Clinical impact of allergy and pre-medication in CT studies with low-osmolality intravenous iodinated contrast media.

AIM: To evaluate the occurrence and severity of allergic reactions to iodinated contrast media (ICM), including associated risk factors and the impact of pre-medication. MATERIALS AND METHODS: Data on patients who had experienced allergic reactions during outpatient computed tomography (CT) examinations between January 2014 and September 2018 were analysed retrospectively. Response severity was assessed according to validated criteria. A control group was selected among individuals who underwent CT during the study period and did not experience allergic reactions. RESULTS: Screening of 36,920 CT studies revealed 74 (0.2%) individuals with systemic reactions to ICM. No significant differences in patient characteristics were found among patients who experienced mild (n=54), moderate (n=17), or severe (n=4) reactions. Previous ICM allergy was reported in 10 patients (13.3%). Patients with a history of ICM allergy had mild (9/10) or moderate (1/10) reactions, with one individual showing decreased intensity of the allergic response compared to a previous event. Within the control group, four patients (4%) had previous ICM allergy. In these individuals, lack of allergic reactions could not be attributed to pre-medication. All patients with severe reactions did not have a prior history of ICM allergy. CONCLUSION: Severe allergic reactions to ICM are rare, lack significant risk factors, and do not appear to be impacted by pre-medication. The findings presented herein highlight the need for prospective work that will re-evaluate the yield of pre-medication protocols.

IL-33 and IgE stimulate mast cell production of IL-2 and regulatory T cell expansion in allergic dermatitis.

BACKGROUND: We have previously shown that mast cells (MCs) suppress chronic allergic dermatitis in mice. The underlying mechanism involves MC-derived IL-2, which supports regulatory T cell (Treg) response at the site of inflammation. However, it is not clear what are the factors that drive MCs to produce IL-2. OBJECTIVE: To understand the mechanisms that lead to IL-2 production from MCs in chronic allergic dermatitis. METHODS: Isolated murine bone marrow-derived MCs (BMMCs) were incubated with various stimulators, and IL-2 production was assessed by RT-PCR and ELISA. The response of signalling pathways was evaluated by MAPK inhibitors and Western blot analysis. The effect of MC-IL-2 on Tregs was studied by incubation of splenic T cells with conditioned media obtained from activated BMMCs. Dermatitis was elicited by repeated exposures of mouse ears to oxazolone. MCs in mouse and human skin samples were evaluated by immunostaining. RESULTS: BMMCs released IL-2 in response to IL-33, and IL-2 production was further enhanced by concomitant FcεRI activation. The effect of IL-33 was mediated by activation of the MAPK family members. IL-2 in conditioned media from IL-33 and IgE-stimulated BMMCs led to considerable expansion of Tregs in vitro. IL-33 levels were elevated in oxazolone-challenged ears along with increased numbers of IL-2-expressing MCs. Human skin with chronic inflammation also contained IL-2-expressing MCs that colocalized with IL-33 staining in the dermis. CONCLUSIONS: IL-33, in collaboration with IgE, is critical for MC-IL-2 production in allergic skin disease, thus leading to Treg stimulation and suppression of allergic dermatitis.

Factor IX oligomerization underlies reduced activity upon disruption of physiological conditions.

Coagulation factor IX (FIX) is a serine protease that plays a pivotal role in the blood coagulation cascade. FIX deficiency leads to a blood clotting disorder known as haemophilia B. FIX, synthesized as a prepro-peptide of 461 amino acids, is processed and secreted into plasma. The protein undergoes numerous modifications, including, but not limited to glycosylation, γ-carboxylation and disulphide bond formation. Upon processing and limited proteolysis, the protein is converted into an active protease. Under physiological conditions, the FIX zymogen is a monomer. The purpose of this work was to analyse the conditions that may affect FIX monomeric state and promote and/or reduce oligomerization. Using native gel electrophoresis and size exclusion chromatography, we found that under decreased pH and ionic strength conditions, the FIX zymogen can oligomerize, resulting in the formation of higher molecular weight species, with a concomitant reduction in specific activity. Similarly, FIX oligomers formed readily with low bovine serum albumin (BSA) concentrations; however, increased BSA concentrations impeded FIX oligomerization. We hypothesize that normal blood physiological conditions are critical for maintaining active FIX monomers. Under conditions of stress associated with acidosis, electrolyte imbalance and low albumin levels, FIX oligomerization is expected to take place thus leading to compromised activity. Furthermore, albumin, which is commonly used as a drug stabilizer, may enhance the efficacy of FIX biological drugs by reducing oligomerization.

