Hormonal Regulation of the MHC Class I Gene in Thyroid Cells: Role of the Promoter “Tissue-Specific” Region

In previous studies we have demonstrated that the expression of the Major Histocompatibility Complex (MHC) class I gene in thyrocytes is controlled by several hormones, growth factors, and drugs. These substances mainly act on two regions of the MHC class I promoter a “tissue-specific” region (−800 to −676 bp) and a “hormone/cytokines-sensitive” region (−500 to −68 bp). In a previous study, we have shown that the role of the “tissue-specific” region in the MHC class I gene expression is dominant compared to that of the “hormone/cytokines-sensitive” region. In the present report we further investigate the dominant role of the “tissue-specific” region evaluating the effect of thyroid stimulating hormone (TSH), methimazole (MMI), phenylmethimazole (C10), glucose and thymosin-α1. By performing experiments of electrophoretic mobility shift assays (EMSAs) we show that TSH, MMI and C10, which inhibit MHC class I expression, act on the “tissue-specific” region increasing the formation of a silencer complex. Glucose and thymosin-α1, which stimulate MHC class I expression, act decreasing the formation of this complex. We further show that the silencer complex is formed by two distinct members of the transcription factors families activator protein-1 (AP-1) and nuclear factor-kB (NF-kB), c-jun and p65, respectively. These observations are important in order to understand the regulation of MHC class I gene expression in thyroid cells and its involvement in the development of thyroid autoimmunity.

Anti-inflammatory Effect of the Peptide LKEKK

We have established that the peptide LKEKK (Np5) corresponding to the sequence 16-20 of thymosin-α1 and to the sequence 131-135 of interferon-α2, in the concentration range 50 300 µg/ear reduces in a dose-dependent manner phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin edema in mice .Tested in parallel peptide with inverted sequence (iNp5, KKEKL, 150-300 µg/ear) was inactive, indicating high specificity of the Np5 action. In the concentration range of 5 20 µM Np5 significantly decrease the TNF-α-induced production by normal human keranocytes of pro-inflammatory mediators IL-6 and IL-1β. Thus, Np5t has a pronounced anti-inflammatory activity in vivo and in vitro.

Anti-inflammatory Effect of the Peptide LKEKK

We have established that the peptide LKEKK (Np5) corresponding to the sequence 16-20 of thymosin-α1 and to the sequence 131-135 of interferon-α2, in the concentration range 50 300 µg/ear reduces in a dose-dependent manner phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin edema in mice .Tested in parallel peptide with inverted sequence (iNp5, KKEKL, 150-300 µg/ear) was inactive, indicating high specificity of the Np5 action. In the concentration range of 5 20 µM Np5 significantly decrease the TNF-α-induced production by normal human keranocytes of pro-inflammatory mediators IL-6 and IL-1β. Thus, Np5t has a pronounced anti-inflammatory activity in vivo and in vitro.

Interferon α2–Thymosin α1 Fusion Protein (IFNα2–Tα1): A Genetically Engineered Fusion Protein with Enhanced Anticancer and Antiviral Effect

Human interferon α2 (IFNα2) and thymosin α1 (Tα1) are therapeutic proteins used for the treatment of viral infections and different types of cancer. Both IFNα2 and Tα1 show a synergic effect in their activities when used in combination. Furthermore, the therapeutic fusion proteins produced through the genetic fusion of two genes can exhibit several therapeutic functions in one molecule. In this study, we determined the anticancer and antiviral effect of human interferon α2–thymosin α1 fusion protein (IFNα2–Tα1) produced in our laboratory for the first time. The cytotoxic and genotoxic effect of IFNα2–Tα1 was evaluated in HepG2 and MDA-MB-231 cells. The in vitro assays confirmed that IFNα2–Tα1 inhibited the growth of cells more effectively than IFNα2 alone and showed an elevated genotoxic effect. The expression of proapoptotic genes was also significantly enhanced in IFNα2–Tα1-treated cells compared to IFNα2-treated cells. Furthermore, the HCV RNA level was significantly reduced in IFNα2–Tα1-treated HCV-infected Huh7 cells compared to IFNα2-treated cells. The quantitative PCR analysis showed that the expression of various genes, the products of which inhibit HCV replication, was significantly enhanced in IFNα2–Tα1-treated cells compared to IFNα2-treated cells. Our findings demonstrate that IFNα2–Tα1 is more effective than single IFNα2 as an anticancer and antiviral agent.

