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Acute graft-versus-host disease (aGvHD) is a severe and often life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). AGvHD is mediated by alloreactive donor T-cells targeting predominantly the gastrointestinal tract, liver, and skin. Recent work in mice and patients undergoing allo-HCT showed that alloreactive T-cells can be identified by the expression of α4β7 integrin on T-cells even before manifestation of an aGvHD. Here, we investigated whether the detection of a combination of the expression of T-cell surface markers on peripheral blood (PB) CD8\(^+\) T-cells would improve the ability to predict aGvHD. To this end, we employed two independent preclinical models of minor histocompatibility antigen mismatched allo-HCT following myeloablative conditioning. Expression profiles of integrins, selectins, chemokine receptors, and activation markers of PB donor T-cells were measured with multiparameter flow cytometry at multiple time points before the onset of clinical aGvHD symptoms. In both allo-HCT models, we demonstrated a significant upregulation of α4β7 integrin, CD162E, CD162P, and conversely, a downregulation of CD62L on donor T-cells, which could be correlated with the development of aGvHD. Other surface markers, such as CD25, CD69, and CC-chemokine receptors were not found to be predictive markers. Based on these preclinical data from mouse models, we propose a surface marker panel on peripheral blood T-cells after allo-HCT combining α4β7 integrin with CD62L, CD162E, and CD162P (cutaneous lymphocyte antigens, CLA, in humans) to identify patients at risk for developing aGvHD early after allo-HCT.

Aspergillus fumigatusis an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment and progression are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are paramount to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in the desired intra-alveolar deposition, as more than 60% of fungal growth occurred outside of the alveolar space. Hence, LSFM allows for more rigorous characterization of murine models of invasive pulmonary aspergillosis and pinpointing their strengths and limitations.IMPORTANCEThe use of animal models of infection is essential to advance our understanding of complex host-pathogen interactions that take place duringAspergillus fumigatuslung infections. As in the case of humans, mice need to be immunosuppressed to become susceptible to invasive pulmonary aspergillosis, the most serious infection caused byA. fumigatus. There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis (IPA). However, the precise consequences that each immunosuppressive model has on the local immune populations and for fungal growth are not completely understood. Here we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution, to pin down the scenario commonly used IPA models. Our results will be valuable to optimize and refine animal models to maximize their use in future research.VISUAL ABSTRACTQuantitative light sheet fluorescence microscopy to dissect local host-pathogen interactions in the lung afterA. fumigatusairway infection.

Objective: Enteric glial cells (EGCs) are recognized as a heterogeneous population capable of interacting with all major intestinal cell types to ensure tissue homeostasis. According to central nervous system (CNS) astrocytes, the subpopulation of EGCs expressing the glial fibrillary acidic protein (GFAP) is assigned to exert immunomodulatory functions. However, the mechanisms of such immunocompetence are largely unknown. Previously, much attention has been paid to changes within the enteric nervous system in chronic inflammatory bowel diseases (IBD). In IBD, an inappropriate mucosal immune response causes a persisting state of tissue inflammation, influencing structure as well as function of the ENS. Here, we focus our attention on acute inflammatory conditions and their impact on ENS function with emphasis on the GFAP-expressing glial subpopulation and their contribution to the inflammatory microenvironment. Method: In our ongoing study we experimentally induced intestinal inflammation in hGFAP-eGFP transgenic mice to specifically study the physiological responses of GFAP-glia. Colon samples were analyzed employing advanced techniques such as 3D light sheet fluorescence microscopy (LSFM). We correlated the in vivo findings of GFAP-glia physiology to in vitro experiments, in which we cultured flow cytometrysorted GFAP-glia as gliospheres and stimulated the mono-cultures with lipopolysaccharide (LPS). Additionally, we are currently setting up co-culture models incorporating primary ENS cells with lamina propria immune cells to develop suitable in vitro test systems for acute inflammatory stimuli. Results: LSFM of inflamed murine colon samples revealed the close proximity of GFAP-glia to lamina propria immune cells. When directly stimulated with LPS in vitro, hGFAP-eGFP transgenic murine EGCs boosted the expression of genes associated with immunomodulatory capacities, such as immune responses, cell locomotion and metabolism. Both, microarray analysis and multiplex protein profiling identified an increased production and secretion of proinflammatory cytokines. Employing the in vivo model we aim to further characterize the physiological potential of EGCs to modulate a pro-inflammatory microenvironment. Conclusion: With a better understanding of the potential of EGCs to fuel the inflammatory microenvironment in intestinal disorders it will be necessary and promising to consider the enteric nervous system in the development of new therapeutic strategies.

