177Lu-DOTA-HH1, a Novel Anti-CD37 Radio-Immunoconjugate: A Study of Toxicity in Nude Mice

Background

CD37 is an internalizing B-cell antigen expressed on Non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia cells (CLL). The anti-CD37 monoclonal antibody HH1 was conjugated to the bifunctional chelator p-SCN-Bn-DOTA and labelled with the beta-particle emitting radionuclide ¹⁷⁷Lu creating the radio-immunoconjugate (RIC) ¹⁷⁷Lu-DOTA-HH1 (¹⁷⁷Lu-HH1, trade name Betalutin). The present toxicity study was performed prior to initiation of clinical studieswith ¹⁷⁷Lu-HH1.

Methodology/Principal Findings

Nude mice with or without tumor xenografts were treated with 50 to 1000 MBq/kg ¹⁷⁷Lu- HH1 and followed for clinical signs of toxicity up to ten months. Acute, life threatening bone marrow toxicity was observed in animals receiving 800 and 1000 MBq/kg ¹⁷⁷Lu-HH1. Significant changes in serum concentrations of liver enzymes were evident for treatment with 1000 MBq/kg ¹⁷⁷Lu-HH1. Lymphoid depletion, liver necrosis and atrophy, and interstitial cell hyperplasia of the ovaries were also observed for mice in this dose group.

Conclusions/Significance

¹⁷⁷Lu-DOTA-HH1 was well tolerated at dosages about 10 times above those considered relevant for radioimmunotherapy in patients with B-cell derived malignancies.The toxicity profile was as expected for RICs. Our experimental results have paved the way for clinical evaluation of ¹⁷⁷Lu-HH1 in NHL patients.     

Interaction of a Partially Disordered Antisigma Factor with Its Partner,the Signaling Domain of the TonB-Dependent Transporter HasR

Bacteria use diverse signaling pathways to control gene expression in response to external stimuli. In Gram-negative bacteria, the binding of a nutrient is sensed by an outer membrane transporter. This signal is then transmitted to an antisigma factor and subsequently to the cytoplasm where an ECF sigma factor induces expression of genes related to the acquisition of this nutrient. The molecular interactions involved in this transmembrane signaling are poorly understood and structural data on this family of antisigma factor are rare. Here, we present the first structural study of the periplasmic domain of an antisigma factor and its interaction with the transporter. The study concerns the signaling in the heme acquisition system (Has) of Serratia marcescens. Our data support unprecedented partially disordered periplasmic domain of an anti-sigma factor HasS in contact with a membrane-mimicking environment. We solved the 3D structure of the signaling domain of HasR transporter and identified the residues at the HasS−HasR interface. Their conservation in several bacteria suggests wider significance of the proposed model for the understanding of bacterial transmembrane signaling.     

Maternal Hypertension after a Low-Birth-Weight Delivery Differs by Race/Ethnicity: Evidence from the National Health and Nutrition Examination Survey (NHANES) 1999–2006

Studies have suggested an increase in maternal morbidity and mortality due to cardiovascular diseases in women with a prior low-birth-weight (LBW, <2,500 grams) delivery. This study evaluated blood pressure and hypertension in women who reported a prior preterm or small-for-gestational-age (SGA) LBW delivery in the National Health and Nutrition Examination Survey 1999–2006 (n = 6,307). This study also aimed to explore if race/ethnicity, menopause status, and years since last pregnancy modified the above associations. A total of 3,239 white, 1,350 black, and 1,718 Hispanics were assessed. Linear regression models were used to evaluate blood pressure by birth characteristics (preterm-LBW, SGA-LBW, and birthweight ≥2,500). Logistic regression models estimated the odds ratios (OR) of hypertension among women who reported a preterm-LBW or SGA-LBW delivery compared with women who reported an infant with birthweight ≥2,500 at delivery. Overall, there was a positive association between a preterm-LBW delivery and hypertension (adjusted OR = 1.39, 95% confidence interval (CI) 1.02–1.90). Prior SGA-LBW also increased the odds of hypertension, but the estimate did not reach statistical significance (adjusted OR = 1.21, 95% CI 0.76–1.92). Race/ethnicity modified the above associations. Only black women had increased risk of hypertension following SGA-LBW delivery (adjusted OR = 2.09, 95% CI 1.12–3.90). Black women were at marginally increased risk of hypertension after delivery of a preterm-LBW (adjusted OR = 1.49, 95% CI 0.93–2.38). Whites and Hispanics had increased, but not statistically significant, risk of hypertension after a preterm-LBW (whites: adjusted OR = 1.39, 95% CI 0.92–2.10; Hispanics: adjusted OR = 1.22, 95% CI 0.62–2.38). Stratified analysis indicated that the associations were stronger among women who were premenopausal and whose last pregnancy were more recent. The current study suggests that in a representative United States population, women with a history of preterm- or SGA-LBW deliveries have increased odds of hypertension and this risk appears to be higher for black women and younger women.     

