Distinct roles of vaccine-induced SARS-CoV-2-specific neutralizing antibodies and T cells in protection and disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific neutralizing antibodies (NAbs) lack cross-reactivity between SARS-CoV species and variants and fail to mediate long-term protection against infection. The maintained protection against severe disease and death by vaccination suggests a role for cross-reactive T cells. We generated vaccines containing sequences from the spike or receptor binding domain, the membrane and/or nucleoprotein that induced only T cells, or T cells and NAbs, to understand their individual roles. In three models with homologous or heterologous challenge, high levels of vaccine-induced SARS-CoV-2 NAbs protected against neither infection nor mild histological disease but conferred rapid viral control limiting the histological damage. With no or low levels of NAbs, vaccine-primed T cells, in mice mainly CD8+ T cells, partially controlled viral replication and promoted NAb recall responses. T cells failed to protect against histological damage, presumably because of viral spread and subsequent T cell-mediated killing. Neither vaccine- nor infection-induced NAbs seem to provide long-lasting protective immunity against SARS-CoV-2. Thus, a more realistic approach for universal SARS-CoV-2 vaccines should be to aim for broadly cross-reactive NAbs in combination with long-lasting highly cross-reactive T cells. Long-lived cross-reactive T cells are likely key to prevent severe disease and fatalities during current and future pandemics.

Proteomic Analysis of Pleural Effusions from COVID-19 Deceased Patients: Enhanced Inflammatory Markers.

Critically ill COVID-19 patients with pleural effusion experience longer hospitalization, multisystem inflammatory syndrome, and higher rates of mortality. Generally, pleural effusion can serve as a diagnostic value to differentiate cytokine levels. This study aimed to evaluate the pleural effusions of COVID-19 deceased patients for 182 protein markers. Olink® Inflammation and Organ Damage panels were used to determine the level of 184 protein markers, e.g., ADA, BTC, CA12, CAPG, CD40, CDCP1, CXCL9, ENTPD2, Flt3L, IL-6, IL-8, LRP1, OSM, PD-L1, PTN, STX8, and VEGFA, which were raised significantly in COVID-19 deceased patients, showing over-stimulation of the immune system and ravaging cytokine storm. The rises of DPP6 and EDIL3 also indicate damage caused to arterial and cardiovascular organs. Overall, this study confirms the elevated levels of CA12, CD40, IL-6, IL-8, PD-L1, and VEGFA, proposing their potential either as biomarkers for the severity and prognosis of the disease or as targets for therapy. Particularly, this study reports upregulated ADA, BTC, DPP6, EDIL3, LIF, ENTPD2, Flt3L, and LRP1 in severe COVID-19 patients for the first time. Pearson's correlation coefficient analysis indicates the involvement of JAK/STAT pathways as a core regulator of hyperinflammation in deceased COVID-19 patients, suggesting the application of JAK inhibitors as a potential efficient treatment.

Morphological changes without histological myocarditis in hearts of COVID-19 deceased patients.

Objective. Patients with underlying heart diseases have a higher risk of dying from Covid-19. It has also been suggested that Covid-19 affects the heart through myocarditis. Despite the rapidly growing research on the management of Covid-19 associated complications, most of the ongoing research is focused on the respiratory complications of Covid-19, and little is known about the prevalence of myocarditis. Design. This study aimed to characterize myocardial involvement by using a panel of antibodies to detect hypoxic and inflammatory changes and the presence of SARS-CoV-2 proteins in heart tissues obtained during the autopsy procedure of Covid-19 deceased patients. Thirty-seven fatal COVID-19 cases and 21 controls were included in this study. Results. Overall, the Covid-19 hearts had several histopathological changes like the waviness of myocytes, fibrosis, contract band necrosis, infiltration of polymorphonuclear neutrophils, vacuolization, and necrosis of myocytes. In addition, endothelial damage and activation were detected in heart tissue. However, viral replication was not detected using RNA in situ hybridization. Also, lymphocyte infiltration, as a hallmark of myocarditis, was not seen in this study. Conclusion. No histological sign of myocarditis was detected in any of our cases; our findings are thus most congruent with the hypothesis of the presence of a circulating endothelium activating factor such as VEGF, originating outside of the heart, probably from the hypoxic part of the Covid-19 lungs.

Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CLpro substrate degradome.

The main viral protease (3CLpro) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CLpro by TAILS substrate-targeted N-terminomics. We identify more than 100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CLpro engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CLpro targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral-autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike in healthy lungs. The 3CLpro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design.

Pulmonary stromal expansion and intra-alveolar coagulation are primary causes of COVID-19 death.

Most COVID-19 victims are old and die from unrelated causes. Here we present twelve complete autopsies, including two rapid autopsies of young patients where the cause of death was COVID-19 ARDS. The main virus induced pathology was in the lung parenchyma and not in the airways. Most coagulation events occurred in the intra-alveolar and not in the intra-vascular space and the few thrombi were mainly composed of aggregated thrombocytes. The dominant inflammatory response was the massive accumulation of CD163 + macrophages and the disappearance of T killer, NK and B-cells. The virus was replicating in the pneumocytes and macrophages but not in bronchial epithelium, endothelium, pericytes or stromal cells. The lung consolidations were produced by a massive regenerative response, stromal and epithelial proliferation and neovascularization. We suggest that thrombocyte aggregation inhibition, angiogenesis inhibition and general proliferation inhibition may have a roll in the treatment of advanced COVID-19 ARDS.

Diagnostic disagreement between clinical standard histopathological- and retrospective assessment of histopathology-based gastrointestinal graft-versus-host disease in children.

BACKGROUND: No previous paediatric study has evaluated the frequency of diagnostic disagreement between clinical standard histopathological assessment (CSHA) and retrospective, independent, histopathological assessment (RIHA) of gastrointestinal Graft-Versus-Host Disease (GI-GVHD) METHODS: In a retrospective cohort study, based on gastrointestinal biopsies collected from allogeneic HSCT-treated children (<18 years) with symptom-based GI-GVHD, we evaluated; disagreement of histopathology-based GI-GVHD diagnosis in CSHA vs RIHA, and potential clinical consequences of differences between the assessments. The CSHA-based diagnoses were retrieved from histopathology reports. The RIHA was performed by one pathologist, blinded to the CSHA outcomes and based on the minimal criteria for histopathology-based GI-GVHD diagnosis by the NIH 2014. RESULTS: Seventy children with 92 endoscopic occasions (including 22 re-endoscopies) were enrolled. GI-GVHD was observed in 73% (67/92) of the endoscopies in the RIHA and in 54% (50/92) in the CSHA (P = .014). The RIHA confirmed 94% (47/50) with GI-GVHD and 52% (22/42) with non-GI-GVHD diagnoses, established in the CSHA. Disagreement, that is endoscopic occasions with GI-GVHD solely detected in RIHA or detection of GI-GVHD in CSHA but not in RIHA, was observed in 20/42 (48%) and 3/50 (6%), respectively (McNemar's test, P = .0008). The risk of a subsequent re-endoscopy was higher in endoscopic occasions with GI-GVHD detected in RIHA but not in CSHA vs if non-GI-GVHD were detected in both readings (P = .005). CONCLUSION: Our results suggest that in children with symptom-based GI-GVHD without histopathological confirmation in CSHA, a second, NIH 2014 based histopathological assessment should be considered before performing a re-endoscopy.

The Effect of Methylselenocysteine and Sodium Selenite Treatment on microRNA Expression in Liver Cancer Cell Lines.