In acute graft-versus-host disease (GVHD) alloreactive donor T cells selectively damage skin, liver, and the gastrointestinal tract while other organs are rarely affected. The mechanism of this selective target tissue infiltration is not well understood. We investigated the importance of alloantigen expression for the selective organ manifestation by examining spatiotemporal changes of cellular and molecular events after allogeneic hematopoietic cell transplantation (allo-HCT). To accomplish this we established a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized protocols for antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule–1 (MAdCAM-1) and T cell responses in Peyer’s patches following allo-HCT. In addition, we proofed that LSFM is suitable to map individual T cell subsets after HCT and detected rare cellular events. We employed this versatile technique to study the role of alloantigen expression for the selective organ manifestation after allo-HCT. Therefore, we used a T cell receptor (TCR) transgenic mouse model of GVHD that targets a single peptide antigen and thereby mimics a major histocompatibility complex (MHC)-matched single antigen mismatched (miHAg-mismatched) HCT. We transplanted TCR transgenic (OT-I) T cells into myeloablatively conditioned hosts that either express the peptide antigen ovalbumin ubiquitously (βa-Ova) or selectively in the pancreas (RIP-mOva), an organ that is normally not affected by acute GVHD. Of note, at day+6 after HCT we observed that OT-I T cell infiltration occurred in an alloantigen dependent manner. In βa-Ova recipients, where antigen was ubiquitously expressed, OT-I T cells infiltrated all organs and were not restricted to gastrointestinal tract, liver, and skin. In RIP-mOva recipients, where cognate antigen was only expressed in the pancreas, OT-I T cells selectively infiltrated this organ that is usually spared in acute GVHD. In conditioned RIP-mOva the transfer of 100 OT-I T cells sufficed to effectively infiltrate and destroy pancreatic islets resulting in 100% mortality. By employing intact tissue LSFM in RIP-mOva recipients, we identified very low numbers of initial islet infiltrating T cells on day+4 after HCT followed by a massive T cell migration to the pancreas within the following 24 hours. This suggested an effective mechanism of effector T cell recruitment to the tissue of alloantigen expression after initial antigen specific T cell encounter. In chimeras that either expressed the model antigen ovalbumin selectively in hematopoietic or in parenchymal cells only, transplanted OT-I T cells infiltrated target tissues irrespective of which compartment expressed the alloantigen. As IFN-γ could be detected in the serum of transplanted ovalbumin expressing recipients (βa-Ova, βa-Ova-chimeras and RIP-mOva) at day+6 after HCT, we hypothesized that this cytokine may be functionally involved in antigen specific OT-I T cell mediated pathology. In vitro activated OT-I T cells responded with the production of IFN-γ upon antigen re-encounter suggesting that IFN-γ might be relevant in the alloantigen dependent organ infiltration of antigen specific CD8+ T cell infiltration after HCT. Based on these data we propose that alloantigen expression plays an important role in organ specific T cell infiltration during acute GVHD and that initial alloreactive T cells recognizing the cognate antigen propagate a vicious cycle of enhanced T cell recruitment that subsequently culminates in the exacerbation of tissue restricted GVHD., In der akuten Graft-Versus-Host Disease (GVHD) infiltrieren allogene Spender T Zellen Haut, Leber und den Magen-Darm-Trakt des Empfängers und attackieren das Gewebe. Andere Organe sind dagegen interessanterweise nur selten betroffen. Die Mechanismen dieser selektiven Organinfliltration sind bisher weitestgehend unbekannt. In meiner Dissertationsarbeit untersuchte ich den Einfluss der Alloantigenexpression auf die selektive Organmanifestation während der GVHD. Um komplexe Immunprozesse die nach allogener Stammzelltransplantation auftreten, besser zu verstehen, entwickelten wir eine Lichtblattmikroskopietechnik (LSFM) die zelluläre und molekulare Veränderungen im intakten Gewebe detektieren kann. Wir etablierten eine neuartigen mehrfarben LSFM-Methodik, die es ermöglicht, Immunprozesse in großen Gewebsstücken von Maus und Mensch in Einzelzellauflösung dreidimensional darzustellen. Dazu kombinierten und optimierten wir Protokolle, um eine Penetration von Antikörpern tief in das Gewebe sowie die Aufklärung und die dreifache Beleuchtung des Gewebes zu ermöglichen. Diese Methode erlaubte uns die erfolgreiche Quantifizierung der Proteinexpression des Adressins mucosal vascular addressin cell adhesion molecule–1 (MAdCAM-1) als auch die Quantifizierung der T Zell Antwort im intakten Peyer’s Plaque nach allogener hämatopoetischer Transplantation (HCT). Weiterhin konnten wir die Methode zur Untersuchung der Migration unterschiedlicher T Zell-Subpopulationen nach HCT erfolgreich einsetzen und konnten einzelne, organinfiltrierende Zellen detektierten und quantifizieren. Wir benutzten die LSFM Methode um den Einfluss der Alloantigenexpression auf die selektive Organmanifestation zu studieren. Dazu verwendeten wir ein Transplantationsmodell, in dem der Haupthistokompatibilitätskomplex übereinstimmt (MHC-matched) und eine Diskrepanz nur in einem einzelnen Peptid Antigen (miHAG-mismatch) zwischen Spender und Empfänger bestand. Wir transplantierten T Zell Rezeptor (TCR) transgene (OT-I) T Zellen in myeloablativ bestrahlte Empfänger, die das Peptidantigen Ovalbumin entweder in allen Geweben (βa-Ova) oder selektiv in der Bauchspeicheldrüse (RIP-mOva) exprimieren. Die