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Introduction: A large number of risk and protective factors have been identified during the SARS-CoV-2 pandemic which may influence the outcome of COVID-19. Among these, recent studies have explored the role of HLA-G molecules and their immunomodulatory effects in COVID-19, but there are very few reports exploring the genetic basis of these manifestations. The present study aims to investigate how host genetic factors, including HLA-G gene polymorphisms and sHLA-G, can affect SARS-CoV-2 infection., Materials and Methods: We compared the immune-genetic and phenotypic characteristics between COVID-19 patients (n = 381) with varying degrees of severity of the disease and 420 healthy controls from Sardinia (Italy)., Results: HLA-G locus analysis showed that the extended haplotype HLA-G*01:01:01:01/UTR-1 was more prevalent in both COVID-19 patients and controls. In particular, this extended haplotype was more common among patients with mild symptoms than those with severe symptoms [22.7% vs 15.7%, OR = 0.634 (95% CI 0.440 - 0.913); P = 0.016]. Furthermore, the most significant HLA-G 3'UTR polymorphism ( rs371194629 ) shows that the HLA-G 3'UTR Del/Del genotype frequency decreases gradually from 27.6% in paucisymptomatic patients to 15.9% in patients with severe symptoms (X 2  = 7.095, P = 0.029), reaching the lowest frequency (7.0%) in ICU patients (X 2  = 11.257, P = 0.004). However, no significant differences were observed for the soluble HLA-G levels in patients and controls. Finally, we showed that SARS-CoV-2 infection in the Sardinian population is also influenced by other genetic factors such as β-thalassemia trait ( rs11549407 C>T in the HBB gene), KIR2DS2/HLA -C C1+ group combination and the HLA-B*58:01, C*07:01, DRB1*03:01 haplotype which exert a protective effect [P = 0.005, P = 0.001 and P = 0.026 respectively]. Conversely, the Neanderthal LZTFL1 gene variant ( rs35044562 A>G) shows a detrimental consequence on the disease course [P = 0.001]. However, by using a logistic regression model, HLA-G 3'UTR Del/Del genotype was independent from the other significant variables [OR M = 0.4 (95% CI 0.2 - 0.7), P M = 6.5 x 10 -4 ]., Conclusion: Our results reveal novel genetic variants which could potentially serve as biomarkers for disease prognosis and treatment, highlighting the importance of considering genetic factors in the management of COVID-19 patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mocci, Littera, Chessa, Campagna, Melis, Ottelio, Piras, Lai, Firinu, Tranquilli, Mascia, Vacca, Schirru, Lecca, Rassu, Cannas, Sanna, Carta, Sedda, Giuressi, Cipri, Miglianti, Perra and Giglio.)

The immunomodulatory effects of HLA-G expression and its role in cancers, human liver infections and liver transplantation are well documented, but so far, there are only a few reports addressing autoimmune liver diseases, particularly autoimmune hepatitis (AIH)., Method and Materials: We analyzed the genetic and phenotypic characteristics of HLA-G in 205 type 1 AIH patients (AIH-1) and a population of 210 healthy controls from Sardinia (Italy)., Results: Analysis of the HLA-G locus showed no substantial differences in allele frequencies between patients and the healthy control population. The HLA-G UTR-1 haplotype was the most prevalent in both AIH-1 patients and controls (40.24% and 34.29%). Strong linkage was found between the HLA-G UTR-1 haplotype and HLA-DRB1*03:01 in AIH-1 patients but not controls ( D' = 0.92 vs D' = 0.50 respectively; P = 1.3x10 -8 ). Soluble HLA-G (sHLA-G) levels were significantly lower in AIH-1 patients compared to controls [13.9 (11.6 - 17.4) U/mL vs 21.3 (16.5 - 27.8) U/mL; P = 0.011]. Twenty-four patients with mild or moderate inflammatory involvement, as assessed from liver biopsy, showed much higher sHLA-G levels compared to the 28 patients with severe liver inflammation [33.5 (23.6 - 44.8) U/mL vs 8.8 (6.1 - 14.5) U/mL; P = 0.003]. Finally, immunohistochemistry analysis of 52 liver biopsies from AIH-1 patients did not show expression of HLA-G molecules in the liver parenchyma. However, a percentage of 69.2% (36/52) revealed widespread expression of HLA-G both in the cytoplasm and the membrane of plasma cells labeled with anti-HLA-G monoclonal antibodies., Conclusion: This study highlights the positive immunomodulatory effect of HLA-G molecules on the clinical course of AIH-1 and how this improvement closely correlates with plasma levels of sHLA-G. However, our results open the debate on the ambiguous role of HLA-G molecules expressed by plasma cells, which are pathognomonic features of AIH-1., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Littera, Perra, Miglianti, Piras, Mocci, Lai, Melis, Zolfino, Balestrieri, Conti, Serra, Figorilli, Firinu, Onali, Matta, Porcu, Pes, Fanni, Manieli, Vacca, Cusano, Trucas, Cipri, Tranquilli, Rassu, Cannas, Carta, Kowalik, Giuressi, Faa, Chessa and Giglio.)

