Pulmonary Embolism in COVID-19 Patients Is Not Related to a Deterioration of Tissue Oxygenation.

BACKGROUND: COVID-19 induces robust systemic inflammation. One of the main complications is the increased coagulation due to endotheliitis. There is an increased incidence of pulmonary embolism (PE) in COVID-19 patients. However, clinical characteristics for a strict analysis are yet to be determined. AIM: We evaluated oxygenation and characteristics in patients with COVID-19 PE (CPE). MATERIAL AND METHODS: We evaluated 215 COVID-19 patients from 1 January to 30 April 2021. We found 18 patients affected by PE (CPE, 50.0% males, aged 67.00 ± 10.86 years). As controls, we used data from patients affected by PE evaluated in our ward between 1 January 2015 and 31 December 2019 (64 patients, 53.1% males, aged 70.88 ± 16.44 years). All patients underwent a complete physical examination, pulmonary computerised tomography, laboratory tests, D-dimers and blood gas analysis at the time of diagnosis. RESULTS: There were no differences in laboratory tests nor in D-dimers between the two groups. In the CPE group we found a significantly increased pO2 (92.83 ± 42.52 vs. 76.11 ± 32.58 mmHg; p < 0.05), difference of oxygen between alveoli and arteries (A-aDO2; 169.3 ± 171.9 vs. 52.97 ± 39.65 mmHg; p < 0.05), and oxygen saturation % (97.06 ± 2.59 vs. 93.77 ± 5.53%; p < 0.05) compared to controls. No difference was found in pCO2 and the ratio between pO2 and percentage of inspired oxygen (P/F). Finally, a significantly decreased urate (3.67 ± 1.49 vs. 5.60 ± 2.10; p < 0.05) was found in CPE compared to controls. In CPE, platelets count presents an inverse correlation to P/F (r = -0.389, p = 0.02) but a direct correlation to A-aDO2 (r = 0.699, p = 0.001). No similar findings were present in controls. DISCUSSION: COVID-19 PE appears to have a different clinical setting. Reduced oxygenation described in PE may not to be considered as a sign of disease. The increased A-aDO2 may indicate that COVID-19 PE involved smaller vessels compared to classical PE. A possible diffuse capillary thrombosis could explain these results.

A different perspective on sofosbuvir-ledipasvir treatment of patients with HCV genotype 1b cirrhosis: The ital-c network study.

The effectiveness of a 12-week course of sofosbuvir-ledipasvir in treatment-experienced HCV genotype 1b-infected patients with cirrhosis is still under debate. Our primary endpoint was to compare the sustained virological response at post-treatment week 12 (SVR12) of sofosbuvir-ledipasvir in combination with ribavirin for 12 weeks, and sofosbuvir-ledipasvir alone for 24 weeks. This was a prospective observational study that enrolled 424 (195 naive, 229 experienced; 164 treated for 12 weeks with Ribavirin and 260 with sofosbuvir-ledipasvir alone for 24 weeks) consecutive HCV genotype 1b-infected patients with cirrhosis. The SVR12 rates were 93.9% and 99.2% in patients treated for 12 and 24 weeks, respectively (P = .002). The baseline characteristics of patients treated for 12 weeks were significantly different from those treated for 24 weeks as regards their younger age (P = .002), prevalence of Child-Pugh class A (P = .002), lower MELD scores (P = .001) and smaller number of nonresponders (P = .04). The shorter treatment was significantly associated with a lower SVR12 in univariate and multivariate analyses (P = .007 and P = .008, respectively). The SVR rate was unaffected by age, gender, BMI, Child-Pugh class, MELD score or previous antiviral treatment. Patients receiving ribavirin experienced more episodes of ascites and headache but less recurrence of hepatocellular carcinoma (HCC), and were prescribed more diuretics and cardiopulmonary drugs. No patient discontinued treatment. The therapeutic regimen of sofosbuvir-ledipasvir plus ribavirin administered for 12 weeks was less effective than sofosbuvir-ledipasvir alone given for 24 weeks. At odds with European guidelines, the recommended 12-week treatment with sofosbuvir-ledipasvir alone might be suboptimal for this setting of patients.

