Clinical and Experimental Medicine

Neutrocyte-to-lymphocyte ratio predicts the presence of a replicative hepatitis C virus strand after therapy with direct-acting antivirals Abstract Residual HCV-RNA can persist in liver tissue and peripheral blood mononuclear cells (PBMCs) long after antiviral therapy of chronic hepatitis C in patients repeatedly negative for viral RNA in serum. This occult infection associates with impaired immune response and the risk of lymphoproliferative disorders or progressive liver disease. There are currently no monitoring strategies for patients after treatment. We investigated if serum inflammation markers and interferon lambda (IFNL) genotype can be predictors of the presence of HCV-RNA and the replicative HCV-RNA (−) strand in patients who reached sustained virological response after interferon-free therapy. Forty-two consecutive patients who remained HCV-RNA negative in serum 24 weeks after the end of treatment (EOT) and during the follow-up were enrolled. Total HCV-RNA and HCV-RNA (−) strand were detected using ultrasensitive RT-PCR in PBMCs collected 12–15 months after EOT. Polymorphisms within IFNL3–IFNL4 region (rs12979860 and ss469415590) were genotyped with allele-specific PCR. Viral RNA was found in PBMCs from 31 (74%) patients, and of those 29 (69%) were also positive for HCV-RNA (−). Neither normalization of alanine aminotransferase nor IFNL genotype predicted the presence of residual HCV-RNA. A significantly higher neutrocyte-to-lymphocyte ratio (NLR) 24 weeks after the start of treatment predicted elimination of replicative HCV-RNA strand (OR 0.23; 95% CI 0.10–0.86; P = 0.019). Patients with no HCV-RNA (−) in PBMCs showed a greater increase in neutrocyte count between EOT and baseline (P = 0.028). Lack of significant elevation of NLR after therapy with direct-acting antivirals could predict the presence of residual replicative HCV-RNA strand in PBMCs.

Ex vivo modelling of the formation of inflammatory platelet-leucocyte aggregates and their adhesion on endothelial cells, an early event in sepsis Abstract Septicaemia is an acute inflammatory reaction in the bloodstream to the presence of pathogen-associated molecular patterns. Whole blood stimulation assays capture endotoxin-induced formation of aggregates between platelets and leucocytes using flow cytometry. We wanted to assess extent of spontaneous aggregate formation in whole blood stimulation assays and compare the effects of endotoxin and heat-killed, clinically relevant, bacterial pathogens on aggregate formation and then on adhesion of aggregates to TNFα-stimulated endothelial cells. We found that endotoxin (from Escherichia coli or Salmonella enteritidis) was not a suitable stimulus to provoke platelet-leucocyte aggregates in vitro, as it did not further increase the extent of aggregates formed spontaneously in stasis of hirudin-anticoagulated blood. Specifically, whole blood samples stimulated with or without LPS produced aggregates with a mean surface area of 140.97 and 117.68 μm2, respectively. By contrast, incubation of whole blood with heat-killed Klebsiella pneumoniae or Staphylococcus aureus produced significantly enhanced and complex cellular aggregates (with a mean surface area of 470.61 and 518.39 μm2, respectively) which adhered more frequently to TNFα (and free fatty acid)-stimulated endothelial cells. These were reliably captured by scanning electron microscopy. Adhesion of cellular aggregates could be blocked by incubation of endothelial cells with a commercial P-selectin antibody and an angiopoietin-2 ligand trap. In conclusion, we have developed an in vitro method that models the acute inflammatory reaction in whole blood in the presence of sepsis-relevant bacterial pathogen surfaces.

Adeno-associated virus as a gene therapy vector: strategies to neutralize the neutralizing antibodies Abstract Adeno-associated virus (AAV)-derived vectors are currently the most common type of viral vectors used in gene therapy clinical trials. The presence of neutralizing antibodies (NAbs) against wild-type AAVs in the host body is one of the limitations for the successful use of AAV vectors. AAV capsid manipulation, by which recombinant vectors lose their ability to interact with NAbs, can help overcome this obstacle. Various methods can be used for this purpose, including directed evolution as well as conjugation of certain chemical groups to AAV epitopes. The present review concisely explains the use of AAV vectors in the clinic for gene therapy of some diseases, their limitations, and solutions to these limitations.

