Innate immune response in COVID-19: single-cell multi-omics profile of NK lymphocytes in a clinical case series.

BACKGROUND: COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) represents the biggest global health emergency in recent decades. The host immune response to SARS-CoV-2 seems to play a key role in disease pathogenesis and clinical manifestations, with Natural Killer (NK) lymphocytes being among the targets of virus-induced regulation. METHODS: This study performed a single-cell multi-omics analysis of transcripts and proteins of NK lymphocytes in COVID-19 patients, for the characterization of the innate immunological response to infection. NK cells were isolated from peripheral blood samples collected from adult subjects divided into 3 study groups: (1) non-infected subjects (Naïve group, n = 3), (2) post COVID-19 convalescent subjects (Healed group, n = 3) and (3) patients that were vaccinated against SARS-CoV-2 (Vaccine group, n = 3). Cells were then analysed by the BD Rhapsody System for the single-cell multi-omics investigation of transcriptome and membrane proteins. RESULTS: The bioinformatic analysis identified 5 cell clusters which differentially expressed gene/protein markers, defining NK cell subsets as "Active NK cells" and "Mature NK cells". Calculating the relative proportion of each cluster within patient groups, more than 40% of the Naïve group cell population was found to belong to Mature NKs, whereas more than 75% of the Vaccine group cell population belonged to the cluster of Active NKs. Regarding the Healed group, it seemed to show intermediate phenotype between Active and Mature NK cells. Differential expression of specific genes, proteins and signaling pathways was detected comparing the profile of the 3 experimental groups, revealing a more activated NK cell phenotype in vaccinated patients versus recovered individuals. CONCLUSIONS: The present study detected differential expression of NK cell markers in relation to SARS-CoV-2 infection and vaccine administration, suggesting the possibility to identify key molecular targets for clinical-diagnostic use of the individual response to viral infection and/or re-infection.

Factors associated with blood cord unit bankability: an analysis of a 15-year-long case series.

Blood banking is a long and complex process requiring an accurate screening of potential donors and high-quality control systems. Previous studies in literature investigated factors potentially determining a higher cell levels with the aim of optimizing donors' selection and improving banking process. This study aims to identify factors associated with the concentration of stem cells in umbilical cord blood, so increasing the probability of bankability, focusing on the possible implications in terms of obstetric and resources management. This is a retrospective study conducted in the Obstetric Units of two Italian Hospitals in Montebelluna and Castelfranco Veneto. Study has been conducted on cord blood units banked between 1999 and 2015. Data on medical histories and clinical characteristics of mother and baby have been retrieved via a retrospective examination of medical records. A total of 869 cord blood units were studied. At multivariable analysis, in agreement with literature, birthweight and placental weight have been found to be associated with higher concentration of total nucleated cells. As additional factor, amount of fluid infused was associated with cord blood units' count. This study is the first one to clearly identify the role of fluid infusion on cord blood units' counts in addition to placental weight and delivery. Some non-modifiable features can help in predicting bankability from pre-natal aspects to factors more related with obstetric management is suggested.

New method to produce hemocomponents for regenerative use from peripheral blood: integration among platelet growth factors monocytes and stem cells.

Recent studies have shown the importance of monocytes/macrophageses and of CD34+ progenitors in tissue regeneration processes. These cells, obtained generally from bone marrow, are seen in damaged tissue. We have studied a method to collect from the peripheral blood, using a cell separator and without stimulation of the patient/donor, a leukocyte platelet concentrated hemocomponent (CLP) for regenerative use which contains platelets, monocytes/macrophages, fibrinogen and CD34+ cells. We appraised the composition and cell functionality of the final hemocomponent during production and cryoconservation. The results show a positive increase in concentration values, in comparison with the pre-collection, of the cells that were involved in regeneration; i.e. the platelets, monocytes and CD34+ cells. These concentrations were also maintained at an effective level during cryoconservation of the hemocomponent. The CLP also demonstrated positive clonogenic potential in culture, showing that the CD34+ progenitors involved in CFU formation are functional in the fresh and thawed product. In brief we have shown that it is possible to produce, in a simple way, a hemocomponent for regenerative use that is standardized, reliable, and is economically feasible.

A polymorphic variant of the insulin-like growth factor 1 (IGF-1) receptor correlates with male longevity in the Italian population: a genetic study and evaluation of circulating IGF-1 from the "Treviso Longeva (TRELONG)" study.

