Inactivation of protozoan parasites in red blood cells using INACTINE PEN110 chemistry

BACKGROUND: The transmission of parasites, including Babesia, plasmodia, and Trypanosoma cruzi, via transfusions is an important public health concern. INACTINE technology is a pathogen‐reduction process that utilizes PEN110, an electrophilic agent that inac‐tivates a wide range of pathogens by disrupting nucleic acid replication. The present study investigated the effect of PEN110 treatment on the viability of protozoa in RBCs. STUDY DESIGN AND METHODS: B. microti‐parasitized RBCs from infected hamsters were treated with PEN110 and inoculated to naïve animals. Parasitemia was detected by blood smears and PCR. Human RBCs infected with P. falciparum were treated with PEN110 and incubated with fresh RBCs. P. falciparum multiplication was detected by blood smears. Human RBCs spiked with T. cruzi and treated with PEN110 were analyzed for the presence of live parasites using in‐vitro infectivity assay or by inoculating susceptible mice. RESULTS: Treatment of RBCs infected with B. microti or P. falciparum with 0.01 to 0.1 percent (vol/vol) PEN110 resulted in parasite inactivation to below the limit of detection during 24 hours. T. cruzi inoculated into human RBCs was inactivated below the limit of detection by 0.1 percent PEN110 after 3 hours. CONCLUSION: The study demonstrates that treatment of blood with PEN110 is highly effective in eradicating transfusion‐transmitted protozoan parasites.     

Reduction of malaria transmission by transgenic mosquitoes expressing an antisporozoite antibody in their salivary glands

We have previously developed a robust salivary gland‐specific expression system in transgenic Anopheles stephensi mosquitoes. To establish transgenic mosquito lines refractory to Plasmodium falciparum using this system, we generated a transgenic mosquito harbouring the gene encoding an anti‐P. falciparum circumsporozoite protein (PfCSP) single‐chain antibody (scFv) fused to DsRed in a secretory form (mDsRed‐2A10 scFv). Fluorescence microscopy showed that the mDsRed‐2A10 scFv was localized in the secretory cavities and ducts of the salivary glands in a secreted form. To evaluate P. falciparum transmission‐blocking in a rodent malaria model, a transgenic Plasmodium berghei line expressing PfCSP in place of PbCSP (PfCSP/Pb) was constructed. The PfCSP/Pb parasites were able to bind to the mDsRed‐2A10 scFv in the salivary glands of the transgenic mosquitoes. Importantly, the infectivity of the transgenic mosquitoes to mice was strongly impaired, indicating that the parasites had been inactivated. These results suggest that salivary gland‐specific expression of antisporozoite molecules could be a promising strategy for blocking malaria transmission to humans.     

Atorvastatin or transgenic expression of TFPI inhibits coagulation initiated by anti‐nonGal IgG binding to porcine aortic endothelial cells

Summary. Background: Intravascular thrombosis remains a barrier to successful xenotransplantation. Tissue factor (TF) expression on porcine aortic endothelial cells (PAECs), which results from their activation by xenoreactive antibodies (Abs) to Galα1,3Gal (Gal) and subsequent complement activation, plays an important role. Objectives: The present study aimed to clarify the role of Abs directed against nonGal antigens in the activation of PAECs to express functional TF and to investigate selected methods of inhibiting TF activity. Methods: PAECs from wild‐type (WT), α1,3‐galactosyltransferase gene‐knockout (GT‐KO) pigs, or pigs transgenic for CD46 or tissue factor pathway inhibitor (TFPI), were incubated with naïve baboon serum (BS) or sensitized BS (with high anti‐nonGal Ab levels). TF activity of PAECs was assessed. Results: Only fresh, but not heat‐inactivated (HI), naïve BS activated WT PAECs to express functional TF. Similarly, PAECs from CD46 pigs were resistant to activation by naïve BS, but not to activation by fresh or HI sensitized BS. HI sensitized BS also activated GT‐KO PAECs to induce TF activity. TF expression on PAECs induced by anti‐nonGal Abs was inhibited if serum was pretreated with (i) an anti‐IgG Fab Ab or (ii) atorvastatin, or (iii) when PAECs were transgenic for TFPI. Conclusions: Anti‐nonGal IgG Abs activated PAECs to induce TF activity through a complement‐independent pathway. This implies that GT‐KO pigs expressing a complement‐regulatory protein may be insufficient to prevent the activation of PAECs. Genetic modification with an ‘anticoagulant’ gene (e.g. TFPI) or a therapeutic approach (e.g. atorvastatin) will be required to prevent coagulation dysregulation after pig‐to‐primate organ transplantation.     