Direct antiviral agents upregulate natural killer cell potential activity in chronic hepatitis C patients Abstract Direct antiviral agents (DAAs) can eliminate hepatitis C virus rapidly and make chronic hepatitis C (CHC) curable. The changes in the innate immune system during treatment with DAAs are still in dispute. To investigate how the functions of natural killer (NK) cells change during and after treatment with DAAs in each NK cell subset. Thirteen CHC patients were treated with sofosbuvir/ledipasvir, and the expression levels of NKp46 and NKG2A were tested via flow cytometry at baseline, at 2, 4, 8 and 12 weeks during the therapy and 12 and 24 weeks after the end of treatment; expression levels were compared between CHC patients and 13 healthy controls. A redirected killing assay was used to detect the cytotoxicity of NK cells. After coculturing NK cells with JFH-Huh7 cells for 72 h, HCV RNA was tested to analyze the inhibition ability of NK cells. All patients achieved sustained virologic response. The expression of the activating receptor NKp46 was decreased first at week 8 during therapy with DAAs and then increased and normalized to levels in healthy controls after treatment with DAAs. The expression of the inhibitory receptor NKG2A was decreased during and after treatment with DAAs. Each NK cell subset has a similar changing trend during and after treatment with DAAs, although some differences can be found earlier and later. The ratio of NKp46 and NKG2A was upregulated after treatment with DAAs. CD56bright NK cells have less amplitude in the frequency ratio changes after treatment with DAAs. The coculture results showed that both the specific lysis and the inhibition of HCV replication were significantly upregulated after treatment with DAAs. DAA treatments can affect patients’ NK cell function. After DAA treatments, the expression of functional markers is downregulated, but the potential activity of NK cells is upregulated. The function of NK cells is normalized to levels in healthy controls. CD56bright NK cells play an important role in this process.

Anti-domain 1 of beta2-glycoprotein I aids risk stratification in lupus anticoagulant-positive patients Abstract Lupus anticoagulant (LA) is considered a risk factor for thromboembolism (TE) and adverse pregnancy outcomes (APOs). However, quite a few patients diagnosed with LA positivity do not suffer these adverse events. Further testing of anticardiolipin (aCL), anti-beta2-glycoprotein I (anti-β2GPI) or anti-domain 1 of β2GPI (anti-D1) may help to assess the occurrence risk of TE and APOs. Therefore, we aimed to study how to stratify LA-positive patients. In our study, 167 LA-positive patients were consecutively enrolled from January 2015 to December 2016. Serum aCL and anti-β2GPI (IgG, IgM and IgA) and anti-D1 IgG were simultaneously measured. Among these patients, 114 (68.3%) were followed for an average of 36.5 months for TE and APOs. The outcomes showed that 105 patients experienced TE and/or APOs, and 62 patients were LA carriers. Anti-D1 had good consistency with triple positivity (LA+, aCL+, anti-β2GPI+) (kappa = 0.742). Elevated anti-D1 was related to increased risks for TE [odds ratio (OR) 29.87, 95% confidence interval (CI) 8.05–110.74] and APOs (OR 8.73, 95% CI 3.41–22.31). Area under curve showed that the diagnostic power of anti-D1 for TE and APOs was 0.856 (95% CI 0.743–0.970) and 0.682 (95% CI 0.599–0.765), respectively. Survival analysis revealed that patients with high anti-D1 titres had a high cumulative incidence of APOs (hazard ratio 4.66, 95% CI 1.46–14.87). In conclusion, anti-D1, based on good consistency with triple positivity in LA-positive patients, has a stronger association with TE and APOs and, to some degree, could predict pregnancy outcomes. Therefore, anti-D1 may aid risk stratification in LA-positive patients.