BACKGROUND: An attenuation of the insulin-like growth factor 1 (IGF-1) signaling has been associated with elongation of the lifespan in simple metazoan organisms and in rodents. In humans, IGF-1 level has an age-related modulation with a lower concentration in the elderly, depending on hormonal and genetic factors affecting the IGF-1 receptor gene (IGF-1R). METHODS: In an elderly population from North-eastern Italy (n = 668 subjects, age range 70-106 years) we investigated the IGF-1R polymorphism G3174A (rs2229765) and the plasma concentration of free IGF-1. Frequency distributions were compared using chi2-test "Goodness of Fit" test, and means were compared by one-way analysis of variance (ANOVA); multiple regression analysis was performed using JMP7 for SAS software (SAS Institute, USA). The limit of significance for genetic and biochemical comparison was set at alpha = 0.05. RESULTS: Males showed an age-related increase in the A-allele of rs2229765 and a change in the plasma level of IGF-1, which dropped significantly after 85 years of age (85+ group). In the male 85+ group, A/A homozygous subjects had the lowest plasma IGF-1 level. We found no clear correlation between rs2229765 genotype and IGF-1 in the females. CONCLUSION: These findings confirm the importance of the rs2229765 minor allele as a genetic predisposing factor for longevity in Italy where a sex-specific pattern for IGF-1 attenuation with ageing was found.

Adrenomedullin in the growth modulation and differentiation of acute myeloid leukemia cells.

Adrenomedullin (ADM) is a regulatory peptide endowed with multiple biological effects, including the regulation of blood pressure, cell growth and innate host defence. In the present study, we demonstrated that ADM signaling could be involved in the impaired cellular differentiation of myeloid leukemia cells to mature granulocytes or monocytes by modulating RAMPs/CRLR expression, PI3K/Akt cascade and the ERK/MAPK signaling pathway. When exogenously administered to in vitro cultures of HL60 promyelocytic leukemia cells, ADM was shown to exert a strong proliferative effect with minimal upregulation in the expression level of monocyte antigen CD14. Notably, the experimental inhibition of ADM signaling with inhibitor ADM22-52 promoted a differentiative stimulation towards monocytic and granulocytic lineages. Moreover, based on the expression of CD31 relative to CD38, we hypothesized that an excess of ADM in bone marrow (BM) niche could increase the transendothelial migration of leukemia cells while any inhibitory event of ADM activity could raise cell retention in hyaluronate matrix by upregulating CD38. Taken into consideration the above evidence, we concluded that ADM and ADM22-52 could differently affect the growth of leukemia cells by autocrine/paracrine mechanisms and may have clinical relevance as biological targets for the intervention of tumor progression.

New human embryo liver cell lines obtained by stabilization and immortalization enhance in vitro clonal growth of cordonal blood cells.

We developed two new cell lines derived from embryo liver and tested their inductive capacity on in vitro clonal growth of cordonal blood (CB) hematopoietic cells. One line was stabilized and named BAEP2-WILD (W), and the other one was immortalized by retroviral transduction with SV40 Large T antigen and called BAEP2-SV40. Southern blot analysis demonstrated the integration of the Large T antigen gene in the BAEP2-SV40 cell genome, but this line did not display the expected growth arrest at the non-permissive temperature of 39 degrees C. Immunocytochemistry showed that BAEP2-SV40 cell line was positive for several cytokeratins and stromal markers (vimentin, desmin and laminin), as well as for epidermal growth factor (EGF), fibroblast growth factor (FGF) and their receptors (Rs). In contrast, BAEP2-W evidenced positivity only for cytokeratin-7 and laminin, and low positivity to EGF, EGF-R, FGF and FGF-R. BAEP2-SV40 cell line, but not BAEP2-W, expressed interleukin (IL)-1, IL-6, granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor, stem cell factor and vascular-cell adhesion molecule-1 mRNAs, and secreted IL-6 and GM-CSF. Taken together, these findings could suggest that BAEP2-W cell line possesses the phenotype of fetal hepatocytes, while BAEP2-SV40 cell line has that of stromal cells. The supernatants conditioned by both cell lines stimulated the clonal growth of CB hematopoietic cells cultured on semisolid media deprived of growth factors and cytokines, the inductive capacity of the BAEP2-SV40 cell line being markedly higher than that of its wild counterpart, conceivably due to its ability to produce cytokines. Our study indicates that these two new cell lines, and especially BAEP2-SV40 one could be used in co-culture systems as feeder-layers for hematopoietic CB SC expansion in vitro.

