Vaccination survey in patients with rheumatoid arthritis: a cross-sectional study

The objective of this study is to evaluate the vaccination status in rheumatoid arthritis (RA) patients during routine clinical practice, data from a German non-interventional cross-sectional study. In this prospective study, patients with rheumatoid arthritis were interviewed using a standardized questionnaire focusing on vaccination. Available vaccination documents were evaluated, and titers for common vaccination antigens (hepatitis B, rubella, mumps, measles, diphtheria, tetanus) were analyzed with special regard to the underlying treatment and age of patients. A total of 301 RA patients treated with conventional DMARDs alone (cohort I, n?=?125), TNF-blocking agents (cohort II, n?=?117), or B-cell depletion with rituximab (cohort III, n?=?59) have been studied. Significantly more patients in the biologic cohorts II and III were aware of an increased risk of infections (I: 67.7%, II: 83.8%*, III: 89.9%*, P?<?0.05). Pneumococcal vaccination rate was significantly higher (I: 20.2%, II 36.8%* and III: 39.0%*, P?<?0.05) compared with cohort I. Differences were less evident for influenza. Significantly more patients ≥60?years of age have been vaccinated against Streptococcus pneumoniae and influenza. An obvious discrepancy existed between vaccination awareness and actual vaccination rates for all cohorts. No significant differences in vaccination titers could be seen between the three cohorts. Awareness of infectious complications was more present in patients treated with biologicals, and also, the rate of patients vaccinated against Streptococcus pneumoniae increased significantly depending on the underlying treatment. Nevertheless, there was a discrepancy between vaccination awareness and actual vaccination rates for all cohorts.     

Clinical correlations of miR-21 expression in colorectal cancer patients and effects of its inhibition on DLD1 colon cancer cells

Purpose

MicroRNA-21 (miR-21) is one of the miRNAs that are frequently and highly overexpressed in tumor tissue of colorectal cancer (CRC) patients; however, only a little is known about its functional role in CRC.

Methods

We examined the expression level of miR-21 in 44 paired samples of tumoral and non-tumoral colon tissues diagnosed for CRC using TaqMan real-time PCR method. Furthermore, we used miR-21 inhibitor (anti-miR-21) to transient knockdown of miR-21 in DLD-1 colon cancer cells and examined the effects of miR-21 silencing on viability, apoptosis, chemosensitivity, cell cycle, and migration of DLD1 cells.

Results

The expression levels of miR-21 were significantly increased in CRC tumor tissue (P?<?0.0001). Significant differences in miR-21 levels were observed also between CRC tissues of patients with CRC in different clinical stages: I vs. II (P?=?0.033) and I vs. IV (P?=?0.021). Kaplan–Meier analysis proved that the miR-21 expression levels are correlated to shorter overall survival of CRC patients (P?=?0.0341). MiR-21 silencing in DLD1 cell line had no effect on the cell viability; however, when combined with chemotherapeutics (5-FU, L-OHP, and SN38), it contributed to the decrease of cell viability. Suppression of miR-21 decreased cell migration ability of DLD-1 cells by nearly 30?% (P?=?0.016).

Conclusion

We have confirmed the overexpression of miR-21 in CRC samples and its correlation with advanced disease and shorter overall survival. These findings could be described in part by the fact that CRC cells with increased expression of miR-21 have higher migration ability.     

Multiple co-infections of rodents with hantaviruses, Leptospira, and Babesia in Croatia