Pharmacokinetics of cysteamine bitartrate following intraduodenal delivery

Cysteamine is approved for the treatment of cystinosis and is being evaluated for Huntington's disease and non‐alcoholic fatty liver disease. Little is known about the bioavailability and biodistribution of the drug. The aim was to determine plasma, cerebrospinal fluid (CSF), and tissue (liver, kidney, muscle) cysteamine levels following intraduodenal delivery of the drug in rats pretreated and naïve to cysteamine and to estimate the hepatic first‐pass effect on cysteamine. Healthy male rats (n = 66) underwent intraduodenal and portal (PV) or jugular (JVC) venous catheterization. Half were pretreated with cysteamine, and half were naïve. Following intraduodenal cysteamine (20 mg/kg), serial blood samples were collected from the PV or the JVC. Animals were sacrificed at specific time points, and CSF and tissue were collected. Cysteamine levels were determined in plasma, CSF, and tissue. The C_max was achieved in 5–10 min from PV and 5–22.5 min from JVC. The PV‐C_max (P = 0.08), PV‐AUC_0–t (P = 0.16), JVC‐C_max (P = 0.02) and JVC‐AUC_0–t (P = 0.03) were higher in naive than in pretreated animals. Plasma cysteamine levels returned to baseline in ≤120 min. The hepatic first‐pass effect was estimated at 40%. Peak tissue and CSF cysteamine levels occurred ≤22.5 min, but returned to baseline levels ≤180 min. There was no difference in CSF and tissue cysteamine levels between naïve and pretreated groups, although cysteamine was more rapidly cleared in the pretreated group. Cysteamine is rapidly absorbed from the small intestine, undergoes significant hepatic first‐pass metabolism, crosses the blood brain barrier, and is almost undetectable in plasma, CSF, and body tissues 2 h after ingestion. Sustained‐release cysteamine may provide prolonged tissue exposure.     

Activation of Aedes aegypti prophenoloxidase‐3 and its role in the immune response against entomopathogenic fungi

Serine protease cascade‐mediated melanization is an important innate immune response in insects and crustaceans, which involves the proteolytic activation of prophenoloxidase (PPO). In this study, we investigated the role of Aedes aegypti PPO3 in antifungal immune defence. We expressed and purified recombinant PPO3 (rPPO3) in Escherichia coli and demonstrated that rPPO3 was activated by ethanol and, to a lesser extent, by cetylpyridinium chloride. In the presence of Cu²⁺, rPPO3 exhibited enzyme activity. Immunoblot results revealed that the rPPO3 was cleaved by the haemolymph from immune‐challenged mosquitoes or purified Ostrinia furnacalis serine protease 105 in vitro. The cleaved rPPO3 converted dopamine to toxic intermediates that killed fungal conidia of Beauveria bassiana in vitro. In mosquitoes challenged with Be. bassiana, cleavage of rPPO3 produced a 50 kDa phenoloxidase (PO) fragment. Further analysis revealed that the survival rate of mosquitoes with fungal infection increased significantly following injection of rPPO3 into the haemocoel. Taken together, our results suggest that proteolytic cleavage of the mosquito PPO3 plays an important role in the antifungal immune response. This has led to a better understanding of the mechanism of PPO activation in the mosquito and the role of melanization in the antifungal immune response.     

