The Association between Immune Checkpoint Proteins and Therapy Outcomes in Acute Myeloid Leukaemia Patients.

The development of novel drugs with different mechanisms of action has dramatically changed the treatment landscape of AML patients in recent years. Considering a significant dysregulation of the immune system, inhibitors of immune checkpoint (ICI) proteins provide a substantial therapeutic option for those subjects. However, use of ICI in haematological malignancies remains very limited, in contrast to their wide use in solid tumours. Here, we analysed expression patterns of the most promising selected checkpoint-based therapeutic targets in AML patients. Peripheral blood of 72 untreated AML patients was used for flow cytometric analysis. Expression of PD-1, PD-L1, CTLA-4, and B7-H3 was assessed within CD4+ (Th) lymphocytes and CD33+ blast cells. Patients were stratified based on therapy outcome and cytogenetic molecular risk. AML non-responders (NR) showed a higher frequency of PD-1 in Th cells compared to those with complete remission (CR). Reduced blast cell level of CTLA-4 was another factor differentiating CR from NR subjects. Elevated levels of PD-1 were associated with a trend for poorer patients' survival. Additionally, prognosis for AML patients was worse in case of a higher frequency of B7-H3 in Th lymphocytes. In summary, we showed the significance of selected ICI as outcome predictors in AML management. Further, multicentre studies are required for validation of those data.

The Role and Place of Antioxidants in the Treatment of Male Infertility Caused by Varicocele.

The inability to become pregnant for at least 1 year despite regular unprotected intercourse may indicate infertility of one or both partners. This problem affects approximately 10-20% of couples worldwide, regardless of race, with male infertility reported to account for 25-60% of cases. Among the most common pathological causes of male infertility is the presence of varicocele and chronic infections of the male reproductive system. This study was performed using data collected at the Genesis Infertility Treatment Clinic, Bydgoszcz, Poland, between 1 January 2015 and 30 June 2017. A total of 163 men meeting the inclusion criteria were selected and divided into the idiopathic infertility group (78 men) and varicocele-related infertility group (85 men). All patients received treatment with a male fertility supplement containing a combination of 1725 mg of L-carnitine fumarate, 500 mg of acetyl-L-carnitine, 90 mg of vitamin C, 20 mg of coenzyme Q10, 10 mg of zinc, 200 µg of folic acid, 50 µg of selenium, and 1.5 µg of vitamin B12 (Proxeed® Plus, Sigma-Tau, Italy) twice a day for a period of 6 months from the time of the diagnosis of infertility. The treatment resulted in significant improvements in general semen parameters, particularly sperm count, sperm concentration, total motility, and progressive motility. This antioxidant therapy produced a particularly marked therapeutic benefit in patients with Grade III varicocele, with a greater improvement in progressive motility than in men with less severe or no varicocele. The use of the antioxidant preparation examined here seems reasonable in men with idiopathic infertility and as an adjuvant in those with varicocele-related infertility in whom surgical treatment has resulted in no improvement. Its use should be considered particularly in patients with Grade III varicocele who do not wish to undergo surgical treatment or in whom such a treatment is not possible for various reasons.

Aptamers-Diagnostic and Therapeutic Solution in SARS-CoV-2.

The SARS-CoV-2 virus is currently the most serious challenge to global public health. Its emergence has severely disrupted the functioning of health services and the economic and social situation worldwide. Therefore, new diagnostic and therapeutic tools are urgently needed to allow for the early detection of the SARS-CoV-2 virus and appropriate treatment, which is crucial for the effective control of the COVID-19 disease. The ideal solution seems to be the use of aptamers-short fragments of nucleic acids, DNA or RNA-that can bind selected proteins with high specificity and affinity. They can be used in methods that base the reading of the test result on fluorescence phenomena, chemiluminescence, and electrochemical changes. Exploiting the properties of aptamers will enable the introduction of rapid, sensitive, specific, and low-cost tests for the routine diagnosis of SARS-CoV-2. Aptamers are excellent candidates for the development of point-of-care diagnostic devices and are potential therapeutic tools for the treatment of COVID-19. They can effectively block coronavirus activity in multiple fields by binding viral proteins and acting as carriers of therapeutic substances. In this review, we present recent developments in the design of various types of aptasensors to detect and treat the SARS-CoV-2 infection.

Comparison of 3- and 6-Month Outcomes of Combined Oral L-Carnitine Fumarate and Acetyl-L-Carnitine Therapy, Included in an Antioxidant Formulation, in Patients with Idiopathic Infertility.

