Employing a systematic approach to biobanking and analyzing clinical and genetic data for advancing COVID-19 research

Within the GEN-COVID Multicenter Study, biospecimens from more than 1000 SARS-CoV-2 positive individuals have thus far been collected in the GEN-COVID Biobank (GCB). Sample types include whole blood, plasma, serum, leukocytes, and DNA. The GCB links samples to detailed clinical data available in the GEN-COVID Patient Registry (GCPR). It includes hospitalized patients (74.25%), broken down into intubated, treated by CPAP-biPAP, treated with O₂ supplementation, and without respiratory support (9.5%, 18.4%, 31.55% and 14.8, respectively); and non-hospitalized subjects (25.75%), either pauci- or asymptomatic. More than 150 clinical patient-level data fields have been collected and binarized for further statistics according to the organs/systems primarily affected by COVID-19: heart, liver, pancreas, kidney, chemosensors, innate or adaptive immunity, and clotting system. Hierarchical clustering analysis identified five main clinical categories: (1) severe multisystemic failure with either thromboembolic or pancreatic variant; (2) cytokine storm type, either severe with liver involvement or moderate; (3) moderate heart type, either with or without liver damage; (4) moderate multisystemic involvement, either with or without liver damage; (5) mild, either with or without hyposmia. GCB and GCPR are further linked to the GCGDR, which includes data from whole-exome sequencing and high-density SNP genotyping. The data are available for sharing through the Network for Italian Genomes, found within the COVID-19 dedicated section. The study objective is to systematize this comprehensive data collection and begin identifying multi-organ involvement in COVID-19, defining genetic parameters for infection susceptibility within the population, and mapping genetically COVID-19 severity and clinical complexity among patients.Subject terms: Genetics research, Viral infection     

Antidepressant response and well-being in pre-, peri- and postmenopausal women with major depressive disorder treated with fluoxetine

BACKGROUND: We assessed the impact of menopausal status on treatment response and well-being in a cohort of outpatient women with major depressive disorder (DSM-III-R criteria), who received treatment with fluoxetine (20 mg/day for 8 weeks). METHODS: Menopausal status was defined based on age, presence of menstrual irregularity or amenorrhea and vasomotor symptoms. Remission and response of depression were defined as a 17-item Hamilton Depression Rating Scale (HAM-D-17) score or=50%, respectively. Well-being was assessed by self-rating with the Symptom Questionnaire. Remitters were followed up for 28 additional weeks. RESULTS: No differences in rates of response and remission as well as in levels of well-being were observed among pre- (n = 121), peri- (n = 28) and postmenopausal (n = 35) women at the endpoint of the acute phase, even after adjustment for baseline depression severity. Residual symptoms, however, were significantly more common in postmenopausal women, except for the continuation phase endpoint. Differences in residual symptoms during the acute phase subsided after adjustment for baseline depression severity. CONCLUSIONS: Overall, menopausal status did not significantly affect the response to fluoxetine treatment and the degree of posttreatment well-being among major depressive disorder patients.     

Efficacy and safety of subcutaneous immunotherapy with a biologically standardized extract of Ambrosia artemisiifolia pollen: a double-blind, placebo-controlled study

BACKGROUND: The allergological relevance of Ambrosia in Europe is growing but the efficacy of the injective immunotherapy for this allergen has been documented only in Northern America. OBJECTIVE: We sought to study the safety and efficacy of injective immunotherapy in European patients sensitized to Ambrosia artemisiifolia. METHODS: Thirty-two patients (18 M/14 F, mean age 36.78, range 23-60 years) suffering from rhinoconjunctivitis and/or asthma and sensitized to Ambrosia were enrolled and randomized in a double-blind, placebo-controlled (DBPC) study lasting 1 year. A maintenance dose corresponding to 7.2 microg of Amb a 1 was administered at 4-week intervals after the build-up. During the second and the third year, all patients were under active therapy in an open fashion. Symptom and medication scores, skin reactivity to Ambrosia (parallel line biological assay), and pollen counts were assessed throughout the trial. RESULTS: Twenty-three patients completed the trial. No severe adverse event was observed. During the DBPC phase, actively treated patients showed an improvement in asthmatic symptoms (P=0.02) and drug (P=0.0068) scores days with asthmatic symptoms (P=0.003), days with rhinitis symptoms (P=0.05), and days with intake of drugs (P=0.0058), as compared to before therapy. No improvement for any of these parameters was detected in the placebo group. Moreover, the number of days with rhinitis and asthma was significantly higher in the placebo as compared to the active group (P=0.048 and P<0.0001, respectively). Patients who switched from placebo to active therapy improved in rhinoconjunctivitis, asthma, and drug intake. The skin reactivity decreased significantly (12.2-fold, P=0.0001) in the active group whereas a slight increase (1.07-fold, P=0.87) was observed in the placebo group after the DBPC phase. After switching to active therapy, patients previously under placebo showed a significant decrease of this parameter (4.78-fold, P=0.002). CONCLUSION: Injective immunotherapy is safe and clinically effective in European patients sensitized to Ambrosia.     

