis of the present knowledge are discussed.

Molecular and functional aspects of anionic channels activated during regulatory volume decrease in mammalian cells.

The ability of cells to readjust their volume after swelling, a phenomenon known as regulatory volume decrease (RVD), is a fundamental biological achievement guaranteeing survival and function of cells under osmotic stress. This article reviews the mechanisms of RVD in mammalian cells with special emphasis on the activation of ion channels during RVD.

A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.

Numerous strains of mice with defined mutations display pronounced abnormalities of hair follicle cycling, even in the absence of overt alterations of the skin and hair phenotype; however, in order to recognize even subtle, hair cycle-related abnormalities, it is critically important to be able to determine accurately and classify the major stages of the normal murine hair cycle. In this comprehensive guide, we present pragmatic basic and auxiliary criteria for recognizing key stages of hair follicle growth (anagen), regression (catagen) and quiescence (telogen) in C57BL/6NCrlBR mice, which are largely based on previous work from other authors. For each stage, a schematic drawing and representative micrographs are provided in order to illustrate these criteria. The basic criteria can be employed for all mouse strains and require only routine histochemical techniques. The auxiliary criteria depend on the immunohistochemical analysis of three markers (interleukin-1 receptor type I, transforming growth factor-beta receptor type II, and neural cell-adhesion molecule), which allow a refined analysis of anatomical hair follicle compartments during all hair cycle stages. In contrast to prior staging systems, we suggest dividing anagen III into three distinct substages, based on morphologic differences, onset and progression of melanogenesis, and the position of the dermal papilla in the subcutis. The computer-generated schematic representations of each stage are presented with the aim of standardizing reports on follicular gene and protein expression patterns. This guide should become a useful tool when screening new mouse mutants or mice treated with pharmaceuticals for discrete morphologic abnormalities of hair follicle cycling in a highly reproducible, easily applicable, and quantifiable manner.

Serological detection of cutaneous T-cell lymphoma-associated antigens.

Cutaneous T-cell lymphomas (CTCL) are a group of skin neoplasms that originate from T lymphocytes and are difficult to treat in advanced stages. The present study is aimed at the identification of tumor-specific antigens from a human testis cDNA library using human sera known as the SEREX (serological identification of recombinantly expressed genes) approach. A cDNA library from normal testicle tissue was prepared and approximately 2 million recombinants were screened with sera from Sézary Syndrome and Mycosis fungoides patients. A total of 28 positive clones belonging to 15 different genes/ORFs were identified, including five hitherto unknown sequences. Whereas control sera did not react with most clones, 11-71% sera from CTCL patients were reactive against the identified clones. Expression analysis on 28 normal control and 17 CTCL tissues by reverse transcription-PCR (RT-PCR) and Northern blotting revealed seven ubiquitously distributed antigens, six differentially expressed antigens (several normal tissues were positive), and two tumor-specific antigens that were expressed only in testis and tumor tissues: (i) A SCP-1-like sequence, which has already been detected in various tumors, has been found in one CTCL tumor and four sera of CTCL patients reacted with various SCP-1-like clones and (ii) a new sequence named cTAGE-1 (CTCL-associated antigen 1) was detected in 35% of CTCL tumor tissues and sera of 6/18 patients reacted with this clone. The present study unravels CTCL-associated antigens independent of the T-cell receptor. The SCP-1-like gene and cTAGE-1 were shown to be immunogenic and immunologically tumor-specific and may therefore be candidates for immunotherapy targeting CTCL.

A comprehensive guide for the recognition and classification of distinct stages of hair follicle morphogenesis.