Bauchspeicheldrüse ist ein Organ, das normalerweise nicht von der akuten GVHD betroffen ist. An Tag 6 nach allogener HCT waren alle Organe die das Alloantigen exprimieren auch von Spender T Zellen infiltriert. In myeloablativ bestrahlten RIP-mOva Empfängern reichten bereits 100 transferierte OT-I T Zellen aus, um Alloantigen-exprimierende pankreatische Inselzellen zu zerstören. Dies führte zu einer Mortalität von 100% der Empfänger und spricht für eine sehr effiziente Alloantigendetektion und Gewebsinfiltration durch die Spender T Zellen. Um die Kinetik der Organinfiltration der Spender T Zellen detailliert zu untersuchen, verwendeten wir die neue Lichtblattmikroskopietechnik, welche die Analyse intakter Organe ermöglicht. In RIP-mOva Empfängern identifizierten wir erste wenige Spender T Zellen im Pankreas an Tag 4 nach Transplantation, gefolgt von einer massiven Pankreasinfiltration durch Spender T Zellen innerhalb von 24 Stunden. Dies deutet auf eine gezielte Rekrutierung der Spender T Zellen nach erstem Antigenkontakt in das Gewebe mit Alloantigenexpression. Um zu untersuchen, ob die Alloantigenexpression vom parenchymalen Gewebe oder aber durch hämatopoetische Zellen zur spezifischen Organinfiltration führt, transplantierten wir OT-I T Zellen in chimäre Empfänger, in denen das Alloantigen entweder nur im Gewebsparenchym oder ausschließlich von hämatopoetischen Zellen exprimiert wird. An Tag 6 nach der allogenen HCT fanden wir Spender T Zellen in allen Geweben, unabhängig davon welches Empfängerzellkompartment das Alloantigen präsentierte. Wir detektierten hohe IFN-γ-Werte im Serum von Ovalbumin exprimierenden Empfänger (βa-Ova, βa- Ova-Chimären und RIP-mOva). Weiterhin fanden wir, dass nach erneutem Kontakt mit dem spezifischen Alloantigen, OT-I T Zellen die in vitro aktiviert wurden, IFN-γ produzierten. Wir schließen aus diesen Beobachtungen, dass für die antigenabhängige Gewebeinfiltration IFN-γ wichtig ist. Zusammenfassend postulieren wir, dass die Alloantigenexpression im Gewebe eine wichtige Rolle in der organspezifischen Infiltration durch Spender T Zellen spielt, und dass T Zellen die Alloantigen spezifisch erkennen, dafür verantwortlich sind, dass weitere Effektor-T Zellen in das Gewebe rekrutiert werden.

Background Acute graft-versus-host disease (aGVHD) poses a major limitation for broader therapeutic application of allogeneic hematopoietic cell transplantation (allo-HCT). Early diagnosis of aGVHD remains difficult and is based on clinical symptoms and histopathological evaluation of tissue biopsies. Thus, current aGVHD diagnosis is limited to patients with established disease manifestation. Therefore, for improved disease prevention it is important to develop predictive assays to identify patients at risk of developing aGVHD. Here we address whether insights into the timing of the aGVHD initiation and effector phases could allow for the detection of migrating alloreactive T cells before clinical aGVHD onset to permit for efficient therapeutic intervention. Methods Murine major histocompatibility complex (MHC) mismatched and minor histocompatibility antigen (miHAg) mismatched allo-HCT models were employed to assess the spatiotemporal distribution of donor T cells with flow cytometry and in vivo bioluminescence imaging (BLI). Daily flow cytometry analysis of peripheral blood mononuclear cells allowed us to identify migrating alloreactive T cells based on homing receptor expression profiles. Results We identified a time period of 2 weeks of massive alloreactive donor T cell migration in the blood after miHAg mismatch allo-HCT before clinical aGVHD symptoms appeared. Alloreactive T cells upregulated α4β7 integrin and P-selectin ligand during this migration phase. Consequently, targeted preemptive treatment with rapamycin, starting at the earliest detection time of alloreactive donor T cells in the peripheral blood, prevented lethal aGVHD. Conclusions Based on this data we propose a critical time frame prior to the onset of aGVHD symptoms to identify alloreactive T cells in the peripheral blood for timely and effective therapeutic intervention.

Astylosternus laticephalus sp. nov. R��del, Hillers, Leach��, Kouam��, Ofori-Boateng, Diaz & Sandberger Figs. 9���12 Holotype. ZMB 75449 (field and tissue #: ATE 10, female, 58.6 mm), Ghana, Atewa Forest Reserve, 6.23375 / - 0.56557, 14 April 2007, forest around stream next to road, coll. C. Ofori-Boateng, A. Hillers & G. Segniagbeto. Paratypes. Ivory Coast: MTD 48026 (field and tissue #: Ba04.24, female, 62.2 mm), ZMB 75454 -75458 (Ba04.26, subadult, 45.5 mm; Ba04.25, female, 62.5 mm; Ba04.21, female, 64.4 mm; Ba04.22, subadult, 43.3 mm; Ba04.23, female, 58.4 mm;), Banco National Park, 5.41667 / -4.10500, rainy season 2004, swampy forest, coll. N.E. Assemian, B. Toh�� & G. Kouam��; MNHN 1993.6073 (male, 53.8 mm), 1999.7333 (female, 54.8 mm), 1999.7334 (male, 48.7 mm), 1999.7335 (female, 59.8 mm), 1999.7736 (female, 52.6 mm), 1999.7337 (female, 51.8 mm), Banco National Park, Abidjan; Ghana: ZMB 75459 (AF 4, juvenile, 25.6 mm), ZMB 75459 -75460 (AF 3 juvenile; 26.7 mm; AF 2, tadpole), Afao Hills Forest Reserve, 6.25461 / - 2.29492, riparian forest around small stream in valley, coll. C. Ofori-Boateng & A. Hillers; ZMB 75450 -75453 (COB 90, juvenile, 34.7 mm; COB 318, subadult, 40.8 mm; COB 119, juvenile, 38.6 mm; COB 123, juvenile, 31.7 mm), Western Province, Ankasa Conservation Area, pristine forest along streams in dry season, coll. C. Ofori-Boateng; MVZ 244910 (female, 67 mm), Ankasa Conservation Area, 5.28173 / - 2.64022, 26 June 2004, wet evergreen forest area, trail adjacent to the bamboo cathedral, coll. A.D. Leach�� & R. Diaz; MVZ 244909 (juvenile, 35 mm), 