Immunogenicity of mAbs in non-human primates during nonclinical safety assessment

The immunogenicity of biopharmaceuticals used in clinical practice remains an unsolved challenge in drug development. Non-human primates (NHPs) are often the only relevant animal model for the development of monoclonal antibodies (mAbs), but the immune response of NHPs to therapeutic mAbs is not considered to be predictive of the response in humans because of species differences. In this study, we accessed the drug registration files of all mAbs registered in the European Union to establish the relative immunogenicity of mAbs in NHPs and humans. The incidence of formation of antidrug-antibodies in NHPs and patients was comparable in only 59% of the cases. In addition, the type of antidrug-antibody response was different in NHP and humans in 59% of the cases. Humanization did not necessarily reduce immunogenicity in humans. Immunogenicity interfered with the safety assessment during non-clinical drug development when clearing or neutralizing antibodies were formed. While important to interpret the study results, immunogenicity reduced the quality of NHP data in safety assessment. These findings confirm that the ability to compare relative immunogenicity of mAbs in NHPs and humans is low. Furthermore, immunogenicity limits the value of informative NHP studies.     

Expression of DNA methyltransferases is involved in Quercus suber cork quality

Cork oak (Quercus suber) is an important Portuguese species, mainly due to the economic value of the cork it produces. Cork results from phellogen, a meristematic tissue, which can locally produce lenticels or have discontinuities, originating “defects”: pores and nail inclusions that are detrimental to cork industrial use. Epigenetic processes control plant development and its deregulation can lead to altered phenotypes; therefore, the study of epigenetic players in the phellogen is important to understand the emergence of cork's defects. DNA methyltransferases (DNMTs) and one protein associated to MET1 (DMAP1) were characterized in Q. suber, and their gene expression was analyzed in phellogen and contiguous differentiating cell layers of trees producing high and low quality cork, after the evaluation of their defects by physical and image analysis methods. All classes of DNMTs (MET, DRM, and CMT) with the respective canonical motifs were identified in Q. suber. The expression analyses of these genes showed that QsDRM2 was the most active methyltransferases in the cells analyzed, and that all the genes were differentially expressed in trees with distinct cork quality, with a tendency for higher expression levels in low quality producers. Interestingly, the global methylation level was higher in cells with low expression of DNA methyltransferases. A positive and significant correlation was obtained between QsDMAP1 gene expression and the percentage of cork defects. This work provides the first evidence that cork quality in Q. suber is likely influenced by epigenetic mechanisms.     

Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

During disease progression to AIDS, HIV-1 infected individuals become increasingly immunosuppressed and susceptible to opportunistic infections. It has also been demonstrated that multiple subsets of dendritic cells (DC), including DC-SIGN(+) cells, become significantly depleted in the blood and lymphoid tissues of AIDS patients, which may contribute to the failure in initiating effective host immune responses. The mechanism for DC depletion, however, is unclear. It is also known that vast quantities of viral envelope protein gp120 are shed from maturing HIV-1 virions and form circulating immune complexes in the serum of HIV-1-infected individuals, but the pathological role of gp120 in HIV-1 pathogenesis remains elusive. Here we describe a previously unrecognized mechanism of DC death in chronic HIV-1 infection, in which ligation of DC-SIGN by gp120 sensitizes DC to undergo accelerated apoptosis in response to a variety of activation stimuli. The cultured monocyte-derived DC and also freshly-isolated DC-SIGN(+) blood DC that were exposed to either cross-linked recombinant gp120 or immune-complex gp120 in HIV(+) serum underwent considerable apoptosis after CD40 ligation or exposure to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines such as TNFα and IL-1β. Furthermore, circulating DC-SIGN(+) DC that were isolated directly from HIV-1(+) individuals had actually been pre-sensitized by serum gp120 for activation-induced exorbitant apoptosis. In all cases the DC apoptosis was substantially inhibited by DC-SIGN blockade. Finally, we showed that accelerated DC apoptosis was a direct consequence of excessive activation of the pro-apoptotic molecule ASK-1 and transfection of siRNA against ASK-1 significantly prevented the activation-induced excessive DC death. Our study discloses a previously unknown mechanism of immune modulation by envelope protein gp120, provides new insights into HIV immunopathogenesis, and suggests potential therapeutic approaches to prevent DC depletion in chronic HIV infection.     

Identification of a Potent Endothelium-Derived Angiogenic Factor

The secretion of angiogenic factors by vascular endothelial cells is one of the key mechanisms of angiogenesis. Here we report on the isolation of a new potent angiogenic factor, diuridine tetraphosphate (Up₄U) from the secretome of human endothelial cells. The angiogenic effect of the endothelial secretome was partially reduced after incubation with alkaline phosphatase and abolished in the presence of suramin. In one fraction, purified to homogeneity by reversed phase and affinity chromatography, Up₄U was identified by MALDI-LIFT-fragment-mass-spectrometry, enzymatic cleavage analysis and retention-time comparison. Beside a strong angiogenic effect on the yolk sac membrane and the developing rat embryo itself, Up₄U increased the proliferation rate of endothelial cells and, in the presence of PDGF, of vascular smooth muscle cells. Up₄U stimulated the migration rate of endothelial cells via P2Y2-receptors, increased the ability of endothelial cells to form capillary-like tubes and acts as a potent inducer of sprouting angiogenesis originating from gel-embedded EC spheroids. Endothelial cells released Up₄U after stimulation with shear stress. Mean total plasma Up₄U concentrations of healthy subjects (N = 6) were sufficient to induce angiogenic and proliferative effects (1.34±0.26 nmol L^-1). In conclusion, Up₄U is a novel strong human endothelium-derived angiogenic factor.     

