Thyroid hormones and platelet activation in COVID-19 patients.

PURPOSE: To retrospectively describe the association between thyroid hormones (TH) and platelet activation, as represented by mean platelet volume (MPV), in a cohort of patients hospitalized for COVID-19 with no known thyroid disease, and to correlate these data with the severity of COVID-19 and the occurrence of death/ARDS (Acute Respiratory Distress Syndrome). METHODS: 103 patients with real-time polymerase chain reaction (RT-PCR) testing-confirmed COVID-19 and hospitalized were enrolled. Serum samples were collected from patients upon admission before starting any treatment. Chi-squared test was used to determine the association between euthyroid sick syndrome (ESS) and COVID-19 severity. Multivariate logistic regression was performed to evaluate the best independent predictors of COVID-19 deaths/ARDS. RESULTS: 39/103 (37.9%) of patients were found to have ESS, and this condition was an independent predictor for the severity of COVID-19 (p = 0.003). Lower TSH and lower FT3/FT4 ratio correlated with higher MPV (p = 0,001 and p = 0.010), with an opposite trend with respect to what has been documented in non-COVID patients. Increasing MPV and lower FT3 significantly increased the risk, in COVID-19 patients, of an adverse outcome of death/ARDS. CONCLUSION: Increased platelet activation, as represented by increased MPV, has already been reported to correlate with COVID-19 severity, possibly as a consequence of cytokine release. We demonstrated, in a cohort of 103 patients with COVID-19, that MPV is inversely correlated to TH levels, in particular in the case of ESS, where downregulation of TH axis may occur in case of systemic cytokine inflammation and more severe outcomes (death/ARDS). That ESS itself may directly cause platelet activation, as demonstrated by higher MPV in these patients, is an interesting hypothesis which deserves further investigation.

Serum 25-hydroxy vitamin D levels in essential hypertension.

Vitamin D may have prognostic value in hypertension patients and, in addition to conventional biomarkers, could be a valuable tool for disease management. The aim of this study was to assess the association of vitamin D status in patients with essential hypertension and to evaluate its prognostic utility. Forty-eight consecutive patients (40 Caucasian and 8 Asian) aged between 30 and 80 years (mean 61.5, range 34-84 years), were enrolled in the study. The main exclusion criteria were age less than 18 years, kidney failure, onco-hematologic disease, hypo-hyperparathyroidism, osteoporosis, treatment with bisphosphonate or 25(OH) vitamin D supplementation. Of the 48 patients included in the study, hyperlipidemia was described in 28, diabetes type 2 in 8, and ischemic heart disease in 14. Serum electrolytes, calcium, sodium, and potassium concentrations were within normal range. Low 25(OH) vitamin D levels inversely correlated with essential hypertension values (p less than 0.001) were considered extremely significant. The determination of 25(OH) vitamin D levels in patients with essential hypertension could improve the research for possible underlying conditions, which should be managed meticulously according to current guidelines.

Performance assessment of a fully automated electro-chemiluminescence immunoassay system for serum S100B protein.

The altered expression levels of S100 proteins can lead to four different categories of diseases: diseases of the heart and of the central nervous system, inflammatory disorders and cancer. Various studies have shown the lack of harmonization of the results obtained with different methods, mainly due to different performances and measurements of S100B. The purpose of this work was to compare quantitatively the fully automated Elecsys® immunoassay with the reference immunoenzimatic method CanAg® EIA for serum S100B protein. In the study serum samples were analyzed of 161 patients: 85 females (aged 22-83 years) and 76 males (aged 16-90 years), affected by oncological and non-oncological pathologies. Passing–Bablok regression was used to analyze the comparison between the assays; it showed a strong interassay correlation: r = 0.9350 (95% CI =0.9122 – 0.9520), with an intercept of 0.02063 (95% CI=-0.02850 – 0.01400) and a slope of 1.1125 (95% CI=1.0200 – 1.2417). Elecsys® S100 assay should be preferred to CanAg® S100 for better standardization, good reliability and precision but also with the aim to reduce costs and obtain results in a shorter time.

Serum 25-hydroxy vitamin D concentration in acute coronary syndrome.

