Children with Kawasaki disease or Kawasaki-like syndrome (MIS-C/PIMS) at the time of COVID-19: are they all the same? Case series and literature review.

Since the coronavirus disease 2019 (COVID-19) outbreak started, children have been considered marginally involved compared to adults, with a quite significant percentage of asymptomatic carriers. Very recently, an overwhelming inflammatory activation, which shares clinical similarities with Kawasaki disease (KD), has been described in children exposed to COVID-19. We report three KD-like cases that occurred during the pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a highly affected area of Northern Italy. The clinical presentation was characterized by the presence of unremitting fever, diarrhea and elevated inflammatory markers. Case #1 and Case #2 occurred one week apart and shared other clinical features: laboratory tests confirmed COVID-19 exposure and high inflammatory activation with myocardial involvement. Case #3 followed a more typical pattern for KD. Interestingly, this patient showed lower levels of procalcitonin, C-reactive protein, D-dimers, and ferritin compared to the other two cases, whereas platelet count was higher. We hypothesize that SARS-CoV-2 might act in children as a trigger, either inducing a classical KD phenotype or causing a systemic inflammatory response leading to a severe KD-like phenotype, eventually characterized by myocardial impairment. We think that bringing these cases and their differences to the attention of the rheumatology community during the COVID-19 pandemic will be beneficial in order to highlight the importance of early diagnosis and to increase awareness of this new phenomenon.

Repression of miR-31 by BCL6 stabilizes the helper function of human follicular helper T cells.

Follicular helper T cells (TFHs) are a key component of adaptive immune responses as they help antibody production by B cells. Differentiation and function of TFH cells are controlled by the master gene BCL6, but it is largely unclear how this transcription repressor specifies the TFH program. Here we asked whether BCL6 controlled helper function through down-regulation of specific microRNAs (miRNAs). We first assessed miRNA expression in TFH cells and defined a TFH-specific miRNA signature. We report that hsa-miR-31-5p (miR-31) is down-regulated in TFH; we showed that BCL6 suppresses miR-31 expression by binding to its promoter; and we demonstrated that miR-31 inhibits the expression of molecules that control T-helper function, such as CD40L and SAP. These findings identify a BCL6-initiated inhibitory circuit that stabilizes the follicular helper T cell program at least in part through the control of miRNA transcription. Although BCL6 controls TFH activity in human and mouse, the role of miR-31 is restricted to human TFH cell differentiation, reflecting a species specificity of the miR-31 action. Our findings highlight miR-31 as a possible target to modulate human T cell dependent antibody responses in the settings of infection, vaccination, or immune dysregulation.

Idiopathic erythrocytosis: a germline disease?

Polycythemia Vera (PV) is typically caused by V617F or exon 12 JAK2 mutations. Little is known about Polycythemia cases where no JAK2 variants can be detected, and no other causes identified. This condition is defined as idiopathic erythrocytosis (IE). We evaluated clinical-laboratory parameters of a cohort of 56 IE patients and we determined their molecular profile at diagnosis with paired blood/buccal-DNA exome-sequencing coupled with a high-depth targeted OncoPanel to identify a possible underling germline or somatic cause. We demonstrated that most of our cohort (40/56: 71.4%) showed no evidence of clonal hematopoiesis, suggesting that IE is, in large part, a germline disorder. We identified 20 low mutation burden somatic variants (Variant allelic fraction, VAF, < 10%) in only 14 (25%) patients, principally involving DNMT3A and TET2. Only 2 patients presented high mutation burden somatic variants, involving DNMT3A, TET2, ASXL1 and WT1. We identified recurrent germline variants in 42 (75%) patients occurring mainly in JAK/STAT, Hypoxia and Iron metabolism pathways, among them: JAK3-V722I and HIF1A-P582S; a high fraction of patients (48.2%) resulted also mutated in homeostatic iron regulatory gene HFE-H63D or C282Y. By generating cellular models, we showed that JAK3-V722I causes activation of the JAK-STAT5 axis and upregulation of EPAS1/HIF2A, while HIF1A-P582S causes suppression of hepcidin mRNA synthesis, suggesting a major role for these variants in the onset of IE.