Intensive Lifestyle Intervention in General Practice to Prevent Type 2 Diabetes among 18 to 60-Year-Old South Asians: 1-Year Effects on the Weight Status and Metabolic Profile of Participants in a Randomized Controlled Trial

Aim

To study 1-year effectiveness of an intensive, culturally targeted lifestyle intervention in general practice for weight status and metabolic profile of South-Asians at risk of type 2 diabetes.

Methods

536 South-Asians at risk of type 2 diabetes were randomized to an intervention (n = 283) or control (n = 253) group. The intervention, which was targeted culturally to the South-Asian population, consisted of individual lifestyle counselling, a family session, cooking classes, and supervised physical activity programme. All components of the intervention were carried out by professionals as part of their daily clinical practice. The control group received generic lifestyle advice. Change in weight status and metabolic profile were assessed after 1 year.

Results

After 1 year, 201 participants were lost to follow-up. Remaining participants in intervention (n = 177) and control (n = 158) group had similar baseline characteristics. Weight loss in the intervention group was 0.2±3.3 kg, weight gain in the control group was 0.4±3.1 kg (p = 0.08). Changes in other weight-related measurements did not differ significantly between groups. Furthermore, there were no differences between groups in changes of metabolic profile. All results remained similar after repeating analyses in a multiple imputed dataset.

Discussion

An intensive, culturally targeted, lifestyle intervention of 1 year did not improve weight status and metabolic profile of South-Asians at risk of type 2 diabetes. The laborious recruitment, high drop-out, and lack of effectiveness emphasise the difficulty of realising health benefits in practice and suggest that this strategy might not be the optimal approach for this population.

Trial Registration

Nederlands Trial Register NTR1499     

Human Hemorrhagic Pulmonary Leptospirosis: Pathological Findings and Pathophysiological Correlations

Background

Leptospirosis is a re-emerging zoonosis with protean clinical manifestations. Recently, the importance of pulmonary hemorrhage as a lethal complication of this disease has been recognized. In the present study, five human necropsies of leptospirosis (Weil‘s syndrome) with extensive pulmonary manifestations were analysed, and the antibodies expressed in blood vessels and cells involved in ion and water transport were used, seeking to better understand the pathophysiology of the lung injury associated with this disease.

Principal Findings

Prominent vascular damage was present in the lung microcirculation, with decreased CD34 and preserved aquaporin 1 expression. At the periphery and even inside the extensive areas of edema and intraalveolar hemorrhage, enlarged, apparently hypertrophic type I pneumocytes (PI) were detected and interpreted as a non-specific attempt of clearence of the intraalveolar fluid, in which ionic transport, particularly of sodium, plays a predominant role, as suggested by the apparently increased ENaC and aquaporin 5 expression. Connexin 43 was present in most pneumocytes, and in the cytoplasm of the more preserved endothelial cells. The number of type II pneumocytes (PII) was slightly decreased when compared to normal lungs and those of patients with septicemia from other causes, a fact that may contribute to the progressively low PI count, resulting in deficient restoration after damage to the alveolar epithelial integrity and, consequently, a poor outcome of the pulmonary edema and hemorrhage.

Conclusions

Pathogenesis of lung injury in human leptospirosis was discussed, and the possibility of primary non-inflammatory vascular damage was considered, so far of undefinite etiopathogenesis, as the initial pathological manifestation of the disease.     

Industrial Scale Isolation,Structural and Spectroscopic Characterization of Epiisopiloturine from Pilocarpus microphyllus Stapf Leaves: A Promising Alkaloid against Schistosomiasis

This paper presents an industrial scale process for extraction, purification, and isolation of epiisopiloturine (EPI) (2(3H)-Furanone,dihydro-3-(hydroxyphenylmethyl)-4-[(1-methyl-1H-imidazol-4-yl)methyl]-, [3S-[3a(R*),4b]]), which is an alkaloid from jaborandi leaves (Pilocarpus microphyllus Stapf). Additionally for the first time a set of structural and spectroscopic techniques were used to characterize this alkaloid. EPI has shown schistomicidal activity against adults and young forms, as well as the reduction of the egg laying adult worms and low toxicity to mammalian cells (in vitro). At first, the extraction of EPI was done with toluene and methylene chloride to obtain a solution that was alkalinized with ammonium carbonate. The remaining solution was treated in sequence by acidification, filtration and alkalinization. These industrial procedures are necessary in order to remove impurities and subsequent application of the high performance liquid chromatography (HPLC). The HPLC was employed also to remove other alkaloids, to obtain EPI purity higher than 98%. The viability of the method was confirmed through HPLC and electrospray mass spectrometry, that yielded a pseudo molecular ion of m/z equal to 287.1 Da. EPI structure was characterized by single crystal X-ray diffraction (XRD), ¹H and ¹³C nuclear magnetic resonance (NMR) in deuterated methanol/chloroform solution, vibrational spectroscopy and mass coupled thermal analyses. EPI molecule presents a parallel alignment of the benzene and the methyl imidazol ring separated by an interplanar spacing of 3.758 Å indicating a π-π bond interaction. The imidazole alkaloid melts at 225°C and decomposes above 230°C under air. EPI structure was used in theoretical Density Functional Theory calculations, considering the single crystal XRD data in order to simulate the NMR, infrared and Raman spectra of the molecule, and performs the signals attribution.     

Measurement,model prediction and uncertainty quantification of plasma clearance of cerium citrate in humans

Radiation and Environmental Biophysics - Double tracer studies in healthy human volunteers with stable isotopes of cerium citrate were performed with the aim of investigating the gastro-intestinal...