Numerous spontaneous and experimentally induced mouse mutations develop a hair phenotype, which is often associated with more or less discrete abnormalities in hair follicle development. In order to recognize these, it is critically important to be able to determine and to classify accurately the major stages of normal murine hair follicle morphogenesis. As an aid, we propose a pragmatic and comprehensive guide, modified after previous suggestions by Hardy, and provide a list of easily recognizable classification criteria, illustrated by representative micrographs. Basic and more advanced criteria are distinguished, the former being applicable to all mouse strains and requiring only simple histologic stains (hematoxylin and eosin, Giemsa, periodic acid Schiff, alkaline phosphatase activity), the latter serving as auxiliary criteria, which require a pigmented mouse strain (like C57BL/6J) or immunohistochemistry (interleukin-1 receptor type I, transforming growth factor-beta receptor type II). In addition, we present simplified, computer-generated schematic drawings for the standardized recording and reporting of gene and antigen expression patterns during hair follicle development. This classification aid serves as a basic introduction into the field of hair follicle morphogenesis, aims at standardizing the presentation of related hair research data, and should become a useful tool when screening new mouse mutants for discrete abnormalities of hair follicle morphogenesis (compared with the respective wild type) in a highly reproducible, easily applicable, and quantifiable manner.

Generation and cyclic remodeling of the hair follicle immune system in mice.

In this immunohistomorphometric study, we have defined basic characteristics of the hair follicle (HF) immune system during follicle morphogenesis and cycling in C57BL/6 mice, in relation to the skin immune system. Langerhans cells and gammadelta T cell receptor immunoreactive lymphocytes were the predominant intraepithelial hematopoietic cells in neonatal mouse skin. After their numeric increase in the epidermis, these cells migrated into the HF, although only when follicle morphogenesis was almost completed. In contrast to Langerhans cells, gammadelta T cell receptor immunoreactive lymphocytes entered the HF only via the epidermis. Throughout HF morphogenesis and cycling, both cell types remained strikingly restricted to the distal outer root sheath. On extremely rare occasions, CD4+ or CD8+ alphabetaTC were detected within the HF epithelium or the sebaceous gland. Major histocompatibility complex class II+, MAC-1+ cells of macrophage phenotype and numerous mast cells appeared very early on during HF development in the perifollicular dermis, and the percentage of degranulated mast cells significantly increased during the initiation of synchronized HF cycling (first catagen). During both depilation- and cyclosporine A-induced HF cycling, the numbers of intrafollicular Langerhans cells, gammadelta T cell receptor immunoreactive lymphocytes, and perifollicular dermal macrophages fluctuated significantly. Yet, no numeric increase of perifollicular macrophages was detectable during HF regression, questioning their proposed role in catagen induction. In summary, the HF immune system is generated fairly late during follicle development, shows striking differences to the extrafollicular skin immune system, and undergoes substantial hair cycle-associated remodeling. In addition, synchronized HF cycling is accompanied by profound alterations of the skin immune system.

Clusters of perifollicular macrophages in normal murine skin: physiological degeneration of selected hair follicles by programmed organ deletion.

In back skin sections from adolescent C57BL/6 mice, regularly distributed, perifollicular inflammatory cell clusters (PICC) were found located around the distal noncycling portion of about 2% of all hair follicles examined. The PICC and the affected hair follicles were characterized during spontaneously developed or induced hair cycle stages, using antibodies against MHC Class II, F4/80, ER-MP23, NLDC 145, CD4, CD8, gammadeltaTCR, IL-1 receptor, and ICAM-1. PICC consisted predominantly of macrophages (MAC), accompanied by a few CD4+ cells, whereas gammadeltaTCR+ and CD8+ cells were absent. During anagen and catagen, some of the PICC+ hair follicles showed variable degenerative phenomena reminiscent of scarring alopecia: thickened basement membrane, ectopic MHC II expression, MAC infiltration into the follicle epithelium, and signs of keratinocyte apoptosis. Loss of distal outer root sheath keratinocytes was detected in 10% of PICC+ hair follicles (0.2% of all hair follicles). Because PICC were located in the vicinity of the bulge region, MAC-dependent damage to follicle stem cells might eventually lead to follicle degeneration. These perifollicular MAC clusters around selected hair follicles may indicate the existence of a physiological program of MAC-dependent controlled follicle degeneration by which damaged or malfunctioning follicles are removed by programmed organ deletion (POD).

