Hydroxychloroquine in recurrent pregnancy loss: data from a French prospective multicenter registry.

STUDY QUESTION: What are the outcomes of pregnancies exposed to hydroxychloroquine (HCQ) in women with a history of recurrent pregnancy loss (RPL), and what factors predict the course of these pregnancies beyond the first trimester? SUMMARY ANSWER: In our cohort of pregnancies in women with a history of RPL exposed to HCQ early in pregnancy, we found that the only factor determining the success of these pregnancies was the number of previous miscarriages. WHAT IS KNOWN ALREADY: Dysregulation of the maternal immune system plays a role in RPL. HCQ, with its dual immunomodulating and vascular protective effects, is a potential treatment for unexplained RPL. STUDY DESIGN, SIZE, DURATION: The FALCO (Facteurs de récidive précoce des fausses couches) registry is an ongoing French multicenter infertility registry established in 2017 that includes women (aged from 18 to 49 years) with a history of spontaneous RPL (at least three early miscarriages (≤12 weeks of gestation (WG)) recruited from several university hospitals. PARTICIPANTS/MATERIALS, SETTING, METHODS: Spontaneous pregnancies enrolled in the FALCO registry with an exposure to HCQ (before conception or at the start of pregnancy) were included. Pregnancies concomitantly exposed to tumor necrosis factor inhibitors, interleukin-1 and -2 inhibitors, intravenous immunoglobulin, and/or intravenous intralipid infusion, were excluded. Concomitant treatment with low-dose aspirin (LDA), low-molecular weight heparin (LMWH), progesterone, and/or prednisone was allowed. All patients underwent the recommended evaluations for investigating RPL. Those who became pregnant received obstetric care in accordance with French recommendations and were followed prospectively. The main endpoint was the occurrence of a pregnancy continuing beyond 12 WG, and the secondary endpoint was the occurrence of a live birth. MAIN RESULTS AND THE ROLE OF CHANCE: One hundred pregnancies with HCQ exposure in 74 women were assessed. The mean age of the women was 34.2 years, and the median number of previous miscarriages was 5. Concomitant exposure was reported in 78 (78%) pregnancies for prednisone, 56 (56%) pregnancies for LDA, and 41 (41%) pregnancies for LMWH. Sixty-two (62%) pregnancies ended within 12 WG, the other 38 (38%) continuing beyond 12 WG. The risk of experiencing an additional early spontaneous miscarriage increased with the number of previous miscarriages, but not with age. The distributions of anomalies identified in RPL investigations and of exposure to other drugs were similar between pregnancies lasting ≤12 WG and those continuing beyond 12WG. The incidence of pregnancies progressing beyond 12 WG was not higher among pregnancies with at least one positive autoantibody (Ab) (i.e. antinuclear Ab titer ≥1:160, ≥1 positive conventional and/or non-conventional antiphospholipid Ab, and/or positive results for ≥1 antithyroid Ab) without diminished ovarian reserve (18/51, 35.3%) than among those without such autoantibody (18/45, 40.0%) (P = 0.63). Multivariate analysis showed that having ≤4 prior miscarriages was the only factor significantly predictive for achieving a pregnancy > 12 WG, after adjustment for age and duration of HCQ use prior to conception (adjusted odds ratio (OR) = 3.13 [1.31-7.83], P = 0.01). LIMITATIONS, REASONS FOR CAUTION: Our study has limitations, including the absence of a control group, incomplete data for the diagnostic procedure for RPL in some patients, and the unavailability of results from endometrial biopsies, as well as information about paternal age and behavioral factors. Consequently, not all potential confounding factors could be considered. WIDER IMPLICATIONS OF THE FINDINGS: Exposure to HCQ in early pregnancy for women with a history of RPL does not seem to prevent further miscarriages, suggesting limited impact on mechanisms related to the maternal immune system. STUDY FUNDING/COMPETING INTEREST(S): The research received no specific funding, and the authors declare no competing interests. TRIAL REGISTRATION NUMBER: clinicaltrial.gov NCT05557201.