Neurological Involvement in Primary Sj?gren Syndrome: A Focus on Central Nervous System

Objectives

Sjögren syndrome is an autoimmune disease involving mainly salivary and lacrimal glands. Beyond widely described PNS involvement, high variable prevalence of CNS manifestations ranging from 2.5 and 60% of all pSS patients has been reported, without specific syndrome definition. The aim of this cohort study was to evaluate the prevalence of CNS signs and symptoms in pSS patients and to identify possible biomarkers of CNS damage.

Methods

120 patients with pSS diagnosis according to the 2002 American-European Consensus Group criteria were enrolled after exclusion of secondary causes. All patients underwent to a wide neurological, neuropsychological, psychiatric, neuroradiological and ultrasonographic evaluation.

Results

Central and peripheral nervous system involvement was observed in 81 patients with a prevalence of 67.5%. The prevalence of CNS involvement was significantly higher than PNS disease (p 0.001). 68 patients (84%) shown non-focal CNS symptoms and 64 (79%) focal CNS deficits with headache as the most common feature (46.9%), followed by cognitive (44.4%) and mood disorders (38.3%). Particularly, we observed a high prevalence of migraine without aura, subcortical frontal executive functions and verbal memory impairment and apathy/alexythimia. MR spectroscopy revealed a reduction of NAA levels or NAA/Cr ratio decrease in subcortical frontal and basal ganglia white matter, while ultrasonography showed an impairment of microvasculature response. At multivariate analysis, headache, cognitive disorders and psychiatric symptoms was significantly associated to serological markers (anti-SSA), MRS and ultrasonographic features.

Conclusions

The higher prevalence of MWO-mimic headache, cognitive dys-esecutive syndrome and mood disorders observed in this series confirmed previous evidences of a higher diffused CNS compromission rather than focal involvement such as SM-like clinical course or NMO-like syndrome. The association with immunological biomarkers, metabolic cerebral dysfunction and microvascular damage suggests a possible endothelial dysfunction of the cerebral microcirculation or a potential inflammation-mediated shift of the neurovascular coupling.     

Genetic Diversity and Spatial Genetic Structure of Chamaedaphne calyculata (Ericaceae) at the Western Periphery in Relation to its Main Continuous Range in Eurasia

A previous phylogeography and genetic diversity study of Chamaedaphne calyculata (Ericaceae) showed that populations over its geographic range were strongly separated into two groups: a Eurasian/NW North American group and a NE North American one corresponding with the disjunct distribution of Sphagnum-dominated peatlands in north-western and central-eastern North America. Here, I have extended the survey and focused on the species’ detailed postglacial origin and the effect of isolation on genetic diversity patterns, particularly within island-like populations at the western periphery of its range in Europe. Using AFLP markers, estimates of genetic diversity within 16 C. calyculata populations in the Eurasian group were low (percentage of polymorphic loci P _PL=14.9–24.8 %, Nei’s gene diversity H=0.060–0.119). Genetic diversity patterns within this species did not support the hypothesis that genetic diversity decreases towards the periphery of the range. Bayesian clustering analysis showed that population-level admixture was present in almost all studied 16 populations, suggesting multi-directional gene flow. On the other hand, the majority of assigned individuals (ca. 98 % of individuals) were offspring of the original residents, confirming that C. calyculata populations in the present day acted as discrete genetic units both in its continuous range and at its western periphery, and that gene flow was historic rather than contemporary in Eurasia. There was no correlation between genetic and geographic distance in the Eurasian group (r=0.02, P>0.05, Mantel test) nor at the western periphery (r=0.15, P>0.05, Mantel test). The isolation-by-distance (IBD) scatterplot matched Hutchinson and Templeton’s interpretation (case III), and geographic distance between populations was not a reliable predictor of the degree of genetic differentiation between populations. It is suggested that the lack of IBD might be a result of random genetic drift in rather disconnected populations that have become increasingly fragmented relatively recently. Positive and significant relationships between genetic and geographic distance on a small population scale was the result of biparental inbreeding of C. calyculata and restricted seed rain. Despite sporadic generative reproduction and limited dispersal, the fine-scale genetic structure within populations has been maintained, even though population sizes have been reduced to small fragments in recent years.     

Stream carbon and nitrogen supplements during leaf litter decomposition: contrasting patterns for two foundation species

Leaf litter decomposition plays a major role in nutrient dynamics in forested streams. The chemical composition of litter affects its processing by microorganisms, which obtain nutrients from litter and from the water column. The balance of these fluxes is not well known, because they occur simultaneously and thus are difficult to quantify separately. Here, we examined C and N flow from streamwater and leaf litter to microbial biofilms during decomposition. We used isotopically enriched leaves (¹³C and ¹⁵N) from two riparian foundation tree species: fast-decomposing Populus fremontii and slow-decomposing Populus angustifolia, which differed in their concentration of recalcitrant compounds. We adapted the isotope pool dilution method to estimate gross elemental fluxes into litter microbes. Three key findings emerged: litter type strongly affected biomass and stoichiometry of microbial assemblages growing on litter; the proportion of C and N in microorganisms derived from the streamwater, as opposed to the litter, did not differ between litter types, but increased throughout decomposition; gross immobilization of N from the streamwater was higher for P. fremontii compared to P. angustifolia, probably as a consequence of the higher microbial biomass on P. fremontii. In contrast, gross immobilization of C from the streamwater was higher for P. angustifolia, suggesting that dissolved organic C in streamwater was used as an additional energy source by microbial assemblages growing on slow-decomposing litter. These results indicate that biofilms on decomposing litter have specific element requirements driven by litter characteristics, which might have implications for whole-stream nutrient retention.     