CMRF35-like molecule 1 (CLM-1) regulates eosinophil homeostasis by suppressing cellular chemotaxis.

Eosinophil accumulation in health and disease is a hallmark characteristic of mucosal immunity and type 2 helper T cell (Th2) inflammation. Eotaxin-induced CCR3 (chemokine (C-C motif) receptor 3) signaling has a critical role in eosinophil chemotactic responses. Nevertheless, the expressions of immunoreceptor tyrosine-based inhibitory motif-bearing receptors such as CMRF35-like molecule-1 (CLM-1) and their ability to govern eosinophil migration are largely unknown. We now report that CLM-1 (but not CLM-8) is highly and distinctly expressed by colonic and adipose tissue eosinophils. Furthermore, Clm1⁻/⁻ mice display elevated baseline tissue eosinophilia. CLM-1 negatively regulated eotaxin-induced eosinophil responses including eosinophil chemotaxis, actin polymerization, calcium influx, and extracellular signal-regulated kinase (ERK)-1/2, but not p38 phosphorylation. Addition of CLM-1 ligand (e.g., phosphatidylserine) rendered wild-type eosinophils hypochemotactic in vitro and blockade of CLM-1/ligand interactions rendered wild-type eosinophils hyperchemotactic in vitro and in vivo in a model of allergic airway disease. Interestingly, suppression of cellular recruitment via CLM-1 was specific to eosinophils and eotaxin, as leukotriene B4 (LTB4)- and macrophage inflammatory protein-1α (MIP-1α)-induced eosinophil and neutrophil migration were not negatively regulated by CLM-1. Finally, peripheral blood eosinophils obtained from allergic rhinitis patients displayed elevated CLM-1/CD300f levels. These data highlight CLM-1 as a novel regulator of eosinophil homeostasis and demonstrate that eosinophil accumulation is constantly governed by CLM-1, which negatively regulates eotaxin-induced eosinophil responses.

Rituximab induces resolution of recurrent diffuse alveolar hemorrhage in a patient with primary antiphospholipid antibody syndrome.

Diffuse alveolar hemorrhage (DAH) is a rare manifestation of primary antiphospholipid antibody syndrome (APS). We describe a patient with primary APS and refractory recurrent episodes of DAH. The patient was admitted 15 times due to recurrent episodes of DAH in a period of 18 months. Multiple immunosuppressive drugs did not improve his condition. Two years after his presentation, he was treated with rituximab (two doses of 1 g, 2 weeks apart). Six months later, the attacks of DAH have gradually disappeared. In a follow-up of more than 2 years after he received rituximab, the patient has had no further admissions due to DAH. Levels of antiphospholipid antibodies were measured during follow-up of 4 years. Anti-ß2 glycoprotein IgG titer decreased to normal 6 months after therapy but anticardiolipin (aCL) antibody titer increased. We conclude that rituximab caused a dramatic clinical response in this patient. Anti-ß2 glycoprotein IgG correlated better with the clinical response in this patient than aCL.

Antibodies against the VRT101 laminin epitope correlate with human SLE disease activity and can be removed by extracorporeal immunoadsorption.

OBJECTIVE: We have previously shown that murine pathogenic lupus autoantibodies bind to VRT101, a 21-mer peptide located at the globular part of the laminin-alpha chain. In this study, we evaluated whether VRT101 also serves as a target for human lupus antibodies, upholding the hypothesis that VRT101 may serve as a potential target in the therapy of lupus. METHODS: Anti-VRT101 and anti-dsDNA reactivity were measured in the serum of lupus patients and compared with that of healthy individuals and patients with other rheumatic disorders. Statistical correlations between disease activity measured by the SLEDAI-2k scale and compatible serum anti-VRT101 and anti-dsDNA levels were defined. A VRT101-coupled sepharose column was assessed for its efficacy in removing serum anti-VRT101 antibody and its safety in extracorporeal apheresis in sheep. RESULTS: Anti-VRT101 and anti-dsDNA antibodies were significantly higher in SLE patients compared with patients with other rheumatic conditions. A high degree of correlation was detected between anti-VRT101 levels and the SLEDAI-2k activity in patients with SLE. Immunoadsorption of lupus patients' sera on the VRT101-sepharose column removed most of the anti-VRT101 antibodies. The column was found to transfer effectively 3l of normal sheep plasma without significant removal of non-specific antibodies or other proteins. CONCLUSIONS: Anti-VRT101 anibodies are abundantly detected in the serum of patients with SLE and correlate with disease activity. Specific removal of serum anti-VRT101 by extracorporeal plasmapheresis with specific immunoadsorption on the VRT101-coupled sepharose columns may serve as a new therapeutic tool for specific immunoadsorption of pathogenic antibodies in SLE patients.