Long-term outcomes of patients with type 1 or 2 autoimmune hepatitis presenting in childhood.

BACKGROUND & AIMS: In children with autoimmune hepatitis, uncertainties include outcomes associated with type 2 hepatitis, the possibility of and criteria for attempting withdrawal of treatment, and long-term outcomes. We report our experience on these issues. METHODS: From 1973 to 2002, 117 children with type 1 (n = 65) or type 2 (n = 52) hepatitis, excluding fulminant hepatitis, were treated, primarily with prednisone and azathioprine. Median follow-up was 20 years in survivors. RESULTS: Normalisation of aminotransferases and prothrombin ratio were observed in 93% and 84% of children, respectively; sustained remission after treatment withdrawal was recorded in 24% of the entire population, with a median follow-up of 7 years. Sustained treatment-free remission was obtained in 11 of 24 children with follow-ups of 4-22 years based on durable normalisation of aminotransferases (without histological assessment). Gastrointestinal bleeding from varices and the emergence of extrahepatic autoimmune disorders occurred in 10 and 22 patients, respectively. Liver transplantation was performed in 23 patients at a median age of 21 years. The 30-year probabilities of overall and native liver survival were 81% and 61%, respectively. No differences were observed between type 1 and 2 hepatitis for any of the component parts of outcome. In the multivariate analysis, a persistent abnormal prothrombin ratio was associated with worse probabilities of overall and native liver survival. CONCLUSIONS: In terms of liver outcome, type 2 hepatitis is not different from type 1. Withdrawal of treatment is possible without prior liver histology. A persistent abnormal prothrombin ratio identifies patients who will require liver transplantation in adolescence or early adulthood. IMPACT AND IMPLICATIONS: In children with autoimmune hepatitis, there are conflicting reports on the differences in outcome between type 1 and type 2 hepatitis, and on the possibility of treatment withdrawal, before which liver histology is required; data concerning >10-year overall and native liver survival rates are limited. In this study, we found no differences in outcomes between type 1 and 2 hepatitis; a durable treatment-free state was achieved in 19% of all patients throughout childhood and early adulthood, and in 45% of children for whom treatment withdrawal was attempted without prior liver histology; prothrombin was found to be predictive of 30-year overall and native liver survival. The results allow for a less-strict approach to treatment withdrawal in children, avoiding the risks of a liver biopsy, and they provide a tool to help anticipate the need for liver transplantation before complications occur.

Autoantibody signatures defined by serological proteome analysis in sera from patients with cholangiocarcinoma.

BACKGROUND: The challenging diagnosis and poor prognosis of cholangiocarcinoma require the determination of biomarkers. Autoantibodies could be used in the clinic as diagnostic markers for the early detection of tumours. By proteomic approaches, several autoantibodies were proposed as potential markers. We tried in this study, to perform a serological proteome analysis, using various antigenic substrates, including tumours and human liver. METHODS: Sera from patients (n = 13) and healthy donors (n = 10) were probed on immunoblots performed using 2-dimensionally separated proteins from cholangiocarcinoma cell lines (CCLP1 and CCSW1), from the liver of healthy subject and interestingly, from tumour and adjacent non-tumour liver tissues from five patients with cholangiocarcinoma and tested with their corresponding serum. Spots of interest were identified using mass spectrometry and classified according gene ontology analysis. RESULTS: A comparison of the whole immunoblotting patterns given by cholangiocarcinoma sera against those obtained with normal control sera enabled the definition of 862 spots. Forty-five different proteins were further analysed, corresponding to (1) spots stained with more than four of 13 (30 %) sera tested with the CCLP1 or the CCSW1 cell line and with the normal liver, and (2) to spots immunoreactive with at least two of the five sera probed with their tumour and non-tumour counter-part of cholangiocarcinoma. Immunoreactive proteins with catalytic activity as molecular function were detected at rates of 93 and 64 % in liver from healthy subjects or cholangiocarcinoma non-tumour tissues respectively, compared to 43, 33, 33 % in tumour tissues, or CCSW1 and CCLP1 cell lines. A second pattern was represented by structural proteins with rates of 7 and 7 % in normal liver or non-tumour tissues compared to 14, 33 and 67 % in tumour tissue, CCSW1 or CCLP1 cell lines. Proteins with a binding function were detected at rates of 7 % in non-tumour tissue and 14 % in tumour tissue. Using the extracted tumour tissue, serotransferrin was targeted by all cholangiocarcinoma-related sera. CONCLUSIONS: Immunological patterns depended on the type of antigen substrate used; i.e. tumour versus non tumour specimens. Nevertheless, a combination of multiple autoantibodies tested with the most appropriate substrate might be more sensitive and specific for the diagnosis of cholangiocarcinoma.

