Cytophagic Histiocytic Panniculitis (CHP) in a Patient with SLE Found after Autopsy: When a Rash Is “Complicated!”

Introduction. Cytophagic histolytic panniculitis (CHP) is a clinical disorder characterized by nodular panniculitis of the subcutaneous adipose tissue. It was first described in 1980 by Winkelmann. Histologically it is described as an infiltration of the adipose tissue by T- lymphocytes and phagocytic macrophages (also known as “bean bag cells”). Most of the cases are reported under the age of 50 and is a rare cause of panniculitis. We report a case of CHP in a young patient who presented to our emergency room (ER). Case Summary. A 39-year-old African American woman who presented to our hospital with lethargy, progressive confusion, and generalized rash involving both lower extremities of 1 week duration. She had a history of pancytopenia and focal proliferative and membranous lupus nephritis classes 3 and 5. Her physical examination was remarkable for bilateral lower extremity pitting edema and a desquamating rash on both of her legs. The Nicolsky sign was positive. She was noted to be hypotensive and was started on intravenous fluids and broad spectrum antibiotics. Routine laboratory tests revealed severe pancytopenia, with a hemoglobin of 3.9 g/dl, white blood cell count 600/ul, and platelet count of 11000/ul. Within an hour of arrival to the ER she developed acute respiratory failure. She was intubated and placed on mechanical ventilation. She developed shock requiring vasopressors. No imaging could be done due to her unstable condition. Four hours after her initial presentation she developed asystole and expired. Postmortem histopathology of the adipose tissue revealed CHP. Conclusion. CHP can be rapidly fatal. The treatment involves high dose of intravenous steroids and immunosuppressants such as cyclosporine.

Phase I/IIb trial: In-situ vaccine for metastatic colorectal cancer.

TPS871 Background: Except for MSI-H tumors, CRC does not respond to immunotherapy. CRC does respond to the graft vs. tumor (GVT) immune effect that occurs after allogeneic stem cell transplantation. GVT is associated with GVHD toxicity which limits the clinical application. A bioengineered allograft (BAG) has been developed which can elicit host-mediated GVT-like effects without GVHD toxicity, chemotherapy conditioning or a HLA-matched donor. BAG are Th1 memory cells derived from blood of healthy donors with CD3/CD28 microbeads attached. These cells have immunomodulatory properties which enable modulation of Th1/Th2 balance and dysregulation of immunosuppressive circuits. We are evaluating the safety and efficacy of BAG in third-line mCRC. Methods: The study uses a standard 3+3 design followed by an expansion phase with the optimal dosing pattern. The protocol has four components: (A) priming; (B) in-situ vaccination; (C) extravasation and trafficking; and (D) counter immune suppression/avoidance. Priming involves intradermal injections of BAG cells which activates NK cells and develops allo-specific Th1/Tc1 immunity. In-Situ vaccination involves tumor cryoablation to release endogenous HSP which chaperone tumor neoantigens, followed immediately by the intralesional injection of BAG cells as adjuvant. Released HSP are engulfed and processed by immature dendritic cells (DC) attracted to the tissue damage. The inflammatory microenvironment created by the BAG and the subsequent allo-rejection response amplified by the priming induces DC maturation. These DC display processed tumor antigens on upregulated MHCI/II and express co-stimulatory CD80/86 enabling priming of a tumor-specific Th1/Tc1 response. CD40L and interferon-gamma expressed by BAG activates allo-specific and tumor-specific memory cells upon intravenous infusion, permitting trafficking to tumor. The host rejection of BAG releases endogenous danger signals creating a sustained systemic inflammatory cytokine release which serves to counter-regulate immunosuppressive mechanisms. Longitudinal CT scans biopsies, PBMC and serum samples are collected for analysis to verify immune events within each phase of the protocol. Clinical trial information: NCT02380443.

Personalized vaccine for advanced HCC.