Sardinia has one of the lowest incidences of hospitalization and related mortality in Europe and yet a very high frequency of the Neanderthal risk locus variant on chromosome 3 (rs35044562), considered to be a major risk factor for a severe SARS-CoV-2 disease course. We evaluated 358 SARS-CoV-2 patients and 314 healthy Sardinian controls. One hundred and twenty patients were asymptomatic, 90 were pauci-symptomatic, 108 presented a moderate disease course and 40 were severely ill. All patients were analyzed for the Neanderthal-derived genetic variants reported as being protective (rs1156361) or causative (rs35044562) for severe illness. The β°39 C>T Thalassemia variant (rs11549407 ) , HLA haplotypes, KIR genes, KIRs and their HLA class I ligand combinations were also investigated. Our findings revealed an increased risk for severe disease in Sardinian patients carrying the rs35044562 high risk variant [OR 5.32 (95% CI 2.53 - 12.01), p = 0.000]. Conversely, the protective effect of the HLA-A*02:01, B*18:01, DRB*03:01 three-loci extended haplotype in the Sardinian population was shown to efficiently contrast the high risk of a severe and devastating outcome of the infection predicted for carriers of the Neanderthal locus [OR 15.47 (95% CI 5.8 - 41.0), p < 0.0001]. This result suggests that the balance between risk and protective immunogenetic factors plays an important role in the evolution of COVID-19. A better understanding of these mechanisms may well turn out to be the biggest advantage in the race for the development of more efficient drugs and vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mocci, Littera, Tranquilli, Provenzano, Mascia, Cannas, Lai, Giuressi, Chessa, Angioni, Campagna, Firinu, Del Zompo, La Nasa, Perra and Giglio.)

Background: The diversity in the clinical course of COVID-19 has been related to differences in innate and adaptative immune response mechanisms. Natural killer (NK) lymphocytes are critical protagonists of human host defense against viral infections. It would seem that reduced circulating levels of these cells have an impact on COVID-19 progression and severity. Their activity is strongly regulated by killer-cell immuno-globulin-like receptors (KIRs) expressed on the NK cell surface. The present study's focus was to investigate the impact of KIRs and their HLA Class I ligands on SARS-CoV-2 infection., Methods: KIR gene frequencies, KIR haplotypes, KIR ligands and combinations of KIRs and their HLA Class I ligands were investigated in 396 Sardinian patients with SARS-CoV-2 infection. Comparisons were made between 2 groups of patients divided according to disease severity: 240 patients were symptomatic or paucisymptomatic (Group A), 156 hospitalized patients had severe disease (Group S). The immunogenetic characteristics of patients were also compared to a population group of 400 individuals from the same geographical areas., Results: Substantial differences were obtained for KIR genes, KIR haplotypes and KIR-HLA ligand combinations when comparing patients of Group S to those of Group A. Patients in Group S had a statistically significant higher frequency of the KIR A/A haplotype compared to patients in Group A [34.6% vs 23.8%, OR = 1.7 (95% CI 1.1-2.6); P = 0.02, Pc = 0.04]. Moreover, the KIR2DS2/HLA C1 combination was poorly represented in the group of patients with severe symptoms compared to those of the asymptomatic-paucisymptomatic group [33.3% vs 50.0%, OR = 0.5 (95% CI 0.3-0.8), P = 0.001, Pc = 0.002]. Multivariate analysis confirmed that, regardless of the sex and age of the patients, the latter genetic variable correlated with a less severe disease course [ORM = 0.4 (95% CI 0.3-0.7), PM = 0.0005, PMC = 0.005]., Conclusions: The KIR2DS2/HLA C1 functional unit resulted to have a strong protective effect against the adverse outcomes of COVID-19. Combined to other well known factors such as advanced age, male sex and concomitant autoimmune diseases, this marker could prove to be highly informative of the disease course and thus enable the timely intervention needed to reduce the mortality associated with the severe forms of SARS-CoV-2 infection. However, larger studies in other populations as well as experimental functional studies will be needed to confirm our findings and further pursue the effect of KIR receptors on NK cell immune-mediated response to SARS-Cov-2 infection., Competing Interests: The authors have declared that no competing interests exist.