Epigenetic control of early neurodegenerative events in diabetic retinopathy by the histone deacetylase SIRT6

Diabetic retinopathy (DR ) is one of the common complications associated with diabetes mellitus and the leading cause of blindness worldwide. Recent research has demonstrated that DR is not only a microvascular disease but may be a result of neurodegenerative processes. Moreover, glucose‐induced neuron and glial cell damage may occur shortly after the onset of diabetes which makes the disease hard to diagnose at early stages. SIRT 6, a NAD ‐dependent sirtuin deacylase, modulates aging, energy metabolism, and neurodegeneration. In previous studies we showed that SIRT 6 deficiency causes major retinal transmission defects, changes in the expression of glycolytic genes, and elevated levels of apoptosis. Given the importance of glucose availability for retinal function and the critical role of SIRT 6 in modulating glycolysis, we aimed to analyze SIRT 6 participation in the molecular machinery that regulates the development of experimental DR . Using non‐obese diabetic mice, we determined by western blot that 2 weeks after the onset of the disease, high glucose concentrations induced retinal increase in a neovascularization promoting factor (vascular endothelial growth factor, VEGF ), and the loss of a neuroprotective factor (brain‐derived neurotrophic factor, BDNF) associated with reduced levels of SIRT 6 and increased acetylation levels of its substrates (H3K9 and H3K56) suggesting a deregulation of key neural factors. Noteworthy, retinas from CNS conditionally deleted SIRT 6 mice showed a resemblance to diabetic retinas exhibiting lower protein levels of BDNF factor and increased protein levels of VEGF . Moreover, cultured Müller glial cells subjected to high glucose concentrations exhibited decreased levels of SIRT 6 and increased levels of H3K56 acetylation. In addition, the increment of VEGF levels induced by high glucose was reverted by the over‐expression of SIRT 6 in this cell type. Accordingly, siRNA experiments showed that, when SIRT 6 was silenced, VEGF levels increased. Our findings suggest that epigenetically regulated neurodegenerative events may occur at an early diabetic stage prior to the characteristic proliferative and vascular changes observed at a later diabetic stage.

     

SNP frequency, haplotype structure and linkage disequilibrium in elite maize inbred lines

Background  

Recent studies of ancestral maize populations indicate that linkage disequilibrium tends to dissipate rapidly, sometimes within 100 bp. We set out to examine the linkage disequilibrium and diversity in maize elite inbred lines, which have been subject to population bottlenecks and intense selection by breeders. Such population events are expected to increase the amount of linkage disequilibrium, but reduce diversity. The results of this study will inform the design of genetic association studies.     

8-Oxoguanine DNA-glycosylase repair activity and expression: a comparison between cryopreserved isolated lymphocytes and EBV-derived lymphoblastoid cell lines

Several lines of evidence suggest an association between oxidative DNA-damage repair capacity and cancer risk. In particular, a DNA-glycosylase assay for removal of 8-oxoguanine (8-oxoG) in peripheral blood mononuclear cells (PBMC) has been successfully applied to identify populations with increased risk for lung cancer and squamous cell carcinomas of head and neck. In order to verify whether EBV-transformed lymphoblastoid cell lines (LCL) are a suitable surrogate for PBMC in specific DNA-repair phenotypic assays, a validation trial was conducted. PBMC from 20 healthy subjects were collected and an aliquot was transformed with EBV to obtain LCL. The ability of cell-free extracts from both cell types to incise a 3'-fluorescently labelled duplex oligonucleotide containing a single 8-oxoG (OGG assay) was evaluated. Since this activity is mediated predominantly by OGG1, the OGG1 gene expression was also measured. 8-oxoG DNA-glycosylase activity and OGG1 expression were significantly higher (p<0.0001) in LCL than in PBMC. However, while this assay was shown to be robust and reproducible when used on PBMC (intra-assay CV=8%), a high intra-culture variability was observed with LCL (intra-culture CV=16.8%). Neither differences on OGG1 gene expression nor the cell-cycle distribution seemed to account for this variability. Inter-individual variability of OGG activity in PBMC and LCL was not associated with OGG1 gene expression. We have therefore established a non-radioactive cleavage assay that can be easily applied to measure OGG activity in human PBMC. The use of LCL for DNA-repair genotype-phenotype correlation studies seems to be inappropriate, at least with cell-free based functional assays.