Induction of factor VIII‐specific unresponsiveness by intrathymic factor VIII injection in murine hemophilia A

Summary. Background: Hemophilia A is a congenital bleeding disorder caused by a deficiency of coagulation factor VIII. Approximately 30% of hemophilia A patients develop inhibitors against FVIII following replacement therapy. We have reported that neonatal exposure of FVIII antigen can induce antigen‐specific immune tolerance by interferon‐γ (IFN‐γ)‐dependent T‐cell anergy in hemophilia A mice. Objective: The thymus plays crucial roles in self‐tolerance, with negative selection of self‐reactive effector T cells and positive selection of self‐reactive regulatory T cells. We investigated the possibility of the induction of antigen‐specific immune tolerance by intrathymic injection of FVIII in hemophilia A mice. Methods: Hemophilia A mice were injected with recombinant FVIII into the thymus under real‐time high‐resolution image guidance. Results: Anti‐FVIII inhibitory antibody titers in mice challenged with intravenous administration of FVIII were significantly lower in mice (n = 22) that had received thymic FVIII injection than in mice (n = 18) without thymic injection (9.4 ± 2.3 vs. 122.5 ± 27.6 BU mL^?1, respectively, P = 0.00078). The CD4⁺ T cells from thymic‐injected mice could not proliferate or produce interleukin (IL)‐2, IL‐12 and IFN‐γ in response to FVIII. The CD4⁺CD25⁺ T cells generated from thymic‐treated mice but not from naïve mice efficiently suppressed the in vitro proliferative response of CD4⁺ T cells and blocked the in vivo development of anti‐FVIII antibodies in the adoptive transfer. Conclusion: These data suggest that intrathymic administration of FVIII could result in immune tolerance by induction of FVIII‐specific regulatory T cells.     

Insulin detemir improves glycaemic control without weight gain in insulin-naïve patients with type 2 diabetes: subgroup analysis from the PREDICTIVE study

Objective:  Predictable Results and Experience in Diabetes through Intensification and Control to Target: an International Variability Evaluation (PREDICTIVE^TM) is a multi‐national, open‐label, prospective, observational study assessing the safety and efficacy of insulin detemir in clinical practice. This post hoc subanalysis evaluates insulin‐naïve patients on oral antidiabetic drugs (OADs) who were initiated on insulin detemir as basal therapy (± OADs). Methods:  The European cohort of the PREDICTIVE study currently includes 20,531 patients (12,981 with type 2 diabetes) who were prescribed insulin detemir and followed up for 12, 26 or 52 weeks. Here, we report data from a subgroup of 2377 OAD‐treated, insulin‐naïve type 2 diabetes patients for a mean follow‐up of 14.4 weeks. Patients were prescribed insulin detemir as basal therapy (± OADs) by their physician, as part of routine clinical care. Results were reported in comparison with baseline observations. Results:  One serious adverse drug reaction was reported, which was a major hypoglycaemic episode. Treatment with insulin detemir (± OADs) significantly reduced mean haemoglobin A_1c (HbA_1c) (?1.3%; p < 0.0001), fasting glucose (?3.7 mmol/l; p < 0.0001), and within‐patient fasting glucose variability (?0.5 mmol/l; p < 0.0001). In the majority of patients (82%), these improvements in glycaemic control were achieved with once daily administration of insulin detemir. There was a small reduction in mean body weight (?0.7 kg; p < 0.0001), which was most apparent in patients with a higher body mass index (BMI) at baseline. A significant negative relationship between weight change and baseline BMI was observed (greater the BMI, greater the weight reduction). Multiple regression analysis showed that BMI and HbA_1c at baseline, and change in HbA_1c, were all predictors for weight change (p < 0.0001 for all), with BMI being the strongest predictor. Conclusions:  Patients with type 2 diabetes naïve to insulin can be effectively treated with once‐daily insulin detemir (± OADs) to achieve improved glycaemic control with no adverse effect on weight and a low risk of hypoglycaemia. These short‐term results are consistent with the findings of clinical trials.     

Investigation of red blood cells from alpha1,3-galactosyltransferase-knockout pigs for human blood transfusion