Vitamin D may have prognostic value in cardiovascular disease (CVD) patients and, in addition to conventional biomarkers, could be a valuable tool for disease management. The aim of this study was to assess the association of vitamin D status in patients with acute coronary syndrome (ACS) and to evaluate its prognostic utility. The levels of 25(OH) vitamin D were correlated with troponin T hs. Forty-eight consecutive outpatients (40 Caucasian and 8 Asian) aged between 40 and 70 years (mean 61.5, range 43-77 years) were enrolled in the study. All patients were admitted to the Emergency Department with chest pain and suspected ACS. The main exclusion criteria were age <18 years, kidney failure, onco-haematological disease, hypo-hyperparathyroidism, hypo/hyperthyroidism, osteoporosis, treatment with bisphosphonate or 25(OH) vitamin D supplementation. Of the 48 subjects included in the study, thoracic pain symptoms were described in 12 patients with unstable angina (UA) and in 6 patients with ST elevation myocardial infarction (STEMI) and in 30 patients with non-ST-elevation myocardial infarction (NSTEMI). Low 25(OH) vitamin D levels correlated with the presence of ACS (p< 0.02) and inversely correlated with Troponin T hs (TnT hs) levels (p< 0.03). The determination of 25(OH) vitamin D levels in combination with TnT hs could improve the research for possible underlying conditions, and these should be managed meticulously according to current guidelines.

Role of biomarkers in adult sepsis and their application for a good laboratory practice: a pilot study.

This study measured Procalcitonin (PCT), Presepsin (PRE-S) and pro-Adrenomedullin (pro-ADM) in intensive care unit (ICU) patient’s blood to assess their contribution to accurate diagnosis of sepsis and potential predictive impact on prognosis. The final aim was to improve the use of infection biomarkers for optimizing the impact of laboratory medicine on clinical outcomes, focusing on the good management of resources designed to produce maximum effectiveness and efficiency. Sixty-four adult patients were studied during their hospitalization in ICU; blood samples were collected and categorized according to their clinical diagnosis and illness severity, and sepsis marker levels were measured on automated immunoassay platforms. PCT, PRE-S and pro-ADM infection markers were significantly lower in controls than in sepsis or septic shock groups. The area under the curve, by ROC curve analysis, was 0.945 for PCT, 0.756 for PRE-S and 0.741 for pro-ADM. Sepsis diagnostic accuracy was not improved by combining PCT, PRE-S and pro-ADM measures. Preliminary data demonstrated that, despite PRE-S and pro-ADM being able to differentiate between septic and non-septic patients with accuracy, PCT remains the most reliable marker available. The results obtained still do not allow us to consider a combination of markers, because it would merely increase laboratory costs without improving diagnostic performance. Furthermore, the results confirm a possible prognostic role of pro-ADM in septic states, but no correlation between biomarker levels and survival at 48 h was detected. Hence PCT, PRE-S, nor pro-ADM can be used to predict short-term prognosis.

Assessing the association between 25-OH vitamin D levels and ROMA score in a population of obese women.

The “Risk of Malignancy Algorithm” (ROMA) combines the diagnostic power of the CA125 and HE4 markers with menopausal status to predict the risk for developing epithelial ovarian cancer (EOC). The aim of this study was to evaluate the association between 25-OH vitamin D levels and ROMA score in obese women. One hundred and eighteen patients with a Body Mass Index (BMI) > 30 kg/m2 (Group 1) and 80 women with a BMI less than 25 kg / m² (Group 2) were studied. The 25-OH vitamin D was quantified with LUMIPULSE® G 1200. As a threshold value, identified by ROC curve analysis, 20.2 ng/ mL (sensitivity 73.3%, specificity 84%) was chosen corresponding to the limit between sufficient and insufficient 25-OH vitamin D according to the World Health Organization (WHO). Low 25-OH vitamin D levels were observed in 64% of obese women and in 11% of normal-weight women (p less than 0.001). ROMA score above 13% was detected only in obese women (19%). An association between low levels of 25-OH vitamin D and ROMA score was observed. Indeed, 64% of obese women with ROMA score >13% had concomitant insufficient levels of 25-OH vitamin D, while only 36% of obese women with ROMA score >13% had sufficient 25-OH vitamin D levels (p less than 0.0001). This study suggests that the deficiency of 25- OH vitamin D in obese women has a possible correlation with high ROMA score.

Transient plasma cell dyscrasia in COVID-19 patients linked to IL-6 triggering.

An unusual clonal gammopathy was reported in COVID-19 patient but whether this anomaly is related or not to the disease has not yet been clarified. To this aim, we selected a cohort of 35 COVID-19 patients swab positive and investigated serological levels of IL-6, immune response to major viral antigens and electrophoretic profile. Elevated levels of IL-6 were accompanied by a significative humoral response to viral Spike protein, revealing an altered electrophoretic profile in the gamma region. We can conclude that elevated levels of IL-6 triggers humoral response inducing a transient plasma cell dyscrasia in severe COVID-19 patients.

PNKP deficiency mimicking a benign hereditary chorea: The misleading presentation of a neurodegenerative disorder.