TPS527 Background: The chronic inflammation and viral infection associated with HCC combined with liver tolerogenic mechanisms creates a profoundly immunosuppressive microenvironment. Modulating the microenvironment with checkpoint blockade benefits ~20% of patients. The non-responders and those with poor liver function are an unmet need. Checkpoint blockade requires effector cells to be resident within tumors. However, the majority of HCC lesions lack effector cells. Therefore, a strategy for amplifying the tumor-specific immune response, while counteracting the immunosuppressive mechanisms may provide an improved immunotherapy. Endogenous heat shock proteins (HSP) chaperone tumor neoantigens. A concentrated composition of calreticulin, hsp70, hsp90 and gr94/gp96 (CRCL) is purified from biopsy samples. In addition, a bioengineered allograft (BAG) derived from healthy blood donors with a Th1 memory phenotype, CD3/CD28-coated microbeads and high expression of CD40L has potent immunomodulatory and counter-regulatory properties. The combination of CRCL with BAG is being evaluated as a strategy to amplify tumor-specific immunity and counter-regulate the immunosuppressive microenvironment. Methods: An open-label Phase II clinical trial evaluating the safety and efficacy of CRCL+BAG vaccine in Child-Pugh A/B advanced/metastatic HCC w/o Sorafenib. Approximately 6-10 cores of tumor are collected at baseline. The tumor is lysed and CRCL purified using an isoelectric focusing technique. Subjects are primed with multiple intradermal injections of BAG cells alone to increase the titer of allo-specific Th1/Tc1 cells. Next, multiple intradermal injections of BAG+CRCL are administered to elicit increased titers of tumor-specific Th1/Tc1 cells. Subsequent intravenous infusions of BAG cells activate allo- and tumor-specific memory cells, enabling their infiltration into tumor lesions. The BAG allo-rejection response creates a type I cytokine storm which serves to down-regulate suppressor circuits. Response is evaluated by mRECIST and experimental biomarkers. Longitudinal CT scans with concurrent biopsies as well as multiple collections of PBMC and plasma permits immunomonitoring of these immune mechanisms. Clinical trial information: NCT02409524.

Persistent Ca2+ Current Contributes to a Prolonged Depolarization in Aplysia Bag Cell Neurons

Neurons may initiate behavior or store information by translating prior activity into a lengthy change in excitability. For example, brief input to the bag cell neurons of Aplysia results in an approximate 30-min afterdischarge that induces reproduction. Similarly, momentary stimulation of cultured bag cells neurons evokes a prolonged depolarization lasting many minutes. Contributing to this is a voltage-independent cation current activated by Ca2+ entering during the stimulus. However, the cation current is relatively short-lived, and we hypothesized that a second, voltage-dependent persistent current sustains the prolonged depolarization. In bag cell neurons, the inward voltage-dependent current is carried by Ca2+; thus we tested for persistent Ca2+ current in primary culture under voltage clamp. The observed current activated between −40 and −50 mV exhibited a very slow decay, presented a similar magnitude regardless of stimulus duration (10–60 s), and, like the rapid Ca2+ current, was enhanced when Ba2+ was the permeant ion. The rapid and persistent Ca2+ current, but not the cation current, were Ni2+ sensitive. Consistent with the persistent current contributing to the response, Ni2+ reduced the amplitude of a prolonged depolarization evoked under current clamp. Finally, protein kinase C activation enhanced the rapid and persistent Ca2+ current as well as increased the prolonged depolarization when elicited by an action potential-independent stimulus. Thus the prolonged depolarization arises from Ca2+ influx triggering a cation current, followed by voltage-dependent activation of a persistent Ca2+ current and is subject to modulation. Such synergy between currents may represent a common means of achieving activity-dependent changes to excitability.

