Hypoalbuminemia in COVID-19: assessing the hypothesis for underlying pulmonary capillary leakage.

BACKGROUND: Since the first observations of patients with COVID-19, significant hypoalbuminaemia was detected. Its causes have not been investigated yet. OBJECTIVE: We hypothesized that pulmonary capillary leakage affects the severity of respiratory failure, causing a shift of fluids and proteins through the epithelial-endothelial barrier. METHODS: One hundred seventy-four COVID-19 patients with respiratory symptoms, 92 admitted to the intermediate medicine ward (IMW) and 82 to the intensive care unit (ICU) at Luigi Sacco Hospital in Milan, were studied. RESULTS: Baseline characteristics at admission were considered. Proteins, interleukin 8 (IL-8) and interleukin 10 (IL-10) in bronchoalveolar lavage fluid (BALF) were analysed in 26 ICU patients. In addition, ten autopsy ultrastructural lung studies were performed in patients with COVID-19 and compared with postmortem findings in a control group (bacterial pneumonia-ARDS and H1N1-ARDS). ICU patients had lower serum albumin than IMW patients [20 (18-23) vs 28 (24-33) g L-1 , P < 0.001]. Serum albumin was lower in more compromised groups (lower PaO2 -to-FiO2 ratio and worst chest X-ray findings) and was associated with 30 days of probability of survival. Protein concentration was correlated with IL-8 and IL-10 levels in BALF. Electron microscopy examinations of eight out of ten COVID-19 lung tissues showed loosening of junctional complexes, quantitatively more pronounced than in controls, and direct viral infection of type 2 pneumocytes and endothelial cells. CONCLUSION: Hypoalbuminaemia may serve as severity marker of epithelial-endothelial damage in patients with COVID-19. There are clues that pulmonary capillary leak syndrome plays a key role in the pathogenesis of COVID-19 and might be a potential therapeutic target.

Co-administration of H-ferritin-doxorubicin and Trastuzumab in neoadjuvant setting improves efficacy and prevents cardiotoxicity in HER2 + murine breast cancer model.

Neoadjuvant chemotherapy has been established as the standard of care for HER2-positive breast cancer since it allows cancer down-staging, up to pathological complete response. The standard of care in the neoadjuvant setting for HER2-positive breast cancer is a combination of highly cytotoxic drugs such as anthracyclines and the anti-HER2 monoclonal antibody. Despite this cocktail allows a pathological complete response in up to 50%, their co-administration is strongly limited by intrinsic cardiotoxicity. Therefore, only a sequential administration of anthracyclines and the anti-HER2 treatment is allowed. Here, we propose the anthracycline formulation in H-Ferritin nanocages as promising candidate to solve this unmet clinical need, thanks to its capability to increase anthracyclines efficacy while reducing their cardiotoxicity. Treating a murine model of HER2-positive breast cancer with co-administration of Trastuzumab and H-Ferritin anthracycline nanoformulation, we demonstrate an improved tumor penetration of drugs, leading to increased anticancer efficacy and reduced of cardiotoxicity.

Induction of anti-DNA antibodies in preautoimmune NZBxNZW F1 mice by immunization with a DNA-DNase I complex.

Recent studies suggest that anti-DNA antibodies may arise from the immune response to a complex of DNA and a DNA-binding protein. One of the protein targets frequently recognized by anti-DNA antibodies is the enzyme DNase I. To investigate the possible role of DNase I in the induction of anti-DNA antibodies, we immunized preautoimmune NZBxNZW F1 mice with a complex of DNA and DNase I emulsified in complete Freund's adjuvant. Control mice received DNA or DNase in adjuvant. IgG anti-dsDNA antibodies were induced in 50% of the mice immunized with DNA-DNase, in 25% of the mice immunized with DNase and in 6% of the mice immunized with DNA. However, immunized mice that produced anti-DNA antibodies did not develop renal disease. These data show that a DNA-binding protein like DNase may act as carrier in the immune response that leads to anti-DNA antibodies production in an autoimmune strain, but the induced anti-dsDNA antibodies have a low pathogenic potential.

Alpha-enolase is a renal-specific antigen associated with kidney involvement in mixed cryoglobulinemia.