Transforming growth factor-beta receptor type I and type II expression during murine hair follicle development and cycling.

Although the TGF-beta family of growth factors probably regulates skin and hair follicle development, its exact role is still quite ill-defined. Here, we characterize the correlative expression pattern of the interdependent high affinity receptor proteins for TGF-beta1 and TGF-beta3, TGF-beta receptor type I (TGF-betaRI) and TGF-beta receptor type II (TGF-betaRII), during hair follicle development and cycling in C57BL/6 mice. During neonatal follicle development, TGF-betaRII immunoreactivity is confined to epithelial cells. Focal epidermal TGF-betaRII expression is seen even before actual hair placode formation. In contrast to the TGF-betaRII immunoreactivity in the outer root sheath, precortical hair matrix and inner root sheath cells were TGF-betaRII negative during hair bulb morphogenesis. TGF-betaRI (Alk-5) immunoreactivity largely overlapped the TGF-betaRII expression pattern, but was more widespread. During hair follicle cycling in adolescent mice, TGF-betaRII immunoreactivity was restricted to follicles, and was strikingly hair cycle dependent (maximal immunoreactivity: anagen VI and early catagen). Again, TGF-betaRI (Alk-5) immunoreactivity co-localized with TGF-betaRII immunoreactivity, but was more extensive. Reverse transcriptase polymerase chain reaction analysis of TGF-betaRII mRNA confirmed peak transcript levels in back skin with most hair follicles in the anagen VI-catagen transformation. mRNA levels of TGF-betaRI (Alk-5) did not vary significantly during the hair cycle, whereas those of TGF-betaRI (threonine-serine kinase 7 L) declined during early anagen, and were maximal during the anagen-catagen transition. This provides a basis for defining the choreography of TGF-beta-related signalling during hair follicle morphogenesis and cycling, introduces intraepidermal TGF-betaRII immunoreactivity as a marker for imminent follicle development, and supports the concept that both TGF-betaRII and TGF-betaRI stimulation is involved in, but not restricted to, the control of catagen induction.

Hair cycle-dependent plasticity of skin and hair follicle innervation in normal murine skin.

The innervation of normal, mature mammalian skin is widely thought to be constant. However, the extensive skin remodeling accompanying the transformation of hair follicles from resting stage through growth and regression back to resting (telogen-anagen-catagen-telogen) may also be associated with alteration of skin innervation. We, therefore, have investigated the innervation of the back skin of adolescent C57BL/6 mice at various stages of the depilation-induced hair cycle. By using antisera against neuronal (protein gene product 9.5 [PGP 9.5], neurofilament 150) and Schwann cell (S-100, myelin basic protein) markers, as well as against neural cell adhesion molecule (NCAM) and growth-associated protein-43 (GAP-43), we found a dramatic increase of single fibers within the dermis and subcutis during early anagen. This was paralleled by an increase in the number of anastomoses between the cutaneous nerve plexuses and by distinct changes in the nerve fiber supply of anagen vs. telogen hair follicles. The follicular isthmus, including the bulge, the seat of epithelial follicle stem cells, was found to be the most densely innervated skin area. Here, a defined subpopulation of nerve fibers increased in number during anagen and declined during catagen, accompanied by dynamic alterations in the expression of NCAM and GAP-43. Thus, our study provides evidence for a surprising degree of plasticity of murine skin innervation. Because hair cycle-associated tissue remodeling evidently is associated with tightly regulated sprouting and regression of nerve fibers, hair cycle-dependent alterations in murine skin and hair follicle innervation offer an intriguing model for studying the controlled rearrangement of neuronal networks in peripheral tissues under physiological conditions.

Identification and measurement of beta-endorphin levels in the skin during induced hair growth in mice.

