Optimising the clinical strategy for autoimmune liver diseases: Principles of value-based medicine

Background

Autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis represent the three major autoimmune liver diseases (AILDs). Their management is highly specialized, requires a multidisciplinary approach and often relies on expensive, orphan drugs. Unfortunately, their treatment is often unsatisfactory, and the care pathway heterogeneous across different centers. Disease-specific clinical outcome indicators (COIs) able to evaluate the whole cycle of care are needed to assist both clinicians and administrators in improving quality and value of care. Aim of our study was to generate a set of COIs for the three AILDs. We then prospectively validated these indicators based on a series of consecutive patients recruited at three tertiary clinical centers in Lombardy, Italy.

Reproductive features of homospecific hybridogenetically-derived stick insects suggest how unisexuals can evolve

Hybridogenetic reproduction has been demonstrated in both vertebrate and invertebrate unisexual hybrids. Its most peculiar feature is the transmission to the progeny of one invariant genome (hemiclone) through the egg and the replacement of the other by host fathering males. Bacillus hybridogens are the only known example of hemiclonal invertebrates; their comparison to Poeciliopsis and Rana systems helps in understanding peculiar and shared features of vertebrate and insect hybridogenesis. In P. monacha-lucida, the experimental production of non-hybrid progeny through the reunion of the maternal hemiclone with a homospecific paternal genome provided by males of the maternal ancestor leads to inviable or severely impaired sterile specimens, whereas in Rana esculenta viable offspring are the rule. The comparable synthetic B. rossius progeny (Rr) embodying the maternal R hemiclone and a paternal r haploset, appear perfectly viable and fertile, clearly demonstrating compatibility between the two homospecific genomes, and also supporting a lack of deterioration of the R hemiclone. This condition can be ascribed to the recent origin of the hemiclones, and also to the absence of lethal recessives, owing to their most likely derivation from an automictic doubling in the parthenogenetic mechanisms of the maternal ancestor. However, the hybridogenetic system breaks down in the gamete production of the majority of Rr females, since normal allele segregation also occurs in their progeny. These reproductive modes suggest a likely evolutionary dynamic for newly originated hybridogens: to achieve stability, an interruption of reproductive interactions with the maternal ancestor seems necessary. In stick insects, this constraint appears to be fulfilled in both areas of sympatry. The microevolutionary pathway suggested by the ecological scenario also supports the possibility that a shift of hemiclonal stick insect strains to clonality has occurred.     

EXPRESSION OF MONOCYTE CHEMOTACTIC PROTEIN-3 IN HUMAN MONOCYTES EXPOSED TO THE MYCOBACTERIAL CELL WALL COMPONENT LIPOARABINOMANNAN

Monocyte chemotactic protein-3 (MCP-3) is a C–C chemokine which interacts with the CCR1, CCR2 (MCP-1) and CCR3 receptors and has a distinct spectrum of action. The present study was designed to assess whether mycobacterial components were able to induce expression and production of MCP-3 in human monocytes. Mycobacterial lipoarabinomannan (LAM) induced expression of MCP-3 mRNA in human peripheral blood mononuclear cells. The non-mannose-capped version of lipoarabinomannan (AraLAM) was considerably more potent than the mannose-capped version ManLAM or the simpler version phosphatidylinositol mannoside (PLM). Among mononuclear cells, monocytes were responsible for LAM-induced MCP-3 mRNA expression. Whole mycobacteria (Mycobacterium bovisBCG) strongly induced MCP-3 expression. Pretreatment with actinomycin D abolished LAM-induced MCP-3 expression, whereas cycloheximide only partially reduced the expression. LAM-induced MCP-3 expression was associated with the production of immunoreactive PTX3. Interleukin 10 (IL-10) and IL-13 inhibited the induction of MCP-3 by LAM. Thus mycobacterial cell wall components induced expression of MCP-3 in human monocytes. MCP-3, a chemokine active on mononuclear phagocytes, NK cells, T cells and dendritic cells, may be relevant to the induction and expression of immunity against mycobacteria.     

