Plasma cell-directed therapies in monoclonal gammopathy-associated scleromyxedema.

Scleromyxedema is a rare skin and systemic mucinosis that is usually associated with monoclonal gammopathy (MG). In this French multicenter retrospective study of 33 patients, we investigated the clinical and therapeutic features of MG-associated scleromyxedema. Skin molecular signatures were analyzed using a transcriptomic approach. Skin symptoms included papular eruptions (100%), sclerodermoid features (91%), and leonine facies (39%). MG involved an immunoglobulin G isotype in all patients, with a predominant λ light chain (73%). Associated hematologic malignancies were diagnosed in 4 of 33 patients (12%) (smoldering myeloma, n = 2; chronic lymphoid leukemia, n = 1; and refractory cytopenia with multilineage dysplasia, n = 1). Carpal tunnel syndrome (33%), arthralgia (25%), and dermato-neuro syndrome (DNS) (18%) were the most common systemic complications. One patient with mucinous cardiopathy died of acute heart failure. High-dose IV immunoglobulin (HDIVig), alone or in combination with steroids, appeared to be quite effective in nonsevere cases (clinical complete response achieved in 13/31 patients). Plasma cell-directed therapies using lenalidomide and/or bortezomib with dexamethasone and HDIVig led to a significant improvement in severe cases (HDIVig refractory or cases with central nervous system or cardiac involvement). The emergency treatment of DNS with combined plasmapheresis, HDIVig, and high-dose corticosteroids induced the complete remission of neurological symptoms in 4 of 5 patients. Quantitative reverse-transcriptase polymerase chain reaction analysis of 6 scleromyxedema skin samples showed significantly higher profibrotic pathway levels (transforming growth factor ß and collagen-1) than in healthy skin. Prospective studies targeting plasma cell clones and/or fibrotic pathways are warranted for long-term scleromyxedema management.

Proteomic and secretomic comparison of young and aged dermal fibroblasts highlights cytoskeleton as a key component during aging.

Crucial for skin homeostasis, synthesis and degradation of extracellular matrix components are orchestrated by dermal fibroblasts. During aging, alterations of component expression, such as collagens and enzymes, lead to reduction of the mechanical cutaneous tension and defects of skin wound healing. The aim of this study was to better understand the molecular alterations underwent by fibroblasts during aging by comparing secretomic and proteomic signatures of fibroblasts from young (<35years) and aged (>55years) skin donors, in quiescence or TGF-stimulated conditions, using HLPC/MS. The comparison of the secretome from young and aged fibroblasts revealed that 16 proteins in resting condition, and 11 proteins after a 24h-lasting TGF-ß1-treatment, were expressed in significant different ways between the two cell groups (fold change>2, p-value <0.05), with a 77% decrease in the number of secreted proteins in aged cells. Proteome comparison between young and aged fibroblasts identified a significant change of 63 proteins in resting condition, and 73 proteins in TGF-ß1-stimulated condition, with a 67% increase in the number of proteins in aged fibroblasts. The majority of the differentially-expressed molecules belongs to the cytoskeleton-associated proteins and aging was characterized by an increase in Coronin 1C (CORO1C), and Filamin B (FLNB) expression in fibroblasts together with a decrease in Cofilin (CFL1), and Actin alpha cardiac muscle 1 (ACTC1) detection in aged cells, these proteins being involved in actin-filament polymerization and sharing co-activity in cell motility. Our present data reinforce knowledge about an age-related alteration in the synthesis of major proteins linked to the migratory and contractile functions of dermal human fibroblasts.

Proteostasis and metabolic dysfunction in a distinct subset of storage-induced senescent erythrocytes targeted for clearance.

Although refrigerated storage slows the metabolism of volunteer donor RBCs, cellular aging still occurs throughout this in vitro process, which is essential in transfusion medicine. Storage-induced microerythrocytes (SMEs) are morphologically-altered senescent RBCs that accumulate during storage and which are cleared from circulation following transfusion. However, the molecular and cellular alterations that trigger clearance of this RBC subset remain to be identified. Using a staining protocol that sorts long-stored SMEs (i.e., CFSE high ) and morphologically-normal RBCs (CFSE low ), these in vitro aged cells were characterized. Metabolomics analysis identified depletion of energy, lipid-repair, and antioxidant metabolites in CFSE high RBCs. By redox proteomics, irreversible protein oxidation primarily affected CFSE high RBCs. By proteomics, 96 proteins, mostly in the proteostasis family, had relocated to CFSE high RBC membranes. CFSE high RBCs exhibited decreased proteasome activity and deformability; increased phosphatidylserine exposure, osmotic fragility, and endothelial cell adherence; and were cleared from the circulation during human spleen ex vivo perfusion. Conversely, molecular, cellular, and circulatory properties of long-stored CFSE low RBCs resembled those of short-stored RBCs. CFSE high RBCs are morphologically and metabolically altered, have irreversibly oxidized and membrane-relocated proteins, and exhibit decreased proteasome activity. In vitro aging during storage selectively alters metabolism and proteostasis in SMEs, targeting these senescent cells for clearance.