Immunoproteomic analysis of potentially severe non-graft-versus-host disease hepatitis after allogenic bone marrow transplantation.

UNLABELLED: The development of potentially severe non-graft-versus-host disease (GVHD) hepatitis resembling autoimmune hepatitis (AIH) has been reported after bone marrow transplantation (BMT). The aim of this study was to better characterize this form of hepatitis, particularly through the identification of autoantigens recognized by patient sera. Five patients who received an allogeneic BMT for the treatment of hematological diseases developed liver dysfunction with histological features suggestive of AIH. Before and during the onset of hepatic dysfunction, sera were tested on immunoblottings performed with cytosolic, microsomal, mitochondrial, and nuclear proteins from rat liver homogenate and resolved by two-dimensional electrophoresis. Antigenic targets were identified by mass spectrometry. During the year that followed BMT, all patients presented with GVHD. Acute hepatitis then occurred after the withdrawal, or during the tapering, of immunosuppressive therapy. At that time, no patients had a history of liver toxic drug absorption, patent viral infection, or any histopathological findings consistent with GVHD. Immunoreactive spots stained by sera collected at the time of hepatic dysfunction were more numerous and more intensely expressed than those stained by sera collected before. Considerable patient-dependent pattern heterogeneity was observed. Among the 259 spots stained exclusively by sera collected at the time of hepatitis, a total of 240 spots were identified, corresponding to 103 different proteins. Twelve of them were recognized by sera from 3 patients. CONCLUSIONS: This is the first immunological description of potentially severe non-GVHD hepatitis occurring after BMT, determined using a proteomic approach and enabling a discussion of the mechanisms that transform an alloimmune reaction into an autoimmune response. Any decision to withdraw immunosuppression after allogeneic BMT should be made with caution.

Twenty-year protocol liver biopsies: Invasive but useful for the management of liver recipients.

BACKGROUND & AIMS: Most liver transplant centres have discontinued the practice of protocol liver biopsies (LB), mainly because of the perceived lack of therapeutic benefit. This study aimed to examine the usefulness of 20-year LBs. METHODS: Ten, 15, and 20-year protocol LBs from 147 patients surviving for >20 years were reviewed. Twenty-year biopsy findings were correlated with clinical data. RESULTS: Twenty-year-biopsy patients (N=91) and 20-year-non-biopsy patients (N=56) were similar in terms of transplant data, adverse events, and liver function tests (LFTs). Twenty-year LBs revealed a 90% prevalence of abnormalities, among which viral chronic hepatitis (VCH) was the most common (46%). Between 15 and 20 years, hepatic structural abnormalities were the only disorder to increase (p=0.008). An individual progression of abnormalities occurred in 56% of patients. At 20 years, the negative and positive predictive values (PV) of LFTs with respect to histological abnormalities were 95% and 18%, respectively; in VCH, Fibrotest and transient elastography displayed poor discriminative ability for fibrosis (80% and 81% discordance, respectively), but were satisfactory regarding significant fibrosis (negative PV of 77.7% and 80%, respectively). A decrease in immunosuppression was less frequent (14/91 vs. 20/56, p=0.008) while an increase was more common (15/91 vs. 2/56, p=0.017) in 20-year-biopsy patients than in non-biopsy patients. Antiviral therapy was administered in seven of the 20-year biopsy patients, but in none of the non-biopsy patients (p=0.04). CONCLUSIONS: Twenty-year LBs provided important histological information on graft function that was available to a limited degree from LFTs and non-invasive markers. They exerted an impact on immunosuppressive and antiviral therapies.