The unique character of selenium compounds, including sodium selenite and Se-methylselenocysteine (MSC), is that they exert cytotoxic effects on neoplastic cells, providing a great potential for treating cancer cells being highly resistant to cytostatic drugs. However, selenium treatment may affect microRNA (miRNA) expression as the pattern of circulating miRNAs changed in a placebo-controlled selenium supplement study. This necessitates exploring possible changes in the expression profiles of miRNAs. For this, miRNAs being critical for liver function were selected and their expression was measured in hepatocellular carcinoma (HLE and HLF) and cholangiocarcinoma cell lines (TFK-1 and HuH-28) using individual TaqMan MicroRNA Assays following selenite or MSC treatments. For establishing tolerable concentrations, IC50 values were determined by performing SRB proliferation assays. The results revealed much lower IC50 values for selenite (from 2.7 to 11.3 µM) compared to MSC (from 79.5 to 322.6 µM). The treatments resulted in cell line-dependent miRNA expression patterns, with all miRNAs found to show fold change differences; however, only a few of these changes were statistically different in treated cells compared to untreated cells below IC50. Namely, miR-199a in HLF, miR-143 in TFK-1 upon MSC treatment, miR-210 in HLF and TFK-1, miR-22, -24, -122, -143 in HLF upon selenite treatment. Fold change differences revealed that miR-122 with both selenium compounds, miR-199a with MSC and miR-22 with selenite were affected. The miRNAs showing minimal alterations included miR-125b and miR-194. In conclusion, our results revealed moderately altered miRNA expression in the cell lines (less alterations following MSC treatment), being miR-122, -199a the most affected and miR-125b, -194 the least altered miRNAs upon selenium treatment.

Clinical relevance of endoscopy with histopathological assessment in children with suspected gastrointestinal graft-versus-host disease.

Endoscopy with histopathological assessment is an established practice to confirm gastrointestinal graft-versus-host disease (GI-GVHD). However, the clinical relevance of this approach in children is incompletely evaluated. In a retrospective cohort study, we investigated the frequency of treatment changes in response to histopathological findings in all children (<18 years) in Sweden who underwent endoscopy for suspected GI-GVHD (2000-2013) after receiving hematopoietic stem cell transplantation. Sixty-eight children with ninety-one endoscopic occasions were enrolled. At the time of endoscopy, anti-GI-GVHD treatment was ongoing in 71% (65/91). In 18% (12/65) with ongoing treatment, no histopathological evidence of GI-GVHD or another cause to justify anti-GI-GVHD treatment was found. In 48% (44/91), endoscopy with histopathological assessment led to changes in the treatment regimen. Re-endoscopy was more frequent among those with treatment changes, versus unchanged treatment, 39% (17/44) and 13% (6/47), respectively (P = .007). Histopathological findings generating treatment changes were as follows: GI-GVHD in 68% (30/44), normal histology in 25% (11/44), and an alternative diagnosis in 7% (3/44). In conclusion, this study supports that endoscopy with histopathological assessment should be considered in all children with suspected GI-GVHD.

Choice of Endoscopic Procedure in Children With Clinically Suspected Gastrointestinal Graft-versus-host Disease.