An Oil-Based Adjuvant Improves Immune Responses Induced by Canine Adenovirus-Vectored Vaccine in Mice.

There is a significant need for highly effective vaccines against emerging and common veterinary infectious diseases. Canine adenovirus type 2 (CAV2) vectors allow rapid development of multiple vaccines and have demonstrated their potential in animal models. In this study, we compared the immunogenicity of a non-replicating CAV2 vector encoding the rabies virus glycoprotein with and without MontanideTM ISA 201 VG, an oil-based adjuvant. All vaccinated mice rapidly achieved rabies seroconversion, which was associated with complete vaccine protection. The adjuvant increased rabies antibody titers without any significant effect on the anti-CAV2 serological responses. An RT2 Profiler™ PCR array was conducted to identify host antiviral genes modulated in the blood samples 24 h after vaccination. Functional analysis of differentially expressed genes revealed the up-regulation of the RIG-I, TLRs, NLRs, and IFNs signaling pathways. These results demonstrate that a water-in-oil-in-water adjuvant can shape the immune responses to an antigen encoded by an adenovirus, thereby enhancing the protection conferred by live recombinant vaccines. The characterization of early vaccine responses provides a better understanding of the mechanisms underlying the efficacy of CAV2-vectored vaccines.

Low-Dose Dietary Fish Oil Improves RBC Deformability without Improving Post-Transfusion Recovery in Mice.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are important modulators of red blood cell (RBC) rheology. Dietary LC-PUFAs are readily incorporated into the RBC membrane, improving RBC deformability, fluidity, and hydration. Female C57BL/6J mice consumed diets containing increasing amounts of fish oil (FO) ad libitum for 8 weeks. RBC deformability, filterability, and post-transfusion recovery (PTR) were evaluated before and after cold storage. Lipidomics and lipid peroxidation markers were evaluated in fresh and stored RBCs. High-dose dietary FO (50%, 100%) was associated with a reduction in RBC quality (i.e., in vivo lifespan, deformability, lipid peroxidation) along with a reduced 24 h PTR after cold storage. Low-dose dietary FO (6.25-12.5%) improved the filterability of fresh RBCs and reduced the lipid peroxidation of cold-stored RBCs. Although low doses of FO improved RBC deformability and reduced oxidative stress, no improvement was observed for the PTR of stored RBCs. The improvement in RBC deformability observed with low-dose FO supplementation could potentially benefit endurance athletes and patients with conditions resulting from reduced perfusion, such as peripheral vascular disease.

Storage-Induced Micro-Erythrocytes Can Be Quantified and Sorted by Flow Cytometry.

Refrigerated storage of red cell concentrates before transfusion is associated with progressive alterations of red blood cells (RBC). Small RBC (type III echinocytes, sphero-echinocytes, and spherocytes) defined as storage-induced micro-erythrocytes (SME) appear during pretransfusion storage. SME accumulate with variable intensity from donor to donor, are cleared rapidly after transfusion, and their proportion correlates with transfusion recovery. They can be rapidly and objectively quantified using imaging flow cytometry (IFC). Quantifying SME using flow cytometry would further facilitate a physiologically relevant quality control of red cell concentrates. RBC stored in blood bank conditions were stained with a carboxyfluorescein succinimidyl ester (CFSE) dye and incubated at 37°C. CFSE intensity was assessed by flow cytometry and RBC morphology evaluated by IFC. We observed the accumulation of a CFSE high RBC subpopulation by flow cytometry that accounted for 3.3 and 47.2% at day 3 and 42 of storage, respectively. IFC brightfield images showed that this CFSE high subpopulation mostly contains SME while the CFSE low subpopulation mostly contains type I and II echinocytes and discocytes. Similar numbers of SME were quantified by IFC (based on projected surface area) and by flow cytometry (based on CFSE intensity). IFC and scanning electron microscopy showed that ≥95% pure subpopulations of CFSE high and CFSE low RBC were obtained by flow cytometry-based sorting. SME can now be quantified using a common fluorescent dye and a standard flow cytometer. The staining protocol enables specific sorting of SME, a useful tool to further characterize this RBC subpopulation targeted for premature clearance after transfusion.