Clinical and prognostic significance of antinuclear antibodies in primary antiphospholipid syndrome: A multicenter retrospective study.

INTRODUCTION: The antiphospholipid syndrome (APS) (1) is defined by the development of vascular thrombosis, or pregnancy morbidity in the presence of persistent antiphospholipid antibodies (aPL). Antinuclear antibodies (ANA) can be detected in primary APS patients without any clinical systemic autoimmune disease. The presence of ANA antibodies could confer a specific phenotype in primary APS. OBJECTIVE: To evaluate the characteristics of APS patients with antinuclear antibodies without other autoimmune disease (ANA positive APS patients) in comparison with primary APS without ANA or secondary APS patients with associated systemic lupus erythematosus (SLE). METHODS: Clinical and biologic data from 195 APS were retrospectively collected and patients were classified as primary APS with positive ANA (ANA-positive APS), primary APS without any ANA (ANA-negative APS), and SLE-associated APS (SLE-APS). RESULTS: Fourty patients (21%) were classified into ANA-positive APS group, 77 (39%) in ANA-negative APS and 78 (40%) in SLE-APS. In ANA-positive APS patients, 20 patients (51%) had arterial thrombosis, 14 (41%) had veinous thrombosis and 19% had obstetrical complications. There was no difference between the three groups for the frequency of thrombotic manifestations and obstetrical complications. ANA-positive APS patients had more non-criteria manifestations than ANA-negative APS (48% versus 25%; P≤0.01). ANA-positive APS had more triple aPL positivity (59% versus 18%; P<0.001) and more thrombosis and obstetrical recurrences (63% versus 36%; P<0.01) in comparison with ANA-negative APS patients. ANA-positive APS had more triple aPL positivity than SLE-APS patients (54% versus 33%; P<0.05). ANA-positive APS and SLE-APS patients had similar clinical manifestations, and recurrences. Despite a limited follow-up (28 months (11-50)) none of the ANA-positive APS develop SLE. Antiplatelet and anticoagulant therapies were similar for the three groups. SLE-APS patients received more immunomodulatory therapies. CONCLUSION: ANA positivity in patients with APS enables to individualize a subset of patients with a more severe phenotype. Whereas the ANA positivity does not seem to be associated with the risk to develop SLE, prospective studies with a longer follow-up are necessary, in particular to evaluate the effect of additional therapies in this subset of APS.

Serum antibodies to Mycobacterium avium subspecies paratuberculosis combined with anti-Saccharomyces cerevisiae antibodies in Crohn's disease patients: prevalence and diagnostic role.

