Neutralizing Antibody Titers in Hospitalized Patients with Acute Puumala Orthohantavirus Infection Do Not Associate with Disease Severity

Nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome (HFRS), is an acute febrile illness caused by Puumala orthohantavirus (PUUV). NE manifests typically with acute kidney injury (AKI), with a case fatality rate of about 0.1%. The treatment and management of hantavirus infections are mainly supportive, although neutralizing monoclonal antibodies and immune sera therapeutics are under investigation. In order to assess the potential use of antibody therapeutics in NE, we sought to determine the relationship between circulating PUUV neutralizing antibodies, PUUV nucleocapsid protein (N) IgG antibodies, and viral loads with markers of disease severity. The study included serum samples of extensively characterized patient cohorts (n = 116) from Tampere University Hospital, Finland. The results showed that upon hospitalization, most patients already had considerable neutralizing and anti-PUUV-N IgG antibody levels. However, contrary to expectations, neutralizing antibody titers from the first day of hospitalization did not appear to protect from AKI or correlate with more favorable disease outcomes. This indicates that further studies are needed to investigate the applicability of neutralizing antibodies as a therapy for hospitalized NE patients.     

Hormonal Defects Are Common during Puumala Hantavirus Infection and Associate with Disease Severity and Biomarkers of Altered Haemostasis

Central and peripheral hormone deficiencies have been documented during and after acute hantavirus infection. Thrombocytopenia and coagulation abnormalities are common findings in haemorrhagic fever with renal syndrome (HFRS). The associations between coagulation and hormonal abnormalities in HFRS have not been studied yet. Forty-two patients diagnosed with Puumala virus (PUUV) infection were examined during the acute phase and on a follow-up visit approximately one month later. Hormonal defects were common during acute PUUV infection. Overt (clinical) hypogonadism was identified in 80% of the men and approximately 20% of the patients had overt hypothyroidism. At the one-month follow-up visit, six patients had central hormone deficits. Acute peripheral hormone deficits associated with a more severe acute kidney injury (AKI), longer hospital stay and more severe thrombocytopenia. Half of the patients with bleeding symptoms had also peripheral hormonal deficiencies. Patients with free thyroxine levels below the reference range had higher D-dimer level than patients with normal thyroid function, but no thromboembolic events occurred. Acute phase hormonal abnormalities associate with severe disease and altered haemostasis in PUUV infection.     

Increased Serum Levels of Growth-Differentiation Factor 3 (GDF3) and Inflammasome-Related Markers in Pregnant Women during Acute Zika Virus Infection

The systemic inflammatory response elicited by acute Zika virus (ZIKV) infection during pregnancy plays a key role in the clinical outcomes in mothers and congenitally infected offspring. The present study aimed to evaluate the serum levels of GDF-3 and inflammasome-related markers in pregnant women during acute ZIKV infection. Serum samples from pregnant (n = 18) and non-pregnant (n = 22) women with acute ZIKV infection were assessed for NLRP3, IL-1β, IL-18, and GDF3 markers through an enzyme-linked immunosorbent assay. ZIKV-negative pregnant (n = 18) and non-pregnant women (n = 15) were used as control groups. All serum markers were highly elevated in the ZIKV-infected groups in comparison with control groups (p < 0.0001). Among the ZIKV-infected groups, the serum markers were significantly augmented in the pregnant women in comparison with non-pregnant women (NLRP3 p < 0.001; IL-1β, IL-18, and GDF3 p < 0.0001). The IL-18 marker was found at significantly higher levels (p < 0.05) in the third trimester of pregnancy. Bivariate and multivariate analyses showed a strong positive correlation between GDF3 and NLRP3 markers among ZIKV-infected pregnant women (r = 0.91, p < 0.0001). The findings indicated that acute ZIKV infection during pregnancy induces the overexpression of GDF-3 and inflammasome-related markers, which may contribute to congenital disorders and harmful pregnancy outcomes.     

