Complementary Effects of Carbamylated and Citrullinated LL37 in Autoimmunity and Inflammation in Systemic Lupus Erythematosus

LL37 acts as T-cell/B-cell autoantigen in Systemic lupus erythematosus (SLE) and psoriatic disease. Moreover, when bound to “self” nucleic acids, LL37 acts as “danger signal,” leading to type I interferon (IFN-I)/pro-inflammatory factors production. T-cell epitopes derived from citrullinated-LL37 act as better antigens than unmodified LL37 epitopes in SLE, at least in selected HLA-backgrounds, included the SLE-associated HLA-DRB1*1501/HLA-DRB5*0101 backgrounds. Remarkably, while “fully-citrullinated” LL37 acts as better T-cell-stimulator, it loses DNA-binding ability and the associated “adjuvant-like” properties. Since LL37 undergoes a further irreversible post-translational modification, carbamylation and antibodies to carbamylated self-proteins other than LL37 are present in SLE, here we addressed the involvement of carbamylated-LL37 in autoimmunity and inflammation in SLE. We detected carbamylated-LL37 in SLE-affected tissues. Most importantly, carbamylated-LL37-specific antibodies and CD4 T-cells circulate in SLE and both correlate with disease activity. In contrast to “fully citrullinated-LL37,” “fully carbamylated-LL37” maintains both innate and adaptive immune-cells’ stimulatory abilities: in complex with DNA, carbamylated-LL37 stimulates plasmacytoid dendritic cell IFN-α production and B-cell maturation into plasma cells. Thus, we report a further example of how different post-translational modifications of a self-antigen exert complementary effects that sustain autoimmunity and inflammation, respectively. These data also show that T/B-cell responses to carbamylated-LL37 represent novel SLE disease biomarkers.     

Asbestos lung burden and asbestosis after occupational and environmental exposure in an asbestos cement manufacturing area: a necropsy study

OBJECTIVE: The largest Italian asbestos cement factory had been active in Casale Monferrato until 1986: in previous studies a substantial increase in the incidence of pleural mesothelioma was found among residents without occupational exposure to asbestos. To estimate exposure to asbestos in the population, this study evaluated the presence of histological asbestosis and the lung burden of asbestos fibres (AFs) and asbestos bodies (ABs). METHODS: The study comprises the consecutive series of necropsies performed at the Hospital of Casale Monferrato between 1985 and 1988. A sample of lung parenchima was collected and stored for 48 out of 55 necropsies. The AF concentration was measured with a TEM electron microscope with x ray mineralogical analysis. The ABs were counted and fibrosis evaluated by optical microscopy. The nearest relative of each subject was interviewed on occupational and residential history. Mineralogical and histological analyses and interviews were conducted in 1993-4. RESULTS: Statistical analyses included 41 subjects with AF, AB count, and interview. Subjects without occupational exposure who ever lived in Casale Monferrato had an average concentration of 1500 AB/g dried weight (gdw); Seven of 18 presented with asbestosis or small airway lung disease (SAL). G2 asbestosis was diagnosed in two women with no occupational asbestos exposure. One of them had been teaching at a school close to the factory for 12 years. Ten subjects had experienced occupational asbestos exposure, seven in asbestos cement production: mean concentrations were 1.032 x 10(6) AF/gdw and 96,280 AB/gdw. Eight of the 10 had asbestosis or SAL. CONCLUSION: The high concentration of ABs and the new finding of environmental asbestosis confirm that high asbestos concentration was common in the proximity of the factory. Subjects not occupationally exposed and ever living in Casale Monferrato tended to have higher AB concentration than subjects never living in the town (difference not significant). The concentrations of ABs and AFs were higher than those found in other studies on nonoccupationally exposed subjects.     