To improve pathogenetic studies in cancer development and reliable preclinical testing of anti-cancer treatments, three-dimensional (3D) cultures, including spheroids, have been widely recognized as more physiologically relevant in vitro models of in vivo tumor behavior. Currently, the generation of uniformly sized spheroids is still challenging: different 3D cell culture methods produce heterogeneous populations in dimensions and morphology, that may strongly influence readouts reliability correlated to tumor growth rate or antitumor natural killer (NK) cell-mediated cytotoxicity. In this context, an increasing consensus claims the integration of microfluidic technologies within 3D cell culture, as the physical characterization of tumor spheroids is unavoidably demanded to standardize protocols and assays for in vitro testing. In this paper, we employed a flow-based method specifically conceived to measure weight, size and focused onto mass density values of tumor spheroids. These measurements are combined with confocal and digital imaging of such samples. We tested the spheroids of four colorectal cancer (CRC) cell lines that exhibit statistically relevant differences in their physical characteristics, even though starting from the same cell seeding density. These variations are seemingly cell line-dependent and associated with the number of growing cells and the degree of spheroid compaction as well, supported by different adenosine-triphosphate contents. We also showed that this technology can estimate the NK cell killing efficacy by measuring the weight loss and diameter shrinkage of tumor spheroids, alongside with the commonly used cell viability in vitro test. As the activity of NK cells relies on their infiltration rate, the in vitro sensitivity of CRC spheroids proved to be exposure time- and cell line-dependent with direct correlation to the cell viability reduction. All these functional aspects can be measured by the system and are documented by digital image analysis. In conclusion, this flow-based method potentially paves the way towards standardization of 3D cell cultures and its early adoption in cancer research to test antitumor immune response and set up new immunotherapy strategies., Competing Interests: The authors of Affiliation 1 (AS, FM, FB, DC, FC, SB, SP, and DG) are employed by Cell Dynamics isrl company. The authors declare that a Patent Application (No. 102020000006031) incorporating parts of this work has been filed. DG, SB, DC, AS, and FM are the inventors of patent No. 102020000006031. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Sargenti, Musmeci, Bacchi, Delprete, Cristaldi, Cannas, Bonetti, Pasqua, Gazzola, Costa, Villa, Zocchi and Poggi.)

Quantitative correlation between capillary morphologic patterns and digital pulp flow in idiopathic acrocyanosis (IA) is as yet unexplained. We studied capillary pattern, scored according mainly to capillary dilatation and tortuosity. Digital flow was recorded by laser Doppler flowmetry during a standardised cooling and rewarming thermal challenge in a controlled thermostatic chamber. A negative correlation was found by linear regression analysis between increasing capillary abnormalities and decreasing flow rates both at rest and during cooling, while a positive correlation was observed among the presence and intensity of the flow stop reaction during cold challenge and the decrease of hyperaemic re-flow in the rewarming phase. Further investigations may be justified to assess the nature of these abnormalities, whether constitutional or induced by prolonged cold exposure. For this purpose, a standardised method, as the cooling-rewarming challenge, could verify the several pathophysiological hypotheses proposed for vascular acrosyndromes. We need to use a subjective method such as capillaroscopy-based scoring (as in our study endorsed by an objective technique) to acquire the necessary reliability and reproducibility in the functional diagnosis. In fact IA patients with lower morphologic scores show better flow properties.

Background: The valuation of the extracranial carotid by echo color-Doppler takes on an extraordinary importance for the prevention of cerebral ictus at geriatric age. In this "naturalistic" study a population of old people (> 65 yrs) of Cagliari's province was considered in order to: 1) discriminate the lesions of the carotid that are imputed to atherosclerotic disease by anatomic changes of the arterial wall caused by aging; 2) study relations between lesions of the carotid and cardiovascular risk factors., Methods: The carotids of 50 old subjects were studied by echo color-Doppler and the lesion classified in different classes of severity, according to the hemodynamic standard, comparing them with the presence of the most important cardiovascular risk factors. Hypercholesterolemia was the most frequent risk factor (76%), followed by hypertension (62%), over-weight (54%) and smoking (42%). Moreover a diffused intima-media thickening (IMT) was constant in all the subjects with values > 0.75 mm; athero-sclerotic plaques were even present in 39 subjects which only in 4 cases could be considered at risk of cerebral ischemia., Results: A significant correlation between the severity of the lesions and levels of total cholesterol and LDL cholesterol for the male sex emerged, while for smoking only a trend of correlation has been obtained., Conclusions: Considering this experience it is suggested that in old subjects the presence of a diffused IMT with values > 0.75 mm must be considered as a marker of aging of the arterial wall of the carotid and not as a cardiovascular risk factor as reported in the literature for the middleaged.