HCV RNA Genomic sequences and HCV-E2 glycoprotein in sural nerve biopsies from HCV-infected patients with peripheral neuropathy.

AIMS: Peripheral neuropathy (PN), the major neurological complication of chronic HCV infection, is frequently associated with mixed cryoglobulinaemia (MC) and small-vessel systemic vasculitis. While humoral and cell-mediated immune mechanisms are suspected to act together in an aberrant immune response that results in peripheral nerve damage, the role of HCV remains largely speculative. The possible demonstration of HCV in peripheral nerve tissue would obviously assume important pathogenic implications. METHODS: We studied sural nerve biopsies from 11 HCV-positive patients with neuropathic symptoms: five with and six without MC. In situ hybridization (ISH) and immunofluorescence studies were carried out to detect genomic and antigenomic HCV RNA sequences and HCV-encoded E2-glycoprotein, respectively. RESULTS: Epineurial vascular deposits of E2-glycoprotein were found in four (80%) MC and in two (33.3%) non-MC patients, respectively. These findings were enhanced by the perivascular deposition of positive-, though not negative-strand replicative RNA, as also found in the nerve extracts of all patients. Mild inflammatory cell infiltrates with no deposits of immunoglobulins and/or complement proteins were revealed around small vessels, without distinct vasculitis changes between MC and non-MC patients. CONCLUSIONS: These results indicate that nerve vascular HCV RNA/E2 deposits associated to perivascular inflammatory infiltrates were similar in chronically HCV-infected patients, regardless of cryoglobulin occurrence. Given the failure to demonstrate HCV productive infection in the examined sural nerve biopsies, nerve damage is likely to result from virus-triggered immune-mediated mechanisms.

T cell receptor variable ß gene repertoire in liver and peripheral blood lymphocytes of chronically hepatitis C virus-infected patients with and without mixed cryoglobulinaemia.

To characterize the repertoire of T lymphocytes in chronically hepatitis C virus (HCV)-infected patients with and without mixed cryoglobulinaemia (MC). T cell receptor (TCR) variable (V) ß clonalities in portal tracts isolated from liver biopsy sections with a laser capture microdissection technique in 30 HCV-positive MC patients were studied by size spectratyping. Complementarity-determining region 3 (CDR3) profiles of liver-infiltrating lymphocytes (LIL) were also compared with those circulating in the blood. The representative results of TCR Vß by CDR3 were also obtained from liver tissues and peripheral blood lymphocytes (PBL) of 21 chronically HCV-infected patients without MC. LIL were highly restricted, with evidence of TCR Vß clonotypic expansions in 23 of 30 (77%) and in 15 of 21 (71%) MC and non-MC patients, respectively. The blood compartment contained TCR Vß expanded clones in 19 (63%) MC and 12 (57%) non-MC patients. The occurrence of LIL clonalities was detected irrespective of the degree of liver damage or circulating viral load, whereas it correlated positively with higher levels of intrahepatic HCV RNA. These results support the notion that TCR Vß repertoire is clonally expanded in HCV-related MC with features comparable to those found in chronically HCV-infected patients without MC.

Double-filtration plasmapheresis in the treatment of leg ulcers in cryoglobulinemia.

Hepatitis C virus (HCV) is the major cause of cryoglobulinemia. Skin lesions are frequent and can be cured from the removal of cryoglobulins by therapeutic apheresis. We describe a case of HCV-positive type I cryoglobulinemia with severe leg ulcers, not responsive to antiviral and immunosuppressive treatment. Thirty sessions of double filtration plasmapheresis were performed, over a period of 6 months, with no other associated treatment. Before and after each session an assessment of immunoglobulins, complement, cryocrit, and fibrinogen was made. HCV RNA levels were determined in serum cryoprecipitate, supernatant before and after each session, and in the collection bag. No differences in pre and postapheresis values were observed in the serum concentrations and the supernatant, whereas the postapheresis cryoprecipitate showed a significantly reduced viral load (P < 0.02) as compared with the preapheresis values. There was improvement in the condition of ulcers in the leg during apheresis and had completely regressed by the end of the cycle.