28 June 2004, approximately 1 km farther from the bamboo cathedral, other data as MVZ 244909. Additional material. ZMB 77461 (field# AF 03), tadpole, Ghana, Afao Hills Forest Reserve, 6.25461 / - 2.29492, riparian forest around small stream in valley, coll. C. Ofori-Boateng & A. Hillers; ATE 7 (tissue only), juvenile, 25.8 mm; ATE 8 (tissue only), juvenile, 28.6 mm; ATE 27 (tissue only), male, 45.6 mm, Ghana, Atewa Forest Reserve, 6.23375 / - 0.56557, 15 April 2007, swampy area next to small stream, in valley in forest, coll. C. Ofori-Boateng, A. Hillers & G. Segniagbeto; COB 2202 (collection Ofori-Boateng, Kumasi, Ghana), Ghana, Atewa Forest Reserve, coll. C. Ofori-Boateng; three adult specimens without number (amphibian reference collection at University Abobo-Adjam��, Ivory Coast), male (51.0 mm), females (60.0, 67.0 mm), Ivory Coast, Banco National Park, 5.41667 / -4.10500, 5 May 2004, forest close to Banco River, coll. N.E. Assemian, N.G. Kouam�� & B. Toh��. Diagnosis. Astylosternus laticephalus sp. nov. has the typical body shape of frogs of the genus Astylosternus, however, with exceptionally broad heads, i.e. broader than in A. occidentalis; males of the new species without spines on throat and belly (always present in A. occidentalis males); males without nuptial black skin layer in pectoral region (present in male A. occidentalis from western Guinea); back brownish to brownish red, always with distinct red dots (red dots only rarely present in A. occidentalis); bicoloured iris, grey with a reddish upper third (A. occidentalis always with uniform grey iris). A. laticephalus sp. nov. differs from A. occidentalis by a mean of 3.2 % (range 2.9-3.7 %) in the investigated part of the 16 S rRNA gene. Genetic divergence to the morphologically most similar Central African species, A. diadematus, was 11.9 %. Description of the holotype [measures in mm]. The holotype is an adult female with a snout-vent length of 58.6; head width 22.7; head as long as broad; interorbital distance narrower than length of upper eyelid; snout rounded in dorsal view, obtuse in lateral view, longer than eye diameter; eyes large (7.9) and protuberant directed anterolaterally; interorbital distance 7.2; pupil vertically elliptical; eye diameter larger than tympanum diameter, 5.3; tympanum vertically elliptical; supratympanal fold from posterior vertical midpoint of orbit to meet with dorsal border of tympanic annulus, where descent is sharp and terminates at half the vertical diameter of the tympanic annulus dorsally to the axilla; nares closer to snout-tip (2.7) than to eye (5.5), nostrils oriented posterolaterally; internarial distance 4.4; canthus rostralis rounded and straight; loreal region slightly concave and gradually sloping laterally; single, small, tooth-like process at lower jaw symphysis, with corresponding socket in between premaxillae; upper premaxillae and maxillae with numerous teeth; vomerine teeth in form of two hemispherical odontophores, perpendicular to body axis, almost being in contact to each other medially, each with row of teeth like tips (5 left, 6 right); distance from odontophores to elliptical choanae slightly larger than maximum length of odontophores; choanae almost equal in size to odontophores; tongue broadly heart shaped, deeply notched anteriorly, densely beset with small papillae, extends over entire length of lower jaw; posterior 2 / 3 of tongue free. Forelimbs robust, fingers slender and long; prepollex absent; first finger 10.3, almost double the length of second finger (5.1); relative finger lengths I>III>II>IV; finger tips broadened without forming discs; subarticular tubercles large, subconical, protruding distally; thenar and palmar tubercles large and elliptical; supernumerary tubercles absent; number of subarticular tubercles on digits I-IV: 1, 1, 2, 2; no digital webbing; hind limbs short and robust; femur length 28.3, almost equal to tibia 28.4; tibia-fibula with longest toe 38.4; relative toe lengths IV>III>V>II>I; shortest toe (4.2) almost double the length of inner metatarsal tubercle (2.3); inner metatarsal tubercle large, elliptical; toe tips broadened without forming discs; number of subconical subarticular tubercles on toes I-V: 1, 1, 2, 3, 2; supernumerary tubercles absent; traces of webbing between toe bases; no skin fringes along toes. Tip of toe IV on left foot missing. Skin on head, back, belly and throat predominantly smooth; small whitish spines scattered on back, dorsal parts of extremities and eyelids; white warts posterior to angle of mouth, ventral from tympanum; posterior region of thighs areolate; irregularly spread narrow low tubercles along flanks; no tubercles or warts on forearms or interocular region; head with white scratches (scars). Base colour of dorsum in preservation is a light brown to reddish brown; back with irregularly arranged and shaped light beige spots; upper eyelids greyish; head laterally with two darker bars, the posterior one below eye; upper and lower lips white; supratympanic fold anteriorly rimmed thin black; warts on flanks beige; forelimbs dorsally light brown, slightly mottled beige, first two fingers almost uniform white; hind limb colour as forelimbs, dark bordered cross bars indicated in thighs (4) and lower legs (5-6); throat, belly, ventral parts of fore- and hind limbs uniform white or creamish; only ventral part of feet reddish brown, metatarsal tubercle and subarticular tubercles creamish white. Variation. Females seem to be larger (up to 67.0 mm; MVZ 244910) than males (53.8 mm), although the small sample size of the latter (N = 2) does not allow for generalization. The tympanum shape varies between vertically elliptical (most