Secretory Phospholipase A2 Is Associated with the Odds of Acute Coronary Syndromes through Elevation of Serum Amyloid-A Protein

In coronary heart disease (CHD), levels of secretory phospholipase A2 (sPLA2) are commonly increased. Serum amyloid-A (SAA) is increased in acute coronary syndromes (ACS) as well. It is needed to verify the hypotheses that sPLA2 is associated with the odds of ACS through elevation of SAA. We conducted a case–control study with 57 male patients with ACS and 30 controls matched by gender category. Levels of sPLA2, SAA, and myeloperoxidase (MPO) were measured by immunoreactive assay on the basis of a double-antibody sandwich technique. Levels of sPLA2, MPO, and SAA were significantly higher in patients than those in controls (11,359.0 ± 10,372.4 pg/mL vs. 1,320.5 ± 654.5 pg/mL, p = 0.00; 438.6 ± 310.7 ng/mL vs. 271.1 ± 176.8 ng/mL, p = 0.01; 10,995.2 ± 2,842.6 ng/mL vs. 3,861.7 ± 3,173.5 ng/mL, p = 0.00). There were significant correlations between age, visceral obesity, MPO, sPLA2, and SAA (r = 0.43; p = 0.00; r = 0.30; p = 0.00; r = 0.28; p = 0.00 and r = 0.53; p = 0.00). On multivariate logistic regression analyses, there were significant and independent associations between sPLA2 and SAA with odds of ACS [OR (95% CI) = 14.2 (2.1 to 98.6), p = 0.00; OR (95% CI) = 44.9 (6.9 to 328.4), p = 0.00]. Our findings suggest that sPLA2 may be associated with the odds of ACS compared with controls through increased inflammation, represented by elevated SAA.     

Iron Related Biomarkers Predict Disease Severity in a Cohort of Portuguese Adult Patients during COVID-19 Acute Infection

Large variability in COVID-19 clinical progression urges the need to find the most relevant biomarkers to predict patients’ outcomes. We evaluated iron metabolism and immune response in 303 patients admitted to the main hospital of the northern region of Portugal with variable clinical pictures, from September to November 2020. One hundred and twenty-seven tested positive for SARS-CoV-2 and 176 tested negative. Iron-related laboratory parameters and cytokines were determined in blood samples collected soon after admission. Demographic data, comorbidities and clinical outcomes were recorded. Patients were assigned into five groups according to severity. Serum iron and transferrin levels at admission were lower in COVID-19-positive than in COVID-19-negative patients. The levels of interleukin (IL)-6 and monocyte chemoattractant protein 1 (MCP-1) were increased in COVID-19-positive patients. The lowest serum iron and transferrin levels at diagnosis were associated with the worst outcomes. Iron levels negatively correlated with IL-6 and higher levels of this cytokine were associated with a worse prognosis. Serum ferritin levels at diagnosis were higher in COVID-19-positive than in COVID-19-negative patients. Serum iron is the simplest laboratory test to be implemented as a predictor of disease progression in COVID-19-positive patients.     

Folliculin-interacting proteins Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn

Folliculin (FLCN)-interacting proteins 1 and 2 (FNIP1, FNIP2) are homologous binding partners of FLCN, a tumor suppressor for kidney cancer. Recent studies have revealed potential functions for Flcn in kidney; however, kidney-specific functions for Fnip1 and Fnip2 are unknown. Here we demonstrate that Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn. We observed no detectable phenotype in Fnip2 knockout mice, whereas Fnip1 deficiency produced phenotypes similar to those seen in Flcn-deficient mice in multiple organs, but not in kidneys. We found that absolute Fnip2 mRNA copy number was low relative to Fnip1 in organs that showed phenotypes under Fnip1 deficiency but was comparable to Fnip1 mRNA copy number in mouse kidney. Strikingly, kidney-targeted Fnip1/Fnip2 double inactivation produced enlarged polycystic kidneys, as was previously reported in Flcn-deficient kidneys. Kidney-specific Flcn inactivation did not further augment kidney size or cystic histology of Fnip1/Fnip2 double-deficient kidneys, suggesting pathways dysregulated in Flcn-deficient kidneys and Fnip1/Fnip2 double-deficient kidneys are convergent. Heterozygous Fnip1/homozygous Fnip2 double-knockout mice developed kidney cancer at 24 mo of age, analogous to the heterozygous Flcn knockout mouse model, further supporting the concept that Fnip1 and Fnip2 are essential for the tumor-suppressive function of Flcn and that kidney tumorigenesis in human Birt–Hogg–Dubé syndrome may be triggered by loss of interactions among Flcn, Fnip1, and Fnip2. Our findings uncover important roles for Fnip1 and Fnip2 in kidney tumor suppression and may provide molecular targets for the development of novel therapeutics for kidney cancer.Germline alteration of the folliculin (FLCN) gene, a novel tumor suppressor, is responsible for Birt–Hogg–Dubé (BHD) syndrome, an inherited kidney cancer syndrome characterized by cutaneous fibrofolliculomas, pulmonary cysts, and an increased risk for the development of kidney cancer (1–4). Genetic studies using Flcn knockout mice have defined important roles for Flcn in metabolism. Kidney-targeted Flcn knockout mice developed enlarged polycystic kidneys with elevated mechanistic target of rapamycin complex 1 (mTORC1) activity (5) and increased mitochondrial biogenesis through up-regulated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a) (6). Muscle-targeted Flcn knockout mice displayed both red-colored muscle with increased mitochondrial biogenesis caused by elevated Ppargc1, and cardiac hypertrophy with up-regulated mTORC1, which were ameliorated by Ppargc1a inactivation, suggesting that Flcn might serve as a critical link connecting mTORC1 with Ppargc1a-driven mitochondrial biogenesis (5–7). Importantly, mice heterozygous for Flcn inactivation develop renal tumors at 24 mo of age with increased mTORC1/2 activity and Ppargc1a expression, which mimics the human BHD tumor phenotype (6, 8–10), suggesting potential therapeutic targets in metabolic pathways for BHD-associated kidney cancer.The first FLCN interacting protein FNIP1 was identified through protein–protein interaction studies of the FLCN protein (11). FNIP1 binds to the C terminus of FLCN and to AMP-activated protein kinase (AMPK) (11), a critical molecule for energy sensing, further underscoring a central role for the FLCN/FNIP1 pathway in cellular metabolism. A second folliculin-interacting protein FNIP2 was discovered through bioinformatics searches for sequences similar to FNIP1 (12, 13). Similar to FNIP1, FNIP2 was found to bind to the C terminus of FLCN and to AMPK (12), suggesting a potential functional redundancy with FNIP1. Recent studies with Fnip1 knockout mouse models have demonstrated that Fnip1 is required for B-cell development (14, 15). Interestingly, a Flcn knockout mouse model using the tamoxifen-inducible ER (mutated form of the ligand-binding domain of the estrogen receptor)-Cre system also displayed defects in B-cell development (14), suggesting Fnip1 knockout mice might develop phenotypes similar to those that develop as a consequence of Flcn deficiency.Furthermore, FLCN has been shown to have a variety of functions that might potentially link AMPK, mTOR, and Ppargc1a with other important pathways. Crystallographic studies have shown that the C terminus of FLCN may be distantly related to Differentially Expressed in Normal Cells and Neoplasia (DENN) domain proteins and may possess guanine nucleotide exchange factor activity toward RAB35 (16). FLCN modulates TFE3 localization (17), which may play an important role in the exit of stem cells from pluripotency (18), and interacts with other signaling pathways including the von Hippel-Lindau–hypoxia inducible factor–vascular endothelial growth factor axis (19–21), the TGF-beta pathway (22, 23), Rho A signaling (24, 25), cell cycle regulation (26, 27), Rag-mediated amino acid sensing (28, 29), and autophagy (30, 31). These findings underscore FLCN as an important molecule, inactivation of which affects multiple pathways.To clarify the function of FLCN-interacting proteins Fnip1 and Fnip2, we inactivated Fnip1 and/or Fnip2 in mouse kidneys, muscle, and heart and investigated the effect on the mTOR pathway and mitochondrial metabolism. The absolute mRNA copy number of Fnip1 and Fnip2 was measured using droplet digital PCR (ddPCR) technology. To evaluate functional synergy of Fnip1 and Fnip2 with Flcn, we also inactivated Flcn, Fnip1, and Fnip2 simultaneously in mouse kidneys. Finally, we searched for latent tumor development in Fnip1 and Fnip2 knockout mice.     