PNKP gene encodes for a kinase/phosphatase involved in DNA damage response, controlled and stabilized by ATM phosphorylation. PNKP deficiency, thus far described in 40 subjects, has been associated with a complex neurological phenotype encompassing microcephaly, seizures, developmental delay, ataxia, oculomotor apraxia and polyneuropathy. We report a new case expanding the clinical phenotype of this rare disorder. This 25 years old girl presented with chorea at the age of 2 years and remained stable up to the adult age when the emergence of fatigability and asthenia of lower limbs prompted a new examination disclosing a sensory-motor axonal demyelinating neuropathy. Clinical exome sequencing revealed two previously described variants in PNKP gene. This case highlights the phenotypic variability of PNKP associated disorders, showing that an early onset apparently non progressive chorea can be the presenting symptoms of this rare condition.

Automated Intelligent Microscopy for the Recognition of Decoy Cells in Urine Samples of Kidney Transplant Patients.

BACKGROUND: BK virus (BKV)-associated nephropathy is definitely involved in allograft failure after kidney transplant. Thus, the need for an early control of viral reactivation in immunocompromised patients is well established. Determination of urinary release of decoy cells (DC) and BK viral load in plasma and urine by polymerase chain reaction (PCR) usually precedes renal biopsy. The aim of the study is to assess viral reactivation by BKV-DNA PCR and DC detection in urinary sediment using automated intelligent microscopy. METHODS: Seventy-eight kidney transplant patients were analyzed for the presence of plasma BKV-DNA by quantitative TaqMan real-time PCR. Additionally, automated intelligent microscopy was used for urine sediment analysis, allowing to count cells with decoy feature, confirmed by phase contrast microscopic review. RESULTS: Plasma BKV-DNA PCR was detected in 14 (17.9%) patients. DC were identified in 19 (24.3%) urine sediments by automated analyzers and confirmed by microscopic observation. Two patients were BKV-DNA-positive/DC-negative; conversely, 7 subjects were DC-positive/BKV-DNA-negative. CONCLUSIONS: Plasma quantification of BK viral load is currently the best noninvasive method for the detection of viral reactivation. Nevertheless, automated methods to screen for the presence of DC in urine could facilitate early BK virus replication diagnosis and patient follow-up by quantitative and visual results.

Human herpesvirus 6 variant A, but not variant B, infects EBV-positive B lymphoid cells, activating the latent EBV genome through a BZLF-1-dependent mechanism.

Human herpesvirus 6, a predominantly T lymphotropic virus, has been recently shown to infect some EBV-positive B cell lines, and to induce in them the activation of the EBV lytic cycle. Here we have confirmed and extended such observations, showing that (1) this phenomenon is restricted to the variant A of HHV-6: in fact two isolates belonging to the HHV-6 variant B (BA92 and Z29) were neither able to infect any B cell line, independently of the EBV status, nor to induce the EBV genome expression. The only exception is represented by the P3HR1 cells, in which, however, the infection by the variant B does not determine induction of EBV antigens; (2) the presence of the EBV genome contributes to the susceptibility of the B cell lines to HHV-6 infection, increasing the binding sites and the percentage of infectable cells, as detected by immunoelectron microscopy; and (3) HHV-6 infected T cells, transfected with plasmids bearing the promoter regions of the EBV early genes BZLF1 and BMRF1, show a strong transactivation of these promoters.

Infection by human herpesvirus 6 (HHV-6) of human lymphoid T cells occurs through an endocytic pathway.

We have analyzed by immunoelectron microscopy the early events of binding and internalization of human herpesvirus 6 (HHV-6, strain GS) on a susceptible T-lymphoblastoid cell line, HSB-2. The virions bound to the cell surface at 4 degrees C were tightly associated with the plasma membrane. Gold immunolabeling of the viral envelope proteins was strong and specific. Warming at 37 degrees C for different times showed viral internalization through smooth surfaced pits and vesicles. Fusion events of the virions with the cell plasma membrane were never observed. Gold immunolabeling performed in parallel experiments before or after viral internalization showed: (1) absence of viral envelope proteins on the cell plasma membranes at all times of internalization, again excluding fusion events; (2) entry of the virions with their envelopes. Treatment of the cells with chloroquine, a drug known to affect the endocytic pathway, led to an almost complete inhibition of viral infectivity, suggesting that the endocytosed virions are responsible for a successful infection. Comparable results were obtained using a second strain of HHV-6 (BA92), with biologic and molecular characteristics similar to the prototype strain Z29. The chloroquine inhibition was effective on two different T cell lines (HSB-2 and J-Jhan), as well as on phytohemagglutinin-stimulated peripheral blood mononuclear cells.

The benign cystic lymphoepithelial lesion of the parotid gland is a viral reservoir in HIV type 1-infected patients.