OBJECTIVES: Gastrointestinal graft-versus-host disease (GI-GVHD) is a potentially life-threatening complication after hematopoietic stem cell transplantation. Symptoms indicating GI-GVHD motivates endoscopy with biopsy sampling and histopathological confirmation. Optimal extent of endoscopy in children is, however, presently unknown. Therefore, we aimed to evaluate whether biopsies from the rectosigmoid area versus the rest of the colon/ileocolon with or without biopsies from simultaneous upper endoscopy, were equally reliable for detection of GI-GVHD and relevant differential diagnoses. METHODS: Retrospective multicenter study based on histopathological re-evaluation of biopsies and hospital record data, collected from children with suspected GI-GVHD. RESULTS: Forty-four children with 51 endoscopic occasions (81 procedures) were included. Thirty-nine of 51 (76.5%) were diagnosed as GI-GVHD, 14 (27.4%) received a differential diagnosis and 7 (13.7%) had normal histology findings. Comorbidity, that is, simultaneous detection of a differential diagnosis and GI-GVHD, was observed in 9 (23.1%) cases. Cytomegalovirus infection was the most frequent differential diagnosis, 6 of 7 were detected in biopsies from rectosigmoid and esophagogastroduodenal areas. Sensitivity for detection of GI-GVHD in biopsies collected from rectosigmoid-ileocolonic-, rectosigmoid-, or esophagogastroduodenal areas were 97.4%, 84.6%, 83.3%, respectively, and 97.4% when the latter 2 were merged. The difference, nondetected GI-GVHD in the rectosigmoid area versus detected elsewhere in the GI tract, was statistically significant (P = 0.03). CONCLUSIONS: Biopsies collected from the rectosigmoid area solely were not optimal for detection of pediatric GI-GVHD. When biopsy sampling from rectosigmoid and upper GI tract areas was combined, the sensitivity for GI-GVHD was, however, equally high as for ileocolonoscopy or full upper and lower endoscopy.

Risk Factors for Invasive Mold Infections and Implications for Choice of Prophylaxis after Allogeneic Stem Cell Transplantation.

Invasive mold infections (IMIs) are major complications after allogeneic hematopoietic stem cell transplantation (HSCT) with high mortality. We retrospectively investigated incidence and risk factors for IMI after 797 HSCTs in a center with high autopsy frequency, trying to identify patient groups that would potentially benefit from mold-active prophylaxis. The cumulative 1-year incidence of IMI was 2.1% in patients aged 21 to 40, 7.1% in patients aged 41 to 60, and 16.4% in patients > 60 years of age (P < .01 for patients aged 21 to 40 versus 41 to 60, P < .001 for patients aged 21 to 40 versus patients > 60). Risk factors for a new IMI in multivariate analysis were older age, grades II to IV acute graft-versus-host disease (GVHD) (risk hazard, 4.1; 95% CI, 1.9 to 8.8; P < .001), treatment with mesenchymal stromal cells (risk hazard, 4.0; 95% CI, 2.1 to 7.8; P < .001), transplantation with female donor to male recipient (risk hazard, 2.2; 95% CI, 1.1 to 4.3; P = .02), and hematopoietic stem cell transplantation-specific comorbidity index over 5 (risk hazard, 2.8; 95% CI, 1.1 to 6.8; P = .03). In patients with grade II acute GVHD, no IMI was seen after onset of acute GVHD in 109 HSCTs performed in patients < 40 years of age, as compared with 14 IMIs in 97 HSCTs (14%) performed in patients > 40 years of age (P < .001). To conclude, older age is an important risk factor for developing IMIs, and patients < 40 years of age with grade II acute GVHD do not appear to need mold-active prophylaxis unless receiving prolonged treatment with corticosteroids.

Epidemiology and outcomes of patients with invasive mould infections: a retrospective observational study from a single centre (2005-2009).

Invasive mould infection (IMI) is an important cause of morbidity and mortality in immunocompromised patients. However, Swedish epidemiology data are lacking. The aim of this study was to investigate the epidemiology and outcome of IMI. Cases of proven/probable IMI at Karolinska University Hospital, Stockholm, from 2005 to 2009, were included. A total of 100 patients with 104 episodes of IMI were enrolled. Identified isolates included 101 mould isolates. The majority of the isolates were Aspergillus spp. (74.3%), followed by Mucorales spp. (13.9%), Fusarium spp. (4.9%) and other mould spp. (6.9%). In 13% of the episodes, more than one mould caused the IMI. The lung was most often affected (88.5%). The most frequent underlying disease was haematological malignancies (70%). Following diagnosis, 83.7% initially received antifungal monotherapy, 9.6% received combination therapy and 6.7% no treatment. The overall 90-day and 1-year overall survival was 49% and 46% respectively. Survival at 90 days post diagnosis was 71.4% in the solid tumour cohort, 62.5% in patients with solid organ transplants, 43.5% in haematological malignancy (HMs) and 37% in those undergoing allogeneic haematopoietic stem cell transplantation (HSCT). Overall survival was poor in the studied cohort, but is variable among different host categories, with particular opportunities for improvement in patients with underlying HMs and allogeneic HSCT.