Aplysia Bag Cells Function as a Distributed Neurosecretory Network

The anatomical organization of many neuroendocrine systems implies multiple sites of hormone release in areas mediating multiple aspects of physiology and behavior, yet this neurosecretory complexity has not often been verified. Here we probe the well-characterized hormonal model, the reproductive bag cell neuroendocrine system of the sea slug Aplysia californica. The bag cell neurons of Aplysia mediate egg-laying behavior through the coordinated secretion of a suite of peptides derived from a single gene product, the egg-laying prohormone (proELH). Although the majority of bag cell neurons are located within two major abdominal bag cell clusters, smaller groups of egg-laying hormone-expressing cells have been observed in specific pleural and cerebral ganglia regions, some of which have been reported to be electrically connected to the abdominal bag cell clusters. Releasates are sampled from discrete locations within the Aplysia CNS before and during stimulation of afterdischarge activity and subsequently analyzed with matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Site-specific release profiles are observed at bag cell cluster, pleural, and genital ganglion sites after site-specific electrophysiological activation of bag cell afterdischarges. These data demonstrate that the bag cell network has multiple neurohemal release sites, exhibits descending activation that travels from the cerebral and pleural ganglia down to the abdominal bag cell clusters, and releases spatially distinct profiles of proELH-derived peptides within the Aplysia nervous system. Such distributed neurosecretory organization may be a common feature of neuroendocrine systems across higher order organisms linking multiple behavioral aspects to a single neuronal network.

Egg-laying hormone peptides in the aplysiidae family

The neuropeptidergic bag cells of the marine mollusc Aplysia californica are involved in the egg-laying behavior of the animal. These neurosecretory cells synthesize an egg-laying hormone (ELH) precursor protein, yielding multiple bioactive peptides, including ELH, several bag cell peptides (BCP) and acidic peptide (AP). While immunohistochemical studies have involved a number of species, homologous peptides have been biochemically characterized in relatively few Aplysiidae species. In this study, a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and electrospray ionization Fourier transform ion cyclotron resonance MS is used to characterize and compare the ELH peptides from related opisthobranch molluscs including Aplysia vaccaria and Phyllaplysia taylori. The peptide profiles of bag cells from these two Aplysiidae species are similar to that of A. californica bag cells. In an effort to characterize further several of these peptides, peptides from multiple groups of cells of each species were extracted, and microbore liquid chromatography was used to separate and isolate them. Several MS-based sequencing approaches are applied to obtain the primary structures of bag cell peptides and ELH. Our studies reveal that (α)-BCPs are 100 % conserved across all species studied. In addition, the complete sequences of (ε)-BCP and ELH of A. vaccaria were determined. They show a high degree of homology to their counterparts in A. californica, with only a few amino acid residue substitutions.

Characterization of Na+ and Ca2+ currents in bag cells of sexually immature aplysia californica