The Elevated De Ritis Ratio on Admission Is Independently Associated with Mortality in COVID-19 Patients

Liver damage in COVID-19 patients was documented as increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels or an elevated AST/ALT ratio, known as the De Ritis ratio. However, the prognostic value of the elevated De Ritis ratio in COVID-19 patients is still unknown. The aim of our study was to evaluate the prognostic value of the De Ritis ratio compared to other abnormal laboratory parameters and its relation to mortality. We selected 322 COVID-19 patients in this retrospective study conducted between November 2020 and March 2021. The laboratory parameters were measured on admission and followed till patient discharge or death. Of the 322 COVID-19 patients, 57 (17.7%) had gastrointestinal symptoms on admission. The multivariate analysis showed that the De Ritis ratio was an independent risk factor for mortality, with an OR of 29.967 (95% CI 5.266–170.514). In ROC analysis, the AUC value of the the De Ritis ratio was 0.85 (95% CI 0.777–0.923, p < 0.05) with sensitivity and specificity of 80.6% and 75.2%, respectively. A De Ritis ratio ≥1.218 was significantly associated with patient mortality, disease severity, higher AST and IL-6 levels, and a lower ALT level. An elevated De Ritis ratio on admission is independently associated with mortality in COVID-19 patients, indicating liver injury and cytokine release syndrome.     

Serum Copeptin and Zinc-α2-glycoprotein Levels Are Novel Biomarkers of Tolvaptan Treatment in Decompensated Cirrhotic Patients with Ascites

Objective The efficacy of tolvaptan, an orally active vasopressin V2-receptor antagonist, has recently been reported in patients with massive ascites unresponsive to conventional diuretics. However, the effect of tolvaptan varies among patients. Recently, the prognostic role of the tolvaptan response in cases of decompensated liver cirrhosis (LC) has been attracting increasing attention. Using serum copeptin (vasopressin precursor), zinc-α2-glycoprotein (ZAG), cystatin C (renal biomarker), neutrophil gelatinase-associated lipocalin (NGAL) and liver-type fatty acid-binding protein (L-FABP), we explored which factors portend a good response to tolvaptan in LC patients with ascites. Methods We enrolled 113 LC patients and divided them into the tolvaptan treatment group and non-treatment group. Tolvaptan (3.75 or 7.5 mg/day) was administrated to 38 LC patients with ascites, and a follow-up assessment was performed after a 7-day tolvaptan treatment regimen. Results We determined the predictive ability for kidney and/or liver damage of serum copeptin, ZAG, cystatin C, NGAL and L-FABP levels in all patients. After 7-day tolvaptan treatment, 19 patients had lost more than 1.5 kg of body weight (Responders), while 19 showed no marked change in their body weight (Non-responders). Basal blood urea nitrogen (BUN) (p=0.0014), serum copeptin (p=0.0265) and serum ZAG levels (p=0.0142) were significantly higher in the Non-responders than in the Responders. BUN (odds ratio 7.43, p=0.0306), copeptin (odds ratio 9.12, p=0.0136) and ZAG (odds ratio 7.43, p=0.0306) were determined to be predictive factors of drug responsiveness using a multivariate logistic regression analysis. Conclusion Serum BUN, copeptin and ZAG levels predict the patient response to tolvaptan, even when measured prior to treatment.