Hantaviruses, Leptospira spp., and Babesia spp. are rodent-borne pathogens present worldwide. We studied multiple co-infections of small rodents in Croatia with all three pathogens. Twenty-eight Apodemus flavicollis and 16 Myodes glareolus were tested for the presence of hantavirus RNA by real-time RT-PCR, Leptospira strains by renoculture method and Babesia DNA by PCR. Anti-hantavirus antibodies and anti-Leptospira antibodies were detected by serological methods. Very high infection rates with each pathogen were found in A. flavicollis: 20 of 28 rodents (71%) were infected with Dobrava virus, 13 rodents (46%) were infected with Leptospira, and 5 rodents (18%) were infected with Babesia. Multiple co-infections with all three pathogens were found in 3 of 28 (11%) A. flavicollis animals, suggesting that the same rodent host can be infected with several pathogens at the same time. Dual infections with both hantaviruses and Leptospira were found in 7 of 44 rodents (16%), with hantaviruses and Babesia in 2 rodents (5%), and double infection with both Leptospira and Babesia were found in 1 rodent (2%). Since hantaviruses, Leptospira, and Babesia have similar geographical distributions, it is to be expected that in other parts of the world multiple co-infections, representing a serious threat to public health, can be found.     

Renal function assessed using cystatin C and antiplatelet efficacy of clopidogrel assessed using the vasodilator-stimulated phosphoprotein index in patients having percutaneous coronary intervention

Renal dysfunction is a strong independent predictor of stent thrombosis. The aim of the present study was to evaluate the strength and direction of the association between kidney function and clopidogrel efficacy. The study group consisted of consecutive patients (n = 275) who underwent stent implantation. Drug efficacy was measured using the vasodilator-stimulated phosphoprotein (VASP) index 20 ± 4 hours after clopidogrel 600 mg. Nonresponse was defined as an VASP index ≥50%. Renal function was determined using serum cystatin C. The upper reference levels are 1.12 mg/L for ≤65 years of age and 1.21 mg/L for >65 years of age. Estimated glomerular filtration was calculated using cystatin C. The median value of cystatin C was 1.16 mg/L (twenty-fifth and seventy-fifth percentiles 0.96 and 1.43); 47.63% of the study population had cystatin C above reference levels and 33.1% of patients were nonresponders to clopidogrel. No correlation was found between clopidogrel efficacy assessed with the VASP index and kidney function assessed with cystatin C (Spearman r = -0.070, p = 0.248). Based on cystatin C the proportion of nonresponders to clopidogrel was 34.4% versus 31.9% (p = 0.702) in patients with impaired renal function compared to normal renal function, respectively. The proportion of clopidogrel nonresponders did not differ (p = 0.902) among groups with normal (28.8%), mildly impaired (34.8%), moderately impaired (32.9%), and severely impaired (34.8%) renal function. In conclusion, renal function assessed by cystatin C does not predict clopidogrel efficacy. Renal dysfunction is a complex entity and its significant relation to stent thrombosis cannot be explained simply by a decrease in clopidogrel efficacy.     

Renin–angiotensin system blockade alone or combined with ETA receptor blockade: effects on the course of chronic kidney disease in 5/6 nephrectomized Ren-2 transgenic hypertensive rats

Background: Early addition of endothelin (ET) type A (ET_A) receptor blockade to complex renin–angiotensin system (RAS) blockade has previously been shown to provide better renoprotection against progression of chronic kidney disease (CKD) in Ren-2 transgenic hypertensive rats (TGR) after 5/6 renal ablation (5/6 NX). In this study, we examined if additional protection is provided when ET_A blockade is applied in rats with already developed CKD. Methods: For complex RAS inhibition, an angiotensin-converting enzyme inhibitor along with angiotensin II type 1 receptor blocker was used. Alternatively, ET_A receptor blocker was added to the RAS blockade. The treatments were initiated 6 weeks after 5/6 NX and the follow-up period was 50 weeks. Results: When applied in established CKD, addition of ET_A receptor blockade to the complex RAS blockade brought no further improvement of the survival rate (30% in both groups); surprisingly, aggravated albuminuria (588 ± 47 vs. 245 ± 38 mg/24 h, p < 0.05) did not reduce renal glomerular injury index (1.25 ± 0.29 vs. 1.44 ± 0.26), did not prevent the decrease in creatinine clearance (203 ± 21 vs. 253 ± 17 µl/min/100 g body weight), and did not attenuate cardiac hypertrophy to a greater extent than observed in 5/6 NX TGR treated with complex RAS blockade alone. Conclusions: When applied in the advanced phase of CKD, addition of ET_A receptor blockade to the complex RAS blockade brings no further beneficial renoprotective effects on the CKD progression in 5/6 NX TGR, in addition to those seen with RAS blockade alone.     

