Maria Addolorata Bonifacio

Autism spectrum disorders (ASD) represent a complex medical and social issue with a multifactorial etiology, often correlated with the host genetic background, particularly with the folate metabolic pathway. This study aimed to investigate the association between MTHFR (C677T and A1298C) and MTRR (A66G) polymorphisms and ASD susceptibility in an Apulian cohort. We analyzed 73 patients diagnosed with ASD and 84 healthy controls recruited at the University Hospital Policlinico, Bari. Genotyping was performed using a Real-time/FRET method. Statistical analysis evaluated genotypic/allelic distributions and haplotype frequencies. While single genotype distributions did not differ significantly between groups, the MTHFR 677T allele frequency was significantly higher in controls (50%) compared to the ASD group (37.67%; p = 0.038), suggesting a potential protective role. Haplotype analysis identified the C-C-G combination as a significant risk factor for ASD (OR = 3.60; p = 0.019). In silico analysis confirmed that the C677T polymorphism (A222V) significantly destabilizes the MTHFR protein structure, in agreement with previous findings. Our findings suggest that an unknown evolutionary advantage may explain the circulation of the MTHFR 677T allele in the general Apulian population, supported by the high folate intake peculiar to the Mediterranean diet. The enzymatic alterations caused by the C-C-G haplotype, selected by the ASD Apulian group, are herein detrimental for folate metabolism.

Background: Acquired haemophilia A (AHA) is a rare autoimmune disorder characterized by the development of autoantibodies against Factor VIII activity, leading to a significant reduction in its functionality. Clinically, AHA presents with an unexpected prolongation of activated partial thromboplastin time (aPTT) and spontaneous bleeding episodes in patients without any personal or family history of haemorrhages. Bleeding manifestations can be severe at presentation, making early diagnosis and prompt treatment essential to reduce morbidity and mortality. Methods: We report on a single-centre cohort of 35 patients with AHA (examined from 1984 to 2024), analysing their demographics, underlying conditions, bleeding characteristics, treatment and outcome. Results: The median age of patients at diagnosis was 69 years (ranging from 18 to 92), 15 were males and 20 females. AHA was idiopathic in 37% of cases, severe bleeding was observed in 54% of patients treated with bypassing agents. Recombinant activated Factor VII (rFVIIa) was administered in 79% of cases and activated prothrombin complex concentrate (aPCC) in 10%, with no significant differences in haemostatic response and no thromboembolic complications. Occurrence of major bleeding showed no significant association with sex, age group, underlying condition, baseline Factor VIII activity or inhibitor titre at diagnosis. A total of 69% of patients were treated with corticosteroids alone, and 23% received a combination of corticosteroids and cyclophosphamide. Two patients died, six were lost to follow-up after partial remission, and one relapsed without bleeds after complete remission. Statistical analyses highlighted that the FVIII inhibitor titre > 20 BU was the only significant prognostic factor affecting time to complete remission. Conclusions: These observations emphasize the critical role of clinical suspicion and timely referral to experienced centres with adequate laboratory support for the effective management of AHA.

Background: Next-generation sequencing (NGS) is gradually replacing Sanger sequencing for HIV genotypic drug resistance testing (GRT). This work evaluated the concordance among different NGS-GRT interpretation tools in a real-life setting. Methods: Routine NGS-GRT data were generated from viral RNA at 11 Italian laboratories with the AD4SEQ HIV-1 Solution v2 commercial kit. NGS results were interpreted by the SmartVir system provided by the kit and by two online tools (HyDRA Web and Stanford HIVdb). NGS-GRT was considered valid when the coverage was >100 reads (100×) at each PR/RT/IN resistance-associated position listed in the HIVdb 9.5.1 algorithm. Results: Among 629 NGS-GRT, 75.2%, 74.2%, and 70.9% were valid according to SmartVir, HyDRA Web, and HIVdb. Considering at least two interpretation tools, 463 (73.6%) NGS-GRT had a valid coverage for resistance analyses. The proportion of valid samples was affected by viremia 10% showed fair concordance among different interpretation tools. Conclusion: This Italian survey on NGS resistance testing suggests that viremia levels and HIV subtype affect NGS-GRT coverage. Within the current routine method for NGS-GRT, only mutations with frequency >10% seem reliably detected across different interpretation tools.

