IL-12-impaired and IL-12-secreting dendritic cells produce IL-23 upon CD154 restimulation

Experimental studies in monkeys on the basis of ex vivo-generated, reinjected dendritic cells (DCs) allow investigations of primate DC biology in vivo. To study in vitro and in vivo properties of DCs with a reduced capacity to produce IL-12, we adapted findings obtained in vitro with human cells to the rhesus macaque model. Following exposure of immature monocyte-derived monkey DCs to the immunomodulating synthetic polypeptide glatiramer acetate (GA) and to dibutyryl-cAMP (d-cAMP; i.e., a cAMP enhancer that activates DCs but inhibits the induction of Th1 immune responses), the resulting DCs displayed a mature phenotype with enhanced Ag-specific T cell stimulatory function, notably also for memory Th1 cells. Phosphorylation of p38 MAPK was not induced in GA/d-cAMP-activated DCs. Accordingly, these cells secreted significantly less IL-12p40 (p < or = 0.001) than did cytokine-activated cells. However, upon restimulation with rhesus macaque CD154, GA/d-cAMP-activated DCs produced IL-12p40/IL-23. Additionally, DCs activated by proinflammatory cytokines following protocols for the generation of cells used in clinical studies secreted significantly more IL-23 upon CD154 restimulation than following prior activation. Two days after intradermal injection, GA/d-cAMP-activated fluorescence-labeled DCs were detected in the T cell areas of draining lymph nodes. When similarly injected, GA/d-cAMP as well as cytokine-activated protein-loaded DCs induced comparable Th immune responses characterized by secretion of IFN-gamma, TNF, and IL-17, and transiently expanded FOXP3(+) regulatory T cells. Reactivation of primate DCs through CD154 considerably influences their immmunostimulatory properties. This may have a substantial impact on the development of innovative vaccine approaches.     

Primary antiphospholipid antibody syndrome—one further aspect of thrombophilia in overweight and obese patients with venous thromboembolism

Objective: Overweight and obesity are established risk factors for venous thromboembolism (VTE). We examined the difference in the frequency of primary antiphospholipid antibody syndrome (PAPS) in VTE patients according to their BMI. Design and Methods: We included 998 VTE patients treated at our institution between 2009 and 2011 in a retrospective data analysis. Thrombophilia screening including evaluation for APS (lupus anticoagulant, anti‐cardiolipin, and anti‐B2‐glycoprotein‐I IgG and IgM antibodies) was performed in all patients. Results: PAPS was diagnosed in 6.8% (24/355) of normal weight (BMI < 24 kg/m²) VTE patients, in 11.1% (50/452) of overweight (BMI 25–30 kg/m²) VTE patients, and in 15.7% (30/191) of obese (BMI > 31 kg/m²) VTE patients. The difference of PAPS occurrence between these groups was statistically significant (P = 0.001). PAPS patients demonstrated higher fibrinogen levels as compared to non‐PAPS patients (median 416.0 md/dl vs. 352.0 mg/dl, P = 0.001). Furthermore, fibrinogen levels increased significantly according to the body weight of patients (median normal weight patients 330.0 mg/dl vs. overweight patients 359.0 mg/dl vs. obese patients 415.0 mg/dl, P = 0.001). Conclusion: PAPS seems to be more frequent in overweight and obese patients. As PAPS patients showed significantly higher fibrinogen levels and as fibrinogen levels increased significantly according to the body weight of patients, an elevated inflammatory state in overweight and obese patients as a reason for the increased PAPS occurrence can be assumed.     

Antibacterial Immune Competence of Honey Bees (Apis mellifera) Is Adapted to Different Life Stages and Environmental Risks

The development of all honey bee castes proceeds through three different life stages all of which encounter microbial infections to a various extent. We have examined the immune strength of honey bees across all developmental stages with emphasis on the temporal expression of cellular and humoral immune responses upon artificial challenge with viable Escherichia coli bacteria. We employed a broad array of methods to investigate defence strategies of infected individuals: (a) fate of bacteria in the haemocoel; (b) nodule formation and (c) induction of antimicrobial peptides (AMPs). Newly emerged adult worker bees and drones were able to activate efficiently all examined immune reactions. The number of viable bacteria circulating in the haemocoel of infected bees declined rapidly by more than two orders of magnitude within the first 4–6 h post-injection (p.i.), coinciding with the occurrence of melanised nodules. Antimicrobial activity, on the other hand, became detectable only after the initial bacterial clearance. These two temporal patterns of defence reactions very likely represent the constitutive cellular and the induced humoral immune response. A unique feature of honey bees is that a fraction of worker bees survives the winter season in a cluster mostly engaged in thermoregulation. We show here that the overall immune strength of winter bees matches that of young summer bees although nodulation reactions are not initiated at all. As expected, high doses of injected viable E.coli bacteria caused no mortality in larvae or adults of each age. However, drone and worker pupae succumbed to challenge with E.coli even at low doses, accompanied by a premature darkening of the pupal body. In contrast to larvae and adults, we observed no fast clearance of viable bacteria and no induction of AMPs but a rapid proliferation of E.coli bacteria in the haemocoel of bee pupae ultimately leading to their death.     