We describe new and effective techniques for extracting proopiomelanocortin (POMC)-derived peptides from mammaliar skin. Using this methodology (hot-acid extraction) and two independent HPLC-controlled RIA systems, we identify beta-endorphin peptide in mammalian skin and demonstrate significant hair cycle-dependent fluctuations in both the skin concentration and the in situ expression pattern of beta-endorphin (sebaceous glands) during the entire murine hair cycle. The observed anagen (growth phase) associated increase in beta-endorphin concentration and its decline during the follicle involution (catagen) or resting (telogen) phase raise the possibility of a regulatory function of this neuropeptide in cyclic changes of skin physiology.

Neural mechanisms of hair growth control.

Clinical and experimental observations have long suggested that skin nerves have "trophic" functions in hair follicle development, growth and/or cycling, even though the molecular and cellular basis of the underlying neuroepithelial interactions has remained obscure. Here, we critically review currently available evidence arguing in favor of or against the existence of neural mechanisms of hair growth control, and outline why the murine hair cycle provides an excellent experimental system for characterizing and manipulating piloneural interactions. Summarizing relevant, recent data from the C57BL/6 mouse model, it is pointed out that the sensory and autonomic innervation of normal pelage hair follicles, the substance P skin content, and cutaneous mast cell-nerve contacts show striking changes during synchronized hair follicle cycling. Furthermore, the murine hair follicle appears to be both a source and a target of neurotrophins, whereas neuropharmacologic manipulations alter murine hair follicle cycling in vivo. For example, anagen is induced by substance P or adrenocorticotropin (ACTH), and by the experimentally triggered release of neuropeptides from sensory nerves and of neurotransmitters from adrenergic nerves. Taken together, this argues in favor of neuroepithelial interactions as regulatory elements in hair growth control and suggests that the study of piloneural interactions promises important insights into general principles of neuroepithelial communication, namely during epithelial morphogenesis and remodeling. We delineate a hypothetical working model of piloneural interactions and propose that targeted manipulations deserve systematic exploration as a novel strategy for managing hair growth disorders.

A simple immunofluorescence technique for simultaneous visualization of mast cells and nerve fibers reveals selectivity and hair cycle--dependent changes in mast cell--nerve fiber contacts in murine skin.

Close contacts between mast cells (MC) and nerve fibers have previously been demonstrated in normal and inflamed skin by light and electron microscopy. A key step for any study in MC-nerve interactions in situ is to simultaneously visualize both communication partners, preferably with the option of double labelling the nerve fibers. For this purpose, we developed the following triple-staining technique. After paraformaldehyde-picric acid perfusion fixation, cryostat sections of back skin from C57BL/6 mice were incubated with a primary rat monoclonal antibody to substance P (SP), followed by incubation with a secondary goat-anti-rat TRITC-conjugated IgG. A rabbit antiserum to CGRP was then applied, followed by a secondary goat-anti-rabbit FITC-conjugated IgG. MCs were visualized by incubation with AMCA-labelled avidin, or (for a more convenient quantification of close MC-nerve fiber contacts) with a mixture of TRITC- and FITC-labelled avidins. Using this simple, novel covisualization method, we were able to show that MC-nerve associations in mouse skin are, contrary to previous suggestions, highly selective for nerve fiber types, and that these interactions are regulated in a hair cycle-dependent manner: in telogen and early anagen skin, MCs preferentially contacted CGRP-immunoreactive (IR) or SP/CGRP-IR double-labelled nerve fibers. Compared with telogen values, there was a significant increase in the number of close contacts between MCs and tyrosine hydroxylase-IR fibers during late anagen, and between MCs and peptide histidine-methionine-IR and choline acetyl transferase-IR fibers during catagen.

Keratin 17 gene expression during the murine hair cycle.