BACKGROUND: Pigs are a potential source of red blood cells (RBCs) for transfusion into humans, but the pre‐sence of galactose‐α1,3‐galactose (Gal) epitopes on their surface, against which humans have anti‐Gal, has been perceived as a major barrier. α1,3‐Galactosyltransferase gene‐knockout pigs, which do not express Gal epitopes on RBCs (Gal–/–), have recently become available. STUDY DESIGN AND METHODS: In vitro, RBCs from Gal–/– pigs were exposed to sera from naïve humans or baboons or from baboons previously sensitized to pig antigens; immunoglobulin binding was measured by flow cytometry, and cytotoxicity, by a hemolytic assay. In vivo, relatively small numbers of Gal–/– RBCs were transfused into two nonsensitized untreated baboons. The survival of pig RBCs was detected by flow cytometry. RESULTS: In vitro, binding of immunoglobulin (Ig) M from naïve human or baboon sera was detected to Gal–/– RBCs but was significantly less than to Gal+/+ RBCs; IgG binding to Gal–/– RBCs was absent or minimal. Sera had minimal cytotoxicity to Gal–/– RBCs compared to Gal+/+ RBCs. Sensitized baboon sera demonstrated much higher IgG binding to Gal–/– RBCs and increased cytotoxicity, but again these were less than to Gal+/+ RBCs. In vivo, the transfusion of relatively small volumes of Gal–/– RBCs was followed by detection of the cells in the baboon's blood for only 5 minutes. CONCLUSION: Pig RBCs are rapidly phagocytosed from the primate circulation by a mechanism not involving anti‐Gal.     

Characterization of circulating and cultured Tfh-like cells in sickle cell disease in relation to red blood cell alloimmunization status

BackgroundPeople living with sickle cell disease (SCD) are prone to red blood cell (RBC) alloimmunization. We hypothesized that subjects with alloantibodies (responders) would have differences in circulating T-follicular helper (Tfh)-like cells compared to subjects without alloantibodies (non-responders).Materials and methodsPeripheral blood mononuclear cells were collected from 28 subjects, including those with SCD and controls. Circulating CD4 T-cell subsets were first evaluated at baseline. CD4 T-cell subsets were also evaluated after naïve CD4 T-cells were differentiated into Tfh-like cells following in vitro culture with CD3/CD28 beads, IL-7, IL-12, and Activin A. Transfusion and alloantibody histories were extracted from the electronic medical record.ResultsNon-responders had a lower percentage of CD45RA negative Tmemory cells than responders or controls (p<0.05). Notably, there were no differences in circulating Tfh-like cells between any group. However, naïve CD4 T-cells from subjects with SCD were more likely to express CXCR5 after in vitro culture than cells from controls. After culture, CXCR5 expressing cells from responders were more likely to express PD1 and ICOS (16.43 %, sd. 20.23) compared to non-responders (3.69 %, s.d. 3.09) or controls (2.78 %, s.d. 2.04).DiscussionThe tendency for naïve CD4 T-cells from responders to differentiate into Tfh-like cells after in vitro culture may suggest these cells are prepared to assist B-cells with antibody production regardless of antigen specificity. Further studies are needed, but it is possible that these results may explain why some responders form RBC alloantibodies with multiple specificities, in addition to RBC autoantibodies and HLA alloantibodies.     

Alterations in serum levels of IL-17 in contrast to TNF-alpha correspond to disease-modifying treatment in relapsing-remitting multiple sclerosis

Cytokines of different types play an important role in multiple sclerosis (MS) pathogenesis as mediators and regulators of the immune processes in the central nervous system. The aim of the study was to determine the effect of interferon-beta and glatiramer acetate on serum concentrations of TNF-alpha and IL-17?A and their correlation with the degree of disability in clinically stable patients with relapsing-remitting MS. A cross-sectional, case-control study of 220 patients (68 treatment naïve; 152 treated with interferon-beta or glatiramer acetate) and 99 clinically healthy age-gender-body mass index-matched subjects were performed. Serum cytokine concentrations were measured during remission of the disease by means of ELISA. Treatment naïve patients showed significantly higher levels of IL-17?A than the treated individuals (p?=?.000109) and controls (p?=?.000044). Within the treated group, only patients with interferon-beta had significantly higher serum IL-17 than the controls (p?=?.023). TNF-alpha concentrations were significantly higher in the treated patients compared to the healthy controls (p?=?.000013), regardless of the type of the therapy. Treatment naïve individuals did not differ from the controls according to their serum TNF-alpha (p?=?.922). No correlation was found between the serum cytokine concentrations and Expanded Disability Status Scale (EDSS) score (p?>?.05). Serum concentrations of these cytokines could not be regarded as reliable predictors for the severity of the residual neurological deficit during disease-modifying treatment. Our data suggest that suppression of IL-17?A production as one of the mechanisms underlying the beneficial effect of first-line disease-modifying treatments is stronger in glatiramer acetate than in interferon-beta.     