NK-active cytokines IL-2, IL-12, and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells.

Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)-2, IL-12, and IL-15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus-infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL-2, IL-12, and IL-15 on PKCalpha and PKCepsilon--a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKCalpha and PKCepsilon activities; 2) whereas PKCepsilon activity is induced by cytokine stimulation, PKCalpha activity is inhibited; and 3) both the induction of PKCepsilon and the inhibition of PKCalpha functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine-induced NK cell stimulation.     

Primary cutaneous large B-cell lymphoma of the leg: histogenetic analysis of a controversial clinicopathologic entity

This study analyzes the pathologic and molecular features of 5 cases of primary cutaneous large B-cell lymphoma of the leg (PCLBCL-leg), recently included in the European Organization for Research and Treatment of Cancer (EORTC) classification of primary cutaneous lymphoma. PCLBCL-leg accounts for 5% to 10% of all primary cutaneous B-cell lymphoma (PCBCL), usually affects elderly patients and carries a worse prognosis than other forms of PCBCL. It has been proposed that the malignant cells of PCLBCL-leg originate from germinal center (GC)-related cells, but their effective normal counterpart is unclear, and the rationale behind the inclusion of this lymphoma as a separate entity is based on its prognosis rather than on its proved histogenesis. All of our cases of PCLBCL-leg morphologically resembled diffuse large B-cell lymphoma (DLBCL), but to better define their histogenesis, we also analyzed various phenotypic and genotypic markers, including mutations of the Ig and of BCL-6 genes, as well as expression of the bcl-6, MUM1, and CD138/syndecan-1 proteins. Immunohistochemically, all of our cases stained for the L-26/CD20cy and CD79a antigens and expressed the bcl-2, bcl-6, and MUM-1 proteins but were negative for both the CD10/CALLA and CD138 antigens. With respect to molecular analysis, the lymphoma population of all PCLBCL-leg carried hypermutation of Ig genes, and all but 1 case also harbored mutations of the BCL-6 gene. Our results indicate that PCLBCL-leg are similar both under the morphofunctional and molecular profiles to most DLBCL of other sites. Thus, caution seems justified before definitely considering PCLBCL of the leg as a distinct entity.     

A patient with novel MBOAT7 variant: The cerebellar atrophy is progressive and displays a peculiar neurometabolic profile

Mutations in the MBOAT7 gene have been described in 43 patients, belonging to 18 families, showing nonspecific clinical features (intellectual disability [ID], seizures, microcephaly or macrocephaly, and mild to moderate cerebellar atrophy) that make the clinical diagnosis difficult. Here we report the first Italian patient, a 22.5‐year‐old female, one of the oldest reported, born to apparently consanguineous parents. She shows severe ID, macrocephaly, seizures, aggressive outbursts, hyperphagia. We also documented progressive atrophy of the cerebellar vermis, that appeared not before the age of 7. The whole‐exome sequencing of the trio identified a novel homozygous variant c.1057_1058delGCinsCA (p.Ala353His) in the MBOAT7 gene. The variant is considered to be likely pathogenic, since it is absent from population database and it lies in a highly conserved amino acid residue. This disorder has a neurometabolic pathogenesis, implicating a phospholipid remodeling abnormalities. A brain hydrogen‐magnetic resonance spectroscopy (H‐MRS) examination in our patient disclosed a peculiar neurometabolic profile in the cerebellar hemispheric region. This new finding could address the clinical suspicion of MBOAT7‐related disorder, among the wide range of genetic conditions associated with ID and cerebellar atrophy. Moreover, the documented progression of cerebellar atrophy and the worsening of the disease only after some years open to the possibility of a therapeutic window after birth.     