Antibodies against lepirudin are polyspecific and recognize epitopes on bivalirudin

Bivalirudin is a synthetic antithrombin sharing a sequence of 11 amino acids with the recombinant hirudin lepirudin. We investigated whether antilepirudin antibodies recognize epitopes on bivalirudin. Antilepirudin antibody-positive sera of 43 patients, treated with lepirudin for heparin-induced thrombocytopenia, were analyzed. Lepirudin- and bivalirudin-coated microtiter plates were used for antibody testing in an enzyme-linked immunosorbent assay (ELISA) system. Of the 43 sera-containing antibodies binding to lepirudin, 22 (51.2%) contained antibodies that also recognized bivalirudin. Binding of these antibodies to bivalirudin was inhibited by more than 70% by preincubation with high doses of bivalirudin. However, if lepirudin-coated microtiter plates were used, high concentrations of bivalirudin inhibited only 2 of the 43 positive sera by more than 30%. Therefore antihirudin antibodies must be polyspecific. The clinical consequences of this cross-reactivity are unknown but bivalirudin, targeted by antibodies of patients treated with lepirudin previously, could potentially boost antibody titers in such patients or even trigger an immune response by itself. Clinically significant antibody formation in response to bivalirudin monotherapy has not been observed, however. Yet, as lepirudin and antilepirudin antibodies have recently been implicated in severe anaphylactic reactions, caution is warranted when using bivalirudin in patients previously treated with lepirudin.     

日本血吸虫和曼氏血吸虫成虫31/32kD蛋白的纯化及在诊断中的应用

本研究以前染蛋白为指示,将制备型SDS—PAGE和电渗析技术相结合,分离、纯化日本血吸虫和曼氏血吸虫成虫31/32kD蛋白,用于ELISA中诊断血吸虫病。结果:纯化日本血吸虫成虫31/32kD蛋白(Psj31/32)与急、慢性日本血吸虫病和曼氏血吸虫病人血清反应的阳性率分别为100％、100％和98.4％;纯化曼氏血吸虫成虫31/32kD蛋白(Psm31/32)与上述病人血清反应的阳性率均为100％。与NHS和其它寄生虫病人血清反应的特异性较高。Psj31/32和Psm31/32检测同种血清,它们的OD值具有高度的正相关关系,但OD值与曼氏血吸虫病人粪便中虫卵数量(EPG)无相关关系。结果提示:两种纯化的31/32kD蛋白在血吸虫病诊断中具有较高的敏感性和特异性,并具有共同抗原决定簇存在,不仅可用于诊断急、慢性日本血吸虫病,而且Psj31/32也可作为诊断曼氏血吸虫病的候选抗原,用于我国输入性曼氏血吸虫病的诊断。     

Herpes Simplex Virus 1 and 2 Infections during Differentiation of Human Cortical Neurons

Herpes simplex virus 1 (HSV-1) and 2 (HSV-2) can infect the central nervous system (CNS) with dire consequences; in children and adults, HSV-1 may cause focal encephalitis, while HSV-2 causes meningitis. In neonates, both viruses can cause severe, disseminated CNS infections with high mortality rates. Here, we differentiated human induced pluripotent stem cells (iPSCs) towards cortical neurons for infection with clinical CNS strains of HSV-1 or HSV-2. Progenies from both viruses were produced at equal quantities in iPSCs, neuroprogenitors and cortical neurons. HSV-1 and HSV-2 decreased viability of neuroprogenitors by 36.0% and 57.6% (p < 0.0001), respectively, 48 h post-infection, while cortical neurons were resilient to infection by both viruses. However, in these functional neurons, both HSV-1 and HSV-2 decreased gene expression of two markers of synaptic activity, CAMK2B and ARC, and affected synaptic activity negatively in multielectrode array experiments. However, unaltered secretion levels of the neurodegeneration markers tau and NfL suggested intact axonal integrity. Viral replication of both viruses was found after six days, coinciding with 6-fold and 22-fold increase in gene expression of cellular RNA polymerase II by HSV-1 and HSV-2, respectively. Our results suggest a resilience of human cortical neurons relative to the replication of HSV-1 and HSV-2.     

Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model

RNA-based cancer therapies are hindered by the lack of delivery vehicles that avoid cancer-induced organ dysfunction, which exacerbates carrier toxicity. We address this issue by reporting modular degradable dendrimers that achieve the required combination of high potency to tumors and low hepatotoxicity to provide a pronounced survival benefit in an aggressive genetic cancer model. More than 1,500 dendrimers were synthesized using sequential, orthogonal reactions where ester degradability was systematically integrated with chemically diversified cores, peripheries, and generations. A lead dendrimer, 5A2-SC8, provided a broad therapeutic window: identified as potent [EC₅₀ < 0.02 mg/kg siRNA against FVII (siFVII)] in dose–response experiments, and well tolerated in separate toxicity studies in chronically ill mice bearing MYC-driven tumors (>75 mg/kg dendrimer repeated dosing). Delivery of let-7g microRNA (miRNA) mimic inhibited tumor growth and dramatically extended survival. Efficacy stemmed from a combination of a small RNA with the dendrimer’s own negligible toxicity, therefore illuminating an underappreciated complication in treating cancer with RNA-based drugs.Since the discovery of RNAi and the recognition of its therapeutic potential, there has been a continuous search for optimal delivery carriers (1–10). Tremendous progress has been made with regard to delivery efficacy of small RNAs to healthy livers, but the clinically required combination of high delivery potency to tumors and low hepatotoxicity is not currently met by existing delivery vehicles. This challenge represents an unappreciated complication in treating cancer with RNA-based drugs. Primary liver cancer, a chronic consequence of liver cirrhosis, is a leading cause of cancer death and a major global health problem (11). Unfortunately, all five phase III human clinical trials of small-molecule drugs for hepatocellular carcinoma (HCC) treatment failed within the past four years in part because debilitating, late-stage liver dysfunction amplifies drug toxicity (11, 12). MicroRNAs (miRNAs) present a promising alternative cancer treatment strategy because they can function as tumor suppressors by concurrently targeting multiple pathways involved in cell differentiation, proliferation, and survival (13–19). In this study, we used a highly aggressive, inducible MYC-driven transgenic liver cancer model where the chance for successful therapy is very limited, in equal part due to the rapidly growing cancer and the compromised function of the overtaken liver. Tumors grow endogenously from within the liver in this genetically engineered model, which is biologically faithful and lethal, in contrast to standard xenograft or orthotopic models derived from cell line implantation (20). The development of dendrimer carriers that can mediate a therapeutic benefit in this difficult-to-treat model was set as the paramount goal of the study. Notably, we find that high potency and low toxicity are of critical importance in the context of aggressive liver cancer models, where the carrier’s own toxicity can negate the benefit of on-target small-RNA therapies.To achieve this balance of low toxicity and high potency, we reasoned that one must more precisely examine the influence of chemical structure by expanding the structural diversity and molecular size of delivery carriers. Dendrimers represented an ideal system for this goal because they possess the same high degree of molecular uniformity as small molecules and the broad theoretical space for chemical tuning as polydisperse polymers (21–23). These intrinsic characteristics enable dendrimers to have unique properties for various biomedical applications (24–26). In gene delivery, most studies have used the limited number of commercial dendrimers for further chemical modification (27–29). However, orthogonal click chemistry has recently made a transformative impact on the synthesis of dendritic materials through protecting group-free methodologies to overcome historical limitations in fidelity and provide a larger toolbox for dendrimer design (30–34). The expansion of dendrimer applications therefore depends on the ability to easily tune the size, chemistry, topology, and, ultimately, dendrimer physical properties through chemical synthesis.We hypothesized that, through introduction of ester bonds and molecular diversity at each expansion step, modular degradable dendrimers would possess a critical balance of low toxicity and high delivery potency (Fig. 1). We further speculated that chemical modulation of cores, peripheries, and generations may be accelerated by utilization of efficient click reactions to enable a large increase in both the total number (>1,500) and chemical diversity of dendrimers. Biocompatible, degradable esters were included as a key design component because all RNAi therapies are transient and require sustained and repeated treatment. This ultimately enabled discovery of well-tolerated small-RNA carriers that could produce a therapeutic benefit in a challenging liver cancer model.Open in a separate windowFig. 1.RNA-based liver cancer therapies are hindered by the lack of potent and nontoxic delivery vehicles that avoid late-stage liver dysfunction that exacerbates toxicity. We envisioned that a modular design would allow discovery of dendrimers that balance low hepatotoxicity and high in vivo small-RNA delivery potency to tumor cells by fine-tuning of the identity and position of functional groups within dendrimer architectures.Efficacious delivery requires overcoming a series of extracellular and intracellular barriers (1). The physicochemical properties of small RNAs (high molecular weight, anionic charge, and hydrophilic nature) prevent passive diffusion across cell membranes. Studies of lipid and polymer carriers have implicated at least two common elements: chemical groups to bind small RNAs and nanoparticle (NP)-stabilizing hydrophobicity, which combine to encapsulate RNA molecules inside stable NPs and release small RNAs into the cytosol after endocytosis (35). Particular tertiary amines with optimal pK_a, alkyl chains, and defined topology/structure have been identified in effective carriers (2–10, 35–37).Despite these advances, demonstration of effective survival benefit in aggressive genetic models of human cancer has not been achieved with synthetic carriers due to the lack of delivery vehicles that avoid cancer-induced organ dysfunction. To address this, we designed a dendrimer library organized through binding, stabilization, and chemical functionality within the cores and peripheries. In vitro and in vivo evaluation identified dendrimers with optimal topological structures and high delivery efficacy (EC₅₀ of FVII knockdown < 0.02 mg/kg). Structure–activity knowledge was used to rationally design additional carriers with predicted in vivo activity. Evaluation of lead dendrimer candidates identified 5A2-SC8 as exhibiting low toxicity after repeated 75 mg/kg dendrimer i.v. dosing in chronically ill, tumor-bearing mice. Finally, we demonstrate that 5A2-SC8 can deliver a let-7g tumor suppressor miRNA with high in vivo potency to suppress tumor growth and result in a dramatic survival benefit of transgenic mice bearing aggressive, MYC-driven liver cancer. These findings suggest that modular degradable dendrimers may greatly expand the scope of chemical discovery research for therapeutic dendrimer delivery applications, ultimately providing new avenues for development of RNA cancer treatments by expansion of the therapeutic window.     

