Giada Rossi

Eosinophilic blast crisis in a case of chronic myeloid leukaemia.

A case of typical chronic myeloid leukaemia (CML) is described, of which the terminal blast crisis was characterized by an impressive proliferation of atypical eosinophils and by the simultaneous complete deficiency of neutrophilic myeloperoxidase. Since eosinophilic cells preserved a strong peroxidase positivity, a high number of immature non‐granular eosinophilic precursors could be recognized among the leukaemic blast cells, thus supporting the diagnosis of eosinophilic blast crisis of CML.

Biosensor analysis of anti-citrullinated protein/peptide antibody affinity

Anti-citrullinated protein/peptide antibodies (ACPAs) are detected in rheumatoid arthritis (RA) sera and because of their strict association with the disease are considered marker antibodies, probably endowed with pathogenic potential. Antibody affinity is one of the parameters affecting pathogenicity. Three diagnostic citrullinated peptides-viral citrullinated peptide 1 (VCP1) and VCP2 derived from Epstein-Barr virus (EBV)-encoded proteins and histone citrullinated peptide 1 (HCP1) derived from histone H4-were synthesized as tetrameric multiple antigen peptides and immobilized on sensor chips CM5 type in a Biacore T100 instrument. Specific binding of purified antibodies from RA patients to the three peptides was analyzed by surface plasmon resonance using two arginine-containing sequences as controls. Employing a 1:1 binding model for affinity constant calculation, ACPAs interacted with VCP1 and VCP2 with lower apparent affinity (10(-6) M>KD>10(-7) M) and interacted with HCP1 with higher apparent affinity (KD=10(-8) M). The results indicate that the binding to citrullinated peptides is characterized by wide differences in affinity, with slower association and faster dissociation rates in the case of antibodies to viral citrullinated peptides as compared with antibodies specific for the histone peptide. This biosensor analysis shows the high cross-reactivity of purified ACPAs that bind other citrullinated peptides besides the one used for purification.

Immune dysfunction in Rett syndrome patients revealed by high levels of serum anti-N(Glc) IgM antibody fraction.

Rett syndrome (RTT), a neurodevelopmental disorder affecting exclusively (99%) female infants, is associated with loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2) and, more rarely, cyclin-dependent kinase-like 5 (CDKL5) and forkhead box protein G1 (FOXG1). In this study, we aimed to evaluate the function of the immune system by measuring serum immunoglobulins (IgG and IgM) in RTT patients (n = 53) and, by comparison, in age-matched children affected by non-RTT pervasive developmental disorders (non-RTT PDD) (n = 82) and healthy age-matched controls (n = 29). To determine immunoglobulins we used both a conventional agglutination assay and a novel ELISA based on antibody recognition by a surrogate antigen probe, CSF114(Glc), a synthetic N-glucosylated peptide. Both assays provided evidence for an increase in IgM titer, but not in IgG, in RTT patients relative to both healthy controls and non-RTT PDD patients. The significant difference in IgM titers between RTT patients and healthy subjects in the CSF114(Glc) assay (P = 0.001) suggests that this procedure specifically detects a fraction of IgM antibodies likely to be relevant for the RTT disease. These findings offer a new insight into the mechanism underlying the Rett disease as they unveil the possible involvement of the immune system in this pathology.

Antibody Recognition in multiple sclerosis and rett syndrome using a collection of linear and cyclic N‐glucosylated antigenic probes

Antibody detection in autoimmune disorders, such as multiple sclerosis (MS) and Rett syndrome (RTT) can be achieved more efficiently using synthetic peptides. The previously developed synthetic antigenic probe CSF114(Glc), a type I′ β‐turn N‐glucosylated peptide structure, is able to recognize antibodies in MS and RTT patients’ sera as a sign of immune system derangement. We report herein the design, synthesis, conformational analysis, and immunological evaluation of a collection of glycopeptide analogs of CSF114(Glc) to characterize the specific role of secondary structures in MS and RTT antibody recognition. Therefore, we synthesized a series of linear and cyclic short glucosylated sequences, mimicking different β‐turn conformations, which were evaluated in inhibition enzyme‐linked immunosorbent assays (ELISA). Calculated IC50 ranking analysis allowed the selection of the candidate octapeptide containing two (S)−2‐amino‐4‐pentynoic acid (L‐Pra) residues Ac‐Pra‐RRN(Glc)GHT‐Pra‐NH2, with an IC50 in the nanomolar range. This peptide was adequately modified for solid‐phase ELISA (SP‐ELISA) and surface plasmon resonance (SPR) experiments. Pra‐RRN(Glc)GHT‐Pra‐NH2 peptide was modified with an alkyl chain linked to the N‐terminus, favoring immobilization on solid phase in SP‐ELISA and differentiating IgG antibody recognition between patients and healthy blood donors with a high specificity. However, this peptide displayed a loss in IgM specificity and sensitivity. Moreover, an analog was obtained after modification of the octapeptide candidate Ac‐Pra‐RRN(Glc)GHT‐Pra‐NH2 to favor immobilization on SPR sensor chips. SPR technology allowed us to determine its affinity (KD = 16.4 nM), 2.3 times lower than the affinity of the original glucopeptide CSF114(Glc) (KD = 7.1 nM).

