Mycobacterium tuberculosis-specific CD8+ T cells preferentially recognize heavily infected cells

Both CD4+ and CD8+ T cells are important for successful immunity to tuberculosis and have redundant effector functions, such as cytolysis and release of potent antimycobacterial cytokines such as interferon-gamma and tumor necrosis factor-alpha. We hypothesized that CD8+ T cells play a unique role in host defense to Mycobacterium tuberculosis infection as well. Possibilities include preferential and/or enhanced release of granular constituents and/or preferential recognition of heavily infected cells. Utilizing human, Mycobacterium tuberculosis-specific, CD4+ and CD8+ T cell clones, we demonstrate that, after recognition of antigen-presenting cells displaying peptide antigen, CD4+ T cells preferentially release interferon-gamma, whereas CD8+ T cells preferentially lyse antigen-presenting cells. Furthermore, utilizing dendritic cells infected with Mycobacterium tuberculosis expressing green fluorescent protein, we show that CD8+ T cells preferentially recognize heavily infected cells that constitute the minority of infected cells. These data support the hypothesis that the central role of CD8+ T cells in the control of infection with Mycobacterium tuberculosis may be that of surveillance; in essence, recognition of cells in which the containment of Mycobacterium tuberculosis is no longer effective.     

Thrombopoietin following transfusion of platelets in preterm neonates

Thrombocytopenia is common in the neonatal intensive care unit. Transfusion of platelets is often required. The purpose of our study was to determine changes in thrombopoietin (Tpo) following transfusion of platelets in preterm neonates. Preterm neonates undergoing platelet transfusion were randomized to receive a transfusion volume of either 10 or 15 ml/kg. Blood was obtained for Tpo measurement pre-transfusion, one and 24 hours post-transfusion. Platelet Factor 4 (PF4) was also measured to quantify platelet activation. Statistical analysis was performed using repeated measures ANOVA, and Mann-Whitney U test as appropriate. Ten infants were enrolled in each group. Gestational age, birth weight, etiology of thrombocytopenia, and timing of transfusion did not differ between the 10 and 15 ml/kg groups. There were no differences between the groups in platelet count prior to and/or following transfusion. Both transfusion volumes were equally well tolerated. Tpo and PF4 did not differ between groups at any of the study time points. When both groups were analysed together, Tpo dropped 43% (95% confidence 37-49%, p = 0.01) 1-hour post compared to pre-transfusion. In conclusion the observed decrease in Tpo following platelet transfusion suggests that Tpo kinetics in neonates is similar to adults following transfusion. PF4 was not affected by transfusion. There was not an increase in platelet count following transfusion volume of 15 ml/kg compared to 10 ml/kg.     

Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity

Despite the beneficial therapeutic effects of intravenous immunoglobulin (IVIg) in inflammatory diseases, consistent therapeutic efficacy and potency remain major limitations for patients and physicians using IVIg. These limitations have stimulated a desire to generate therapeutic alternatives that could leverage the broad mechanisms of action of IVIg while improving therapeutic consistency and potency. The identification of the important anti-inflammatory role of fragment crystallizable domain (Fc) sialylation has presented an opportunity to develop more potent Ig therapies. However, translating this concept to potent anti-inflammatory therapeutics has been hampered by the difficulty of generating suitable sialylated products for clinical use. Therefore, we set out to develop the first, to our knowledge, robust and scalable process for generating a well-qualified sialylated IVIg drug candidate with maximum Fc sialylation devoid of unwanted alterations to the IVIg mixture. Here, we describe a controlled enzymatic, scalable process to produce a tetra-Fc–sialylated (s4-IVIg) IVIg drug candidate and its qualification across a wide panel of analytic assays, including physicochemical, pharmacokinetic, biodistribution, and in vivo animal models of inflammation. Our in vivo characterization of this drug candidate revealed consistent, enhanced anti-inflammatory activity up to 10-fold higher than IVIg across different animal models. To our knowledge, this candidate represents the first s4-IVIg suitable for clinical use; it is also a valuable therapeutic alternative with more consistent and potent anti-inflammatory activity.Intravenous immunoglobulin (IVIg) is a therapeutic blood product prepared from the pooled plasma of 3,000–60,000 healthy donors per batch (1–3). It is a complex heterogeneous mixture of IgG subclasses and low amounts of IgA, IgM, and other plasma proteins (4). IVIg contains a wide array of antibodies expected to be present in human serum, and the large number of donors ensures diversity in the Ig repertoire that far exceeds that of an individual donor (5).IVIg has been used for more than 30 years for the treatment of a variety of acute and chronic autoimmune and systemic inflammatory diseases (2–5). Although efficacious in these diseases, the precise mechanism of action of IVIg is not well-understood. Various studies have documented a series of nonmutually exclusive mechanisms modulating components of the innate and adaptive immune system (4, 6). For example, IVIg has been shown to mediate anti-inflammatory responses through its action on dendritic cells, natural killer cells, regulatory T cells, B cells, and the monocyte/macrophage system, and through its suppression or neutralization of soluble factors, such as inflammatory cytokines, chemokines, and pathogenic autoantibodies (5–7).Although beneficial in numerous indications, IVIg preparations have distinct limitations, such as variable efficacy, clinical risks, high costs, and finite supply (2, 3, 8). Different IVIg preparations are frequently treated as interchangeable products clinically, but it is well-known that significant differences in product preparations exist that may impact tolerability and activity in selected clinical applications (9). At the current maximal dosing regimens, only partial and unsustained responses are obtained in many instances (2, 4). In addition, the long infusion times (4–6 h) associated with the high volume of IVIg treatment consume significant resources at infusion centers (8) and negatively affect patient-reported outcomes, such as convenience and quality of life (10). Developing therapeutic alternatives that could leverage the broad biological activities of IVIg and simultaneously, provide more consistent and potent anti-inflammatory activity with minimal inconvenience would be highly valuable to clinicians and patients.The IgGs in IVIg are composed of a fragment antigen-binding domain (Fab fragment) that facilitates the selective interaction with specific antigens and a fragment crystallizable domain (Fc fragment) that interacts with cellular receptors (Fc receptors) known to play critical functions in modulating the activation state of immune cells (11). The IgG Fc fragment contains a conserved N-linked glycan at position N297. The core of the N297 N-linked glycan is composed of two GlcNAc and three mannose residues. Typically, this core can be further extended with fucose, galactose, sialic acid, and bisecting GlcNAc monosaccharides through selective enzymatic glycosylation reactions (12, 13). Alterations to the N297 glycan composition have been shown to have a significant impact in modulating the interaction between the IgG Fc fragment and the Fc receptors. For example, removal of fucose from the IgG N-glycan core has been shown to increase its affinity for Fc gamma receptor IIIa (FcγRIIIa), leading to enhanced antibody-dependent cellular cytotoxicity (14). Sialylation, the addition of terminal sialic acid to N297 glycan, has also been shown to decrease the affinity for type I Fc receptors and increase the affinity for type II Fc receptors (11).In the past three decades, a wealth of reports has documented alterations in antibody glycosylation associated with different diseases in humans. Among these reports, changes in antibody sialylation have been associated with the evolution of autoimmune and inflammatory diseases. For example, rheumatoid arthritis and juvenile idiopathic arthritis are associated with decreased levels of IgG sialylation (15, 16). Additional studies have shown that this translates particularly to the pathogenic antibodies in inflamed joints of arthritis patients (17). It has also been shown that IgG sialylation increases during pregnancy and that this increase may be associated with the remission of rheumatoid arthritis during pregnancy (18, 19). In addition, pathogenic proteinase 3 autoantibodies are less sialylated in patients with active Wegener’s vasculitis (granulomatosis with polyangiitis) (20).Alterations in endogenous IgG sialylation have been associated with treatment response in inflammatory/autoimmune diseases. For example, in patients with Kawasaki disease treated with IVIg, increased levels of endogenous human IgG sialylation decreased the likelihood of IVIg treatment resistance (defined as persistent or recrudescent fever at least 36 h after the completion of IVIg infusion) (21). Similarly, in patients with Guillain–Barré syndrome, those with more severe forms of the disorder showed a lower level of IgG sialylation, despite IVIg treatment (22).Translation of these natural observations in humans to therapeutic options was first realized in 2006, when it was proposed that high doses (>1 g/kg) of unfractionated IVIg were required to elicit sufficient anti-inflammatory activity because of a limited concentration of sialylated IgG in the total IVIg preparation. This theory was substantiated in a mouse model of arthritis that showed similar levels of anti-inflammatory activity when using 1 g/kg IVIg and 0.1 g/kg sialic acid-enriched IVIg, therefore indicating a 10-fold enhancement with the addition of terminal sialic acids (23, 24). Additional results supporting this theory were subsequently reported in other independent studies and other animal models (25, 26). It was further shown that the anti-inflammatory activity of sialylation can be recapitulated using a sialylated Fc fragment derived from IVIg or an IgG1 recombinant antibody (after in vitro sialylation of the Fc) at a 30-fold lower dose than IVIg (24, 27). These results opened the possibility of developing sialylated IVIg and other sialylated antibodies with enhanced anti-inflammatory properties.In recent years, different reports have debated the anti-inflammatory benefits of sialylation. For example, studies using desialylated IVIg in immune thrombocytopenic purpura (ITP) models (28) and Sambucus nigra agglutinin (SNA) -enriched IVIg in arthritis models (29) have shown that sialylation does not enhance IVIg activity or that it may even be dispensable for its therapeutic effects. On the contrary, more comprehensive studies performed by several independent laboratories testing desialylated or hypersialylated IVIg across different animal models under preventive and therapeutic treatment modalities have shown that sialylation is critical for the anti-inflammatory activity of IVIg (24–27, 30). Furthermore, T cell-independent vaccinations resulted in the generation of hypersialylated immunomodulatory antibodies that were able to modulate other immune responses, establishing a broad relevance of these sialylated IgG glycoforms as modulators of immune responses (31). Notably, none of the previous studies used precisely the same protocols for enriching or depleting sialic acid-containing IgG glycoforms, which may partially explain the discrepancies of these studies. Thus, the major aim of this study was to use rigorous, controlled processes and quality controls to investigate the potential of hypersialylated IVIg as a drug candidate with enhanced therapeutic activity.We describe a robust, controlled sialylation process to generate tetra-Fc–sialylated IVIg and show that this process yields a product with consistent enhanced anti-inflammatory activity. Specifically, we first observed that the sialylated IVIg was at least 10 times more potent than the parent IVIg product in a model of collagen antibody-induced arthritis (CAIA) using a prophylactic dose. We further confirmed this enhanced anti-inflammatory activity with therapeutic dosing in models of K/BxN serum-induced arthritis and ITP, and a prophylactic model of skin-blistering disease. Importantly, we noted that, without a tightly controlled process of sialylation, unwanted side products can accumulate that may be responsible for the inconsistent activity observed in previous studies. Therefore, the process that we describe combined with sensitive controls to generate a tetra-Fc–sialylated IVIg (s4-IVIg) devoid of undesired modifications has been critical to obtain the consistent enhanced anti-inflammatory activity and serves as the first example, to our knowledge, of a therapeutic candidate for product development.     