Deuterated Linoleic Acid Attenuates the RBC Storage Lesion in a Mouse Model of Poor RBC Storage.

Background: Long-chain polyunsaturated fatty acids (PUFAs) are important modulators of red blood cell (RBC) rheology. Dietary PUFAs are readily incorporated into the RBC membrane, improving RBC deformability, fluidity, and hydration. However, enriching the lipid membrane with PUFAs increases the potential for peroxidation in oxidative environments (e.g., refrigerated storage), resulting in membrane damage. Substitution of bis-allylic hydrogens with deuterium ions in PUFAs decreases hydrogen abstraction, thereby inhibiting peroxidation. If lipid peroxidation is a causal factor in the RBC storage lesion, incorporation of deuterated linoleic acid (DLA) into the RBC membrane should decrease lipid peroxidation, thereby improving RBC lifespan, deformability, filterability, and post-transfusion recovery (PTR) after cold storage. Study Design and Methods: Mice associated with good (C57BL/6J) and poor (FVB) RBC storage quality received diets containing 11,11-D2-LA Ethyl Ester (1.0 g/100 g diet; deuterated linoleic acid) or non-deuterated LA Ethyl Ester (control) for 8 weeks. Deformability, filterability, lipidomics, and lipid peroxidation markers were evaluated in fresh and stored RBCs. Results: DLA was incorporated into RBC membranes in both mouse strains. DLA diet decreased lipid peroxidation (malondialdehyde) by 25.4 and 31% percent in C57 mice and 12.9 and 79.9% in FVB mice before and after cold storage, respectively. In FVB, but not C57 mice, deformability filterability, and post-transfusion recovery were significantly improved. Discussion: In a mouse model of poor RBC storage, with elevated reactive oxygen species production, DLA attenuated lipid peroxidation and significantly improved RBC storage quality.

Failed Disruption of Tick Feeding, Viability, and Molting after Immunization of Mice and Sheep with Recombinant Ixodes ricinus Salivary Proteins IrSPI and IrLip1.

To identify potential vaccine candidates against Ixodes ricinus and tick-borne pathogen transmission, we have previously sequenced the salivary gland transcriptomes of female ticks infected or not with Bartonella henselae. The hypothesized potential of both IrSPI (I. ricinus serine protease inhibitor) and IrLip1 (I. ricinus lipocalin 1) as protective antigens decreasing tick feeding and/or the transmission of tick-borne pathogens was based on their presumed involvement in dampening the host immune response to tick feeding. Vaccine endpoints included tick larval and nymphal mortality, feeding, and molting in mice and sheep. Whether the antigens were administered individually or in combination, the vaccination of mice or sheep elicited a potent antigen-specific antibody response. However, and contrary to our expectations, vaccination failed to afford protection against the infestation of mice and sheep by I. ricinus nymphs and larvae, respectively. Rather, vaccination with IrSPI and IrLip1 appeared to enhance tick engorgement and molting and decrease tick mortality. To the best of our knowledge, these observations represent the first report of induction of vaccine-mediated enhancement in relation to anti-tick vaccination.

Multiple Antigenic Peptide-Based Vaccines Targeting Ixodes ricinus Neuropeptides Induce a Specific Antibody Response but Do Not Impact Tick Infestation.

Synthetic peptide vaccines were designed to target the neuropeptides innervating Ixodes ricinus salivary glands and hindgut and they were tested for their capacity to afford protective immunity against nymphs or larvae and Anaplasma phagocytophilum-infected nymph infestation, in mice and sheep, respectively. In both models, the assembly of SIFamide (SIFa) or myoinhibitory peptide (MIP) neuropeptides into multiple antigenic peptide constructs (MAPs) elicited a robust IgG antibody response following immunization. Nevertheless, no observable detrimental impact on nymphs was evidenced in mice, and, unfortunately, the number of engorged nymphs on sheep was insufficient for firm conclusions to be drawn, including for bacterial transmission. Regarding larvae, while vaccination of the sheep did not globally diminish tick feeding success or development, analyses of animals at the individual level revealed a negative correlation between anti-SIFa and MIP antibody levels and larva-to-nymph molting success for both antigens. Our results provide a proof of principle and precedent for the use of MAPs for the induction of immunity against tick peptide molecules. Although the present study did not provide the expected level of protection, it inaugurates a new strategy for protection against ticks based on the immunological targeting of key components of their nervous system.