We studied a sample of adult population over 20 years old of Donori (totally 2049 inhabitants), a small town near Cagliari, South Sardinia, to: (1) Evaluate the prevalence of peripheral arterial occlusive disease of lower limbs (PAOD) diagnosed by standard questionnaire and US CW Doppler examination and associated risk factors in South Sardinia. (2) Compare the reliability of these two diagnostic methods. Our study included the answers to a standard anamnestic questionnaire (according to the WHO recommendations), physical examination and CW Doppler study of the lower limb arteries, the determination of the arterial systolic and diastolic pressure, ankle/arm pressure ratio, Body Mass Index, blood glucose, total and HDL-cholesterol, triglycerides and fibrinogen (the hematochemical examination only on 50% of the sample). The surveyed sample was of 577 subjects (37.96% of 1520, the eligible subjects over 20 years old), 237 males and 340 females. An arteriopathy was diagnosed by means of CW Doppler in 27 subjects, 20 M and 7 F. The overall prevalence of PAOD was 4.67% of the sample (2.06% of females, 8.43% of males). Prevalence steadily increased with age, and, surprisingly, the disease was not absent in young people (2 cases within males with age < or = 40 years). Conversely only 18.5% of PAOD patients were symptomatic. The overall prevalence of associated risk factors was: diabetes 3.6%, hypercholesterolaemia 59.1%, smoking 21.3%, arterial hypertension 21.6%, obesity 17.9%, hypertriglyceridaemia 9.3%, hyperfibrinogenaemia 4.67%. Among the males a significant correlation has been found, among the values of BMI vs age and total cholesterol, age vs total cholesterol, systolic blood pressure, diastolic pressure, fibrinogenaemia. Among the females, the systolic and diastolic pressure, BMI, total cholesterol, LDL-cholesterol, triglycerides and fibrinogen are significantly correlated with age; BMI correlates with systolic and diastolic blood pressure, fibrinogen; a nearly significant correlation has been found between BMI and triglycerides.

Chronic venous insufficiency (CVI) is characterised by stasis which may lead to an imbalance in the cutaneous microcirculation resulting in a wide spectrum of complications. The use of capillaroscopy on the toenail folds of patients with CVI allows the morphological and microhemorrheological aspects of microcirculatory disorders to be studied. The aim of this study was to clarify the correlation between the extent of abnormalities of the nutritional capillary and the degree of severity of CVI according to Widmer's classification. Capillaroscopy of the toenail fold was used to study 100 patients suffering from essential varicose disease (EVD) and post-thrombotic syndrome (PTS) with CVI of various degrees, giving a total of 110 limbs grouped as follows: 30 limbs at stage 0; 49 at stage 1; 20 at stage 2 and 12 at stage 3. In addition, a further 30 limbs of normal subject without a family history of varicose disease were included in the study. All patients and control subjects were examined clinically and using ultrasound c.w. Doppler and eco-color-Doppler of the lower limbs. The results highlighted a progressive deterioration of the capillaroscopic conditions in relation to the severity of CVI. In particular, during stage 1 microcirculatory alterations found in patients with primary varicose syndromes appeared to be more severe than those with PTS. This apparent contradiction may be attributed to the unique topography of this hemodynamic disorders since the subpapillary circulation in EVD is topographically in direct continuity with the site of venous hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Thirty subjects with a diagnosis of isolated Raynaud's phenomenon, according to anamnestic and objective criteria, were cross-evaluated by various methods: laser Doppler flowmetry (LDF) during a standardized cold and rewarming test, nailfold capillaroscopy, immunological and other laboratory parameters, to assess the diagnostic and prognostic significance of each method. Correlations were also assessed among disease duration, capillaroscopic pattern score, quantitative digital flow values and laboratory parameters. Nearly 30% of patients showed immunologic abnormalities (ANA positivity at variable titres, ENA, hypocomplementemia, immunocomplexes); 16% of patients had a pathologic capillaroscopic pattern, not well correlated with immunologic findings; a characteristic cold stop reaction of digital flow (partial or total) was detected in 86% of the subjects; in ANA+ pts., ANA titers were positively correlated with the intensity and length of stop reaction (Trec). A significant correlation between digital flow parameters and the capillaroscopic score was also found in each considered group. Our results outline the relevance of LDF, during a standardized thermic test, to evaluate apparently primary RP, because even if a definite scleroderma-like capillaroscopic pattern is absent, this flowmetric method may detect potentially secondary RP patients.

Twenty-seven subjects with vasospastic diseases: 13 primary and 5 secondary Raynaud phenomenon (RP), 3 primary RP treated with raubasin 60 mg/day, 6 acrocyanosis and 9 normal subjects were studied in a standardized experimental set (thermostatic chamber), recording cutaneous digital micro-vascular reactions to cold and heat exposure by laser Doppler flowmetry, a relatively new method for the objective and reliable assessment of blood flow in the cutaneous microvasculature. The data suggest that patients with vasospastic disease have a defect in local microvascular flow regulation, that is revealed by low temperature exposure. The major difference between primary and secondary RF was recovery time (stop reaction) after cold test, that is easily recorded by this instrumental set. The morphology of the recording and the flow recovery time of acrocyanosis were found to be similar to those of secondary RF.