BACKGROUND: Because Mycobacterium avium subspecies paratuberculosis (MAP), the etiologic agent of Johne's disease in ruminant, has been identified in the mucosal layer and deeper bowel wall in CD patients, the seroactivity against MAP may define a distinct subset of patients requiring individual treatment. The aim of this study was to assess the performance of anti-MAP antibodies in the diagnostic strategy for CD. METHODS: Two hundred seventy-two individuals were included: 81 with CD, 36 with ulcerative colitis, 35 with coeliac diseases and 120 healthy blood donors. Anti-MAP were detected by ELISA using a purified protein derivative from MAP. Anti-Saccharomyces cerevisiae antibodies (ASCA) were detected by indirect immunofluorescence. RESULTS: The sensitivity and specificity of anti-MAP and ASCA for CD diagnosis were similar (sensitivity: 0.33 ± 0.10 and 0.31 ± 0.10; specificity: 0.96 ± 0.03 and 0.98 ± 0.02, respectively). A combination of these two tests enabled an increase in sensitivity (0.53 ± 0.10), although specificity remained unchanged (0.95 ± 0.04). No correlation was found between anti-MAP positivity and clinical features such as age at onset and the duration of CD, disease location, or intestinal complications. Conversely, extra-intestinal manifestations of CD were statistically associated with a positivity of anti-MAP (48% vs. 24%, P = 0.028), mostly with respect to arthritis (44.5% vs. 13%, P < 0.002). Interestingly, anti-MAP and ASCA were also found in an active form of coeliac disease. CONCLUSION: Our results suggest a complementary role of ASCA and anti-MAP for CD diagnosis and a possible common role of bacteria in small intestinal mucosal damage in CD and coeliac disease.

Weak immunogenicity of SARS-CoV-2 vaccine in patients with hematologic malignancies.

This study evaluated the safety and immunogenicity of BNT162b2 vaccine in patients with hematological malignancies. Antibodies blocking spike binding to immobilized ACE-2 (NAb) correlated with anti-Spike (S) IgG d42 titers (Spearman r = 0.865, p < 0.0001), and an anti-S IgG d42 level ≥3100 UA/mL was predictive of NAb ≥ 30%, the positivity cutoff for NAb (p < 0.0001). Only 47% of the patients achieved an anti-S IgG d42 level ≥3100 UA/mL after the two BNT162b2 inocula, compared to 87% of healthy controls. In multivariable analysis, male patients, use of B-cell targeting treatment within the last 12 months prior to vaccination, and CD19+ B-cell level <120/uL, were associated with a significantly decreased probability of achieving a protective anti-S IgG level after the second BNT162b2 inoculum. Finally, using the IFN-γ ELISPOT assay, we found a significant increase in T-cell response against the S protein, with 53% of patients having an anti-S IgG-positive ELISPOT after the second BNT162b2 inoculum. There was a correlation between the anti-S ELISPOT response and IgG d42 level (Spearman r = 0.3026, p = 0.012). These findings suggest that vaccination with two BNT162b2 inocula translates into a significant increase in humoral and cellular response in patients with hematological malignancies, but only around half of the patients can likely achieve effective immune protection against COVID-19.

Identification of plasma membrane autoantigens in autoimmune hepatitis type 1 using a proteomics tool.

UNLABELLED: Autoimmune hepatitis (AIH) is a liver disease with circulating autoantibodies predominantly directed against widely held cellular components. Because AIH is a liver-specific disease, autoantibodies against plasma membrane antigens may be involved in its pathogenesis and have been reported; however, no definite identification has been described. We thus investigated the fine specificity of anti-hepatocyte plasma membrane autoantibodies in type 1 AIH (AIH-1) using a proteomic tool. A plasma membrane-enriched fraction was validated using enzymatic activity and western blot analysis experiments. Sera from AIH-1 patients (n = 65) and from 90 controls, that is, healthy blood donors (n = 40) and patients with systemic diseases (n = 20) or other liver diseases (n = 30), were studied by immunoblot performed with plasma membrane proteins resolved by either sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or 2-dimensional (2D) electrophoresis. Proteins contained in the immunoreactive spots were identified by sequences provided by ion-trap mass spectrometry. Hepatocytes probed with sera were also studied using confocal immunofluorescence and immunoelectron microscopy. The more prominent bands stained by patient sera were located at 38 kDa, 48, 50, 52 kDa, 62 kDa, 70 kDa, and a 95-kDa double band. Six proteins with known potential plasma membrane expression were identified: liver arginase (38 kDa), cytokeratins (CK) 8 and 18 (48-52 kDa), heat shock proteins (HSP) of 60, 70, 90 kDa, and valosin-containing protein (VCP) of 92 kDa. The presence of anti-membrane antibodies was confirmed by immunofluorescence and immunoelectron microscopy. CONCLUSION: Overall, our data demonstrate that liver arginase, CK 8/18, HSP 60, HSP 70, HSP 90, and VCP represent potential candidate targets on liver membrane for autoantibodies in AIH-1.