Highly immunogenic and protective recombinant vaccine candidate expressed in transgenic plants

Vaccine development has been hampered by difficulties in developing new and safe adjuvants, so alternative technologies that offer new avenues forward are urgently needed. The goal of this study was to express a monoclonal recombinant immune complex in a transgenic plant. A recombinant protein consisting of a tetanus toxin C fragment-specific monoclonal antibody fused with the tetanus toxin C fragment was designed and expressed. Immune complex formation occurred between individual fusion proteins to form immune complex-like aggregates that bound C1q and FcgammaRIIa receptor and could be targeted to antigen-presenting cells. Unlike antigen alone, the recombinant immune fusion complexes were highly immunogenic in mice and did not require coadministration of an adjuvant (when injected subcutaneously). Indeed, these complexes elicited antibody titers that were more than 10,000 times higher than those observed in animals immunized with the antigen alone. Furthermore, animals immunized with only 1 mug of recombinant immune complex without adjuvant were fully protected against lethal challenge. This the first report on the use of a genetic fusion between antigen and antibody to ensure an optimal expression ratio between the two moieties and to obtain fully functional recombinant immune complexes as a new vaccine model.     

Evidence that amyloid beta-peptide-induced lipid peroxidation and its sequelae in Alzheimer's disease brain contribute to neuronal death

Amyloid beta-peptide [Abeta(1-42)] is central to the pathogenesis of Alzheimer's disease (AD), and the AD brain is under intense oxidative stress, including membrane lipid peroxidation. Abeta(1-42) causes oxidative stress in and neurotoxicity to neurons in mechanisms that are inhibited by Vitamin E and involve the single methionine residue of this peptide. In particular, Abeta induces lipid peroxidation in ways that are inhibited by free radical antioxidants. Two reactive products of lipid peroxidation are the alkenals, 4-hydroxynonenal (HNE) and 2-propenal (acrolein). These alkenals covalently bind to synaptosomal protein cysteine, histidine, and lysine residues by Michael addition to change protein conformation and function. HNE or acrolein binding to proteins introduces a carbonyl to the protein, making the protein oxidatively modified as a consequence of lipid peroxidation. Immunoprecipitation of proteins from AD and control brain, obtained no longer than 4h PMI, showed selective proteins are oxidatively modified in the AD brain. Creatine kinase (CK) and beta-actin have increased carbonyl groups, and Glt-1, a glutamate transporter, has increased binding of HNE in AD. Abeta(1-42) addition to synaptosomes also results in HNE binding to Glt-1, thereby coupling increased Abeta(1-42) in AD brain to increased lipid peroxidation and its sequelae and possibly explaining the mechanism of glutamate transport inhibition known in AD brain. Abeta also inhibits CK. Implications of these findings relate to decreased energy utilization, altered assembly of cytoskeletal proteins, and increased excitotoxicity to neurons by glutamate, all reported for AD. The epsilon-4 allele of the lipid carrier protein apolipoprotein E (APOE) allele is a risk factor for AD. Synaptosomes from APOE knock-out mice are more vulnerable to Abeta-induced oxidative stress (protein oxidation, lipid peroxidation, and ROS generation) than are those from wild-type mice. Further, synaptosomes from allele-specific APOE knock-in mice have tiered vulnerability to Abeta(1-42)-induced oxidative stress, with APOE4 more vulnerable to Abeta(1-42) than are those from APOE2 or APOE3 mice. These results are consistent with the notion of a coupling of the oxidative environment in AD brain and increased risk of developing this disorder. Taken together, the findings from in-vitro studies of lipid peroxidation induced by Abeta(1-42) and postmortem studies of lipid peroxidation (and its sequelae) in AD brain may help explain the APOE allele-related risk for AD, some of the functional and structural alterations in AD brain, and strongly support a causative role of Abeta(1-42)-induced oxidative stress in AD neurodegeneration.     

Idiopathic CD4+ lymphocytopenia may be due to decreased bone marrow clonogenic capability

BACKGROUND: Idiopathic CD4+ lymphocytopenia is defined by a stable decrease of CD4+ T cells in the absence of any known cause of immune deficiency. The mechanisms responsible for the immunological impairment are still unknown, but a regenerative failure of hematopoietic stem/progenitor cells has been hypothesized. METHODS: We evaluated in the bone marrow (BM) of 5 patients with idiopathic CD4+ lymphocytopenia the phenotype of BM progenitor cells, their differentiation capacity with colony-forming cells and long-term culture-initiating cell assays, in parallel with the spontaneous IL-7 production in the patient sera. RESULTS: Compared with controls, a regenerative failure of hematopoietic stem cells has been observed, both in 'committed' and in 'uncommitted' progenitor cells, despite high IL-7 serum levels. The percentage of phenotypically primitive CD34+CD38-DR+ cells (this includes the lymphoid precursor cells) was decreased, suggesting an involvement of the more primitive BM compartment in the de novo T cell generation. CONCLUSIONS: Despite the low number of patients, due to the low incidence of the disease, the decrease of primitive precursors sustains the possibility that diminished stem cell precursors might contribute to the development of CD4+ T cell depletion.