The male factor is responsible for infertility in about 35-40% of all cases. Idiopathic oligo- and/or astheno- and/or therato-zoospermia is one of the most common male fertility disorders and remains a significant therapeutic challenge. The primary cause of idiopathic male infertility remains unknown but seems to be associated with oxidative stress. Objective: The use of antioxidative formulation to improve qualitative and quantitative deficiencies in the male gametes.In total, 78 subjects were treated with a combination of 1,725 mg L-carnitine fumarate, 500 mg acetyl-L-carnitine, 90 mg vitamin C, 20 mg coenzyme Q10, 10 mg zinc, 200 µg folic acid, 50 µg selenium, and 1.5 µg vitamin B12 (Proxeed® Plus, Sigma-Tau, Italy) for 6 months; the preparation was taken twice daily from the time idiopathic infertility was diagnosed. Basic seminal parameters were evaluated by a European Society of Human Reproduction and Embryology (ESHRE) -certified embryologist following the fifth edition of the World Health Organisation (2010) guidelines at three time points: at baseline and 3 and 6 months of treatment.Improvements in semen parameters (differing in terms of dynamics) were evident at 3 months and gradually improved over the 6 months of treatment. Each parameter: sperm concentration, total sperm count, sperm total and progressive motility improved significantly after treatment except for the percentage of sperm of abnormal morphology and ejaculate volume.Proxeed Plus was effective for patients with idiopathic infertility; however, a long treatment period is needed to achieve optimal results.

The Latest Achievements in the Construction of Influenza Virus Detection Aptasensors.

Aptamers are short fragments of nucleic acids, DNA or RNA that have the ability to bind selected proteins with high specificity and affinity. These properties allow them to be used as an element of biosensors for the detection of specific proteins, including viral ones, which makes it possible to design valuable diagnostic tools. The influenza virus causes a huge number of human and animal deaths worldwide every year, and contributes to remarkable economic losses. In addition, in 2020, a new threat appeared-the SARS-Cov-2 pandemic. Both disease entities, especially in the initial stage of infection, are almost identical in terms of signs and symptoms. Therefore, a diagnostic solution is needed that will allow distinguishing between both pathogens, with high sensitivity and specificity; it should be cheap, quick and possible to use in the field, for example, in a doctor's office. All the mentioned properties are met by aptasensors in which the detection elements are specific aptamers. We present here the latest developments in the construction of various types of aptasensors for the detection of influenza virus. Aptasensor operation is based on the measurement of changes in electric impedance, fluorescence or electric signal (impedimetric, fluorescence and electrochemical aptasensors, respectively); it allows both qualitative and quantitative determinations. The particularly high advancement for detecting of influenza virus concerns impedimetric aptasensors.

Coronaviruses fusion with the membrane and entry to the host cell.

Coronaviruses (CoVs) are positive-strand RNA viruses with the largest genome among all RNA viruses. They are able to infect many host, such as mammals or birds. Whereas CoVs were identified 1930s, they became known again in 2003 as the agents of the Severe Acute Respiratory Syndrome (SARS). The spike protein is thought to be essential in the process of CoVs entry, because it is associated with the binding to the receptor on the host cell. It is also involved in cell tropism and pathogenesis. Receptor recognition is the crucial step in the infection. CoVs are able to bind a variety of receptors, although the selection of receptor remains unclear. Coronaviruses were initially believed to enter cells by fusion with the plasma membrane. Further studies demonstrated that many of them involve endocytosis through clathrin-dependent, caveolae-dependent, clathrin-independent, as well as caveolae-independent mechanisms. The aim of this review is to summarise current knowledge about coronaviruses, focussing especially on CoVs entry into the host cell. Advances in understanding coronaviruses replication strategy and the functioning of the replicative structures are also highlighted. The development of host-directed antiviral therapy seems to be a promising way to treat infections with SARS-CoV or other pathogenic coronaviruses. There is still much to be discovered in the inventory of pro- and anti-viral host factors relevant for CoVs replication. The latest pandemic danger, originating from China, has given our previously prepared work even more of topicality.

Increased levels of interleukin 27 in patients with early clinical stages of non-small cell lung cancer.

