Anti‐DNA cell‐penetrating autoantibodies have been extensively studied in autoimmune but not in normal sera. We investigated herein the presence and properties of cell‐penetrating antibodies (CPAbs) in intravenous immunoglobulin (IVIg), a blood product of pooled normal human IgG. IVIg cell penetration was observed into various cell lines, as well as cells from several organs of mice injected intravenously with IVIg therapeutic dose. In all cell types examined in vitro and in vivo, intracellular IgG localized in the cytoplasm, in contrast to the nuclear accumulation of disease‐related CPAbs. IVIg was found to rapidly enter cells via an energy‐independent mode. The CPAb‐fraction was isolated and found to be polyreactive to nuclear and cytoplasmic components; although it corresponded to ~2% of IVIg, it accounted for its inhibitory effect on splenocyte activation. Investigation of IVIg cell penetration capacity provides insight into its mechanisms of action and may account for some of its beneficial effects in numerous diseases.

Studies on the humoral response to homologous BNT162b2 mRNA-vaccination focus mainly on IgG antibody dynamics, while long-term IgA kinetics are understudied. Herein, kinetics of IgG and IgA levels against trimeric-Spike (S) and Receptor-Binding-Domain (RBD) were evaluated by in-house ELISAs in 146 two-dose vaccinated Greek healthcare workers (HCWs) in a 9-month period at six time points (up to 270 days after the first dose). The effect of a homologous booster third dose was also studied and evaluated. The peak of immune response was observed 21 days after the second dose; 100% seroconversion rate for anti-S and anti-RBD IgG, and 99.7% and 96.3% respectively for IgA. IgG antibody levels displayed higher increase compared to IgA. Declining but persistent anti-SARS-CoV-2 antibody levels were detected 9 months after vaccination; IgG and IgA anti-S levels approached those after the first dose, while a more rapid reduction rate for anti-RBD antibodies led to significantly lower levels for both classes, supporting the need for a booster dose. Indeed, a homologous booster third dose resulted in enhanced levels of anti-S of both classes, whereas anti-RBD didn’t exceed the peak levels after the second dose. Previous SARS-CoV-2 infection, flu vaccination, BMI<35 and the occurrence of an adverse event upon vaccination, were associated with higher IgG antibody levels over time, which however were negatively affected by age increase and the presence of chronic diseases. Overall, after concurrently using the S and RBD target-antigens in in-house ELISAs, we report in addition to IgG, long-term persistence of IgA antibodies. Regarding antibody levels, homologous mRNA vaccination gives rise to an effective anti-viral protection up to 9 months negatively correlated to age. Considering that COVID-19 is still a matter of public concern, booster vaccine doses remain critical to vulnerable individuals.

Naturally occurring antibodies (NAbs), which are major components of innate immunity, exist in circulation under healthy conditions without prior antigenic stimulation and are able to recognize both self- and non-self-constituents. The present study aimed at identifying potential immunological differences between commercial fast- and slow-growth broilers (n = 555) raised in conventional and free-range systems, respectively, through the use of the specificity, isotypes and levels of circulating NAbs. The possible beneficial effect of oregano-based dietary supplementation was also evaluated. To this end, serum IgM and IgY NAbs against self- (actin and DNA) and non-self- antigens (trinitrophenol and lipopolysaccharide) were measured by ELISA and further correlated with genotype, season and performance. Significantly higher levels of IgM NAbs against all antigens were found in slow-growth compared to fast-growth broilers. IgM NAb levels were also significantly increased in dietarily supplemented slow-growth broilers versus those consuming standard feed. Moreover, significantly elevated levels of anti-DNA IgY NAbs were found in fast-growth compared to slow-growth broilers, whereas the opposite was observed for anti-LPS IgY NAbs. Multivariate linear regression analysis confirmed multiple interactions between NAb levels, genotype, season and performance. Overall, serum NAbs have proven to be valuable innovative immunotools in the poultry industry, efficiently differentiating fast-growing versus slow-growing broilers, and dietary supplementation of plant extracts can enhance natural immunity.

The global demand for improvement in the welfare conditions of broilers has generated the necessity to implement alternative rearing systems as well as less intensive growth hybrids. The majority of the data on alternative farming methods, notwithstanding their abundance, are the result of small-scale experiments. The present extended field study examined the effect of two different industrial farming systems on broilers’ antioxidant status, performance, and meat quality, including 13 replicates of each industrial breeding system (intensive conventional; free range) and two different chicken genotypes (fast growth; slow growth). The duration of the study was 51 months, and the total number of broilers was 260.000 for the conventional and 78.000 for the free-range system. The results showed that fast-growth chicks demonstrated a more satisfactory performance (in terms of body weight gain (BWG) and feed conversion ratio (FCR) with p ≤ 0.001), reduced serum lipid oxidation (p ≤ 0.05), and more tender meat. Contrarily, slow-growth chickens presented significantly higher total antioxidant capacity (TAC) in serum and thigh muscle (p ≤ 0.001), significantly lower (p ≤ 0.05) thigh muscle oxidation (in terms of thiobarbituric acid reactive substances, TBARS), increased protein and decreased fat content (p ≤ 0.05), and better smell, taste, color, and texture. In conclusion, the free-range farming system for slow-growth chickens may result in an overall higher nutritional value, sensory score, and serum and thigh muscle antioxidant profile than the conventional farming system for fast-growth broilers. However, fast-growth broilers exhibit better performance and might undergo less stress.

Subset #201 is a clinically indolent subgroup of patients with chronic lymphocytic leukemia defined by the expression of stereotyped, mutated IGHV4-34/IGLV1-44 BCR Ig. Subset #201 is characterized by recurrent somatic hypermutations (SHMs) that frequently lead to the creation and/or disruption of N-glycosylation sites within the Ig H and L chain variable domains. To understand the relevance of this observation, using next-generation sequencing, we studied how SHM shapes the subclonal architecture of the BCR Ig repertoire in subset #201, particularly focusing on changes in N-glycosylation sites. Moreover, we profiled the Ag reactivity of the clonotypic BCR Ig expressed as rmAbs. We found that almost all analyzed cases from subset #201 carry SHMs potentially affecting N-glycosylation at the clonal and/or subclonal level and obtained evidence for N-glycan occupancy in SHM-induced novel N-glycosylation sites. These particular SHMs impact (auto)antigen recognition, as indicated by differences in Ag reactivity between the authentic rmAbs and germline revertants of SHMs introducing novel N-glycosylation sites in experiments entailing 1) flow cytometry for binding to viable cells, 2) immunohistochemistry against various human tissues, 3) ELISA against microbial Ags, and 4) protein microarrays testing reactivity against multiple autoantigens. On these grounds, N-glycosylation appears as relevant for the natural history of at least a fraction of Ig-mutated chronic lymphocytic leukemia. Moreover, subset #201 emerges as a paradigmatic case for the role of affinity maturation in the evolution of Ag reactivity of the clonotypic BCR Ig.