Platelet-to-Lymphocyte Ratio: A Novel Marker for Critical Limb Ischemia in Peripheral Arterial Occlusive Disease Patients

Background

Platelet-to-Lymphocyte Ratio (PLR) is an easily applicable blood test. An elevated PLR has been associated with poor prognosis in patients with different oncologic disorder. As platelets play a key role in atherosclerosis and atherothrombosis, we investigated PLR and its association with critical limb ischemia (CLI) and other vascular endpoints in peripheral arterial occlusive disease (PAOD) patients.

Methods and Findings

We evaluated 2121 PAOD patients treated at our institution from 2005 to 2010. PLR was calculated and the cohort was categorized into tertiles according to the PLR. An optimal cut-off value for the continuous PLR was calculated by applying a receiver operating curve analysis to discriminate between CLI and non-CLI. In our cohort occurrence of CLI significantly increased with an increase in PLR. As an optimal cut-off value, a PLR of 150 was identified. Two groups were categorized, one containing 1228 patients (PLR≤150) and a second group with 893 patients (PLR>150). CLI was more frequent in PLR>150 patients (410(45.9%)) compared to PLR≤150 patients (270(22.0%)) (p<0.001), as was prior myocardial infarction (51(5.7%) vs. 42(3.5%), p = 0.02). Regarding inflammatory parameters, C-reactive protein (median 7.0 mg/l (3.0–24.25) vs. median 5.0 mg/l (2.0–10.0)) and fibrinogen (median 457 mg/dl (359.0–583.0) vs. 372 mg/dl (317.25–455.75)) also significantly differed in the two patient groups (both p<0.001). Finally, a PLR>150 was associated with an OR of 1.9 (95%CI 1.7–2.1) for CLI even after adjustment for other well-established vascular risk factors.

Conclusions

An increased PLR is significantly associated with patients at high risk for CLI and other cardiovascular endpoints. The PLR is a broadly available and cheap marker, which could be used to highlight patients at high risk for vascular endpoints.     

Physiology and pathophysiology of nitrosative and oxidative stress in osteoarthritic joint destruction

Osteoarthritis (OA) is one of the most common chronic diseases, with increasing importance due to increased life expectancy. On a cellular level, the pathophysiology of joint function impairment and ultimate destruction associated with OA remains poorly understood. Free radicals are highly reactive molecules involved in both normal intracellular signal transduction and degenerative cellular processes. An imbalance between the free radical burden and cellular scavenging mechanisms, defined as oxidative stress, has been identified as a relevant factor in OA pathogenesis. This literature review elucidates the involvement of nitrosative and oxidative stress in cellular ageing in joints, cell senescence, and apoptosis. Free radical exposure is known to promote cellular senescence and apoptosis, and the involvement of radical oxygen species (ROS) in inflammation, fibrosis control, and pain nociception has been proven. A relatively novel approach to OA pathophysiology considers the joint to be a dynamic system consisting of 3, continuously interacting compartments, cartilage, synovial tissue, and subchondral bone. Current knowledge concerning free radical involvement in paracrine signalling in OA is reviewed. The interrelationship between oxidative imbalances and OA pathophysiology may provide a novel approach to the comprehension, and therefore modification, of OA disease progression and symptom control.     

Peptide-based pharmacomodulation of a cancer-targeted optical imaging and photodynamic therapy agent

We designed and synthesized a folate receptor-targeted, water-soluble, and pharmacomodulated photodynamic therapy (PDT) agent that selectively detects and destroys the targeted cancer cells while sparing normal tissue. This was achieved by minimizing the normal organ uptake (e.g., liver and spleen) and by discriminating between tumors with different levels of folate receptor (FR) expression. This construct (Pyro-peptide-Folate, PPF) is composed of three components: (1) pyropheophorbide a (Pyro) as an imaging and therapeutic agent, (2) peptide sequence as a stable linker and modulator improving the delivery efficiency, and (3) Folate as a homing molecule targeting FR-expressing cancer cells. We observed an enhanced accumulation of PPF in KB cancer cells (FR+) compared to HT 1080 cancer cells (FR-), resulting in a more effective post-PDT killing of KB cells over HT 1080 or normal CHO cells. The accumulation of PPF in KB cells can be up to 70% inhibited by an excess of free folic acid. The effect of Folate on preferential accumulation of PPF in KB tumors (KB vs HT 1080 tumors 2.5:1) was also confirmed in vivo. In contrast to that, no significant difference between the KB and HT 1080 tumor was observed in case of the untargeted probe (Pyro-peptide, PP), eliminating the potential influence of Pyro's own nonspecific affinity to cancer cells. More importantly, we found that incorporating a short peptide sequence considerably improved the delivery efficiency of the probe--a process we attributed to a possible peptide-based pharmacomodulation--as was demonstrated by a 50-fold reduction in PPF accumulation in liver and spleen when compared to a peptide-lacking probe (Pyro-K-Folate, PKF). This approach could potentially be generalized to improve the delivery efficiency of other targeted molecular imaging and photodynamic therapy agents.     

Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL

Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the determinants of tumor susceptibility to this ligand are not fully defined. mRNA expression of the peptidyl O-glycosyltransferase GALNT14 correlated with Apo2L/TRAIL sensitivity in pancreatic carcinoma, non-small-cell lung carcinoma and melanoma cell lines, and up to 30% of samples from various human malignancies showed GALNT14 overexpression. RNA interference of GALNT14 reduced cellular Apo2L/TRAIL sensitivity, whereas overexpression increased responsiveness. Biochemical analysis of DR5 identified several ectodomain O-(N-acetyl galactosamine-galactose-sialic acid) structures. Sequence comparison predicted conserved extracellular DR4 and DR5 O-glycosylation sites; progressive mutation of the DR5 sites attenuated apoptotic signaling. O-glycosylation promoted ligand-stimulated clustering of DR4 and DR5, which mediated recruitment and activation of the apoptosis-initiating protease caspase-8. These results uncover a new link between death-receptor O-glycosylation and apoptotic signaling, providing potential predictive biomarkers for Apo2L/TRAIL-based cancer therapy.     

Adjunctive passive immunotherapy in human immunodeficiency virus type 1-infected individuals treated with antiviral therapy during acute and early infection

Three neutralizing monoclonal antibodies (MAbs), 2G12, 2F5, and 4E10, with activity in vitro and in vivo were administered in an open-label, nonrandomized, proof-of-concept study to attempt to prevent viral rebound after interruption of antiretroviral therapy (ART). Ten human immunodeficiency virus type 1-infected individuals identified and treated with ART during acute and early infection were enrolled. The first six patients were administered 1.0 g of each of the three MAbs per infusion. The remaining four patients received 2G12 at 1.0 g/infusion and 2.0 g/infusion of 2F5 and 4E10. The MAbs were well tolerated. Grade I post-partial thromboplastin time prolongations were noted. Viral rebound was observed in 8/10 subjects (28 to 73 days post-ART interruption), and 2/10 subjects remained aviremic over the course of the study. In seven of eight subjects with viral rebound, clear resistance to 2G12 emerged, whereas reductions in the susceptibilities of plasma-derived recombinant viruses to 2F5 and 4E10 were neither sustained nor consistently measured. Viral rebound was associated with a preferential depletion of CD4(+) T cells within the gastrointestinal tract. Though safe, the use of MAbs generally delayed, but did not prevent, virologic rebound. Consideration should be given to further pilot studies with alternative combinations of MAbs and perhaps additional novel treatment modalities.     

Mechanisms of gastrointestinal CD4+ T-cell depletion during acute and early human immunodeficiency virus type 1 infection

During acute and early human immunodeficiency virus type 1 (HIV-1) infection (AEI) more than 50% of CD4+ T cells are preferentially depleted from the gastrointestinal (GI) lamina propria. To better understand the underlying mechanisms, we studied virological and immunological events within the peripheral blood (PB) and GI tract during AEI. A total of 32 AEI subjects and 18 uninfected controls underwent colonic biopsy. HIV-1 viral DNA and RNA levels were quantified in CD4+ T cells derived from the GI tract and PB by using real-time PCR. The phenotype of infected cells was characterized by using combinations of immunohistochemistry and in situ hybridization. Markers of immunological memory, activation, and proliferation were examined by flow cytometry and immunohistochemistry, and the host-derived cytotoxic cellular response was examined by using immunohistochemistry. GI CD4+ T cells harbored, on average, 13-fold higher HIV-1 viral DNA levels and 10-fold higher HIV-1 RNA levels than PB CD4+ T cells during AEI. HIV-1 RNA was detected in both "activated" and "nonactivated" mucosal CD4+ T cells. A significantly higher number of activated and proliferating T cells were detected in the GI tract compared to the PB, and a robust cytotoxic response (HIV-1 specificity not determined) was detected in the GI tract as early as 18 days postinfection. Mucosal CD4+ T-cell depletion is multifactorial. Direct viral infection likely accounts for the earliest loss of CD4+ T cells. Subsequently, ongoing infection of susceptible CD4+ T cells, along with activation-induced cellular death and host cytotoxic cellular response, are responsible for the persistence of the lesion.