Stefan B. Eichmüller

A vaccine targeting mutant IDH1 induces antitumour immunity

Monoallelic point mutations of isocitrate dehydrogenase type 1 (IDH1) are an early and defining event in the development of a subgroup of gliomas and other types of tumour. They almost uniformly occur in the critical arginine residue (Arg 132) in the catalytic pocket, resulting in a neomorphic enzymatic function, production of the oncometabolite 2-hydroxyglutarate (2-HG), genomic hypermethylation, genetic instability and malignant transformation. More than 70% of diffuse grade II and grade III gliomas carry the most frequent mutation, IDH1(R132H) (ref. 3). From an immunological perspective, IDH1(R132H) represents a potential target for immunotherapy as it is a tumour-specific potential neoantigen with high uniformity and penetrance expressed in all tumour cells. Here we demonstrate that IDH1(R132H) contains an immunogenic epitope suitable for mutation-specific vaccination. Peptides encompassing the mutated region are presented on major histocompatibility complexes (MHC) class II and induce mutation-specific CD4+ T-helper-1 (TH1) responses. CD4+ TH1 cells and antibodies spontaneously occurring in patients with IDH1(R132H)-mutated gliomas specifically recognize IDH1(R132H). Peptide vaccination of mice devoid of mouse MHC and transgenic for human MHC class I and II with IDH1(R132H) p123-142 results in an effective MHC class II-restricted mutation-specific antitumour immune response and control of pre-established syngeneic IDH1(R132H)-expressing tumours in a CD4+ T-cell-dependent manner. As IDH1(R132H) is present in all tumour cells of these slow-growing gliomas, a mutation-specific anti-IDH1(R132H) vaccine may represent a viable novel therapeutic strategy for IDH1(R132H)-mutated tumours.

Development and experience lead to increased volume of subcompartments of the honeybee mushroom body

The mushroom bodies of insects are believed to be involved in higher order sensory integration and learning. In the honeybee, the mushroom body can be separated into three different, modality-specific input compartments and several morphologically inseparable output regions. By means of morphometric analysis we show that the volumes of these subcompartments depend on both the age of the adult bee and its experience. For the most part a significant, age-dependent increase in neuropile volume is observed. Additionally, the olfactory and visual input regions show experience-related differences. Unlike other subcompartments, the visual input region does not change in volume with age, but only with experience. We thus suggest that experience is an important factor in the structural development of higher order brain regions of an insect, the honeybee.

Alkaline phosphatase activity and localization during the murine hair cycle.

For unknown reasons, the pilosebaceous unit displays prominent alkaline phosphatase (AP) activity, and alterations in AP activity are seen in alopecia areata. The role of AP in hair biology and pathology has been obscured by contradictory reports on the localization and activity of AP during the hair cycle, and by a paucity of instructive models for studying AP functions. Using the C57 BL‐6 mouse model for hair research, we have characterized endogenous AP with a simple histochemical developing solution routinely employed for AP immunohistology. This method was selective for AP, and revealed distinctive hair cycle‐dependent changes in AP activity and localization. Although the dermal papilla displays unusually strong AP activity during the entire hair cycle, the outer root sheath is AP‐positive only during late anagen and early catagen. Strong, rather homogeneous AP activity is seen in the sebaceous gland (SG) only during catagen and telogen. This AP staining pattern indicates hair cycle‐dependent changes in SG functions, and differs to some extent from the previously reported AP activity during the hair cycle of various species. We propose a simple and effective technique for follicle classification based on the AP histochemistry of dermal papilla and sebaceous gland, and discuss uses of the C57 BL‐6 mouse model for functional AP studies.

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. J. Comp. Neurol. 386:379‐395, 1997.

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.

miR-137 Inhibits the Invasion of Melanoma Cells through Downregulation of Multiple Oncogenic Target Genes

MicroRNAs are small noncoding RNAs that regulate gene expression and have important roles in various types of cancer. Previously, miR-137 was reported to act as a tumor suppressor in different cancers, including malignant melanoma. In this study, we show that low miR-137 expression is correlated with poor survival in stage IV melanoma patients. We identified and validated two genes (c-Met and YB1) as direct targets of miR-137 and confirmed two previously known targets, namely enhancer of zeste homolog 2 (EZH2) and microphthalmia-associated transcription factor (MITF). Functional studies showed that miR-137 suppressed melanoma cell invasion through the downregulation of multiple target genes. The decreased invasion caused by miR-137 overexpression could be phenocopied by small interfering RNA knockdown of EZH2, c-Met, or Y box-binding protein 1 (YB1). Furthermore, miR-137 inhibited melanoma cell migration and proliferation. Finally, miR-137 induced apoptosis in melanoma cell lines and decreased BCL2 levels. In summary, our study confirms that miR-137 acts as a tumor suppressor in malignant melanoma and reveals that miR-137 regulates multiple targets including c-Met, YB1, EZH2, and MITF.