Molecular mechanism of thioredoxin regulation in photosynthetic A2B2-glyceraldehyde-3-phosphate dehydrogenase.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-regulated, NAD(P)H-dependent enzyme involved in plant photosynthetic carbon reduction. Unlike lower photosynthetic organisms, which only contain A(4)-GAPDH, the major GAPDH isoform of land plants is made up of A and B subunits, the latter containing a C-terminal extension (CTE) with fundamental regulatory functions. Light-activation of AB-GAPDH depends on the redox state of a pair of cysteines of the CTE, which can form a disulfide bond under control of thioredoxin f, leading to specific inhibition of the NADPH-dependent activity. The tridimensional structure of A(2)B(2)-GAPDH from spinach chloroplasts, crystallized in the oxidized state, shows that each disulfide-containing CTE is docked into a deep cleft between a pair of A and B subunits. The structure of the CTE was derived from crystallographic data and computational modeling and confirmed by site-specific mutagenesis. Structural analysis of oxidized A(2)B(2)-GAPDH and chimeric mutant [A+CTE](4)-GAPDH revealed that Arg-77, which is essential for coenzyme specificity and high NADPH-dependent activity, fails to interact with NADP in these kinetically inhibited GAPDH tetramers and is attracted instead by negative residues of oxidized CTE. Other subtle changes in catalytic domains and overall conformation of the tetramers were noticed in oxidized A(2)B(2)-GAPDH and [A+CTE](4)-GAPDH, compared with fully active A(4)-GAPDH. The CTE is envisioned as a redox-sensitive regulatory domain that can force AB-GAPDH into a kinetically inhibited conformation under oxidizing conditions, which also occur during dark inactivation of the enzyme in vivo.

X-ray diffraction study of bovine lens capsule collagen.

The wide angle X-ray diffraction pattern of air-dried lens capsule collagen under tension is the same as the tendon collagen diffraction pattern with regard to the main reflections, and indicates that lens capsule collagen has the characteristic three-stranded helical structure with an axial repeat of 0.29 nm as tendon collagen. The low angle X-ray diffraction pattern shows several weak diffraction maxima corresponding to the meridional reflections of capsule collagen which show orders of 63.0 nm periodicity. This is an evidence of quarter staggered molecular assembly typical of tendon collagen even if less ordered. The results are consistent with the existence in lens capsule collagen of clearly defined molecular units, which can be oriented by stress and are packed in a poor-ordered fibrillar assembly.

Crystal structure of the non-regulatory A(4 )isoform of spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase complexed with NADP.

Here, we report the first crystal structure of a photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complexed with NADP. The enzyme, purified from spinach chloroplasts, is constituted of a single type of subunit (A) arranged in homotetramers. It shows non-regulated NADP-dependent and NAD-dependent activities, with a preference for NADP. The structure has been solved to 3.0 A resolution by molecular replacement. The crystals belong to space group C222 with three monomers in the asymmetric unit. One of the three monomers generates a tetramer using the space group 222 point symmetry and a very similar tetramer is generated by the other two monomers, related by a non-crystallographic symmetry, using a crystallographic 2-fold axis. The protein reveals a large structural homology with known GAPDHs both in the cofactor-binding domain and in regions of the catalytic domain. Like all other GAPDHs investigated so far, the A(4)-GAPDH belongs to the Rossmann fold family of dehydrogenases. However, unlike most dehydrogenases of this family, the adenosine 2'-phosphate group of NADP does not form a salt-bridge with any positively charged residue in its surroundings, being instead set in place by hydrogen bonds with a threonine residue belonging to the Rossmann fold and a serine residue located in the S-loop of a symmetry-related monomer. While increasing our knowledge of an important photosynthetic enzyme, these results contribute to a general understanding of NADP versus NAD recognition in pyridine nucleotide-dependent enzymes. Although the overall structure of A(4)-GAPDH is similar to that of the cytosolic GAPDH from bacteria and eukaryotes, the chloroplast tetramer is peculiar, in that it can actually be considered a dimer of dimers, since monomers are bound in pairs by a disulphide bridge formed across Cys200 residues. This bridge is not found in other cytosolic or chloroplast GAPDHs from animals, bacteria, or plants other than spinach.

Metabolic fate of zetidoline, a new neuroleptic agent, in man.

Healthy volunteers administered orally a single dose (20 mg) of [2-14C]zetidoline, a new dopamine antagonist, exhibited rapid absorption of radioactivity with peak plasma levels of 250-300 ng/ml achieved in 1 h. The compound underwent intensive metabolic first-pass so that plasma radioactivity was represented mostly by two products, metabolite B endowed with neuroleptic activity, and metabolite D inactive, while unchanged zetidoline was not detected. Disappearance of radioactivity from plasma was rapid with a half-life of 1.78 +/- 0.20 h. The simultaneous assay of plasma prolactin showed increased levels of the hormone (+ 464% at the peak time) up to the 6th h after dosing, with plasma concentration profile which mimic those of metabolite B. The radioactive test-dose was eliminated mainly via the kidneys with an average urinary recovery of 84.7 +/- 1.7% in 4 days (73.4 +/- 1.1% within 8 h). The main urinary metabolite (metabolite G) and two minor ones (metabolites B and D) were purified and their structures assigned by IR, MS and NMR spectroscopy, they are: 1-(3-chloro-4-hydroxyphenyl)-3 [2-(3,3-dimethyl-1-azetidinyl)ethyl]imidazolidin-2-one, metabolite B; 1-[2-(3,3-dimethyl-1-azetidinyl)ethyl]-imidazolidin-2-one, metabolite D and the 4'-O-sulphate ester of metabolite B, metabolite G. The metabolic fate of zetidoline in man follows the same phase I reactions demonstrated in rats and dogs, while the phase II reaction is sulphoconjugation instead of the glucuronidation observed in animals.