Significance of antibodies to soluble liver antigen/liver pancreas: a large French study.

BACKGROUND: Antibodies to soluble liver antigen (SLA)/liver pancreas (LP) are generally considered as highly specific diagnostic markers of type 1 auto-immune hepatitis (AIH-1), and are particularly useful in patients without conventional antibodies. However, the presence of anti-SLA/LP in type 2 auto-immune hepatitis (AIH-2), primary sclerosing cholangitis (PSC) and hepatitis C has recently been reported. The aim was thus to describe the characteristics of anti-SLA/LP-positive patients in the largest series reported to date. METHODS: Sera were selected from the period between 1998 and 2005, based on the presence of antibodies to SLA/LP detected by two methods. The clinical status of patients was determined from their medical records. RESULTS: Eighty-one anti-SLA/LP-positive patients with available clinical data were included: 89% (72/81) had a diagnosis of AIH-1, including 10 (12%) associated with cholestatic diseases (primary biliary cirrhosis in seven cases and PSC in three cases). Six patients (7%) suffered from another liver disease: hepatitis C (n=3) and drug-induced hepatitis (n=3). No specific diagnosis was made in three patients. CONCLUSIONS: Antibodies to SLA/LP are of a major diagnostic value for AIH-1, including paediatric forms and overlap syndromes with cholestatic diseases, but are not found in association with anti-liver/kidney/microsome type 1 or antibodies to liver cytosol type 1. They are rarely present in other liver diseases such as hepatitis C and drug-induced hepatitis.

Identification by proteomic tool of atypical anti-liver/kidney microsome autoantibodies targets in de novo autoimmune hepatitis after liver transplantation.

De novo autoimmune hepatitis (AIH) occurs after liver transplantation for nonautoimmune disorders. Autoantibodies so-called atypical anti-liver/kidney microsome antibodies (LKMA) with an unusual liver/kidney cytoplasmic staining as judged by indirect immunofluorescence, can be detected in some patients' sera. Few studies investigated their molecular targets, and the aim of this work was to identify the atypical anti-LKMA targets by proteomic tool. This proteomic approach consisted of (a) two-dimensional gel electrophoresis of cytosolic and microsomal proteins obtained by differential centrifugations of rat liver and rat kidney, followed by (b) two-dimensional immunoblotting with sera of patients with de novo AIH (n = 8, including 2 with anti-LKMA antibodies) and then (c) identifications of interest spots performed by ion trap mass spectrometry. By this way several proteins at 25 kDa were unambiguously identified: isoforms of carbonic anhydrase III, members of different glutathione S-transferase (GST) families, and subunit beta1 of proteasome. This is the first report of proteasome and carbonic anhydrase III as autoantigens in de novo AIH. These results could lead to a better diagnosis of this disease using identified autoantigens in diagnostic tests, and strengthen proteomic approach as a new way of autoantigens investigation.

Remission with ursodeoxycholic acid of type 1 autoimmune hepatitis resistant to azathioprine and steroids.

Combination therapy with steroids and azathioprine is the reference treatment for autoimmune hepatitis, but potential adverse effects are numerous and intolerance can occur. We report a patient with a well-documented type 1 autoimmune hepatitis intolerant to corticosteroids and azathioprine therapy, in whom eight years of ursodeoxycholic acid monotherapy was associated with biochemical and histological remission.