vouchers) to round; the supratympanic fold is sometimes thin to absent; sometimes tympanic membrane prominent within faint tympanic annulus. Other measures and indices identical between the two sexes; values are summarized in Tabs. 1 and 2. Dorsal and lateral skin texture can consist of many irregularly spaced small roundish warts, not arranged in rows, sometimes fused to small ridge like structures (Fig. 11). Males have paired subgular vocal sacs, visible in preserved frogs as slightly loose, granular black skin near the angles of the mouth; males with large brown nuptial pads on the external side of the thumb. Canthus rostralis in males more prominent bulging and rounded; males with more massive forearms than females. In contrast to other western West African Astylosternus, A. laticephalus sp. nov. males in breeding condition, lack spines on throat and belly. The dorsal base colouration in life consists of a light grey, brown, reddish brown or almost black; all specimens exhibit a more or less dense pattern of smaller to larger, irregularly shaped red spots on back; smaller black or brown spots may be present, in particular in juveniles; iris greyish, upper third reddish to orange; the anterior part of the eyelids bright yellow to red blotches, connected by a very conspicuous interorbital stripe of the same colour; snout tip and area posterior to interorbital stripe, including posterior part of eyelid, darker than rest of back, this pattern sometimes consisting of a symmetrical pair of triangular shaped black spots (Fig. 11); frenal area dark as snout tip; loreal area distinctly lighter coloured than snout tip; light infraorbital batch; supratympanal ridge usually bordered by thin black line; fore and hind limbs with or without distinct black cross bars; lateral colour gradually fainting from back colour to whitish venter, with irregularly scattered black points; ventral coloration (throat and abdomen) creamish-white; ventral surface of thighs and arms creamish-yellow with reddish brown mottling around lateral margins of limbs. In preservative all patterns and coloration almost as in life, but faded; the red dorsal spots remain as light beige spots. Tadpole [measures in mm; description based on ZMB 75460]. Exotrophic, lentic tadpole; Gosner stage 39; total length 62.1; body broad almost as long as wide, slightly elliptical in dorsal, slightly depressed in lateral view (possibly a preservation artefact); body length 23.8; body width 24.2; sides of body slightly concave, snout in dorsal view broadly rounded, a bit more pointed in lateral view; large lateral sacs originating posterior to eyes run along flanks; small eyes, positioned dorsolaterally, pupil round; nares small, positioned dorsolaterally, closer to snout trip than to eyes; oral apparatus in anteroventral position; dorsal lip wide and smooth, with large anterior gap between marginal papillae; lateral papillae ventral lip with large, uni- or biserial marginal papillae; upper jaw sheath massif, broad U-shaped, strongly serrated margin, most central ���tooth��� most prominent; lower jaw sheath massif, V-shaped, margin strongly serrated; labial tooth-row formula 1: 1 + 1 / 3; all keratodont rows on skin sheaths; labial keratodonts unidenticulate, connected by a hyaline skin; vent tube dextral; spiracle sinistral (almost invisible); very long tail axis (> 2.5 times body length); tail axis height largely exceeding height of dorsal and ventral fin, both fins very narrow; dorsal fin originates slightly posterior to tail body junction; dorsal fin almost parallel to tail axis up to rounded tip; no pores (neuromast canals) visible (possibly due to poor preservation or advanced developmental stage); body more or less beige, mottled with dark brown, tail fin brown to almost black in last third of tail. Newly metamorphosed froglets measured 25.6���28.7 mm (N = 2). Natural history. Not much is known about the biology of the new species. Astylosternus laticephalus sp. nov. lives in lowland rainforest habitats (Fig. 13), mostly close to small or mid sized streams (R��del et al. 2005; Assemian et al. 2006). In Banco National Park, Ivory Coast we recorded the new species in the leaf litter of swampy areas in closed canopy forest, open forest and in natural forest gaps, both in close vicinity to the River Banco and in larger distance (about 1 km) from the river (Assemian et al. 2006). A total of 12 specimens were only observed during the rainy season, 11 of them during the night. In the Ankasa Conservation Area we found the new species in wet evergreen forest on a trail adjacent to the Bamboo Cathedral (Fig. 13 b). Distribution. So far A. laticephalus sp. nov. is known from one forest in eastern Ivory Coast, the Banco National Park (Assemian et al. 2006), and various sites in Ghana: Afao Hills Forest Reserve, Draw River Forest Reserve and Ankasa Conservation Area in south-western Ghana (R��del et al. 2005; this paper), and Atewa Range in southern-central Ghana (Fig. 8). The latter site was chosen as type locality of the new taxon to underline the outstanding importance of this mountainous region for the Ghanaian biodiversity. Conservation status. The distribution of the species from eastern Ivory Coast to southern-central Ghana, including various protected areas (i.e. Banco National Park, Afao Hills Forest Reserve, Ankasa Conservation Area), seems to imply a IUCN RedList classification of ���Least Concern���. However, rainforests in south-eastern Ivory Coast are under intense logging pressure and highly fragmented. Forests in south-western Ghana are better protected but likewise fragmented and the Atewa Range is currently under threat because of small scale mining activities and