Generation and flow cytometric quality control of clinical-scale TCRαß/CD19-depleted grafts.

BACKGROUND: The depletion of TCRαß+ T cells and CD19+ B cells is a graft purification method for haploidentical stem cell transplantation (HSCT) retaining stem cells, NK cells and TCRγδ+ T cells. To avoid treatment-related occurrence of severe GvHD a precise quantification of residual TCRαß+ T cells in the graft is of essential importance. METHODS: Nine stem cell grafts were purified immunomagnetically on a CliniMACS device and flow cytometric quality control (QC) was performed before and after TCRαß/CD19-depletion. RESULTS: As a challenge a new 10-color QC-panel was established, which enables accurate quantification of the graft composition. The binding sites of residual TCRαß+ T and CD19+ B cells were at least partly occupied by depletion antibodies impeding flow cytometric analysis. Based on respective controls and an assumed variation coefficient of 18%, the detection limit of residual TCRαß T cells was 1 cell/µl. and 0.002% of CD45+ cells. Log-depletion of TCRαß and CD19 cells was -3.9 and -3.3, respectively. The recovery was 82.1%, 67.1% and 72.7% for stem cells, NK cells and for TCRγδ+ T cells. CONCLUSIONS: In clinical use this method may help to improve transplantation outcome, due to the correct application of the desired stem cell and the limited T cell dose. The panel is designed for the QC following TCRαß/CD19-depletion but is adaptable to other depletion strategies as well. © 2015 International Clinical Cytometry Society.

Functions of corazonin and histamine in light entrainment of the circadian pacemaker in the Madeira cockroach, Rhyparobia maderae.

The circadian pacemaker of the Madeira cockroach, Rhyparobia (Leucophaea) maderae, is located in the accessory medulla (AME). Ipsi- and contralateral histaminergic compound eyes are required for photic entrainment. Light pulses delay locomotor activity rhythm during the early night and advance it during the late night. Thus, different neuronal pathways might relay either light-dependent delays or advances to the clock. Injections of neuroactive substances combined with running-wheel assays suggested that GABA, pigment-dispersing factor, myoinhibitory peptides (MIPs), and orcokinins (ORCs) were part of both entrainment pathways, whereas allatotropin (AT) only delayed locomotor rhythms at the early night. To characterize photic entrainment further, histamine and corazonin were injected. Histamine injections resulted in light-like phase delays and advances, indicating that the neurotransmitter of the compound eyes participates in both entrainment pathways. Because injections of corazonin only advanced during the late subjective night, it was hypothesized that corazonin is only part of the advance pathway. Multiple-label immunocytochemistry in combination with neurobiotin backfills demonstrated that a single cell expressed corazonin in the optic lobes that belonged to the group of medial AME interneurons. It colocalized GABA and MIP but not AT or ORC immunoreactivity. Corazonin-immunoreactive (-ir) terminals overlapped with projections of putatively light-sensitive interneurons from the ipsi- and contralateral compound eye. Thus, we hypothesize that the corazonin-ir medial neuron integrates ipsi- and contralateral light information as part of the phase-advancing light entrainment pathway to the circadian clock. J. Comp. Neurol. 525:1250-1272, 2017. © 2016 Wiley Periodicals, Inc.

Peptidergic circadian clock circuits in the Madeira cockroach.

Circadian clocks control physiology and behavior of organisms in synchrony with external light dark cycles in changing photoperiods. The Madeira cockroach Rhyparobia maderae was the first model organism in which an endogenous circadian clock in the brain was identified. About 240 neurons constitute the cockroach circadian pacemaker network in the accessory medulla. The expression of high concentrations of neuropeptides, among them the most prominent circadian coupling factor pigment-dispersing factor, as well as their ability to generate endogenous ultradian and circadian rhythms in electrical activity and clock gene expression distinguish these pacemaker neurons. We assume that entrainment to light-dark cycles and the control of 24h rest-activity rhythms is achieved via peptidergic circuits forming autoreceptive labeled lines.