Hepatitis C virus productive infection in mononuclear cells from patients with cryoglobulinaemia.

The relationship between the occurrence of cryoglobulins and hepatitis C virus (HCV) productive infection in peripheral blood and bone marrow-derived lymphocytes was explored. HCV minus strand RNA, the viral replicative intermediate, was searched for by a polyA(+) tract strand-specific Tth-based reverse transcriptase-polymerase chain reaction (RT-PCR) in lymphoid cells of 46 patients with acute and chronic infection. The HCV minus strand was demonstrated in RNA extracted from six (13%) and five (11%) peripheral blood and bone marrow-derived lymphocytes, respectively. The HCV replicating form in lymphoid cells was associated strictly with mixed cryoglobulinaemia (MCG), in that it was found in six of 13 (46%) MCG patients, including two with B cell non-Hodgkin's lymphoma (NHL). No traces of HCV-negative strand RNA were found in four patients with acute hepatitis C, in 15 with chronic active hepatitis without extrahepatic disorders, in seven with monoclonal gammopathy of undetermined significance, and in seven with B-NHL without MCG. These results emphasize the direct role of the virus in the pathogenesis of MCG and support the contention that HCV is not specifically lymphotropic, its entry and replication in lymphoid cells being determined largely by selective interactions.

Virological analysis and phenotypic characterization of peripheral blood lymphocytes of hepatitis C virus-infected patients with and without mixed cryoglobulinaemia.

In clinical and pathological terms hepatitis C virus (HCV)-infected patients can be subdivided into two main groups with and without mixed cryoglobulinaemia (MC). Involvement of blood mononuclear cells by HCV has potentially important implications. To this end, HCV-RNA levels in peripheral blood lymphocytes (PBL) preparations of 20 chronically HCV-infected patients with MC were measured and compared with those found in a group of 20 patients without MC matched for age, serum HCV-RNA, infectious genotype, source and presumable duration of infection. Phenotypic abnormalities of PBL subsets in each group of patients were determined by cell surface marker expression and compared. Results showed a significant enrichment of HCV-RNA in PBL of MC patients compared with a non-MC group (P = 0.01). Different distribution of HCV-RNA was accompanied by evidence of an increased frequency of circulating B cells. These data indicate that MC patients are characterized distinctly by a higher quota of cell-associated viral load.

Hepatitis C virus RNA and core protein in kidney glomerular and tubular structures isolated with laser capture microdissection.

The role of hepatitis C virus (HCV) in the production of renal injury has been extensively investigated, though with conflicting results. Laser capture microdissection (LCM) was performed to isolate and collect glomeruli and tubules from 20 consecutive chronically HCV-infected patients, namely 6 with membranoproliferative glomerulonephritis, 4 with membranous glomerulonephritis, 7 with focal segmental glomerulosclerosis and 3 with IgA-nephropathy. RNA for amplification of specific viral sequences was provided by terminal continuation methodology and compared with the expression profile of HCV core protein. For each case two glomeruli and two tubular structures were microdissected and processed. HCV RNA sequences were demonstrated in 26 (65%) of 40 glomeruli, but in only 4 (10%) of the tubules (P < 0.05). HCV core protein was concomitant with viral sequences in the glomeruli and present in 31 of the 40 tubules. HCV RNA and/or HCV core protein was found in all four disease types. The immunohistochemical picture of HCV core protein was compared with the LCM-based immunoassays of the adjacent tissue sections. Immune deposits were detected in 7 (44%) of 16 biopsy samples shown to be positive by extraction methods. The present study indicates that LCM is a reliable method for measuring both HCV RNA genomic sequences and HCV core protein in kidney functional structures from chronically HCV-infected patients with different glomerulopathies and provides a useful baseline estimate to define the role of HCV in the production of renal injury. The different distribution of HCV RNA and HCV-related proteins may reflect a peculiar 'affinity' of kidney microenvironments for HCV and point to distinct pathways of HCV-related damage in glomeruli and tubules.