New experimental models for the in vitro reconstitution of human bladder mucosa.

This study describes two experimental models for the in vitro reconstitution of the human bladder mucosa (neo-bladder): human urothelial stabilized cell lines were cultured on three-dimensional matrices, collagen or platelet-fibrin gels, containing murine fibroblast 3T3-J2 cells. Low-density seeding (2x10(4) cells/ml) of both normal (TCA-48) and neoplastic cell lines (TCA-47) on collagen matrix gave rise to isolated papillar colonies, while high-density seeding (3.75x10(6) cells/ml) led to the formation of wide pluristratified epithelial sheets, resembling the normal transitional epithelium. In contrast, high-density seeding (5x10(5) cells/ml) on platelet-fibrin matrix did not allow the formation of epithelial sheets: only isolated voluminous colonies of normal TCA-48 cells, and sparse and small colonies of neoplastic TCA-47 could be observed. Growth assays and cytotoxicity reduction tests showed that the growth inhibitory effect of platelet-fibrin gel on urothelial cells was probably due to the aspecific activation of the complement contained in the plasmatic fraction, whose precipitation forms fibrin-glue. Collectively, these findings allow us to draw the following conclusions: i) neobladders obtained by culturing urothelial cells on collagen matrix reproduce normal bladder mucosa and could be utilized in pharmacological studies; and ii) platelet-fibrin gels, that specifically inhibit neoplastic urothelial cell growth, could be used as scaffolds in surgical bladder reconstitution.

Effect of cryopreservation on in vitro clonal growth of cordonal blood cells.

Cordonal blood (CB) is today recognized as a potentially important source of hematopoietic stem cells (SCs) for allogeneic transplantation, and to this task it would be of extreme importance to have the possibility of using cryopreserved CB units. Hence we investigated whether freezing and thawing alter the viability of CB hematopoietic cells. Mononuclear cells, recovered from fresh CB units by density-gradient centrifugation, were partly frozen and then thawed and partly immediately utilized for clonogenic tests. The cells were cultured in H4330 (C group) or H4434 (H4330 added with SC clonogenic growth factors) (C+ group) semisolid media added or not with serum-free Dulbecco modified Eagle medium (DMEM/sf). Cells seeded on H4330 were exposed to the conditioned supernatants from two human-embryo liver cell lines, which have been previously found to stimulate clonal growth of fresh CB hematopoietic cells. As expected, the number of colony-forming units (CFU) was higher in C+ than C group, and was not influenced by the addition of DMEM/sf. CFU number was higher in cryopreserved than fresh cells in both C and C+ culture groups. Conditioned supernatants from both cell lines stimulated clonal growth in both fresh and cryopreserved cell cultures. These findings indicate that cryopreservation and thawing do not alter the viability of CB SCs, but, on the contrary, improve their basal and cytokine-stimulated clonal growth, probably by negatively selecting SCs among the mononuclear CB cell population.

Adrenomedullin is expressed in cord blood hematopoietic cells and stimulates their clonal growth.

Adrenomedullin (AM) is a hypotensive peptide, which originates from the proteolytic cleavage of pro(p)AM and acts via AM22-52-sensitive receptors. Reverse transcription polymerase chain reaction allowed the detection of the specific mRNAs of pAM in the mononuclear hematopoietic cells of the cord blood and immunocytochemistry demonstrated their abundant AM-immunoreactivity. AM (10(-8) M) markedly enhanced clonal growth of cord blood hematopoietic cells cultured on semisolid media added with stem cell-growth promoting cytokines, and this effect was abolished by AM22-52 (10(-6) M). Collectively, these findings indicate that AM is expressed in and stimulates the proliferation of cord blood hematopoietic stem cells. Cord blood has been proposed as a source of stem cells alternative to bone marrow for allogeneic transplantation, and our study suggests that AM may be used, in addition to the classic cytokines, to expand in vitro cord blood stem cells in advance of their clinical use.

Adrenomedullin and endothelin-1 stimulate in vitro expansion of cord blood hematopoietic stem cells.