Kidney protective effects of baroreflex activation therapy in patients with resistant hypertension

Baroreflex activation therapy (BAT) is approved for the treatment of resistant hypertension. In addition to blood pressure (BP) reduction, pilot studies suggested several organoprotective effects of BAT. Thirty‐two patients with resistant hypertension were prospectively treated with BAT. Besides office BP and 24‐hour ambulatory BP (ABP) measurements, detection of a urinary proteome‐based classifier (CKD273), which has been shown to predict chronic kidney disease (CKD) progression, was carried out at baseline and after 6 months of BAT. Office BP significantly decreased from 170 ± 25/90 ± 18 to 149 ± 29/82 ± 18 mm Hg. Analysis of CKD273 score and eGFR with CKD‐EPI equation at baseline revealed strong correlation (r = 0.568, P < 0.001). After 6 months of BAT, there was no significant change in CKD273 score (−0.061 [95% CI: −0.262 to 0.140], P = 0.601). However, by stratification of the data regarding ABP response, there was a statistically significant (P = 0.0113) reduction in the CKD273 score from a mean of 0.161 [95% CI: −0.093 to 0.414] to −0.346 [95% CI: −0.632 to −0.060] after BAT in patients with systolic ABP decrease of ≥5 mm Hg. These data emphasized potential nephroprotective effects of BAT in patients with sufficient BP response.