Detection and quantitation of HCV core protein in single hepatocytes by means of laser capture microdissection and enzyme-linked immunosorbent assay.

Immunohistochemistry provides valuable information concerning the localization and distribution of hepatitis C virus (HCV)-related proteins in histological sections of liver tissue, but does not readily permit their quantitation in individual cells and the staining intensity of cell immunodeposits cannot be calibrated with the current number of antigen molecules. We specifically detected and quantitated HCV core protein in single hepatocytes by coupling laser capture microdissection (LCM) with a sensitive enzyme-linked immunosorbent assay (ELISA). Quantitation of HCV core protein per cell was carried out on liver tissue cells obtained by LCM from fixed and stained frozen sections of 10 HCV-positive patients with chronic active hepatitis (CAH). Macromolecules from captured cells were solubilized in an extraction buffer and directly assayed for core protein using a sandwich ELISA. Calibration was achieved by developing a standard curve based on known concentrations of HCV core protein. Precision, linearity and sensitivity were verified for known numbers of microdissected tissue cells. In this study, the concentration of HCV core protein in single hepatocytes ranged from 7 to 56 pg/cell. Specificity was verified on 10 replicates of 10 HCV-negative liver tissues. Immunohistochemical staining of HCV core protein was compared with the results of the soluble immunoassay for the adjacent liver tissue sections. Independent scoring of HCV immunostaining failed to parallel the LCM quantitative immunoassay. LCM-based immunoassay significantly expands our ability to investigate function-related antigens in apparently pure cell populations in HCV infection.

The lymphoid system in hepatitis C virus infection: autoimmunity, mixed cryoglobulinemia, and Overt B-cell malignancy.

Like other hepatotropic viruses, hepatitis C virus (HCV) shares the property of inducing hepatocellular damage, possibly through induction of immune mechanisms that lead to hepatocellular necrosis. After infection of hepatocytes, and possibly other cells, humoral and cellular responses occur aimed at prevention of virus dissemination and elimination of infected cells. The early activated mechanisms include production of nonspecific and specific antibodies that represent the first-line of defense against invading foreign pathogens. As a consequence, circulating immune complexes are promptly formed, and antigen uptake and processing by specialized cells are enhanced. A major fraction of circulating immunoglobulins (Igs) are part of the spectrum of the so-called natural antibodies, which include anti-idiotypic antibodies and molecules with rheumatoid factor (RF) activity. They mainly belong to the IgM class, are polyclonal, and have no intrinsic pathogenetic potential. In 20-30% of HCV-infected patients, RFs share characteristics of high affinity molecules, are monoclonal in nature, and result in the production of cold-precipitating immune complexes and mixed cryoglobulinemia. It has been shown that anti-idiotypic antibodies and polyclonal and monoclonal RF molecules have the same cross-reactive idiotype, called WA, suggesting that their production is highly restricted. This strongly indicates that they arise from stimulation with the same antigen, likely HCV. It has also been speculated that B-1 (CD5+) and B-2 (CD5-) B-cell subsets, which use a limited number of VH germline genes, underlie the production of low-affinity polyclonal and high-affinity monoclonal antibodies, respectively. The persistent production of monoclonal RF molecules implies the existence of a further mechanism capable of restricting the reactivity and reflects a distinct selection of a cell population that can be maintained throughout life because they are continuously exposed to antigen pressure. Either polyclonal or monoclonal profiles of B-cell expansion are demonstrable in the liver of most HCV-infected patients. The occurrence of B-cell clonal expansion is strictly related to intrahepatic production of RF molecules, and this suggests that liver is a microenvironment, other than lymphoid tissue, in which a germinal centerlike reaction is induced. The frequent detection of oligoclonal B-cell expansion may, indeed, represent a key pathobiologic feature that sustains nonmalignant B-cell lymphoproliferation. The preferential expansion of one clone would in turn lead to a monoclonal pattern that could favor stochastic oncogenic events. It can be postulated that HCV is the stimulus not only for the apparent benign lymphoproliferative process underlying a wide spectrum of clinical features, but also for the progression to frank lymphoid malignancy in a subgroup of patients. Current data indicate a higher prevalence of overt B-cell non-Hodgkin's lymphoma in HCV-infected patients, especially in some geographic areas.