SPRi-based strategy to identify specific biomarkers in systemic lupus erythematosus, rheumatoid arthritis and autoimmune hepatitis.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is a target for antinuclear autoantibodies in systemic Lupus erythematosus (SLE), rheumatoid arthritis (RA), and autoimmune hepatitis (AIH). AIM: To monitor molecular interactions between peptides spanning the entire sequence of hnRNP A2/B1 and sera from patients and healthy controls. METHODS: Sera from 8 patients from each pathology and controls were passed across a surface plasmon resonance Imagery (SPRi) surface containing 39 overlapping peptides of 17 mers covering the human hnRNP B1. Interactions involving the immobilised peptides were followed in real time and dissociation rate constants k(off) for each interaction were calculated. RESULTS: Several significant interactions were observed: i) high stability (lower k(off) values) between P55₋70 and the AIH sera compared to controls (p= 0.003); ii) lower stability (higher k(off) values) between P118₋133 and P262₋277 and SLE sera, P145₋160 and RA sera compared to controls (p=0.006, p=0.002, p=0.007). The binding curves and k(off) values observed after the formation of complexes with anti-IgM and anti-IgG antibodies and after nuclease treatment of the serum indicate that i) IgM isotypes are prevalent and ii) nucleic acids participate in the interaction between anti-hnRNAP B1 and P55₋70 and also between controls and the peptides studied. CONCLUSIONS: These results indicate that P55₋70 of hnRNP B1 is a potential biomarker for AIH in immunological tests and suggest the role of circulating nucleic acids, (eg miRNA), present or absent according to the autoimmune disorders and involved in antigen-antibody stability.

Anti-mitochondrial-2 antibodies (anti-PDC-E2): a marker for autoimmune hepatitis of children?

In an 8-year-old boy with biochemical hepatic disorders, an histological examination of a liver biopsy showed a severe chronic hepatitis without cirrhosis. The biliary tract was normal and no toxic or infectious etiologies were found. Spontaneous improvement of the clinical status was observed in the following weeks but biochemical abnormalities were persistent and a second episode occurred 3 years after. Immunological studies showed anti-mitochondrial-2 antibodies (AMA-2) confirmed by an immunoblot performed with rat mitochondrial proteins resolved by two-dimensional electrophoresis. We described here the second case in the literature of paediatric autoimmune hepatitis associated with well documented AMA-2.

Proteomic analysis of NME1/NDPK A null mouse liver: evidence for a post-translational regulation of annexin IV and EF-1Bα.

NME/NDPK family proteins are involved in the control of intracellular nucleotide homeostasis as well as in both physiological and pathological cellular processes, such as proliferation, differentiation, development, apoptosis, and metastasis dissemination, through mechanisms still largely unknown. One family member, NME1/NDPK-A, is a metastasis suppressor, yet the primary physiological functions of this protein are still missing. The purpose of this study was to identify new NME1/NDPK-A-dependent biological functions and pathways regulated by this gene in the liver. We analyzed the proteomes of wild-type and transgenic NME1-null mouse livers by combining two-dimensional gel electrophoresis and mass spectrometry (matrix-assisted laser desorption/ionization time of flight and liquid chromatography-tandem mass spectrometry). We found that the levels of three proteins, namely, phenylalanine hydroxylase, annexin IV, and elongation factor 1 Bα (EF-1Bα), were strongly reduced in the cytosolic fraction of NME1(-/-) mouse livers when compared to the wild type. This was confirmed by immunoblotting analysis. No concomitant reduction in the corresponding messenger RNAs or of total protein level was observed, however, suggesting that NME1 controls annexin IV and EF-1Bα amounts by post-translational mechanisms. NME1 deletion induced a change in the subcellular location of annexin IV in hepatocytes resulting in enrichment of this protein at the plasma membrane. We also observed a redistribution of EF-1Bα in NME1(-/-) hepatocytes to an intracytoplasmic compartment that colocalized with a marker of the reticulum endoplasmic. Finally, we found reduced expression of annexin IV coincident with decreased NME1 expression in a panel of different carcinoma cell lines. Taken together, our data suggest for the first time that NME1 might regulate the subcellular trafficking of annexin IV and EF-1Bα. The potential role of these proteins in metastatic dissemination is discussed.