Efficacy Evaluation of Thymosin Alpha 1 in Non-severe Patients With COVID-19: A Retrospective Cohort Study Based on Propensity Score Matching

Objective: Thymosin alpha 1 (Thymosin-α1) is a potential treatment for patients with COVID-19. We aimed to determine the effect of Thymosin-α1 in non-severe patients with COVID-19.Methods: We retrospectively enrolled 1,388 non-severe patients with COVID-19. The primary and secondary clinical outcomes were evaluated with comparisons between patients treated with or without Thymosin-α1 therapy.Results: Among 1,388 enrolled patients, 232 patients (16.7%) received both Thymosin-α1 therapy and standard therapy (Thymosin-α1 group), and 1,156 patients (83.3%) received standard therapy (control group). After propensity score matching (1:1 ratio), baseline characteristics were well-balanced between the Thymosin-α1 group and control group. The proportion of patients that progressed to severe COVID-19 is 2.17% for the Thymosin-α1 group and 2.71% for the control group (p = 0.736). The COVID-19-related mortality is 0.54% for the Thymosin-α1 group and 0 for the control group (p = 0.317). Compared with the control group, the Thymosin-α1 group had significantly shorter SARS-CoV-2 RNA shedding duration (13 vs. 16 days, p = 0.025) and hospital stay (14 vs. 18 days, p < 0.001). No statistically significant difference was found between the Thymosin-α1 group and control group in duration of symptoms (median, 4 vs. 3 days, p = 0.843) and antibiotic utilization rate (14.1% vs. 15.2%, p = 0.768).Conclusion: For non-severe patients with COVID-19, Thymosin-α1 can shorten viral RNA shedding duration and hospital stay but did not prevent COVID-19 progression and reduce COVID-19-related mortality rate.

The right immune-modulation at the right time: thymosin α1 for prevention of severe COVID-19 in cancer patients

We presented the rationale for the use of thymosin α1 as prophylaxis of severe COVID-19 in cancer patients undergoing active treatment, constituting the background for the PROTHYMOS study, a prospective, multicenter, open-label, Phase II randomized study, currently in its start-up phase (Eudract no. 2020-006020-13). We aim to offer new hope for this incurable disease, especially to frail patient population, such as patients with cancer. The hypothesis of an effective prophylactic approach to COVID-19 would have immediate clinical relevance, especially given the lack of curative approaches. Moreover, in the ‘COVID-19 vaccine race era’ both clinical and biological results coming from the PROTHYMOS trials could even support the rationale for future combinatorial approaches, trying to rise vaccine efficacy in frail individuals.

A critical COVID-19 patient managed with timely evaluation, early prone positioning ventilation, and a multi-pronged pharmacotherapy

There is not yet a standard drug regimen for the treatment of coronavirus disease 2019 (COVID-19) patients. Here, we summarize our experience and successful treatment plan with a critical COVID-19 patient who required mechanical ventilation (MV). A 56-year-old man presented with a fever, cough, and dyspnea. He had not been to a medium/high risk epidemic area in the past year and had no family history of a disease cluster. COVID-19 was suspected based on clinical symptoms and radiologically detected ground-glass lung changes in the context of a normal white blood cell count (WBCC) and lymphocyte fraction (L%). A diagnosis of COVID-19 was confirmed by nucleic acid testing. Initially, he was started on noninvasive ventilation (NIV). Because his respiratory distress worsened over the following 2 h, he was transitioned to mechanical ventilation (MV), placed in prone positioning 12 h/day, and given a multi-pronged pharmacotherapy regimen that included an antiviral cocktail (lopinavir/ritonavir plus α-interferon), an immunity enhancer (thymosin α1), an anti-coagulant to prevent thrombosis (heparin). He was given an antibiotic to treat an opportunistic nosocomial infection. The patient has recovered well. The regimen applied in this case of timely evaluation, early prone positioning with MV, and a multi-pronged pharmacotherapy may be an effective strategy for patients with critical COVID-19, particularly with respect to preventing life-threatening worsening of the illness.

PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.

Thymosin α1; Potential Therapeutic Implications of an Immunoregulatory Drug in the Treatment of Lymphocytopenia Associated with COVID-19

It is not unbeknownst to us that since the very onset of the COVID-19 outbreak, many patients from different age groups have suffered greatly, and in a remarkable number of cases, succumbed to their untimely demise as a result of infection with the novel coronavirus or SARS-CoV- -2. The elderly are perhaps the most vulnerable community, who stand at the pinnacle of morbidity and mortality rates due to contracting severe forms of COVID-19. Hopefully, based on the recent findings and the present evidence, there might be a number of medications that would possibly be of great prophylactic and therapeutic value to the elderly patients diagnosed with COVID-19. According to an interventional study, Thymosin α1 is arguably one such medication that has recently been indicated to be an effective therapeutic agent for inpatient management of lymphocytopenia and T cell exhaustion caused by COVID-19.