INTRODUCTION    Interleukin 27 (IL­27) is a cytokine secreted mostly by antigen­presenting cells. It is important for the immune polarization of T helper­1 (Th1) cells, and its role in interstitial lung diseases (ILDs) and lung cancer has been investigated. OBJECTIVES    We assessed IL­27 expression in the lower airways of patients with selected ILDs and early­stage non-small cell lung cancer (NSCLC). PATIENTS AND METHODS    IL­27 concentrations were examined by an enzyme­linked immunosorbent assay in bronchoalveolar lavage (BAL) fluid supernatants collected from patients with pulmonary sarcoidosis (PS; n = 30), extrinsic allergic alveolitis (EAA; n = 14), idiopathic pulmonary fibrosis (IPF; n = 12), nonspecific interstitial pneumonia (NSIP; n = 14), and NSCLC stages I to IIa (n = 16) with peripheral localization, and in controls (n = 14). The major lymphocyte subsets in BAL fluid were phenotyped, and intracellular IL­27 expression was evaluated by flow cytometry.  RESULTS    IL­27 concentrations in BAL fluid supernatants were significantly increased in Th1­mediated conditions such as EAA and PS, but not in IPF or NSIP. The highest IL­27 levels (median [SEM], 16.9 [17.5] pg/ml) were reported for NCSLC, and the lowest-for controls (median [SEM], 0.4 [0.2] pg/ml). IL­27 was undetectable in corticosteroid­treated patients with PS. Both CD4+ and CD8+ lymphocytes were positive for IL­27; they were a possible local source of IL­27 because the cytokine levels were positivelysignificantly correlated with the total number of lymphocytes, including CD4+ cells. CONCLUSIONS    Our results support the Th1­linked activity of IL­27in ILDs. Early­stageNSCLC is characterizedby high IL­27expression in the lower airways. IL­27 is produced by a high percentage of CD4+ and CD8+ cells in BAL fluid, both in patients and controls.

Challenges and Prospects for Alpha-1 Antitrypsin Deficiency Gene Therapy.

Alpha-1 antitrypsin (AAT) is a protease inhibitor belonging to the serpin family. A number of identified mutations in the SERPINA1 gene encoding this protein result in alpha-1 antitrypsin deficiency (AATD). A decrease in AAT serum concentration or reduced biological activity causes considerable risk of chronic respiratory and liver disorders. As a monogenic disease, AATD appears to be an attractive target for gene therapy, particularly for patients with pulmonary dysfunction, where augmentation of functional AAT levels in plasma might slow down respiratory disease development. The short AAT coding sequence and its activity in the extracellular matrix would enable an increase in systemic serum AAT production by cellular secretion. In vitro and in vivo experimental AAT gene transfer with gamma-retroviral, lentiviral, adenoviral, and adeno-associated viral (AAV) vectors has resulted in enhanced AAT serum levels and a promising safety profile. Human clinical trials using intramuscular viral transfer with AAV1 and AAV2 vectors of the AAT gene demonstrated its safety, but did not achieve a protective level of AAT >11 µM in serum. This review provides an in-depth critical analysis of current progress in AATD gene therapy based on viral gene transfer. The factors affecting transgene expression levels, such as site of administration, dose and type of vector, and activity of the immune system, are discussed further as crucial variables for optimizing the clinical effectiveness of gene therapy in AATD subjects.

Aptamers in diagnostics and treatment of viral infections.

Aptamers are in vitro selected DNA or RNA molecules that are capable of binding a wide range of nucleic and non-nucleic acid molecules with high affinity and specificity. They have been conducted through the process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). It serves to reach specificity and considerable affinity to target molecules, including those of viral origin, both proteins and nucleic acids. Properties of aptamers allow detecting virus infected cells or viruses themselves and make them competitive to monoclonal antibodies. Specific aptamers can be used to interfere in each stage of the viral replication cycle and also inhibit its penetration into cells. Many current studies have reported possible application of aptamers as a treatment or diagnostic tool in viral infections, e.g., HIV (Human Immunodeficiency Virus), HBV (Hepatitis B Virus), HCV (Hepatitis C Virus), SARS (Severe Acute Respiratory Syndrome), H5N1 avian influenza and recently spread Ebola. This review presents current developments of using aptamers in the diagnostics and treatment of viral diseases.

[Viral transfer of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in gene therapy]. / Wirusowy transfer liganda czynnika martwicy nowotworu indukujacego apoptoze (TRAIL) w terapii genowej.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces carcinoma cell death through the extrinsic pathway of apoptosis. Preclinical trials of gene therapy have been conducted using viral transfer of the TRAIL transgene into prostate, bladder, breast, kidney, liver, non-small cell lung cancer and also glioblastoma cells. Experiments in vitro demonstrated the extensive apoptosis of target cells as well as frequent disease regression or remission. TRAIL transfer did not show any side effects, opposite to chemotherapy. Encouraging results of TRAIL-related gene therapy were observed in rheumatoid arthritis and type 1 diabetes. Adenoviral vectors (AdV) encoding TRAIL are the most promising tool in anti-tumor therapy. They have undergone numerous modifications by increasing transfection efficiency and transgene expression in target cells. However, only one clinical phase I trial has been performed. AdV encoding the TRAIL transgene caused local inflammation and apoptosis in patients with prostate cancer.