further plans to mine for gold, diamonds and bauxite on an industrial scale. We thus suggest classifying the new species as ���Near Threatened���. Etymology. The name derives from the Latin words latus for wide and the Greek K������� for head, referring to the conspicuous wide head of the new species., Published as part of R��del, Mark-Oliver, Barej, Michael F., Hillers, Annika, Leach��, Adam D., Kouam��, N'Goran G, Ofori-Boateng, Caleb, Assemian, Emmanuel, Toh��, Blayda, Penner, Johannes, Hirschfeld, Mareike, Doumbia, Joseph, Gonwouo, Legrand Nono, Nopper, Joachim, Brede, Christian, Diaz, Raul, Fujita, Matthew K., Gil, Marlon & H, Gabriel, 2012, The genus Astylosternus in the Upper Guinea rainforests, West Africa, with the description of a new species (Amphibia: Anura: Arthroleptidae), pp. 1-29 in Zootaxa 3245 on pages 16-22, DOI: 10.5281/zenodo.280506, {"references":["Rodel, M. - O., Gil, M., Agyei, A. C., Leache, A. D., Diaz, R. E., Fujita, M. K. & Ernst, R. (2005) The amphibians of the forested parts of south-western Ghana. Salamandra, 41, 107 - 127.","Assemian, N. E., Kouame, N. G., Tohe, B., Gourene, G. & Rodel, M. - O. (2006) The anurans of the Banco National Park, Cote d'Ivoire, a threatened West African rainforest. Salamandra, 42, 41 - 51."]}

Rödel, Mark-Oliver, Barej, Michael F., Hillers, Annika, Leaché, Adam D., Kouamé, N'Goran G, Ofori-Boateng, Caleb, Assemian, Emmanuel, Tohé, Blayda, Penner, Johannes, Hirschfeld, Mareike, Doumbia, Joseph, Gonwouo, Legrand Nono, Nopper, Joachim, Brede, Christian, Diaz, Raul, Fujita, Matthew K., Gil, Marlon, H, Gabriel (2012): The genus Astylosternus in the Upper Guinea rainforests, West Africa, with the description of a new species (Amphibia: Anura: Arthroleptidae). Zootaxa 3245: 1-29, DOI: 10.5281/zenodo.280506

Astylosternus laticephalus sp. nov. Rödel, Hillers, Leaché, Kouamé, Ofori-Boateng, Diaz & Sandberger Figs. 9–12 Holotype. ZMB 75449 (field and tissue #: ATE 10, female, 58.6 mm), Ghana, Atewa Forest Reserve, 6.23375 / - 0.56557, 14 April 2007, forest around stream next to road, coll. C. Ofori-Boateng, A. Hillers & G. Segniagbeto. Paratypes. Ivory Coast: MTD 48026 (field and tissue #: Ba04.24, female, 62.2 mm), ZMB 75454 -75458 (Ba04.26, subadult, 45.5 mm; Ba04.25, female, 62.5 mm; Ba04.21, female, 64.4 mm; Ba04.22, subadult, 43.3 mm; Ba04.23, female, 58.4 mm;), Banco National Park, 5.41667 / -4.10500, rainy season 2004, swampy forest, coll. N.E. Assemian, B. Tohé & G. Kouamé; MNHN 1993.6073 (male, 53.8 mm), 1999.7333 (female, 54.8 mm), 1999.7334 (male, 48.7 mm), 1999.7335 (female, 59.8 mm), 1999.7736 (female, 52.6 mm), 1999.7337 (female, 51.8 mm), Banco National Park, Abidjan; Ghana: ZMB 75459 (AF 4, juvenile, 25.6 mm), ZMB 75459 -75460 (AF 3 juvenile; 26.7 mm; AF 2, tadpole), Afao Hills Forest Reserve, 6.25461 / - 2.29492, riparian forest around small stream in valley, coll. C. Ofori-Boateng & A. Hillers; ZMB 75450 -75453 (COB 90, juvenile, 34.7 mm; COB 318, subadult, 40.8 mm; COB 119, juvenile, 38.6 mm; COB 123, juvenile, 31.7 mm), Western Province, Ankasa Conservation Area, pristine forest along streams in dry season, coll. C. Ofori-Boateng; MVZ 244910 (female, 67 mm), Ankasa Conservation Area, 5.28173 / - 2.64022, 26 June 2004, wet evergreen forest area, trail adjacent to the bamboo cathedral, coll. A.D. Leaché & R. Diaz; MVZ 244909 (juvenile, 35 mm), 28 June 2004, approximately 1 km farther from the bamboo cathedral, other data as MVZ 244909. Additional material. ZMB 77461 (field# AF 03), tadpole, Ghana, Afao Hills Forest Reserve, 6.25461 / - 2.29492, riparian forest around small stream in valley, coll. C. Ofori-Boateng & A. Hillers; ATE 7 (tissue only), juvenile, 25.8 mm; ATE 8 (tissue only), juvenile, 28.6 mm; ATE 27 (tissue only), male, 45.6 mm, Ghana, Atewa Forest Reserve, 6.23375 / - 0.56557, 15 April 2007, swampy area next to small stream, in valley in forest, coll. C. Ofori-Boateng, A. Hillers & G. Segniagbeto; COB 2202 (collection Ofori-Boateng, Kumasi, Ghana), Ghana, Atewa Forest Reserve, coll. C. Ofori-Boateng; three adult specimens without number (amphibian reference collection at University Abobo-Adjamé, Ivory Coast), male (51.0 mm), females (60.0, 67.0 mm), Ivory Coast, Banco National Park, 5.41667 / -4.10500, 5 May 2004, forest close to Banco River, coll. N.E. Assemian, N.G. Kouamé & B. Tohé. Diagnosis. Astylosternus laticephalus sp. nov. has the typical body shape of frogs of the genus Astylosternus, however, with exceptionally broad heads, i.e. broader than in A. occidentalis; males of the new species without spines on throat and belly (always present in A. occidentalis males); males without nuptial black skin layer in pectoral region (present in male A. occidentalis from western Guinea); back brownish to brownish red, always with distinct red dots (red dots only rarely present in A. occidentalis); bicoloured iris, grey with a reddish upper third (A. occidentalis always with uniform grey iris). A. laticephalus sp. nov. differs from A. occidentalis by a mean of 3.2 % (range 2.9-3.7 %) in the investigated part of the 16 S rRNA gene. Genetic divergence to the morphologically most similar Central African species, A. diadematus, was 11.9 %. Description of the holotype [measures in mm]. The holotype is an adult female with a snout-vent length of 58.6; head width 22.7; head as long as broad; interorbital distance narrower than length of upper eyelid; snout rounded in dorsal view, obtuse in lateral view, longer than eye diameter; eyes large (7.9) and protuberant directed anterolaterally; interorbital distance 7.2; pupil vertically elliptical; eye diameter larger than tympanum diameter, 5.3; tympanum vertically elliptical; supratympanal fold from