Keratin 17 (K17) expression is currently considered to be associated with hyperplastic or malignant growth of epithelial cells. The functions of this keratin in normal skin physiology and the regulation of its gene expression, however, are still unclear. As one possible approach to further explore K17 functions, we have studied the differential patterns of mouse K17 (MK17) transcription during the murine hair cycle by means of in situ hybridization, using a digoxigenin-labeled riboprobe. Cycling hair follicles in the skin of C57BL/6 mice were found to be the only skin structures expressing MK17 under physiologic conditions. MK17 transcripts were constantly observed throughout all hair cycle stages in the suprainfundibular outer root sheath (ORS). The MK17 expression was also evident in the isthmus part of the ORS, where it was expressed weakly and was spatially restricted during telogen, with an increase in early anagen and stable expression during mid- and late anagen, localizing to the zone of so-called trichilemmal keratinization. In addition, in early anagen, a group of epithelial cells in or next to the bulge region stained weakly for MK17. With progressing anagen development, MK17 expression in this region increased and was consistently localized to keratinocytes at the advancing front of the emerging epithelial hair bulb. In mid- and late anagen, this zone of MK17 expression spread along the proximal ORS, with a maximal level of expression in the innermost cell layer of the ORS. Overall, these findings provide data on the MK17 expression profile of normal murine skin and demonstrate hair-cycle-dependent regulation of MK17 expression.

A new method for double immunolabelling with primary antibodies from identical species.

There are several double immunolabelling methods but each has its drawbacks. More often than not, antibodies with the required specificities are available in only one species and their use normally produces false labels due to cross-reactivity. We describe a new and reliable technique for staining with primary antibodies from the same species, that can even be employed on tissues of the donor species. The protocol avoids cross-reactivities without loss in sensitivity, uses commercially available reagents and takes advantage of enzymatic detection, although it can be adapted for fluorescent labelling. Briefly, tissue is incubated with one primary antibody, followed by a peroxidase-coupled secondary antibody which is detected using amino ethyl carbazol to give a red reaction product. Meanwhile, the next primary antibody is coupled in vitro to a biotinylated secondary antibody and excess binding sites quenched with normal immune serum from the same species as the primary antibody. This complex is applied to tissue and detected by the avidin-biotin/alkaline phosphatase technique using naphthol-AS-MX-phosphate/Fast Blue BB to produce a blue label. In addition to extensive controls, the reliability and broad applicability of this method has been confirmed in (1) murine skin cryostat sections to co-visualize antigen-presenting cells (MHC class II-immunoreactive; "-ir') with either antigen detecting T lymphocytes (CD4-ir) or Langerhans cells (NLDC-145-ir) and (2) locust (Insecta) abdominal ganglion paraffin sections, where it is known that immunoreactivities for octopamine and a FMRFamide-related peptide are colocalized in only one, uniquely identifiable neuron.

Sensory neuron development revealed by taurine immunocytochemistry in the honeybee.

The formation of ommatidia in the compound eyes and sensilla on the antennae of the honeybee was followed and the development of their sensory neurons was traced using an antiserum against taurine as a marker. Taurine-like immunoreactivity (Tau-IR) is expressed in sensory neurons of several modalities, namely visual, olfactory, gustatory, and mechanosensory. Staining intensity is very high in the larva and in the first half of the pupal stage and gradually decreases towards the end of metamorphosis. In the photoreceptor cells of the compound eyes, Tau-IR can be detected from the fifth larval instar onwards, prior to differentiation of other components of the ommatidium. Already in the midstage larvae, when the antennal primordia of the adult still lie within the peripodial cavity, a few presumably mechanosensory neurons are labelled in the pedicellus of the developing antenna. The majority of the antennal sensory neurons which are located on the flagellum start to exhibit Tau-IR upon pupation, long before any cuticular specializations such as sensory hairs or plates are detectable. All known types of antennal sensilla were identified and it could be shown that all of them are innervated by Tau-IR sensory neurons. Thus, taurine immunocytochemistry can be applied as a useful label for developing sensory neurons. Functional implications of taurine during development are discussed.