Relationship between Solid State NMR Parameters and X-ray Structural Data in Tricadmium Phosphates.

31P and (113)Cd MAS NMR spectra of solid beta'-tricadmium phosphate (beta'-TCdP) show a number of highly resolved resonances that agree well with the number of independent crystallographic sites indicated by the results of X-ray diffraction studies. A correlation of the (31)P chemical shifts with the crystallographic sites for the six different PO(4)(3)(-) groups in the unit cell of beta'-TCdP has been obtained by a method based on the computation of bond strength at oxygen atoms in phosphate moieties. The assignment of the (113)Cd resonances has been carried out on the basis of the relationship between the asymmetry of the chemical shift tensor (evaluated by analysis of the spinning side bands intensities in the MAS spectrum) and a geometric parameter related to the distortion from the bipyramidal trigonal coordination at each cadmium center. Samples of tricadmium phosphate with different degrees of magnesium substitution for cadmium were investigated by (31)P MAS NMR, (113)Cd MAS NMR, and X-ray diffraction. The results of these investigations showed that the magnesiums distribute randomly in the cadmium sites, inducing a marked decrease in the order of the structure.

Oriented crystallization of octacalcium phosphate into beta-chitin scaffold.

Composites of beta-chitin with octacalcium phosphate (OCP) or hydroxylapatite (HAP) were prepared by precipitation of the mineral into a chitin scaffold by means of a double diffusion system. The beta-chitin was obtained from the pen of the Loligo sp. squid. Only oriented precipitation of OCP was observed. The OCP crystals with the usual form of (001) blades grow inside chitin layers preferentially oriented with the [100] faces parallel to the surface of the squid pen and were more stable to the hydrolysis to HAP with respect to that precipitated in solution. Reasons are given why mechanical factors are thought to be the predominant cause for the orientation of the OCP crystals with the a-axis almost normal to the chitin fibers. We conclude that in these in vitro experiments the compartmentalized space in the chitin governs the orientation of the crystals, even if epitaxial factors may play a role in the nucleation processes.

Thermal behavior of bone and synthetic hydroxyapatites submitted to magnesium interaction in aqueous medium.

The thermal behavior of the products obtained from magnesium interaction with powdered femoral bone and carbonate containing synthetic hydroxyapatite under conditions of pH fluctuation in aqueous medium has been investigated. The products, heat treated at different temperatures from 100 to 1300 degrees C, have been characterized by infrared spectroscopy and X-ray diffraction technique. The results show that the interaction with magnesium ion destabilizes the apatitic structure and favours its thermal conversion into beta-tricalcium phosphate (beta-TCP). The replacement of magnesium with calcium in the beta-TCP crystal lattice hinders its subsequent thermal conversion into the alpha form. The influence of magnesium on the thermal stability is much more evident for carbonate-containing synthetic hydroxyapatite than for bone apatite.

Thermal conversion of octacalcium phosphate into hydroxyapatite.

The thermal conversion of octacalcium phosphate into hydroxyapatite has been investigated by a crystallographic, thermogravimetric, and calorimetric study. The conversion of octacalcium phosphate takes place through the remotion of three of its five water molecules and yields a poor crystalline apatitic phase. The three water molecules are lost in two steps. The first one, which is reversible, corresponds to the remotion of one water molecule and induces a slight contraction of the unit cell of OCP. The successive remotion of two water molecules, which provokes the structural conversion of OCP into apatite, is in irreversible process. The mechanism of the water loss of OCP is explained in terms of its crystal structure.

Structural and chemical characterization of inorganic deposits in calcified human mitral valve.

X-ray diffraction, i.r. absorption, and chemical analyses have been carried out on the mineral deposits of calcified human mitral valves and glutaraldehyde-preserved porcine aortic grafts. The mineral deposits isolated from highly calcified mitral valves and porcine aortic grafts are constituted of type B-carbonate apatite. Magnesium substituted beta-tricalcium phosphate is present, together with an apatitic phase similar to dahllite, in the ashes of poorly calcified mitral valves. The contraction of the unit cell of beta-tricalcium phosphate due to magnesium incorporation is compared with the variation of the lattice constants of synthetic beta-tricalcium phosphate at different degree of magnesium substitution for calcium. The results reveal the important role of magnesium on the calcification of human valves. In fact, the apatitic phase deposited at the beginning of the calcification process, when there is a high magnesium content, converts completely into beta-tricalcium phosphate by heat treatment at 1,000 degrees C. On the other hand, when the calcification becomes massive, magnesium content appears highly reduced, and the deposited apatitic phase is characterized by a high thermal stability.