posterior vertical midpoint of orbit to meet with dorsal border of tympanic annulus, where descent is sharp and terminates at half the vertical diameter of the tympanic annulus dorsally to the axilla; nares closer to snout-tip (2.7) than to eye (5.5), nostrils oriented posterolaterally; internarial distance 4.4; canthus rostralis rounded and straight; loreal region slightly concave and gradually sloping laterally; single, small, tooth-like process at lower jaw symphysis, with corresponding socket in between premaxillae; upper premaxillae and maxillae with numerous teeth; vomerine teeth in form of two hemispherical odontophores, perpendicular to body axis, almost being in contact to each other medially, each with row of teeth like tips (5 left, 6 right); distance from odontophores to elliptical choanae slightly larger than maximum length of odontophores; choanae almost equal in size to odontophores; tongue broadly heart shaped, deeply notched anteriorly, densely beset with small papillae, extends over entire length of lower jaw; posterior 2 / 3 of tongue free. Forelimbs robust, fingers slender and long; prepollex absent; first finger 10.3, almost double the length of second finger (5.1); relative finger lengths I>III>II>IV; finger tips broadened without forming discs; subarticular tubercles large, subconical, protruding distally; thenar and palmar tubercles large and elliptical; supernumerary tubercles absent; number of subarticular tubercles on digits I-IV: 1, 1, 2, 2; no digital webbing; hind limbs short and robust; femur length 28.3, almost equal to tibia 28.4; tibia-fibula with longest toe 38.4; relative toe lengths IV>III>V>II>I; shortest toe (4.2) almost double the length of inner metatarsal tubercle (2.3); inner metatarsal tubercle large, elliptical; toe tips broadened without forming discs; number of subconical subarticular tubercles on toes I-V: 1, 1, 2, 3, 2; supernumerary tubercles absent; traces of webbing between toe bases; no skin fringes along toes. Tip of toe IV on left foot missing. Skin on head, back, belly and throat predominantly smooth; small whitish spines scattered on back, dorsal parts of extremities and eyelids; white warts posterior to angle of mouth, ventral from tympanum; posterior region of thighs areolate; irregularly spread narrow low tubercles along flanks; no tubercles or warts on forearms or interocular region; head with white scratches (scars). Base colour of dorsum in preservation is a light brown to reddish brown; back with irregularly arranged and shaped light beige spots; upper eyelids greyish; head laterally with two darker bars, the posterior one below eye; upper and lower lips white; supratympanic fold anteriorly rimmed thin black; warts on flanks beige; forelimbs dorsally light brown, slightly mottled beige, first two fingers almost uniform white; hind limb colour as forelimbs, dark bordered cross bars indicated in thighs (4) and lower legs (5-6); throat, belly, ventral parts of fore- and hind limbs uniform white or creamish; only ventral part of feet reddish brown, metatarsal tubercle and subarticular tubercles creamish white. Variation. Females seem to be larger (up to 67.0 mm; MVZ 244910) than males (53.8 mm), although the small sample size of the latter (N = 2) does not allow for generalization. The tympanum shape varies between vertically elliptical (most vouchers) to round; the supratympanic fold is sometimes thin to absent; sometimes tympanic membrane prominent within faint tympanic annulus. Other measures and indices identical between the two sexes; values are summarized in Tabs. 1 and 2. Dorsal and lateral skin texture can consist of many irregularly spaced small roundish warts, not arranged in rows, sometimes fused to small ridge like structures (Fig. 11). Males have paired subgular vocal sacs, visible in preserved frogs as slightly loose, granular black skin near the angles of the mouth; males with large brown nuptial pads on the external side of the thumb. Canthus rostralis in males more prominent bulging and rounded; males with more massive forearms than females. In contrast to other western West African Astylosternus, A. laticephalus sp. nov. males in breeding condition, lack spines on throat and belly. The dorsal base colouration in life consists of a light grey, brown, reddish brown or almost black; all specimens exhibit a more or less dense pattern of smaller to larger, irregularly shaped red spots on back; smaller black or brown spots may be present, in particular in juveniles; iris greyish, upper third reddish to orange; the anterior part of the eyelids bright yellow to red blotches, connected by a very conspicuous interorbital stripe of the same colour; snout tip and area posterior to interorbital stripe, including posterior part of eyelid, darker than rest of back, this pattern sometimes consisting of a symmetrical pair of triangular shaped black spots (Fig. 11); frenal area dark as snout tip; loreal area distinctly lighter coloured than snout tip; light infraorbital batch; supratympanal ridge usually bordered by thin black line; fore and hind limbs with or without distinct black cross bars; lateral colour gradually fainting from back colour to whitish venter, with irregularly scattered black points; ventral coloration (throat and abdomen) creamish-white; ventral surface of thighs and arms creamish-yellow with reddish brown mottling around lateral margins of limbs. In preservative all patterns and coloration almost as in life, but faded; the red dorsal spots remain as light beige spots. Tadpole [measures in mm; description based on ZMB 75460]. Exotrophic, lentic tadpole; Gosner stage 39; total length 62.1; body broad almost as long as wide, slightly elliptical