Functional polymorphisms of the receptor for the advanced glycation end product promoter gene in inflammatory bowel disease: a case–control study Abstract The receptor for the advanced glycation end products (RAGE) is a multiligand transmembrane receptor involved in chronic inflammation whose specific polymorphisms of the promoter gene were found to increase its transcriptional activity. We investigated the association of both allelic and genotypic -374T/A and -429T/C polymorphisms with inflammatory bowel disease. The STREGA guidelines were applied for planning and reporting. We enrolled 133 patients with Crohn’s disease (CD), 149 with ulcerative colitis (UC), and 128 blood donors. Genomic DNA was extracted from peripheral blood leukocytes collected from each patient and control. RAGE polymorphisms were analyzed by PCR-restriction fragment length polymorphism assay. The Hardy–Weinberg equilibrium was first assessed, and then, the Kruskal–Wallis test and the Fisher exact test were used for etiologic group comparisons. Distribution of patients’ characteristics across genotypes was evaluated by the Fisher exact test, while that across alleles was analyzed with a probit model. A 2-sided value of p < 0.05 was considered significant. Following the evidence of the Hardy–Weinberg equilibrium, we found a higher prevalence of the allele A of the -374T/A haplotype in UC (p = 0.043), and of the allele C of the -429T/C haplotype in CD (p < 0.001) with respect to the other groups. Moreover, the homozygous AA genotype of the -374T/A polymorphism resulted associated with late onset of CD, while its TT genotype with early onset (p = 0.049). The allele C of the 429T/C haplotype was associated with early onset of UC (p = 0.03), while a higher frequency of the heterozygous TC haplotype was found in those with pancolitis (p = 0.026). The differing distribution of these polymorphisms in healthy donors and CD/UC patients suggests a role in the development and outcome of these pathological conditions.

Iloprost use and medical management of systemic sclerosis-related vasculopathy in Italian tertiary referral centers: results from the PROSIT study Abstract Vasculopathy is a crucial feature of systemic sclerosis (SSc), and Raynaud’s phenomenon (RP) and digital ulcers (DU) have a deep impact on the quality of patients’ life. The management of vascular disease can be challenging for the clinician because of the suboptimal tolerability of the treatments and lack of consensus on the best therapeutic approach. Intravenous iloprost, a synthetic analogue of prostacyclin, is broadly used for the treatment of RP and ischemic ulcers secondary to SSc. However, no standardized protocol on iloprost use is currently available and, consequently, the management of this treatment is largely based on the experience of each single center. The PROSIT project is an observational, multicenter study aiming to investigate the current treatments for SSc vasculopathy, the use of prostanoids, with special regard to iloprost, and the perception of the treatment from a patient’s perspective. The study was conducted on a cohort of 346 patients from eight Italian centers and included a structured survey addressed to physicians, data collected from patient’s medical records and two patient-administered questionnaires assessing the level of satisfaction, tolerability and perception of the efficacy of Iloprost. PROSIT data confirmed that in the contest of SSc iloprost represents the first-line choice for the management of severe RP and DU. Moreover, it is a well-tolerated treatment as reported by patients’ experience. Although a standard protocol for the treatment of SSc-related vasculopathy is lacking, PROSIT study identified different therapeutic approaches largely supported by tertiary Italian centers. Further studies are needed in order to optimize the best treatment for SSc vascular diseases, in particular to improve the best iloprost schedule management.

Mutation analysis of POLE gene in patients with early-onset colorectal cancer revealed a rare silent variant within the endonuclease domain with potential effect on splicing Abstract The colorectal cancer harbor germline, somatic or epimutations in mismatch repair genes, MUTYH or POLE gene, which lead to the hypermutated and ultramutator phenotypes with increased immune response. The mutations in POLE gene were reported to occur more frequently in early-onset colorectal cancer (EOCRC), and the patients are strong candidates for checkpoint inhibitor therapy. Here, we report mutation analysis within the endonuclease domain of the POLE gene in the cohort of patients with EOCRC in order to identify recurrent or new mutations and evaluate their association with the presence of tumor-infiltrating lymphocytes (TILs) and peritumoral lymphoid reaction. We have shown a significant association between MSI tumors and TILs (p = 0.004). Using sensitive single-tube nested PCR with subsequent Sanger sequencing, we have found in one female patient diagnosed at age 48 with rectal adenocarcinoma with mucinous elements staged pT3pN2pM1 a silent variant within the exon 9 NM_006231.3 c.849 C > T, NP_00622.2 p.Leu283 = recorded in dSNP as rs1232888774 with MAF = 0.00002. In silico prediction, result showed possible involvement into splicing; therefore, this rare variant can be involved into EOCRC pathogenesis. In the time of precise medicine, it is important to develop screening strategies also for less common conditions such as EOCRC allowing to predict tailored therapy for younger patients suffering from CRC that harbor mutations in the POLE gene.