The improvement of techniques for in vitro expansion of cord blood (CB) hematopoietic stem cells (SCs) is, at present, one main task of tissue engineering. Hence, we investigated whether endothelin-1 (ET-1) and adrenomedullin (AM), two regulatory peptides exerting growth promoting action on several cell systems, favor the in vitro expansion of CB SCs in liquid culture. CB hematopoietic cell middle-term expansion was carried out in a stroma-free liquid culture medium in the presence of ET-1, AM and three different cytokine combinations. After two weeks of incubation, aliquots of expanded-cell suspension were seeded on semisolid medium and clonogenic tests were carried out by counting the number of colony forming units (CFUs) after 14 days of culture. Neither ET-1 nor AM (2.5 x 10(-8) M) were per se able to significantly increase the CFU number, but both peptides magnified the pro-expansive effects of some cytokine cocktails. In light of these findings, we conclude that ET-1 and AM are to be considered novel promising molecules that, in association with cytokines, can be utilized as pro-expansive factors of CB SCs in prevision of their clinical use in allogeneic transplantation.

New immortalized human stromal cell lines enhancing in vitro expansion of cord blood hematopoietic stem cells.

One of the most promising technique for the in vitro expansion of cord blood (CB) hematopoietic stem cells (SCs) seems to be their co-culture with stromal feeder-layers. Hence, we developed and immortalized by retroviral transduction with the temperature-sensitive SV40 large T antigen three new human cell lines, two derived from bone marrow (HM1-SV40 and HM2-SV40) and one from umbilical cord (HCB1-SV40), and investigated the inductive capacity of their conditioned culture media on clonal growth of CB hematopoietic SCs. Immunocytochemistry showed that cell lines were positive to either cytokeratins or stromal markers, as well as to epidermal growth factor (EGF), fibroblast growth factor (FGF) and adrenomedullin. Moreover, cell lines expressed interleukin (IL)-1 beta, IL-6, granulocyte macrophage-colony stimulating factor (GM-CSF), G-CSF and stem cell factor (SCF), and secreted variable amount of IL-1 beta, IL-6 and GM-CSF. Collectively, these findings indicate that cell lines possess the stromal-cell phenotype. The conditioned supernatants of the three cell lines induced similar increases in the clonal growth of both fresh and cryopreserved-thawed CB hematopoietic SCs cultured on semisolid media deprived of growth factors and cytokines. However, the inductive capacity was significantly higher in the case of cryopreserved cells, where the rise in clonal growth reached that induced by the addition to the culture media of IL-3, GM-CSF and SCF. Our findings allow us to conclude that our new human stromal cell lines could be used as feeder-layers for CB hematopoietic SC expansion in vitro.

Interleukin-6 plasma level increases with age in an Italian elderly population ("The Treviso Longeva"-Trelong-study) with a sex-specific contribution of rs1800795 polymorphism.

The transcription rate of interleukin-6 (IL-6) can be reduced by the C-allele of a polymorphism (rs1800795) located in the 5'-flanking region of the IL-6 gene (NM_000600), and IL-6 plasma levels increase with age. We assembled an elderly Italian population ["The Treviso Longeva (Trelong) study", age range 70-106 years, n = 668 subjects] and assessed rs1800795 genotype and plasma IL-6 concentrations. The rs1800795 genotype was also assessed in an independent Italian study ("Milan" study, age range 70-96, n = 245 subjects). To verify an age- or sex-specific effect of rs1800795 genotype we compared people younger (70-85) and older (85+) than 85 years of age. We found a significant reduction in the frequency of rs1800795 C/C genotype in 85+ men from the Trelong study, while in the Milan study this data did not reach significance. However, considering the two studies together, the frequency of the rs1800795 C/C genotype was significantly lower in 85+ than in 70-85 males (4.0% and 10.7%, respectively), while it remained unchanged in females. As for IL-6 plasma levels, after a multivariate analysis to control for confounders, a correlation between age and plasma IL-6 concentrations was revealed (P < 0.0001). An increase in circulating IL-6 levels in the entire 85+ group compared to the 70-85 group (P < 0.05, Tukey's test) was also noticed. We suggest a sex-specific pattern for genetic variability linked to inflammatory response and longevity, consistent with the age-related increase in IL-6.