Non-enveloped HCV core protein as constitutive antigen of cold-precipitable immune complexes in type II mixed cryoglobulinaemia.

Hepatitis C virus (HCV) infection has been detected in a large proportion of patients with mixed cryoglobulinaemia (MC). Circulating 'free' non-enveloped HCV core protein has been demonstrated in HCV-infected patients, and this suggests its possible involvement in the formation of cryoprecipitable immune complexes (ICs). Thirty-two anti-HCV, HCV RNA-positive patients with type II MC were evaluated. Non-enveloped HCV core protein, HCV RNA sequences, total IgM, rheumatoid factor (RF) activity, IgG and IgG subclasses, C3 and C4 fractions, C1q protein and C1q binding activity were assessed in both cryoprecipitates and supernatants. Non-enveloped HCV core protein was demonstrated in 30 of 32 (93.7%) type II MC patients. After separation of cold-precipitable material, the protein was removed completely from supernatant in 12 patients (40%), whereas it was enriched in the cryoprecipitates of the remaining 18. In addition, HCV RNA and IgM molecules with RF activity were concentrated selectively in the cryoprecipitates. Differential precipitation was found for both total IgG and IgG subclasses, as they were less represented in the cryoglobulins and no selective enrichment was noted. Immunological characterization of HCV core protein-containing cryoprecipitating ICs after chromatographic fractionation showed that the IgM monoclonal component had RF activity, whereas anti-HCV core reactivity was confined to the IgG fraction. C1q enrichment in addition to high avidity of ICs for C1q binding in the cryoprecipitates suggest that complement activation may occur through the C1q protein pathway. The present data demonstrate that non-enveloped HCV core protein is a constitutive component of cryoprecipitable ICs in type II MC patients.

Immunochemical and biomolecular studies of circulating immune complexes isolated from patients with acute and chronic hepatitis C virus infection.

Circulating immune complexes (ICs) were isolated by affinity chromatography and sucrose density gradient fractionation during acute and chronic hepatitis C virus (HCV) infection. Immunochemical and biomolecular studies showed that they basically consist of the virus component, IgG with specific anti-HCV activity and IgM bearing 17.109 epitope (IgM 17.109), an antigenic determinant common to rheumatoid factors (RFs) with WA cross-idiotype (XId). An antigen-specific IC assay was used to demonstrate IgG anti-HCV/IgM 17.109 ICs (IgG-IgM ICs) in five out of the five patients with acute and in 8 out of the 10 patients with chronic hepatitis C who mounted an IgG anti-HCV immune response. They were not detected in patients with no IgG anti-HCV response. IgG-IgM ICs appeared in step with IgG anti-HCV seroconversion and remained detectable for a long period irrespective of clinical outcome, in that they were demonstrated over a 4-year follow-up of patients with chronic hepatitis C. Their presence was unrelated to the severity and progression of liver histology. Despite similar serum levels of IgM 17.109 XId, antigen-specific IgG-IgM ICs were not found in acute and chronic hepatitis B or in acute hepatitis A. Thus, these ICs appear to be uniquely associated with HCV infection, supporting the view that IgM 17.109 XId derive from an antigen-driven response strictly related to the involved antigen. Even although they have no apparent effects on the progression of HCV-related liver disease, their presence may help to explain the immunological abnormalities and extrahepatic disorders observed in HCV infection.

Natural interferon-alpha versus its combination with 6-methyl-prednisolone in the therapy of type II mixed cryoglobulinemia: a long-term, randomized, controlled study.