Stimulatory effect of octopamine on juvenile hormone biosynthesis in honey bees (Apis mellifera) : Physiological and immunocytochemical evidence

Abstract The effect of octopamine on the activity of corpora allata of adult worker honey bees has been examined in vitro and correlated to the local distribution of this biogenic amine in brain and retrocerebral complex as studied immunocytochemically by means of an highly specific antiserum. Octopamine causes a dose-dependent increase in juvenile hormone release from corpora allata. Maximum increase is obtained with concentrations of 10−6 M in nurse and foraging bees by 45.3 or 32.3%, respectively. Octopamine-like immunoreactivity occurs in about 45 somata of the median neurosecretory cells in the pars intercerebralis of the bee brain. They project via immunopositive nervus corporis cardiaci I into the corpora cardiaca, where interspersed varicose structures and 8–10 cell bodies in the ventral part of this gland are stained. A network of immunoreactive fine varicose nerve fibres surrounds each gland cell of the corpora allata. Immunoreactivity in these neuronal structures is detectable if bees were starved over night, a condition in which corpora allata elicit the highest juvenile hormone production ever observed in bees. Both, the stimulatory effect of octopamine and the presence of immunoreactive nerve fibers in the corpora allata, strongly indicate a physiological role of this biogenic amine in the regulation of juvenile hormone biosynthesis in adult honey bees.

Distribution and changing density of gamma-delta T cells in murine skin during the induced hair cycle.

Summary Gamma‐delta T cells (gdTC) are recognized as the predominant intraepidermal T‐cell population in murine skin, although their physiological functions are still unclear. Little is known of the exact distribution of gdTC in the other epithelial skin compartments of normal mice. Using selective gdTC‐receptor antibodies in immunohistology (alkaline phosphatase technique), the distribution and density of gdTC was analysed morphometrically in cryostat sections of full‐thickness back skin of normal, adolescent C57 BL‐6 mice in all the different stages of the depilation‐induced hair cycle. We found that, during the entire hair cycle, V gamma 3‐TCR‐bearing lymphocytes are restricted to the epidermis, and to the epithelial hair bulb in, and distal to, the bulge area. No gdTC were seen in the sebaceous glands. During early anagen development, the number of pan‐gdTC receptor‐positive cells increased significantly (P<0·005) in the interfollicular epidermis and the supainfundibular portion of the hair bulb, whereas the number decreased in the infrainfundibular region (P≤0.005). As gdTC are thought to migrate into the skin only during embryogenesis, this finding suggests hair cycle‐dependent, differential intraepithelial proliferation of gdTC in murine skin. We advocate employing only skin of defined hair cycle stages in immunological studies on murine skin, and discuss the value of the C57 BL‐6 model for assessing the functions of gdTC in skin and hair biology.

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, γδTCR, IL-1 receptor, and ICAM-1. PICC consisted predominantly of macrophages (MAC), accompanied by a few CD4+ cells, whereas γδTCR+ 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).

Expression of classical and non‐classical MHC class I antigens in murine hair follicles

Not all keratinocytes in human and rat hair follicles express MHC class I antigens (MHC I). In the present study, we report the first immunohistological profile of classical and non‐classical MHC I expression in the skin of adolescent C57 BL‐6 mice during the induced hair cycle. MHC I immunoreactivity (H‐2b, H‐2Db) is absent in the matrix and inner root sheath of growing (=anagen) hair follicles, and the dermal papillae are H‐2b negative during catagen and telogen. This lack of normal MHC I expression may serve to sequester potentially damaging autoantigens from immune recognition. In addition, we present the first evidence of non‐classical MHC class I antigen expression in normal mammalian skin: during the entire hair cycle, the distal hair follicle shows strong Qa‐2 immunoreactivity, which appears to be restricted to an epithelial follicle compartment densely populated by gamma‐delta T cells with which Qa‐2 molecules may interact as part of a primitive antibacterial defense system of the follicle. The murine hair cycle is an attractive model for dissecting the functional roles of H‐2b and Qa‐2 molecules in hair biology and in related tissue‐interaction systems.