Endothelium damage triggers the multimeric protein von Willebrand factor (VWF) release and subsequent binding to platelets, which are recruited at sites of vascular injury. A complex and fragile equilibrium between circulating levels of von Willebrand factor and its metalloprotease, ADAMTS13, is responsible for the hemostatic balance. However, the presence of autoantibodies targeting ADAMTS13 results in an increase in von Willebrand factor, mainly in its ultra-large multimers. The latter lead to platelet aggregation, the formation of thrombi and microangiopathic hemolytic anemia. This pathologic condition, known as immune-mediated thrombotic thrombocytopenic purpura (iTTP), occurs with high morbidity and a high rate of relapses. In this work, the long-term follow-up of 40 patients with iTTP is reported. We assessed ADAMTS13 activity, plasmatic VWF levels and the ADAMTS13/VWF ratio, comparing iTTP relapsing patients with remitting ones. A decrease in the ADAMTS13/VWF ratio, along with a reduced ADAMTS13 activity, could serve as predictive and sensitive biomarkers of incoming relapses.

The antiphospholipid antibodies (aPL) increase the risk of developing thrombotic events and may coexist with a variety of autoimmune diseases. They can be detected chronically or temporarily in patients with infectious diseases, during drug therapy, or in cases of cancer. A thrombotic event with aPL detection is known as antiphospholipid syndrome (APS) and the diagnostic criteria include the presence of lupus anticoagulant (LA), anticardiolipin (aCL) and β₂-glycoprotein-1(aβ₂GPI) antibodies. Other autoantigens recognized in APS are phosphatidylserine (aPS), prothrombin (aPT) and Annexin-5 (aA5). This real life study aimed to explore the connections between laboratory criteria and the prevalence of “non-criteria aPL” in APS. This study followed 300 patients with thrombosis and employed two phospholipid sensitivity assays for LA detection, chemiluminescence assays for aCL and aβ₂GPI and enzyme-linked immunoassays for aPS, aPT and aA5. A significant association was found between aPS and aCL (r = 0.76) as well as aβ₂GPI (r = 0.77), while the association with LA was less significant (r = 0.33). The results of the aPT and aA5 test did not correlate with criteria-antiphospholipid antibodies (r < 0.30). Since the risk of thrombotic complications increases with the intensity and the number of positive autoantibodies, measuring aPT and aA5 autoantibodies may be useful, particularly in aCL/aβ₂GPI-negative patients or in cases of isolated LA positivity.

After Rudolf Virchow’s pioneering works, technological advances boosted the scientific interest in this research field, which nowadays is still far from extinguished

The treatment of bone injuries must be timely and effective to improve the chances of full recovery. In this respect, a mix of hyaluronic acid and an amino acidic pool has been marketed to promote soft tissue healing, fastening recovery times. Several studies have reported the in vitro and in vivo influence of hyaluronic acid and amino acids on fibroblasts and keratinocytes, highlighting the enhancement of cell proliferation, motility and cytokines synthesis. Even though the effectiveness of this combination of molecules on bone repair has been described in vivo, to the best of our knowledge, its in vitro effects on osteoblasts still need to be investigated. Therefore, this work describes for the first time osteoblast metabolism, proliferation and in vitro differentiation in the presence of hyaluronic acid and amino acids, aiming at understanding the mechanisms underlying their effectiveness in injured tissue repair. The reported results demonstrate the enhancement of osteoblasts’ metabolic activity and the fastening of cell cycle progression. Furthermore, gene expression studies show a significant increase in differentiation markers, i.e., osteoprotegerin and osteonectin. Finally, alkaline phosphatase activity is also boosted by the combination of hyaluronic acid and aminoacids, confirming the ability of in vitro cultured cells to properly differentiate through the osteogenic lineage.

Serological assays are useful in investigating the development of humoral immunity against SARS-CoV-2 in the context of epidemiological studies focusing on the spread of protective immunity. The plaque reduction neutralization test (PRNT) is the gold standard method to assess the titer of protective antibodies in serum samples. However, to provide a result, the PRNT requires several days, skilled operators, and biosafety level 3 laboratories. Therefore, alternative methods are being assessed to establish a relationship between their outcomes and PRNT results. In this work, four different immunoassays (Roche Elecsys^® Anti SARS-CoV-2 S, Snibe MAGLUMI^® SARS-CoV-2 S-RBD IgG, Snibe MAGLUMI^® 2019-nCoV IgG, and EUROIMMUN^® SARS-CoV-2 NeutraLISA assays, respectively) have been performed on individuals healed after SARS-CoV-2 infection. The correlation between each assay and the reference method has been explored through linear regression modeling, as well as through the calculation of Pearson’s and Spearman’s coefficients. Furthermore, the ability of serological tests to discriminate samples with high titers of neutralizing antibodies (>160) has been assessed by ROC curve analyses, Cohen’s Kappa coefficient, and positive predictive agreement. The EUROIMMUN^® NeutraLISA assay displayed the best correlation with PRNT results (Pearson and Spearman coefficients equal to 0.660 and 0.784, respectively), as well as the ROC curve with the highest accuracy, sensitivity, and specificity (0.857, 0.889, and 0.829, respectively).