Type II mixed cryoglobulinemia (MC) is an often progressive vasculitis characterized by circulating cold-precipitable proteins that usually consists of polyclonal IgG and monoclonal IgM kappa with rheumatoid factor (RF) activity. Its etiology is unknown, although recent evidence strongly suggests that hepatitis C virus (HCV) plays a major role. Plasmapheresis, corticosteroids, and cytotoxic drugs have been used in the therapy of MC patients. Recently, favorable results with recombinant interferon-alpha (rIFN alpha) have been reported. To further assess its effectiveness, we studied the effects of natural human interferon-alpha (nIFN alpha), alone and in combination with 6-methyl-prednisolone (PDN), in a prospective, randomized, controlled trial in patients with symptomatic MC. Sixty-five patients were enrolled onto the trial, 52 (80%) of whom presented serum anti-HCV antibodies and specific genomic RNA sequences. Fifteen patients received nIFN alpha (3 MU) intramuscularly (IM) three times weekly, whereas 17 patients also received 16 mg/d of PDN orally on non-IFN days. Moreover, 18 patients received 16 mg/d of PDN only, and 15 were untreated. Treatment was discontinued after 1 year and patients were monitored for 8 to 17 months (mean, 13). A complete response was achieved in eight of 15 patients (53.3%) treated with nIFN alpha and nine of 17 (52.9%) treated with nIFN alpha plus PDN, as compared with three of 18 patients (16.7%) who received PDN only (P < .05) and one of 15 (6.7%) untreated controls (P < .01). Partial response occurred in two of 15 (13.3%) patients treated with nIFN alpha, three of 17 (17.6%) who received nIFN alpha plus PDN, one of 18 (5.5%) who received PDN only, and one of 15 (6.7%) controls. A complete response in six patients (66.7%) was achieved within 3 months in the group that received nIFN alpha plus PDN, as compared with two patients (25%) of those who received nIFN alpha alone (P < .02). In anti-HCV-positive patients, the clinical response occurred in step with reduced or undetectable levels of HCV RNA and transaminase normalization. Quantification of circulating HCV RNA represented a good predictive response marker. The probability of relapse within 3 months after treatment was 100% (three of three patients) and 75% (six of eight patients), respectively, in patients who received PDN alone or nIFN alpha alone as compared with none of those who received nIFN alpha plus PDN (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatitis C virus infection and mixed cryoglobulinemia: a striking association.

The high frequency of liver involvement in cryoglobulinemia is well established. Although both etiology and pathogenesis have remained so far undefined, recent studies suggest an association of mixed cryoglobulinemia with hepatitis C virus infection. To explore this hypothesis further, we assessed the prevalence of hepatitis C virus antibodies and RNA in a large group of patients, including: (1) 35 patients with cryoglobulinemia without clinical evidence of liver involvement (group 1), (2) 15 patients with symptomatic cryoglobulinemia associated with chronic liver disease (group 2) and (3) 12 patients with asymptomatic cryoglobulinemia associated with chronic liver disease (group 3). Anti-hepatitis C virus antibodies were detected by a second-generation enzyme-linked immunosorbent assay and third-generation immunoblot (SIA Prototype RIBA), whereas the polymerase chain reaction was used for the detection of viral RNA. Anti-hepatitis C virus antibodies, as detected by enzyme-linked immunosorbent assay, were demonstrated in 21 (60%) patients from group 1, 11 (73.3%) from group 2 and 10 (83.3%) from group 3. The immunoblot identified as positive 3 further patients in group 1 (giving a prevalence of 68.6%) and all patients in groups 2 and 3. Hepatitis C virus RNA was demonstrated in cryoprecipitates from 21 of 24 immunoblot-positives and from 6 of 11 immunoblot-negatives, indicating an actual active viral replication in 77.1% of group 1. This was also found in 13 (86.7%) and 10 (83.3%) cryoprecipitates of groups 2 and 3, respectively. Type II cryoglobulinemia was the prevalent form in group 1 (88.6%) and group 2 (73.3%), whereas type III was found in group 3 (58.3%) and in 26.7% of group 2.(ABSTRACT TRUNCATED AT 250 WORDS)