The neuropeptide SIFamide in the brain of three cockroach species.

The sequence as well as the distribution pattern of SIFamide in the brain of different insects is highly conserved. As a general rule, at least four prominent SIFamide-immunoreactive somata occur in the pars intercerebralis. They arborize throughout the brain and the ventral nerve cord. Whereas SIFamide is implicated in mating and sleep regulation in Drosophila, other functions of this peptide remain largely unknown. To determine whether SIFamide plays a role in the circadian system of cockroaches, we studied SIFamide in Rhyparobia (= Leucophaea) maderae (Blaberidae), Periplaneta americana (Blattidae), and Therea petiveriana (Polyphagidae). Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry revealed identical SIFamide sequences (TYRKPPFNGSIFamide) in the three species. In addition to four large immunoreactive cells in the pars intercerebralis (group 1), smaller SIFamide-immunoreactive somata were detected in the pars intercerebralis (group 2), in the superior median protocerebrum (group 3), and in the lateral protocerebrum (group 4). Additional cells in the optic lobe (group 5) and posterior protocerebrum (group 6) were stained only in P. americana. Almost the entire protocerebrum was filled with a beaded network of SIFamide-immunoreactive processes that especially strongly invaded the upper unit of the central body. Double-label experiments did not confirm colocalizations with γ-aminobutyric acid (GABA) or the circadian coupling peptide pigment-dispersing factor (PDF). In contrast to locusts, colocalization of SIFamide and histamine immunoreactivity occurred not in group 1, but in group 4 cells. Because the accessory medulla displayed SIFamide immunoreactivity and injections of SIFamide delayed locomotor activity rhythms circadian time-dependently, SIFamide plays a role in the circadian system of cockroaches. J. Comp. Neurol. 524:1337-1360, 2016. © 2015 Wiley Periodicals, Inc.

Bimodal oscillations of cyclic nucleotide concentrations in the circadian system of the Madeira cockroach Rhyparobia maderae.

Pigment-dispersing factor (PDF) is the most important coupling factor of the circadian system in insects, comparable to its functional ortholog vasoactive intestinal polypeptide of the mammalian circadian clock. In Drosophila melanogaster, PDF signals via activation of adenylyl cyclases, controlling circadian locomotor activity rhythms at dusk and dawn. In addition, PDF mediates circadian rhythms of the visual system and is involved in entrainment to different photoperiods. We examined whether PDF daytime-dependently elevates cAMP levels in the Madeira cockroach Rhyparobia maderae and whether cAMP mimics PDF effects on locomotor activity rhythms. To determine time windows of PDF release, we searched for circadian rhythms in concentrations of cAMP and its functional opponent cGMP in the accessory medulla (AMe), the insect circadian pacemaker controlling locomotor activity rhythms, and in the optic lobes, as the major input and output area of the circadian clock. Enzyme-linked immunosorbent assays detected PDF-dependent increases of cAMP in optic lobes and daytime-dependent oscillations of cAMP and cGMP baseline levels in the AMe, both with maxima at dusk and dawn. Although these rhythms disappeared at the first day in constant conditions (DD1), cAMP but not cGMP oscillations returned at the second day in constant conditions (DD2). Whereas in light-dark cycles the cAMP baseline level remained constant in other optic lobe neuropils, it oscillated in phase with the AMe at DD2. To determine whether cAMP and cGMP mimic PDF-dependent control of locomotor activity rhythms, both cyclic nucleotides were injected at different times of the circadian day using running-wheel assays. Whereas cAMP injections generated delays at dusk and advances at dawn, cGMP only delayed locomotor activity at dusk. For the first time we found PDF-dependent phase advances at dawn in addition to previously described phase delays at dusk. Thus, we hypothesize that PDF release at dusk and dawn controls locomotor activity rhythms and visual system processing cAMP-dependently.