Posaconazole concentrations in human tissues after allogeneic stem cell transplantation.

Few data have been published regarding posaconazole tissue concentrations in humans. We analyzed tissue concentrations in biopsy specimens taken at autopsy from seven patients who received posaconazole prophylaxis because of graft-versus-host disease. The results were compared to plasma concentrations collected before death. Tissue concentrations suggestive of an accumulation of posaconazole were found in the heart, lung, liver, and kidney but not in the brain.

Improved survival after allogeneic hematopoietic stem cell transplantation in recent years. A single-center study.

We analyzed the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) over the past 2 decades. Between 1992 and 2009, 953 patients were treated with HSCT, mainly for a hematologic malignancy. They were divided according to 4 different time periods of treatment: 1992 to 1995, 1996 to 2000, 2001 to 2005, and 2006 to 2009. Over the years, many factors have changed considerably regarding patient age, diagnosis, disease stage, type of donor, stem cell source, genomic HLA typing, cell dose, type of conditioning, treatment of infections, use of granulocyte-colony stimulating factor (G-CSF), use of mesenchymal stem cells, use of cytotoxic T cells, and home care. When we compared the last period (2006-2009) with earlier periods, we found slower neutrophil engraftment, a higher incidence of acute graft-versus-host disease (aGVHD) of grades II-IV, and less chronic GVHD (cGHVD). The incidence of relapse was unchanged over the 4 periods (22%-25%). Overall survival (OS) and transplant-related mortality (TRM) improved significantly in the more recent periods, with the best results during the last period (2006-2009) and a 100-day TRM of 5.5%. This improvement was also apparent in a multivariate analysis. When correcting for differences between the 4 groups, the hazard ratio for mortality in the last period was 0.59 (95% confidence interval [CI]: 0.44-0.79; P < .001) and for TRM it was 0.63 (CI: 0.43-0.92; P = .02). This study shows that the combined efforts to improve outcome after HSCT have been very effective. Even though we now treat older patients with more advanced disease and use more alternative HLA nonidentical donors, OS and TRM have improved. The problem of relapse still has to be remedied. Thus, several different developments together have resulted in significantly lower TRM and improved survival after HSCT over the last few years.

Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease.

BACKGROUND: Mesenchymal stem cells (MSC) have immunomodulatory effects. The aim was to study the effect of MSC infusion on graft-versus-host disease (GVHD). METHODS: We gave MSC to eight patients with steroid-refractory grades III-IV GVHD and one who had extensive chronic GVHD. The MSC dose was median 1.0 (range 0.7 to 9)x10(6)/kg. No acute side-effects occurred after the MSC infusions. Six patients were treated once and three patients twice. Two patients received MSC from HLA-identical siblings, six from haplo-identical family donors and four from unrelated mismatched donors. RESULTS: Acute GVHD disappeared completely in six of eight patients. One of these developed cytomegalovirus gastroenteritis. Complete resolution was seen in gut (6), liver (1) and skin (1). Two died soon after MSC treatment with no obvious response. One of them had MSC donor DNA in the colon and a lymph node. Five patients are still alive between 2 months and 3 years after the transplantation. Their survival rate was significantly better than that of 16 patients with steroid-resistant biopsy-proven gastrointestinal GVHD, not treated with MSC during the same period (P = 0.03). One patient treated for extensive chronic GVHD showed a transient response in the liver, but not in the skin and he died of Epstein-Barr virus lymphoma. CONCLUSION: MSC is a very promising treatment for severe steroid-resistant acute GVHD.