Cytotoxicity,genotoxicity and mechanism of action (via gene expression analysis) of the indole alkaloid aspidospermine (antiparasitic) extracted from Aspidosperma polyneuron in HepG2 cells

Aspidospermine is an indole alkaloid with biological properties associated with combating parasites included in the genera Plasmodium, Leishmania and Trypanossoma. The present study evaluated the cytotoxicity (resazurin test), genotoxicity (comet assay) and mechanism of action (gene expression analysis via qRT-PCR) of this alkaloid in human HepG2 cells. The results demonstrated that treatment with aspidospermine was both cytotoxic (starting at 75 μM) and genotoxic (starting at 50 μM). There was no significant modulation of the expression of the following genes: GSTP1 and GPX1 (xenobiotic metabolism); CAT (oxidative stress); TP53 and CCNA2 (cell cycle); HSPA5, ERN1, EIF2AK3 and TRAF2 (endoplasmic reticulum stress); CASP8, CASP9, CASP3, CASP7, BCL-2, BCL-XL BAX and BAX (apoptosis); and PCBP4, ERCC4, OGG1, RAD21 and MLH1 (DNA repair). At a concentration of 50 μM (non-cytotoxic, but genotoxic), there was a significant increase in the expression of CYP1A1 (xenobiotic metabolism) and APC (cell cycle), and at a concentration of 100 μM, a significant increase in the expression of CYP1A1 (xenobiotic metabolism), GADD153 (endoplasmic reticulum stress) and SOD (oxidative stress) was detected, with repression of the expression of GR (xenobiotic metabolism and oxidative stress). The results of treatment with aspidospermine at a 100 μM concentration (the dose indicated in the literature to achieve 89 % reduction of the growth of L. amazonensis) suggest that increased oxidative stress and an unfolded protein response (UPR) occurred in HepG2 cells. For the therapeutic use of aspidospermine (antiparasitic), chemical alteration of the molecule to achieve a lower cytotoxicity/genotoxicity in host cells is recommended.     

Detection of Anti-Pentraxin-3 Autoantibodies in ANCA-Associated Vasculitis

Objectives

Pentraxin 3 (PTX3), in common with myeloperoxidase and proteinase 3, is stored in human neutrophil granules and is expressed on apoptotic neutrophil surface. We therefore investigated the presence of anti-PTX3 autoantibodies (aAbs) in the sera of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients.

Methods

Presence of anti-PTX3 autoantibodies was analysed by a specific enzyme-linked immunosorbent assay in sera from 150 patients with microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), and eosinophilic granulomatosis with polyangiitis (EGPA), and in sera of 227 healthy subjects (HS), 40 systemic sclerosis (SSc) patients, and 25 giant cell arteritis patients (GCA). Using indirect immunofluorescence on fixed human neutrophils, we also analyzed the staining pattern associated with the presence of anti-PTX3 aAbs.

Results

Anti-PTX3 aAbs were detected in 56 of 150 (37.3%) of the AAV patients (versus 12 of 227 (5.3%) of HS, p<0.001) and, interestingly, in 7 of 14 MPO and PR3 ANCA negative AAV patients. Moreover, by indirect immunofluorescence on fixed neutrophils, anti-PTX3 aAbs gave rise to a specific cytoplasmic fluorescence pattern distinct from the classical cytoplasmic (c-ANCA), perinuclear (p-ANCA), and atypical (a-ANCA) pattern. Anti-PTX3 aAbs levels were higher in patients with active AAV as compared to patients with inactive disease.

Conclusion

Our work suggests that PTX3 is as a novel ANCA antigen. Anti-PTX3 aAbs appear thus as a promising novel biomarker in the diagnosis of AAV, including in patients without detectable MPO and PR3 ANCA.     