The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle.

Owing to the intensive research activity on protein synthesis, little attention was paid in the 1950s and 1960s to protein degradation. However, work by my group and others between 1970 and 1990 led to the identification of the ubiquitin-dependent degradation system. We found that this system contains three types of enzymes: E1 ubiquitin--activating enzyme, E2 ubiquitin--carrier enzyme and E3 ubiquitin--protein ligase. The sequential action of these enzymes leads to conjugation of ubiquitin to proteins and then in most cases to their degradation. This review briefly tells the story of how this pathway was discovered describing the main findings that during the years allowed us to draw the complex picture we have now.

Cornea recipients: are their opinions and attitudes toward organ donation different from those of the general population?

BACKGROUND: Cornea transplantation provides a second chance for people with poor visual function. Unfortunately, there is a major shortage of donor cornea tissue. The purpose of this study was to evaluate the attitudes and willingness to donate organs among cornea transplant recipients. METHODS: Sixty-eight patients who underwent cornea transplantation between January 2002 and May 2003 were asked to complete a questionnaire dealing with their attitudes toward cornea and organ donation, and willingness to donate an organ. RESULTS: Religion was a contributing factor for a negative decision to donate organs. Only 29% of participants, most of whom were nonreligious were carrying a signed donation card. Fifty-eight percent of the patients knew that the cornea graft is derived from a deceased person; most of these patients were of European or American origin. Seventy-three percent knew that donation requires the agreement of a family member. Age, gender, marital status, and education were not significantly associated with attitude toward donation. CONCLUSION: Stronger efforts are needed by transplant coordinators, physicians, and nurses to improve the education and knowledge of patients and their families about the basic aspects of transplantation. Greater public awareness may increase the willingness to donate organs.

Paroxetine associated hepatotoxicity: a report of 3 cases and a review of the literature.

We recently encountered 3 patients who had developed reversible paroxetine-associated hepatotoxicity. Two of the patients were over 80 years old and their hepatitis was accompanied by hyponatremia. In the third case, hepatitis was associated with multiple organ failure and the co-administration of trazodone. Here, we will discuss the possible role of preexisting risk factors in the development of paroxetine hepatotoxicity and review the relevant literature.

The mouse Snrpn minimal promoter and its human orthologue: activity and imprinting.

BACKGROUND: Microdeletions in chromosome 15q13-15 of Prader-Willi (PWS) and Angelman Syndrome (AS) patients suggested that SNRPN promoter/exon 1, together with a short sequence located approximately 35 kb upstream, constitute an imprinting control centre that regulates the entire 2 Mb PWS/AS imprinted domain. We have recently shown that a minitransgene composed of the human upstream sequence and mouse Snrpn promoter/exon 1 harbours all the elements necessary for establishing and maintaining an imprinted state. RESULTS: Here we describe, using transfection experiments, the Snrpn minimal promoter (SMP), being composed of the entire 76 bp exon 1 and 84 bp of upstream sequence. A 7 bp element (SBE) within SMP that, in its unmethylated state binds a specific protein, is absolutely required for promoter activity. The orthologous human sequence, in spite of the fact that it possesses an identical SBE, failed to display promoter activity in transfection experiments and failed to create a methylated state of the maternal allele. Transgenic experiments reveal that a mutation in SBE of the mouse sequence did not completely abolish methylation of the maternal allele, indicating that sequences outside SBE participate in this process. Replacement of human exon 1 with the mouse orthologue replenished promoter activity, but left the maternal allele in the transgenic experiment unmethylated. The reciprocal chimera, in which mouse exon 1 was replaced by the human orthologue resulted in loss of promoter activity and did not support differential methylation. CONCLUSIONS: The observations made by in vitro and in vivo experiments suggest that several cis elements which are involved in Snrpn promoter activity and the imprinting process are present in the mouse promoter and absent in the human orthologous sequence.

Constitutive hyperhistaminaemia: a possible mechanism for recurrent anaphylaxis.

The process underlying anaphylaxis involves an uncontrolled elevation in blood levels of mediators, including histamine. Usually, these abnormal levels are attributed to the degranulation of basophils and mast cells. Few reports have assessed the contribution of defects in histamine pharmacodynamics to allergic responses. In this report we describe a patient with recurrent anaphylaxis who was initially suspected to have enhanced histamine intolerance. We evaluated urine and blood samples collected from this patient and from control individuals using an ELISA test. Our data clearly show constitutive hyperhistaminaemia and a markedly impaired urinary histamine clearance ratio in the index patient. It is suggested that this defect facilitates anaphylaxis.