Chemical and structural characterization of the mineral phase from cortical and trabecular bone.

X-ray diffraction, infrared spectroscopy and chemical investigations have been carried out on the inorganic phases from rat cortical and trabecular bone. Although both inorganic phases consist of poorly crystalline B carbonated apatite, several significant differences have been observed. In particular, trabecular bone apatite displays reduced crystallite sizes, Ca/P molar ratio, and carbonate content, and exhibits a greater extent of thermal conversion into beta-tricalcium phosphate than cortical bone apatite. These differences can be related to the different extents of collagen posttranslational modifications exhibited by the two types of bone, in agreement with their different biological functions.

Structural analysis of turkey tendon collagen upon removal of the inorganic phase.

Calcified leg flexor tendons in which the inorganic phase content had been lowered by progressive demineralization were studied by small angle X-ray diffraction and thermogravimetry. The X-ray diffraction results agree very well with the data previously obtained on calcified turkey tendon indicating that the method used to decalcify tendons provides good correspondence with the process of calcification. Up to five thermal processes can be detected in the thermogravimetric scans: (1) water release; (2) collagen decomposition; (3 and 4) combustion of the residual organic components; (5) carbonate removal from the apatitic phase. The temperature of collagen decomposition decreases at lower inorganic phase content in agreement with the higher thermal stability of calcified collagen fibrils compared with uncalcified ones. The decrease of collagen thermal stability upon decalification is paralleled by a decrease of the structural order of the collagen fibrils as indicated by small angle X-ray diffraction data. Decalcification down to about 40% wt of inorganic phase does not significantly alter the inorganic blocks that are regularly arranged inside the gap zone of the collagen. Further removal of inorganic phase down to about 15% wt provokes a variation of the intensity distribution of the small angle meridional reflections that can be ascribed to a reduction of the mean height of the inorganic blocks. At inorganic phase contents below 15% wt the gap region is more free to contract upon air drying as a result of the reduction of the mean length of the inorganic blocks.

Pharmacokinetics and metabolism of tomoxiprole, a new analgesic antiinflammatory agent, in the rat.

The pharmacokinetics and the metabolic profile of tomoxiprole, a new analgesic antiinflammatory agent belonging to the class of 3-alkyl-2-aryl-3H-naphth (1,2-d)imidazoles, were studied in the rat. After oral administration (5 mg/kg) to male rats, tomoxiprole was rapidly absorbed, mostly by the gut, and reached maximum plasma levels of about 0.5 microgram/ml in 0.25-2 h. A metabolic first pass reduced the extent of oral bioavailability of the parent compound to about half, while absorption (total 14C data) was estimated to be complete. After intravenous injection (2.5 mg/kg), the plasma kinetics of tomoxiprole in male rats showed a bi-exponential profile, and the terminal elimination half-life was 4.2 h. The apparent volume of distribution was high, suggesting a wide distribution of the drug. Increasing the oral dose by ten times (50 mg/kg), resulted in linear kinetics with a proportional increase of the C max and AUC values and the same value of terminal elimination half-life. In females given a 5 mg/kg dose, the plasma levels of 14C, tomoxiprole and AUC values were somewhat higher than in males. The plasma levels of total 14C after iv or po treatments were higher and more sustained than those of tomoxiprole. The kinetic profile after iv administration was described by a three exponential terms equation and the terminal elimination half-life was 38.7 h. Upon iv administration, total 14C was rapidly distributed in highly vascularized tissues while in others, like the bone, fat, gonads, pancreas and skin the equilibrium with the central compartment was attained later. Target organs were the adrenals, liver, lungs, pancreas, thyroid, stomach and above all the fat tissue. Elimination from tissues was almost complete 48 h after the treatment. 14C was eliminated mainly in the feces (80% of dose) as metabolites. In the bile, five polar metabolites were detected; one of them, desmethyl tomoxiprole glucuronide, accounting alone for more than 80% of the total biliary radioactivity; was purified and its structure assigned.

NMR monitoring of the anaerobic metabolism of frog muscle at rest.

The time dependence of Lactate (Lac), H+, Adenosine-triphosphate (ATP), Phosphocreatine (PCr), Hexose-monophosphate (PME), and Inorganic Phosphate (Pi) levels has been obtained for frog muscle at rest in anaerobic conditions by multinuclear NMR. All information has been collected on the same samples alternatively tuning the probehead on 1H- and 31P-NMR frequencies. ATP, PCr and H+ levels show the same time dependence for all the samples, while PME, Pi and Lac levels vary in time differently from one sample to another. No direct correlation between the Lac appearance and the H+ concentration has been found.