OBJECTIVE: To study the specificity of antibodies reactive with renal antigens in mixed cryoglobulinemia. METHODS: Sera from mixed cryoglobulinemia (MC) patients were tested on human kidney extracts by immunoblot. A partially purified renal antigen was subjected to N-terminal sequencing. RESULTS: Antibodies reactive with a renal antigen of 48 kD were detected in 7 out of 11 patients with MC and renal involvement. N-terminal sequencing of this antigen showed that it was identical with alpha-enolase. This result was confirmed by the reactivity of the renal antigen with a rabbit anti-serum specific for alpha-enolase. CONCLUSIONS: The results indicate that antibodies specific for alpha-enolase are frequently produced by mixed cryoglobulinemia patients with renal involvement.

An early diagnosis of kidney involvement in immunologically-mediated multisystem diseases.

Kidney involvement in immuno-mediated diseases is a life threatening complication to be early detected. Glomerulo-tubular functional indices, kidney-released enzymes and metabolic profiles were assessed in 21 patients with systemic lupus erythematosus, progressive systemic sclerosis and mixed cryoglobulinaemia, without overt nephropathy at a current laboratory examination, and in 31 age-sex-matched healthy controls. All patients had a urinary total protein excretion rate higher than controls (353.6 +/- 182.4 vs 243.0 +/- 108.2 mg/24 h, p < 0.01); 12 of them resulted albuminuric (775.5 +/- 1192.4 mg/24 h), while 9 were normoalbuminuric (16.6 +/- 7.6 mg/24 h). Urinary enzyme excretion rates (GGT and NAG) were significantly heightened compared to healthy subjects, both in albuminuric and in normoalbuminuric patients. Serum albumin resulted significantly lower in all patients, independent of their urinary albumin leakage. Finally, all subjects with connective tissue diseases had significantly higher triglycerides, lower HDL cholesterol and double serum fasting insulin than normals. In conclusion, all patients with collagen diseases show signs of subclinical nephropathy, not always detectable by albuminuria. They also provide evidence of insulin-resistance, a conceivable forerunner of cardiovascular complications.

Anti-collagen antibodies in systemic sclerosis and in primary Raynaud's phenomenon.

The frequency and specificity of antibodies to native and denatured collagens were evaluated in systemic sclerosis (SSc) and in primary Raynaud's phenomenon (PRP) by direct and competitive ELISA. Antibodies reactive with denatured collagen type I (CI) were found in 43% of the SSc sera, and anti-CIV and anti-CV in 31%. In PRP, anti-CI, anti-CIV and anti-CV antibodies were detected in 8% of patient sera. Anti-CI, anti-CIV and anti-CV antibodies reacted with determinants expressed on the native as well as on the denatured molecule. Anti-CI and anti-CIV were cross-reactive; a reactivity with CII and a lower one with CV were detected. Anti-CV antibodies also reacted with CI and CII and, in a smaller proportion of cases, with CIV. Anti-collagen antibodies, affinity-purified from blotted collagen IV and V and cyanogen bromide (CBr)-digested CI, displayed the cross-reactivities shown by inhibition studies on sera. Moreover, antibodies eluted from a CBr fragment of CI reacted with the other CBr fragments as well. These data show that one-third of SSc sera contain antibodies that react with epitopes expressed on native as well as on heat-denatured CI, CII, CIV and CV, and therefore have the potential to bind collagens in vivo.

Induction of anti-DNA antibodies in non autoimmune mice by immunization with a DNA-DNAase I complex.

Recent studies suggest that anti-DNA antibodies may arise from the immune response to a complex of DNA and a DNA-binding protein. One of the protein targets frequently recognized by anti-DNA antibodies is the enzyme DNAase I. To investigate the possible role of DNAase I in the induction of anti-DNA antibodies, we immunized mice with a complex of DNA and DNAase I. Mammalian double strand DNA was crosslinked with DNAase I by ultraviolet light (UV) treatment and emulsified in complete Freund's adjuvant. BALB/c mice were immunized at the base of the tail with the DNA-DNAase complex, boosted after 2 weeks with the immunogen in incomplete adjuvant and bled one week after the boost. Control mice received UV treated DNA in adjuvant. In one-third of the mice immunized with the DNA-DNAase complex, IgG anti-DNA antibodies were detectable in serum; the antibodies reacted with single and double strand DNA. No anti-DNA response was elicited by immunization with DNA alone. These data show that immunization with a DNA-DNAase complex can induce anti-DNA antibodies in non-autoimmune mice strains and suggest that DNA-binding proteins may act as carriers in the immune response that leads to anti-DNA antibody production.