in dorsal, slightly depressed in lateral view (possibly a preservation artefact); body length 23.8; body width 24.2; sides of body slightly concave, snout in dorsal view broadly rounded, a bit more pointed in lateral view; large lateral sacs originating posterior to eyes run along flanks; small eyes, positioned dorsolaterally, pupil round; nares small, positioned dorsolaterally, closer to snout trip than to eyes; oral apparatus in anteroventral position; dorsal lip wide and smooth, with large anterior gap between marginal papillae; lateral papillae ventral lip with large, uni- or biserial marginal papillae; upper jaw sheath massif, broad U-shaped, strongly serrated margin, most central “tooth” most prominent; lower jaw sheath massif, V-shaped, margin strongly serrated; labial tooth-row formula 1: 1 + 1 / 3; all keratodont rows on skin sheaths; labial keratodonts unidenticulate, connected by a hyaline skin; vent tube dextral; spiracle sinistral (almost invisible); very long tail axis (> 2.5 times body length); tail axis height largely exceeding height of dorsal and ventral fin, both fins very narrow; dorsal fin originates slightly posterior to tail body junction; dorsal fin almost parallel to tail axis up to rounded tip; no pores (neuromast canals) visible (possibly due to poor preservation or advanced developmental stage); body more or less beige, mottled with dark brown, tail fin brown to almost black in last third of tail. Newly metamorphosed froglets measured 25.6–28.7 mm (N = 2). Natural history. Not much is known about the biology of the new species. Astylosternus laticephalus sp. nov. lives in lowland rainforest habitats (Fig. 13), mostly close to small or mid sized streams (Rödel et al. 2005; Assemian et al. 2006). In Banco National Park, Ivory Coast we recorded the new species in the leaf litter of swampy areas in closed canopy forest, open forest and in natural forest gaps, both in close vicinity to the River Banco and in larger distance (about 1 km) from the river (Assemian et al. 2006). A total of 12 specimens were only observed during the rainy season, 11 of them during the night. In the Ankasa Conservation Area we found the new species in wet evergreen forest on a trail adjacent to the Bamboo Cathedral (Fig. 13 b). Distribution. So far A. laticephalus sp. nov. is known from one forest in eastern Ivory Coast, the Banco National Park (Assemian et al. 2006), and various sites in Ghana: Afao Hills Forest Reserve, Draw River Forest Reserve and Ankasa Conservation Area in south-western Ghana (Rödel et al. 2005; this paper), and Atewa Range in southern-central Ghana (Fig. 8). The latter site was chosen as type locality of the new taxon to underline the outstanding importance of this mountainous region for the Ghanaian biodiversity. Conservation status. The distribution of the species from eastern Ivory Coast to southern-central Ghana, including various protected areas (i.e. Banco National Park, Afao Hills Forest Reserve, Ankasa Conservation Area), seems to imply a IUCN RedList classification of “Least Concern”. However, rainforests in south-eastern Ivory Coast are under intense logging pressure and highly fragmented. Forests in south-western Ghana are better protected but likewise fragmented and the Atewa Range is currently under threat because of small scale mining activities and further plans to mine for gold, diamonds and bauxite on an industrial scale. We thus suggest classifying the new species as “Near Threatened”. Etymology. The name derives from the Latin words latus for wide and the Greek Kεφάλɩ for head, referring to the conspicuous wide head of the new species.

BackgroundMultiple myeloma (MM) is a B-cell malignancy, where malignant plasma cells clonally expand in the bone marrow of older people, causing significant morbidity and mortality. Typical clinical symptoms include increased serum calcium levels, renal insufficiency, anemia, and bone lesions. With standard therapies, MM remains incurable; therefore, the development of new drugs or immune cell-based therapies is desirable. To advance the goal of finding a more effective treatment for MM, we aimed to develop a reliable preclinical MM mouse model applying sensitive and reproducible methods for monitoring of tumor growth and metastasis in response to therapy.Material and methodsA mouse model was created by intravenously injecting bone marrow-homing mouse myeloma cells (MOPC-315.BM) that expressed luciferase into BALB/c wild type mice. The luciferase in the myeloma cells allowed in vivo tracking before and after melphalan treatment with bioluminescence imaging (BLI). Homing of MOPC-315.BM luciferase+ myeloma cells to specific tissues was examined by flow cytometry. Idiotype-specific myeloma protein serum levels were measured by ELISA. In vivo measurements were validated with histopathology.ResultsStrong bone marrow tropism and subsequent dissemination of MOPC-315.BM luciferase(+) cells in vivo closely mimicked the human disease. In vivo BLI and later histopathological analysis revealed that 12 days of melphalan treatment slowed tumor progression and reduced MM dissemination compared to untreated controls. MOPC-315.BM luciferase(+) cells expressed CXCR4 and high levels of CD44 and α4β1 in vitro which could explain the strong bone marrow tropism. The results showed that MOPC-315.BM cells dynamically regulated homing receptor expression and depended on interactions with surrounding cells.ConclusionsThis study described a novel MM mouse model that facilitated convenient, reliable, and sensitive tracking of myeloma cells with whole body BLI in living animals. This model is highly suitable for monitoring the effects of different treatment regimens.