Changes in APRI and FIB-4 in HBeAg-negative treatment-naive chronic hepatitis B patients with significant liver histological lesions receiving 5-year entecavir therapy Abstract According to guidelines, antiviral therapy for adults with immune-active chronic hepatitis B (CHB) should be adopted to decrease the risk of liver-related complications. Fibrosis assessment during antiviral treatment is a key step in antiviral therapy evaluation. Liver biopsy is the gold standard for assessing the degree of liver necroinflammation and fibrosis. However, because of its cost and the risk of life-threatening complications, performing a liver biopsy more than once after long-term effective treatment is difficult. In this study, we aimed to evaluate changes in liver fibrosis during 5 years of entecavir (ETV) treatment using noninvasive fibrosis markers in hepatitis B e-antigen (HBeAg)-negative treatment-naive CHB patients who require antiviral therapy. A total of 303 HBeAg-negative treatment-naive patients were enrolled in this study. Liver biopsy was performed before initiation of antiviral therapy. The diagnosis of CHB was made according to Chinese guidelines for the management of CHB. Patients requiring antiviral therapy (liver fibrosis stage ≥ F2, METAVIR scoring system) were treated with ETV for at least 5 years. These patients were followed up at 6-month intervals. A clinical and virological evaluation was performed at baseline and again at 12, 24, 36, 48, and 60 months during ETV treatment. Aspartate Aminotransferase to Platelet Ratio Index (APRI) and Fibrosis-4 (FIB-4) index were used to assess dynamic changes in liver fibrosis in HBeAg-negative CHB patients after 1, 2, 3, 4, and 5 years of ETV treatment. All enrolled patients underwent liver biopsy at baseline. Using the METAVIR fibrosis stages, there were 107, 125, 54, and 17 patients in F1, F2, F3, and F4 stages, respectively. The APRI and FIB-4 indexes enabled the correct identification of patients with severe fibrosis (METAVIR F3–F4), with an area under the receiver operating characteristic curve of 0.77 (95% confidence interval [CI] 0.72–0.83) and 0.76 (95% CI 0.70–0.82), respectively. The APRI values decreased significantly in F2 and F3 patients after 1 year of ETV therapy (P < 0.01). However, for F4 patients, APRI values decreased significantly at year 3 (P < 0.05). The FIB-4 values of F2, F3, and F4 patients who received ETV treatment were significantly decreased after 1, 3, and 5 years of ETV therapy, respectively (P < 0.05). APRI and FIB-4 values decreased significantly during 5-year ETV treatment in HBeAg-negative CHB patients, indicating that these noninvasive fibrosis tests might be useful for monitoring improvement in liver fibrosis and assessing treatment efficacy during long-term ETV treatment.

Eculizumab in refractory catastrophic antiphospholipid syndrome: a case report and systematic review of the literature Abstract Catastrophic antiphospholipid syndrome (CAPS) is a rare disorder, characterized by the development of multiple vascular thrombosis over a short period of time, in patients with persistently detectable antiphospholipid antibodies (aPLs). The vascular occlusions predominantly affect small vessels. The overall mortality is 36.9%, despite the recent progress in the therapeutic approach. It has been shown that aPLs are able to induce a hypercoagulability state through different mechanisms of action, including complement activation, which in turn plays a key role in the pathogenesis of some thrombotic microangiopathies. Consequently, complement inhibition may be proposed as a targeted intervention to effectively prevent the progression of the microthrombotic storm. The employment of the complement inhibitor eculizumab has been proposed in CAPS on the basis of occasional reports and expert opinion. We report the case of a 54-year-old woman with a CAPS refractory to conventional therapies, who was successfully treated with eculizumab. The administration of this anti-C5 monoclonal antibody aborted the acute progressive thrombotic events and prevented further clinical episodes of thrombosis in the following year. We also faced our case to a systematic literature review, by analyzing all reported cases of CAPS in which eculizumab was added to conventional therapy. Even if further investigation is needed, our results suggest that the inhibition of one mechanism of aPL-induced organ damage may be an add-on treatment for this condition.