Extracorporeal photopheresis in refractory chronic graft-versus-host disease: the influence on peripheral blood T cell subpopulations. A study by the Hellenic Association of Hematology

Extracorporeal photopheresis (ECP) has been established as an effective treatment modality for patients with chronic extensive graft-versus host disease (GVHD). In the present study, we evaluated the influence of ECP on the numbers of CD4+, CD8+, CD20+, CD56+ cells, and on T-regulatory (Tregs), as well as on the numbers of naïve, central memory (CM), and effector memory (EM) T-cells in patients treated for refractory chronic GVHD. Flow cytometric analysis of peripheral blood lymphocytes was performed for the calculation of the different T-cell subsets. Patients with GVHD had a higher percentage of EM-CD4+ cells in comparison with healthy donors (p = 0.046). The percentages of naïve-CD8+, naïve-CD4+, CM-CD8+, CM-CD4+, EM-CD8+, and Tregs were not different between patients with GVHD and healthy donors. Similarly there was no statistical difference in the percentages of naïve, CM, and EM CD4+ and CD8+ cells before and after 3 months of treatment with ECP. However, in the subset of Tregs a statistically significant increase was observed after 3 months of treatment with ECP (p = 0.015). Responders to ECP had statistically significantly higher absolute numbers of CD4+, and CD8+ cells, in comparison with non-responders. These data further support the concept that ECP does not cause immune-suppression, but should be better considered as an immune-modulating treatment.     

Temporomandibular joint inflammation decreases the voltage‐gated K+ channel subtype 1.4‐immunoreactivity of trigeminal ganglion neurons in rats

Voltage‐gated K⁺ (Kv) channels are one of the important physiological regulators of the membrane potentials in excitable cells, including sensory ganglion neurons. The aim of the present study was to investigate whether temporomandibular joint (TMJ) inflammation alters expression of Kv channel subtype 1.4 (Kv1.4) of trigeminal ganglion (TRG) neurons innervating TMJ relating allodynia (pain caused by normally innoxious stimulation), by using both behavioral and immunohistochemical techniques. TMJ inflammation was induced by injection of Complete Freund's Adjuvant (CFA) into the rat TMJ. The threshold for escape from mechanical stimulation applied to the orofacial area in TMJ inflamed rats was significantly lower than that in naïve rats. TMJ afferents were identified by fluorogold (FG) labeling. The mean numbers of Kv1.4‐/neurofilament (NF) 200(myelinated fiber marker) positive‐ and negative‐immunoreactivities FG‐labeled small‐/medium‐diameter TRG neurons in inflamed rats were significantly decreased when compared with those in the naïve rats. These findings suggest that TMJ inflammation reduces the expression of Kv1.4 subunits in the small‐/medium sized (Aδ‐/C‐) TRG neurons and this may contribute to trigeminal inflammatory allodynia in TMJ disorder. These results lead us to suggest that Kv channel openers may be a potential therapeutic agents for prevention of mechanical allodynia.     

Bortezomib delays the onset of factor VIII inhibitors in experimental hemophilia A,but fails to eliminate established anti‐factor VIII IgG‐producing cells

Summary. Background: Replacement therapy with exogenous factor VIII to treat hemorrhages induces inhibitory anti‐FVIII antibodies in up to 30% of patients with hemophilia A. Current approaches to eradicate FVIII inhibitors using high‐dose FVIII injection protocols (immune tolerance induction) or anti‐CD20 depleting antibodies (Rituximab) demonstrate limited efficacy; they are extremely expensive and/or require stringent compliance from the patients. Objectives: To investigate whether the proteasome inhibitor bortezomib, which depletes plasmocytes, modulates the anti‐FVIII immune response in FVIII‐deficient mice. Methods and results: Preventive 4‐week treatment of naïve mice with bortezomib at the time of FVIII administration delayed the development of inhibitory anti‐FVIII IgG, and depleted plasma cells as well as different lymphoid cell subsets. Conversely, curative treatment of inhibitor‐positive mice for 10 weeks, along with FVIII administration, failed to eradicate FVIII inhibitors to extents that would be clinically relevant if achieved in patients. Accordingly, bortezomib did not eradicate anti‐FVIII IgG‐secreting plasmocytes that had homed to survival niches in the bone marrow, despite significant elimination of total plasma cells. Conclusions: The data suggest that strategies for the efficient reduction of anti‐FVIII IgG titers in patients with hemophilia A should rely on competition for survival niches for plasmocytes in the bone marrow rather than the mere use of proteasome inhibitors.     