The neurosecretory bag cells of sexually mature Aplysia californica release egg-laying hormones as part of the reproductive process after a train of action potentials termed afterdischarge. Whole-cell voltage-clamp experiments were performed in cultured cells from sexually immature A. californica to characterize the inward voltage-gated currents for Na+ and Ca2+. The goal of these experiments was to investigate the regulation of excitability during sexual maturation. Na+ currents in bag cells of immature A. californica were similar in several ways to those of mature animals. The Na+ currents activated at voltages less negative than -30 mV and peaked at 10-20 mV in artificial sea water. The time course and pharmacology of bag cell Na+ currents were similar to those of bag cells from mature A. californica, although the Na+ current density was lower in immature A. californica. Na+ currents were inhibited by tetrodotoxin (50 nmol l-1). The Na+ current was relatively insensitive to depolarized holding potentials (Vh), maintaining approximately 50 % of peak current amplitude present at Vh=-70 mV throughout the activation range at Vh=-30 mV. In experiments using a 1 s depolarized Vh prior to a test pulse, the half-inactivation voltage (V1/2) was -27 mV. Recovery of immature Na+ current from steady-state inactivation at Vh=-30 mV had a time constant (<IMG src="/images/symbols/tau.gif" WIDTH="8" HEIGHT="12" ALIGN= "BOTTOM" NATURALSIZEFLAG="3">) of 9.5 ms, significantly slower than in mature animals. Ca2+ currents of immature A. californica activated at approximately -30 mV and peaked at approximately 20 mV with 11 mmol l-1 Ba2+ as the charge carrier. The principle differences from mature Ca2+ currents were the low density of the immature Ca2+ currents and their 'run-down' in whole-cell recordings. The pharmacology and V1/2 of bag cell Ca2+ currents were similar to those of L-type Ca2+ currents in mature cells. The Ca2+ currents were inhibited 61+/-10 % by nifedipine (10 micromol l-1) and were unaffected by <IMG src="/images/symbols/omega.gif" WIDTH="9" HEIGHT="12" ALIGN= "BOTTOM" NATURALSIZEFLAG="3">-conotoxin GVIA (10 micromol l-1). The Ca2+ currents were relatively insensitive to depolarized Vh, activating maximally at Vh=-90, -70 and -50 mV, and maintaining 50 % of this peak current amplitude throughout the activation range at Vh=-30 mV. The V1/2 was -23 mV in experiments in which cells were subjected to a 1 s depolarized Vh prior to a test pulse. Na+ current amplitudes were maintained or increased during 1 min of 4 Hz test pulses in bag cells at Vh=-70 mV and Vh=-30 mV. In contrast, Ca2+ current run-down occurred during 1 min of 4 Hz test pulses in seven of 10 cells at Vh=-70 mV and in 12 of 12 cells at Vh=-30 mV. The observed scarcity of Na+ and Ca2+ currents in immature bag cells as well as the specific characteristics of immature bag cell Ca2+ currents make repetitive action potential firing and hormone release less likely than in mature bag cells.

Bovine adrenal glomerulosa cells express such a low level of functional B2 receptors that bradykinin does not significantly increase their aldosterone production

It was recently demonstrated that bradykinin (BK) stimulates aldosterone secretion in bovine adrenal glomerulosa (BAG) cells. The aim of the present study was to characterize the mechanism of action of BK on these cells. Binding experiments with the radioligand 125I-[Tyr8]BK revealed the presence of a relatively small amount (Bmax = 180 +/- 55 fmol/mg of protein) of high affinity (Kd = 0.65 +/- 0.17 nM) binding sites. BK induced a time- and concentration-dependent increase of [3H]inositol trisphosphate ([3H]IP3) in myo-[3H]inositol-labeled BAG cells. A maximal response was obtained with 10 nM BK and the EC50 value was 1.0 +/- 0.5 nM. 125I-[Tyr8]BK binding and BK-induced IP3 production were inhibited by the selective B2 receptor antagonist Icatibant (1 microM) and unaffected by the selective B1 receptor antagonist [DesArg9, Leu8]BK (1 microM). In fura-2 loaded BAG cells, BK (100 nM) induced a typical biphasic Ca2+ response composed of a rapid and transient increase of intracellular Ca2+ concentration [Ca2+]i which slowly declined to a level that remained above basal level for about 5 min. In the presence of EGTA (2 mM), the rapid and transient calcium increase was unaffected whereas the plateau phase was abolished. Angiotensin II (Ang II, 100 nM) also elicited a typical biphasic response in BAG cells. However the rapid and transient elevation of [Ca2+]i was followed by a sustained plateau phase which remained above the basal level for more than 10 min. Although BAG cells express functional B2 receptors, no secretion of aldosterone was observed after stimulation with 100 nM BK for 120 min. Under the same conditions Ang II increased by about 10-fold the basal level of aldosterone. The lack of effect of BK is probably attributable to its very transient effect on IP3 production. Pretreatment of BAG cells with 100 nM BK for 20 min reduced by 70 +/- 10% their total binding capacity. These results suggest a rapid and very efficient desensitization process. We conclude that BAG cells express functional B2 receptors. The weak production of second messengers and the rapid desensitization process could explain why BK fails to increase aldosterone production in these cells. Since functional B2 receptors are expressed in BAG cells it is likely that under some specific physiological or pathological conditions these receptors may play a significant role in aldosterone secretion. However these conditions remain to be determined.