Immunologic assessment determined by response to IL-2 and immunophenotyping of tumor-infiltrating lymphocytes, of invaded and non invaded lymph nodes and of peripheral blood lymphocytes from twenty-one patients with primary laryngeal cancer

The aim of the study was to identify the lymphocyte sets and/or subsets possibly involved in the response to malignant cells. For this purpose we have investigated both the cells at the tumor site, i.e. tumor-infiltrating lymphocytes (TIL), and the cells present in the draining lymph nodes, either invaded or non invaded, as well as the peripheral blood lymphocytes from twenty-one patients with primary laryngeal epidermoid carcinoma. The functional assay was carried out by the proliferative response to mitogens, to Interleukin 2 and to their association, the surface immunophenotyping was performed with a large panel of monoclonal antibodies. TIL are the most responsive cells to mitogens, while the responsiveness of TIL and of LN cells to IL-2 was in the same range. PHA-activated TIL are the most responsive cells to IL-2. Our data indicate that TIL do show in vitro, and probably also in vivo, "activation" with elevated responsiveness to IL-2. The surface phenotype showed a strikingly increased proportion of T8+ cells in TIL as compared to T8+ cells in all types of LN, thus confirming within TIL variable, but high, proportions of clones which display cytolytic activity, possibly induced by IL-2. Our data seem to support the perspective for a therapeutic approach in vivo with IL-2, which via its influence on TIL, may act on tumor cells.     

Production of yeast killer toxin in experimentally infected animals

The ability of a killer yeast (Pichia anomala, UCSC 25F) to produce toxin in vivo was demonstrated, for the first time, in tissues of normal and immunosuppressed experimentally infected mice by means of a fluorescent antibody technique and a killer toxin specific monoclonal antibody. The possible significance of the findings is discussed.     

Effect of acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on humoral antibody production in mice

The effect of single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 1.2, 6 or 30 μg/kg i.p.) on primary humoral antibody production was studied in young adult C57 BL/6J mice. TCDD profoundly suppressed the primary response to thymus-dependent (sheep erythrocytes) and independent (type III pneumococcal polysaccharide) antigens. The inhibitory effect of TCDD was still detectable 42 days after treatment. In contrast, under these experimental conditions, in vitro lymphoproliferative responses to Concanavalin A (Con A) and bacterial lypopolysaccharides and the ability to mediate graft versus host reaction were not significantly affected per unit number of lymphoid cells.     

Monoclonal antibody-defined surface markers of effector cells involved in human monocyte cytotoxicity

Human adherent peripheral blood mononuclear cells were cytotoxic in vitro against the murine TU5 line in a 48-hr [3H]thymidine-release assay. Monocyte-enriched adherent cell preparations contain a small and variable (usually less than 5%) contamination with large granular lymphocytes as assessed by morphology and staining with monoclonal antibody markers B73.1 and HNK1. To assess whether killing was in fact mediated by monocytes, mononuclear cells or monocyte-enriched preparations were separated using monoclonal antibodies directed against mononuclear phagocytes (Mo2, UCHM1, B44.1) or natural killer (NK) cells (B73.1 and HNK1), and a fluorescence-activated cell sorter. Cells positive for monocyte markers were highly cytotoxic against TU5, whereas negative cells were not. B73.1+ or HNK1+ cells had little or no activity. Cytotoxicity of cells positive for monocyte markers (Mo2, UCHM1, B44.1) was augmented by in vitro exposure to lymphokines or less frequently to interferon (IFN). However, cells negative for these monocytes markers were also stimulated to kill TU5 by lymphokine or IFN to an extent similar or greater than that of positive ones. IFN or lymphokines induced killing of TU5 by monocyte-depleted, B73.1-positive, lymphoid cells. These observations demonstrate that human monocytes do kill tumor cells, either in the absence of deliberate stimulation or after exposure to agents such as lymphokines. However, the possible contribution to "monocyte" cytotoxicity of minor NK cell contaminants must be taken into account particularly when agents such as IFN and lymphokines are applied, even when a relatively NK-cell-resistant target such as TU5 is used.