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The iPTH upper reference limit (URL) reported by our laboratory provider (Abbott Laboratories) at Tor Vergata University Hospital was evaluated by internal verification procedures as not representative of our population and resulting as underestimated. In this study, a new reference interval has been investigated and established by comparing a direct and an indirect method based on a statistical reduction from results stored in the laboratory database.For reference interval calculation from the healthy population, we analyzed a cohort of 100 blood donors (84% males and 16% females) screened with no bone-related and malabsorption diseases. We analyzed a cohort of 495 patients retrieved from more than 800 iPTH results by excluding subjects with pathological measurement for calcium, phosphorus, and creatinine for the reference interval evaluation. Patients with vitamin D results were included in the analysis. Vitamin D sufficiency status during the period from January to September 2020 was also evaluated by investigating 3,050 patients.The iPTH reference interval of a healthy blood donor population was measured as 25.2-109.1 pg/mL (2.7-11.6 pmol/L) at 2.5 and 97.5 distribution percentile. The iPTH reference interval from data stored in the laboratory database was 19.3-112.5 pg/mL (2.0-11.9 pmol/L). Furthermore, 60% of the whole population had prevalently insufficient vitamin D concentration (30 ng/dL;75 nmol/L). The impact of vitamin D concentration on the iPTH reference interval was measured for insufficient vitamin D (30 ng/dL;75 nmol/L) as 15.2-127.7 pg/mL (1.6-13.5 pmol/L), desirable vitamin D (30-40 ng/ml; 75-100 nmol/L) as 25.6-105 pg/mL (2.7-10.7 pmol/L) and optimal vitamin D (40 ng/ml;100 nmol/L) as 26.2-89.2 pg/mL (2.8-9.4 pmol/L), respectively.The URL reported in manufacturer datasheets likely refers to a normal population with non-pathological vitamin D levels. On the contrary, the considered population was mostly vitamin D insufficient, resulting in a URL shift. On this basis, we suggest describing in medical reports the iPTH range for vitamin D deficiency for diagnosis of primary hyperparathyroidism even when a specific vitamin D request is lacking. On the other hand, reporting optimal vitamin D-based iPTH reference interval could be clinically relevant in supplemented patients as a marker of treatment efficacy.

Cardiac calcifications are a frequent finding in hemodialysis for chronic renal failure. Several factors may play a role in the intimal and medial calcification of coronary arteries such as age and some known atherogenetic factors. In addition, Fetuin-A has been proposed as a protective agent through solubilization of calcium phosphate salt. Fetuin-A is also a marker of inflammatory-nutritional state, and its changes could be an expression of this condition. The aim of this cross-sectional study is to evaluate the relative importance of risk factors of calcifications with special regard to Fetuin-A. The study was conducted with 132 hemodialysis patients. They were subjected to multislice computed tomography for evaluation of calcium deposits in the heart. In addition, the patients were sampled for evaluation of calcium-phosphate parameters, lipid profile, nutritional and inflammatory markers, and also Fetuin-A. There was a wide variability of the extent of calcium deposits expressed as Agatston score, with only 9.3% of patients without calcifications. Age, hemodialysis age, sex, calcium-phosphate parameters, and lipid profile were important risk factors, together with nutritional and inflammatory status of the patients. An inverse correlation between coronary calcium score and Fetuin-A emerged from a multiple regression analysis. However, there was no significant difference in serum Fetuin-A among different grades of calcium score. By dividing the patients in tertiles of serum Fetuin-A, an association between low levels of Fetuin-A and high calcification score was found. Fetuin-A as dependent variable was strictly linked to prealbumin serum levels. In addition, there was a clear link between cardiac calcification scores and inflammatory-nutritional markers. Serum calcium and treatment with calcitriol emerged as predictive variables of coronary score.Fetuin-A could be involved in the process of calcification both in the case of markedly low serum levels, due to decreased prevention of calcium phosphate precipitation, and also as a marker of inflammation, a well-known risk factor of atherogenesis. Treatment with intravenous calcitriol could marginally enhance cardiac calcifications, probably through its hypercalcemic effect.

The recombinant SA35 peptide has been described as an antigenic portion of a larger Cryptosporidium parvum protein. We identified and characterized the encoding Cpa135 gene and the entire protein, Cpa135. The Cpa135 gene was found to consist of a single exon of 4671 bp, and the mRNA transcribed in the sporozoites was identified. The predicted 1556 amino-acid protein showed the presence of domains which are widely conserved also in other unrelated phylogenetic groups (i.e. a ricin B and a LCCL motif). Comparison of Cpa135 sequence with genomic and protein databases revealed many related genes in other apicomplexan species and high homology with CCP2 protein from Plasmodium yoelii and Plasmodium berghei. The Cpa135 protein was identified and localized by using a monoclonal antibody (Mab) directed against the SA35 antigen (anti-SA35). In oocyst-sporozoite lysate, the anti-SA35 MAb recognized a 135 kDa protein that forms a protein complex larger than 200 kDa, which is mediated by disulfide bridges. Cpa135 synthesis was up-regulated during the excystation process. After host-cell invasion, Cpa135 gene expression was undetectable up to 48 h, whereas mRNA synthesis was newly observed at 72 h post-infection. The Cpa135 protein was localized in the apical complex, and it was found to be secreted by sporozoites during their gliding. Cpa135 persisted during the intracellular stages of the parasite, and it defined the boundaries of the parasitophorous vacuole in the infected cells. The unique array of domains and the homology with other apicomplexan proteins indicate that the Cpa135 protein is representative of a new family of proteins.

Publisher Summary This chapter focuses on the identification and characterization of the antigenic Cpal35 protein. Three antigenic peptides that represent portions of larger sporozoite proteins have been identified previously. One of those peptides (SA35) was specifically recognized by human IgG and the objective of the present study was to determine the origin of that peptide. The search for homologies at the protein level reveals that Cpal35 has a composite structure with regions of similarity with different unrelated proteins. At its N-terminus, Cpal35 shows a possible leader peptide flanked by a region with homology to yeast coronins and animal tropomyosins. The coronins are the proteins involved in cytoskeleton re-modelling that promote actin polymerization; tropomyosins are also actin binding proteins. In the middle of the protein, there is a large region without homology with other proteins and two short repetitive amino acidic sequences. The C-terminus of this protein is a region with high-cystein content that shows a homology to a soybean protein of unknown function. Histidine-tagged peptides are a useful way of obtaining specific antibodies raised against antigenic proteins; thus, the monoclonal antibody specific for SA35 permitted the identification and the characterization of the Cpal35 protein. This protein is localized in the apical complex of the sporozoites, suggesting that Cpal35 can play a role in the host-cell invasion. The presence of a putative leader peptide in the protein sequence suggests that Cpal35 can be a protein secreted through specialized organelles, Tmicronemes. The composite arrangement of that protein suggests that Cpal35 is a multi-domain protein with different motifs to exert different molecular interactions. Further studies will be dedicated to ascribing a functional role to the Cpal35 putative domains and to determine the potential role as a protective immunizing antigen.