Enhanced expression of Fas Ligand (FasL) in the lower airways of patients with fibrotic interstitial lung diseases (ILDs).

The exact role of FasL, and particularly its soluble and membrane-bound forms, in the development of chronic ILDs and lung fibrosis has not been extensively explored. We aimed at analyzing membrane-bound FasL expression on alveolar macrophages (AM) and lymphocytes (AL) as well as soluble FasL (sFasL) levels in bronchoalveolar lavage (BAL) from ILDs patients, incl. pulmonary sarcoidosis (PS), hypersensitivity pneumonitis (HP), silicosis, asbestosis, idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), and healthy subjects (n = 89, 12, 7, 8, 23, 6, 17, respectively). In IPF, significantly increased percentage of AM FasL(+) and CD8(+)FasL(+) cells as well as sFasL levels in BAL were found. Increased sFasL levels were also observed in HP. NSIP and asbestosis were characterized by higher AM FasL(+) relative number; CD8(+)FasL(+) population was expanded in asbestosis only. There was a significant decline in AL FasL(+) percentage in PS and HP. Vital capacity was negatively correlated with sFasL levels, AM FasL(+) and CD8(+)FasL(+) cell relative count. CD4(+)FasL(+) and CD8(+)FasL(+) percentage strongly correlated with BAL neutrophilia, an unfavorable prognostic factor in lung fibrosis. The concurrent comparative BAL analysis of FasL expression indicates that FasL(+) AM and AL (mainly Tc cells) comprise an important element of the fibrotic process, mostly in IPF. FasL might play a crucial role in other fibrosis-complicated ILDs, like NSIP and asbestosis.

[Modifications of the cytoimmunological pattern of bronchoalveolar lavage (BAL) material caused by cigarette smoking in selected lower air-way diseases]. / Modyfikacje obrazu cytoimmunologicznego materialu z plukania oskrzelowo- pecherzykowego (bronchoalveolar lavage, BAL) powodowane przez palenie papierosów w wybranych chorobach dolnych dróg oddechowych.

INTRODUCTION: In the last years we have used flow cytometry as an auxiliary diagnostic tool in alveolar lymphocyte (i.e. originating from BAL) phenotyping in more than 500 persons suspected for lower airways pathology. MATERIAL AND METHODS: In the study we compared the results of 1) BAL lymphocyte typing by flow cytometry, 2) cytological examination, respectively, in nonsmoking/smoking (NS/S) patients with lung sarcoidosis, n = 56/31, extrinsic allergic alveolitis (EAA), n = 9/5, silicosis, n = 15/18, idiopathic pulmonary fibrosis (IPF), n = 20/7, and pulmonary tuberculosis (TBC), n = 7/6. The results were related to the volume of BAL fluid recovery (higher value reflects the dominance of lower airways content versus bronchial content). RESULTS: In smoking patients, in comparison with respective NS, significantly higher total BAL cell numer (except TBC), higher macrophage percentage, lower lymphocyte percentage and lower CD4/CD8 ratio (except EAA) was found. CD4/CD8 results: 8.26 +/- 0.52 (NS) vs 4.29 +/- 0.65 (S) in sarcoidosis (p < 0.001), 1.18 +/- 0.44 (NS) vs 0.99 +/- 0.43 (S) in IPF (p < 0.05), 1.79 +/- 0.22 (NS) vs 0.73 +/- 0.11 (S) in silicosis (p < 0.001) and 1.64 +/- 0.57 vs 0.88 +/- 0.1 in TBC (p < 0.05). Additionally, cigarette smoking modified BAL pattern: 1. in sarcoidosis and silicosis lower CD4+ cell and higher CD8+ cell percentage; 2. in IPF increase in neutrophil percentage; 3. in TBC higher neutrophil and eosinophil percentage. Both in NS and S, BAL fluid recovery rate is significantly positively correlated with CD4/CD8 ratio and total BAL CD3+ cell number and negatively with BAL CD8+ cell percentage. CONCLUSIONS: Interpreting of BAL material cytoimmunology pattern should take into account data on cigarette smoking and BAL fluid recovery rate. The results obtained in the study may reflect more severe disease course in IPF and TBC.