Effect of whey concentration on protein recovery in fresh ovine ricotta cheese

Ricotta cheese, particularly the ovine type, is a typical Italian dairy product obtained by heat-coagulation of the proteins in whey. The aim of this work was to investigate the influence of whey protein concentration, obtained by ultrafiltration, on yield of fresh ovine ricotta cheese. Ricotta cheeses were obtained by thermocoagulation of mixtures with protein content of 1.56, 3.10, 4.16, and 7.09 g/100 g from the mixing of skim whey and ultrafiltered skim whey. A fat-to-protein ratio of 1.1 (wt/wt) was obtained for all mixtures by adding fresh cream. The initial mixtures, as well as the final ricotta cheeses, were analyzed for their composition and by SDS-PAGE. Protein bands were quantified by QuantityOne software (Bio-Rad, Hercules, CA) and identified by liquid chromatography-tandem mass spectrometry. Significant differences in the composition of the ricotta cheese were observed depending on protein concentration. Particularly, ricotta cheese resulting from the mixture containing 7.09 g/100 g of protein presented higher moisture (72.88 ± 1.50 g/100 g) and protein (10.18 ± 0.45 g/100 g) contents than that prepared from the mixture with 1.56 g/100 g of protein (69.52 ± 1.75 and 6.70 ± 0.85 g/100 g, respectively), and fat content was lower in this sample (12.20 ± 1.60 g/100 g) compared with the other treatments, with mean values between 15.72 and 20.50 g/100 g. Each protein fraction presented a different behavior during thermocoagulation. In particular, the recovery of β-lactoglobulin and α-lactalbumin in the cheese increased as their content increased in the mixtures. It was concluded that concentrating ovine rennet whey improved the extent of heat-induced protein aggregation during the thermal coagulation process. This resulted in a better recovery of each protein fraction in the product, and in a consequent increase of ricotta cheese yield.     

Toward the discovery of novel anti-HIV drugs. Second-generation inhibitors of the cellular ATPase DDX3 with improved anti-HIV activity: synthesis, structure-activity relationship analysis, cytotoxicity studies, and target validation

A hit optimization protocol applied to the first nonnucleoside inhibitor of the ATPase activity of human DEAD-box RNA helicase DDX3 led to the design and synthesis of second-generation rhodanine derivatives with better inhibitory activity toward cellular DDX3 and HIV-1 replication. Additional DDX3 inhibitors were identified among triazine compounds. Biological data were rationalized in terms of structure-activity relationships and docking simulations. Antiviral activity and cytotoxicity of selected DDX3 inhibitors are reported and discussed. A thorough analysis confirmed human DDX3 as a valid anti-HIV target. The compounds described herein represent a significant advance in the pursuit of novel drugs that target HIV-1 host cofactors.     

Identification of Hck Inhibitors As Hits for the Development of Antileukemia and Anti‐HIV Agents

Hematopoietic cell kinase (Hck) is a member of the Src family of non‐receptor protein tyrosine kinases. High levels of Hck are associated with drug resistance in chronic myeloid leukemia. Furthermore, Hck activity has been connected with HIV‐1. Herein, structure‐based drug design efforts were aimed at identifying novel Hck inhibitors. First, an in‐house library of pyrazolo[3,4‐d]pyrimidine derivatives, which were previously shown to be dual Abl and c‐Src inhibitors, was analyzed by docking studies within the ATP binding site of Hck to select the best candidates to be tested in a cell‐free assay. Next, the same computational protocol was applied to screen a database of commercially available compounds. As a result, most of the selected compounds were found active against Hck, with K_i values ranging from 0.14 to 18.4 μM , confirming the suitability of the computational approach adopted. Furthermore, selected compounds showed an interesting antiproliferative activity profile against the human leukemia cell line KU‐812, and one compound was found to block HIV‐1 replication at sub‐toxic concentrations.     

Kidney transplantation and pregnancy. Report of a case

The aim of this study was to evaluate the extent to which the presence of a transplanted kidney conditions the physiological renal and maternal cardiocirculatory adaptation to pregnancy. For this purpose, we compared the trend of cardiocirculatory and renal hematochemical parameters in a kidney-transplant patient to a group of 100 physiological pregnant women followed longitudinally. M. G., aged 36, primigravida, who underwent renal transplantation ten years before, was carefully monitored throughout gestation. Pregnancy was free of complications and ended at 38 weeks with an elective caesarian section. The trend of the parameters mirrored the physiological pattern, even if the values for some renal parameters were completely different. This type of comparison enabled us to evaluate the adaptation of the transplanted organ and the body to pregnancy and to formulate a prognostic judgement halfway through pregnancy regarding the outcome.