The presence of HIV-1 in cystic fluid aspirates from six cases of benign cystic lymphoepithelial lesion (BLL) of the parotid gland, a rare disorder affecting HIV-1-infected patients, has been investigated. HIV-1 p24 protein was present at a concentration ranging from 3 to 15 ng/ml, while it was undetectable in the peripheral blood of the same patients. The number of RNA copies of HIV-1 in the cystic fluids was high, ranging from 0.5 x 10(7) to 7.2 x 10(7) RNA copies/ml. BLL cystic fluid aspirates, despite the high level of HIV-1 RNA, were found to contain only a few infectious virions. The low infectivity correlated with the infrequent detection by electron microscopy of complete HIV-1 particles. The pathogenic mechanism leading to virus accumulation in the cystic fluid was studied by immunohistochemistry of tissue sections. p24 protein was associated with DRC-1+/S-100+ follicular dendritic reticulum cells, which were also present within the cystic cavities. Our findings are consistent with the possibility that the large amounts of virus present in the fluid derive from continuous shedding of HIV-1-infected cells from the surrounding lymphoid tissue.

Neuramide inhibits Epstein-Barr virus-induced transformation activity and proliferation of transformed B lymphocytes.

Neuramide, in Italy, is a prescription drug and contains various polypeptides, with molecular weights ranging between 10,000 and 1,000. It is commonly used in Italy for the clinical therapy of varicella-zoster and other viral herpetic diseases. In the present study, we investigated its effect on the in vitro transformation activity and transformed status of the Epstein-Barr virus (EBV), ab herpesvirus closely associated with infectious mononucleosis (IM), nasopharyngeal carcinoma (NPC) and Burkitt's lymphoma (BL). Antiviral effects of neuramide were evaluated on viral-induced immortalization, transformed status and DNA synthesis. Our results show that simultaneous and/or post-treatment of EBV-infected lymphocytes with neuramide, at a concentration of 2 inhibiting units (IU)/ml, blocks EBV-mediated transformation and related events, whereas pretreatment does not show any inhibiting activity against EBV. Neuramide was also found to be a potent inhibitor of the proliferation of EBV-transformed B lymphocytes in vitro. These results suggest that the molecular mechanisms of action of the drug should be investigated more closely and also support the necessity to further purify the active polypeptides, in order to improve its possible in vivo efficacy in EBV-associated diseases, such as infectious mononucleosis and other lymphoproliferative diseases.

A novel heart/trunk simulator for the study of electromagnetic interference with active implantable devices.

This paper describes a portable heart simulator for the study of electromagnetic interference with active implantable devices. The simulator consists of plexiglas box divided into three chambers simulating the left atrium and the ventricles, plus a lateral compartment for the implantable device. The box is linked to a laptop computer by an analogue-to-digital convertor board, and the three chambers are monitored and driven by dedicated hardware and software interfaces. Synthetic endocardial atrial and ventricle signals for 13 cardiac rhythms are stored in the computer. They are applied to the cardiac chambers by AgCl plates. Sensing electrodes are in the form of AgCl needles inserted in saline. The simulator was able to demonstrate the behaviour of three pacemakers tested in the absence and presence of electromagnetic interference, generated by mobile phones (European GSM 900 and 1800 MHz) that emitted up to 2W (1 W at 1800 MHz). Pacemakers can be programmed with sensitivity from 0.1 mV to 5 mV, pulse width from 0.1 ms to 1.5 ms and pulse amplitude from 0.5 V to 5 V. The structural separation in three cardiac chambers (plus the one for the device) allowed a fast analysis procedure for dual- and tri-chamber implantable devices.

Lymphocyte function tests in cirrhotic patients under treatment with spironolactone and potassium canrenoate.

This controlled study in cirrhotic patients investigated whether two antialdosteronic steroids, spironolactone (100-200 mg/day; n = 12 patient pairs) and potassium canrenoate (50-100 mg/day, n = 32 patient pairs) which are reported to bind to intracellular membranes and modify cytochrome P-450, could also produce nuclear changes. The model used was the response of peripheral lymphocytes to blastogenic agents by studying lymphocyte sub-populations. No changes occurred in the B- and T-lymphocyte sub-populations or in the helper and suppressor sub-types. The response to the blastogenic agents, phytohaemagglutinin and purified protein derived from mycobacteria, did not change significantly from before entry into the study to the follow-up (18.1 +/- 2.9 months). All control patients (n = 44 patient pairs) had slightly greater mitogenic activity compared with patients treated with spironolactone; no difference was found when control patients were compared with patients given potassium canrenoate. The difference between spironolactone and potassium canrenoate might be due to toxicity caused by the thio group of spironolactone. Overall, however, both drugs may be regarded as safe, in terms of effects on lymphatic tissue, occurring during the course of cirrhosis.