Eplerenone, a selective aldosterone blocker, improves diastolic function in aged rats with small-to-moderate myocardial infarction

BACKGROUND: The incidence of cardiovascular diseases increases rapidly with age, and the elderly suffer higher morbidity and mortality. Aldosterone blockers have shown benefits in patients with left ventricular (LV) dysfunction and heart failure after myocardial infarction (MI). However, aldosterone blockade efficacy has not been explored in aged animals with MI.Methods and results Small-to-moderate MI was induced by coronary artery ligation in 16-month old rats, divided into 3 groups: sham-operated (control, n = 9), MI (n = 9), and MI fed a diet containing eplerenone (120 mg/kg/day, MI+Eplerenone, n = 9) given 18 days postsurgery and up to sacrifice 3 months later. At sacrifice, untreated MI rats did not show overt systolic dysfunction but they had (1) echocardiographic evidences of impaired relaxation (increase of E wave deceleration time and of isovolumic relaxation time, decrease of peak E wave velocity), (2) hemodynamically impaired LV relaxation (LV -dP/dt from 7413 +/- 720 to 4956 +/- 475 mm Hg/s, P < .05), and (3) significant increase of collagen content in LV interstitium (from 4.27 +/- 0.23 to 5.34 +/- 0.24%, P < .01) and in aorta (from 19 +/- 1 to 24 +/- 2%, P < .05). Eplerenone normalized echocardiographic and hemodynamic evidences of diastolic dysfunction, as well as myocardial interstitial collagen and aortic fibrosis (all parameters statistically different from untreated MI). CONCLUSION: In aged rats with small to moderate MI, eplerenone normalized diastolic relaxation, possibly through a reduction of interstitial fibrosis.     

Effects of bilateral and unilateral ophthalmectomy on plasma melatonin in Rana tadpoles and froglets under various experimental conditions

The effect of ophthalmectomy (enucleation) on plasma melatonin in Rana tadpoles and froglets was studied under various experimental conditions to determine if ocular melatonin is released into the circulation from the eyes and to study the factors which might affect this process. Where operations occurred in early or mid-photophase on a 12 light:12 dark (12L:12D) cycle (light onset at 08:00 h), sampling in mid-light and mid-dark revealed that scotophase plasma melatonin was reduced at all developmental stages, with the more significant effects occurring before metamorphic climax. Experiments sampling prometamorphic tadpoles six times in a 24h period on 18L:6D, 12L:12D, or 6L:18D five days after enucleation also showed a significant lowering of plasma melatonin in the dark, so that the scotophase peak was virtually eliminated on all the LD cycles. These findings indicated that the reduction in plasma melatonin after bilateral eye removal was independent of the LD cycle and the metamorphic stage, and that it abolished the diel melatonin rhythm at the expense of the scotophase peak. Experiments carried out for 5 weeks suggested that compensatory secretion of melatonin by other organs after eye removal might partially restore the plasma melatonin level over time. Unilateral ophthalmectomy tended to reduce, but not eliminate, the night peak of plasma melatonin, and did not result in a compensatory increase in ocular melatonin in the remaining eye. Ophthalmectomized tadpoles exhibited darkening of the skin after the operation, which was not associated with a significant change in pituitary alpha-melanotropin. The findings overall indicate that the eyes in Rana tadpoles and froglets contribute up to somewhat over one-half of the circulating melatonin, particularly during the scotophase, and provide experimental evidence for ocular secretion into the blood for the first time in the Amphibia.