Autoantibodies from mixed cryoglobulinaemia patients bind glomerular antigens.

Mixed cryoglobulinaemia (MC) is a disorder characterized by the presence of large amounts of cryoprecipitating IgM-IgG complexes. An immune complex glomerulonephritis develops in one third of all patients, but its occurrence does not seem related to the amount of cryoglobulins in the sera, nor to their complement-fixing ability. In this study we investigated the presence of IgG antibodies reactive with kidney antigens in 33 MC patients (11 with glomerulonephritis, 22 without renal involvement). A total glomerular extract was run on a 10% acrylamide gel, blotted to nitrocellulose and probed with the patients' sera. Sera from half of the patients without renal involvement reacted with several glomerular antigens whose molecular weight ranged between 200 and 29 kD. In the group with renal involvement, sera from 7/11 patients reacted with an antigen of 50 kD, which is also expressed in thymus, but not in the heart or liver. In a follow-up study of four patients with renal involvement, the amount of serum antibody specific for the 50-kD antigen fluctuated, either spontaneously or in response to therapy. These results show that antibodies specific for glomerular antigens are detectable in MC sera. The immune response against a 50-kD antigen expressed in the kidney and thymus seems to be restricted to a subset of MC patients with renal involvement. Circulating autoantibodies specific for glomerular antigens might contribute to the induction of glomerulonephritis in MC forming immune complexes in situ.

Immune response to different sequences of the EBNA I molecule in Epstein-Barr virus-related disorders and in autoimmune diseases.

Epstein-Barr virus (EBV) infection is associated with production of autoantibodies. The N-terminal 35-58 sequence of EBNA I, one of the nuclear antigens encoded by EBV, is highly homologous to the C-terminal 95-119 region of the ribonucleoprotein SmD. Autoantibodies specific for SmD are present only in systemic lupus (SLE) sera and are therefore considered a serological marker of SLE. We measured antibodies to the EBNA I 35-58 sequence in EBV-related diseases and in autoimmune disorders. Antibodies to the EBNA I 35-58 peptide were present in 30% of normal sera, 12% Burkitt lymphoma, 22% infectious mononucleosis, 25% rheumatoid arthritis, 38% SLE and 33% Sjogren's syndrome. Antibodies to the SmD 95-119 peptide were detectable in 32% of SLE sera, 17% infectious mononucleosis and 12% Burkitt lymphoma. The specificity of anti-EBNA I 35-58 antibodies affinity-purified from nine sera was analysed by means of an inhibition assay. Only anti-EBNA I 35-58 antibodies affinity-purified from SLE sera have a similar affinity for the viral peptide and the SmD C-terminal one; they also bind the recombinant SmD in western blot. The results indicate that antibodies to EBNA I 35-58 are produced in normals, in EBV-related diseases and in autoimmune disorder, but only SLE sera contain anti-viral antibodies cross-reactive with an autoantigen.

Mapping of epitopes on the SmD molecule: the use of multiple antigen peptides to measure autoantibodies in systemic lupus erythematosus.

Autoantibodies against the ribonucleoproteins B, B' and D are a serological marker of systemic lupus erythematosus (SLE). We mapped the epitopes recognized by autoantibodies on the SmD molecule by means of 7 synthetic peptides corresponding to the entire length of the protein. By ELISA assay, 25% of the lupus sera contained IgG antibodies specific for the C-terminal SmD sequence 95-119. This reactivity was confirmed by synthesizing the sequence as a multiple antigen peptide (MAP): antibodies reactive with the MAP 95-119 were present only in SLE and not in other connective tissue disorders. Sera containing high titers of anti-MAP 95-119 antibodies reacted in immunoblot with the SmD protein. These results indicate the presence of a dominant epitope in the C-terminal region of SmD, which is highly homologous to the Epstein-Barr virus induced nuclear protein EBNA I.