Comparison of pathologic and normal sera by immune complex determination: five disease groups within 190 samples are discriminated by computer-selected combinations of 13 methods. Report of the Italian committee for the study of immune complexes (WIC).

Pathological (190) and normal (33) sera were tested for their content of circulating immune complexes (CIC) by a battery of 13 assays performed in 11 laboratories. Statistical processing was done both by pooling all pathological samples and by extracting those falling into well-defined disease groups, i.e., rheumatoid arthritis, diabetes, lupus, melanoma, and glomerulonephritis. Highly significant correlations between methods--taken two at a time--for each disease differed in proportion (ranging from 6 to 30%) and in the pattern displayed on a checkerboard. Disease-linked patterns were also found when a function maximizing discrimination between pathological and normal samples was derived by combining the information from all methods. Here the order and the weight attributed by the computer to the methods differed for each of the disease groups. Taken together these results are interpreted as an indication that all assays may not determine the same classes of CIC, and thus vary in sensitivity depending on the prevailing properties of the complexes present in the serum, which in turn may depend on the etiology, pathogenesis, and stage of the disease.

Label-free method for anti-glucopeptide antibody detection in Multiple Sclerosis

Graphical abstract

Antibodies from multiple sclerosis patients preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus influenzae.

In autoimmune diseases, there have been proposals that exogenous “molecular triggers”, i.e., specific this should be ‘non-self antigens’ accompanying infectious agents, might disrupt control of the adaptive immune system resulting in serious pathologies. The etiology of the multiple sclerosis (MS) remains unclear. However, epidemiologic data suggest that exposure to infectious agents may be associated with increased MS risk and progression may be linked to exogenous, bacterially-derived, antigenic molecules, mimicking mammalian cell surface glycoconjugates triggering autoimmune responses. Previously, antibodies specific to a gluco-asparagine (N-Glc) glycopeptide, CSF114(N-Glc), were identified in sera of an MS patient subpopulation. Since the human glycoproteome repertoire lacks this uniquely modified amino acid, we turned our attention to bacteria, i.e., Haemophilus influenzae, expressing cell-surface adhesins including N-Glc, to establish a connection between H. influenzae infection and MS. We exploited the biosynthetic machinery from the opportunistic pathogen H. influenzae (and the homologous enzymes from A. pleuropneumoniae) to produce a unique set of defined glucosylated adhesin proteins. Interestingly we revealed that a hyperglucosylated protein domain, based on the cell-surface adhesin HMW1A, is preferentially recognized by antibodies from sera of an MS patient subpopulation. In conclusion the hyperglucosylated adhesin is the first example of an N-glucosylated native antigen that can be considered a relevant candidate for triggering pathogenic antibodies in MS.

Surface Plasmon Resonance Method to Evaluate Anti-citrullinated Protein/Peptide Antibody Affinity to Citrullinated Peptides.

Surface plasmon resonance (SPR) technique is extremely interesting in immunology because it has the potential to directly visualize biomolecular interactions in real-time monitoring antibody affinity, one of the parameters affecting pathogenicity in autoimmune diseases. Herein we describe the affinity evaluation of anti-citrullinated peptide antibodies (ACPA) to a peptide-based biosensor by SPR. The method describes the purification of ACPA isolated from rheumatoid arthritis (RA) patients using affinity columns, the strategy employed for the immobilization of citrullinated peptides onto a sensor chip, and the evaluation of the specific binding of purified ACPA to immobilized peptides.

Circulating immune complexes in human acute leukaemia.

Summary. Circulating immune complexes (CIC) in the sera of 60 newly diagnosed leukaemic patients were investigated by two methods, 125I‐C1q binding test (C1q‐BA) and conglutinin binding assay (KgB‐SP). Positivity percentages were respectively 20.0% (C1q‐BA) and 28.3% (KgB‐SP). The small overlap between the results of the two methods suggests the occurrence of different types of CIC. The presence of CIC was found to be related only to clinical haemorrhage and thrombocytopenia; it did not prove to affect the prognosis and the survival of leukaemic patients.

Human recombinant domain antibodies against multiple sclerosis antigenic peptide CSF114(Glc)

Multiple sclerosis (MS) is a chronic auto‐immune disease characterized by a damage to the myelin component of the central nervous system. Self‐antigens created by aberrant glycosylation have been described to be a key component in the formation of auto‐antibodies. CSF114(Glc) is a synthetic glucopeptide detecting in vitro MS‐specific auto‐antibodies, and it is actively used in diagnostics and research to monitor and quantify MS‐associated Ig levels. We reasoned that antibodies raised against this probe could have been relevant for MS. We therefore screened a human Domain Antibody library against CSF114(Glc) using magnetic separation as a panning method. We obtained and described several clones, and the one with the highest signals was produced as a 6×His‐tagged protein to properly study the binding properties as a soluble antibody. By surface plasmon resonance measurements, we evidenced that our clone recognized CSF114(Glc) with high affinity and specific for the glucosylated peptide. Kinetic parameters of peptide–clone interaction were calculated obtaining a value of KD in the nanomolar range. Harboring a human framework, this antibody should be very well tolerated by human immune system and may represent a valuable tool for MS diagnosis and therapy, paving the way to new research strategies. Copyright