Costs and health resources utilization following switching to pregabalin in individuals with gabapentin-refractory neuropathic pain: a post hoc analysis

Purpose: To analyze the changes in pain severity and associated costs resulting from resource utilization and reduced productivity in patients with gabapentin‐refractory peripheral neuropathic pain who switched to pregabalin therapy in primary care settings in Spain. Patients and Methods: This is a post hoc analysis of a 12‐week, multicentre, noninterventional cost‐of‐illness study. Patients were included in the study if they were over 18 years of age and had a diagnosis of chronic, treatment‐refractory peripheral neuropathic pain. The analysis included all pregabalin‐naïve patients who had previously shown an inadequate response to gabapentin and switched to pregabalin. Severity of pain before and after treatment with pregabalin, alone or as an add‐on therapy, was assessed using the Short‐Form McGill Pain Questionnaire (SF‐MPQ) and its related visual analogue scale (VA). Healthcare resource utilization, productivity (including lost‐workday equivalents [LWDE]), and related costs were assessed at baseline and after pregabalin treatment. Results: A total of 174 patients switched to pregabalin had significant and clinically relevant reductions in pain severity (mean [SD] change on SF‐MPQ VA scale, ?31.9 [22.1]; P < 0.05 vs. baseline; effect size, 1.87). Reduction in pain was similar with both pregabalin monotherapy and add‐on therapy. Significant reductions in healthcare resource utilization (concomitant drug use [in pregabalin add‐on group], ancillary tests, and unscheduled medical visits) were observed at the end of trial. Additionally, there were substantial improvements in productivity, including a reduction in the number of LWDE following pregabalin treatment (?18.9 [26.0]; P < 0.0001). These changes correlated with substantial reductions in both direct (?652.9 ± 1622.4 €; P < 0.0001) and indirect healthcare costs (?851.6 [1259.6] €; P < 0.0001). Conclusions: The cost of care in patients with gabapentin‐refractory peripheral neuropathic pain appeared to be significantly reduced after switching to pregabalin treatment, alone or in combination with other analgesic drugs, in a real‐life setting.     

Induction of partial immune tolerance to factor VIII through prior mucosal exposure to the factor VIII C2 domain

Summary. Background: The development of anti‐factor VIII (FVIII) neutralizing antibodies (inhibitors) is a significant obstacle to FVIII replacement therapy. Objective: As mucosal administration of an antigen may induce immune tolerance we have evaluated the efficacy of mucosal antigen exposure to achieve tolerance to FVIII. Methods: We investigated the effects of oral and nasal administration of the purified FVIII C2 domain (FVIII‐C2) to FVIII‐deficient BALB/c mice prior to FVIII protein challenge. Mice received oral or nasal doses of FVIII‐C2, followed by a subcutaneous challenge of either FVIII‐C2 or FVIII. The development of anti‐FVIII inhibitors, cytokine production by splenocytes in vitro, and adoptive transfer assays were analyzed. Results and Conclusions: Mucosal administration of FVIII‐C2 decreases the titer of anti‐FVIII‐C2 inhibitors after FVIII‐C2 challenge, and decreases the percentage of FVIII‐C2 specific antibodies after challenge with full‐length FVIII. Tolerance induction to FVIII‐C2 is associated with increased IL‐10 production by splenocytes in vitro, and can be adoptively transferred to naïve mice. This study is the first to demonstrate that tolerance to the FVIII‐C2 domain can be induced via the mucosal route. Based on these results, the potential use of FVIII‐specific mucosal tolerance induction as an immunotherapy treatment for anti‐FVIII inhibitor development warrants further investigation.