Inverse relation between levels of p27(Kip1) and of its ubiquitin ligase subunit Skp2 in colorectal carcinomas.

BACKGROUND: Previous studies have shown that low levels of p27(Kip1), an inhibitor of G1 cyclin-dependent kinases, are associated with high aggressiveness and poor prognosis in a variety of cancers. Decreased levels of p27 are caused, at least in part, by acceleration of the rate of its ubiquitin-mediated degradation. In cultured cells and cell-free biochemical systems, it has been shown that p27 is targeted for degradation by a ubiquitin ligase complex that contains Skp2 (S-phase kinase-associated protein 2) as the specific substrate-recognizing and rate-limiting subunit. This investigation was undertaken to examine the possible relation between levels of p27 and of its specific ubiquitin ligase subunit Skp2 in human cancers. METHODS: Quick-frozen colorectal tumor samples from 20 patients were homogenized at 0 degrees C in buffer containing a mixture of protease inhibitors. Samples were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose, and probed with highly specific monoclonal antibodies directed against Skp2 and p27. The expression of Skp2 also was examined by immunohistochemistry using formalin fixed, paraffin embedded tissue sections from the same cases. RESULTS: A strongly significant inverse correlation was found between levels of Skp2 and p27 (r = -0.812; P < 0.0001). Thus, decreased levels of p27 were associated with strongly increased levels of Skp2, whereas high levels of p27 coincided with low levels of Skp2. Immunohistochemical examination of Skp2 expression agreed with immunoblot analysis in 89% of cases. CONCLUSIONS: The results are compatible with the notion that increased expression of Skp2 may have a causative role in decreasing the levels of p27 in aggressive colorectal carcinomas.

The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27.

The cyclin-dependent kinase (CDK) inhibitor p27 is degraded in late G1 phase by the ubiquitin pathway, allowing CDK activity to drive cells into S phase. Ubiquitinylation of p27 requires its phosphorylation at Thr 187 (refs 3, 4) and subsequent recognition by S-phase kinase associated protein 2 (Skp2; refs 5-8), a member of the F-box family of proteins that associates with Skp1, Cul-1 and ROC1/Rbx1 to form an SCF ubiquitin ligase complex. However, in vitro ligation of p27 to ubiquitin could not be reconstituted by known purified components of the SCFSkp2 complex. Here we show that the missing factor is CDK subunit 1 (Cks1), which belongs to the highly conserved Suc1/Cks family of proteins that bind to some CDKs and phosphorylated proteins and are essential for cell-cycle progression. Human Cks1, but not other members of the family, reconstitutes ubiquitin ligation of p27 in a completely purified system, binds to Skp2 and greatly increases binding of T187-phosphorylated p27 to Skp2. Our results represent the first evidence that an SCF complex requires an accessory protein for activity as well as for binding to its phosphorylated substrate.

The imprinting box of the Prader-Willi/Angelman syndrome domain.

A subset of mammalian genes is monoallelically expressed in a parent-of-origin manner. These genes are subject to an imprinting process that epigenetically marks alleles according to their parental origin during gametogenesis. Imprinted genes can be organized in clusters as exemplified by the 2-Mb domain on human chromosome 15q11-q13 and its mouse orthologue on chromosome 7c (ref. 1). Loss of this 2-Mb domain on the paternal or maternal allele results in two neurogenetic disorders, Prader-Willi syndrome (PWS) or Angelman syndrome (AS), respectively. Microdeletions on the paternal allele share a 4.3-kb short region of overlap (SRO), which includes the SNRPN promoter/exon1, cause PWS and silence paternally expressed genes. Microdeletions on the maternal allele share a 0.88-kb SRO located 35 kb upstream to the SNRPN promoter, cause AS and alleviate repression of genes on the maternal allele. Individuals carrying both AS and PWS deletions on the paternal allele show a PWS phenotype and genotype. These observations suggest that cis elements within the AS-SRO and PWS-SRO constitute an imprinting box that regulates the entire domain on both chromosomes. Here we show that a minitransgene composed of a 200-bp Snrpn promoter/exon1 and a 1-kb sequence located approximately 35 kb upstream to the SNRPN promoter confer imprinting as judged by differential methylation, parent-of-origin-specific transcription and asynchronous replication.

Phosphorylation of Cdc20/fizzy negatively regulates the mammalian cyclosome/APC in the mitotic checkpoint.

The cyclosome/anaphase promoting complex (APC) is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. It is activated at the end of mitosis by phosphorylation and association with the WD-40 protein Cdc20/Fizzy and is then kept active in the G1 phase by association with Cdh1/Hct1. The mitotic checkpoint system that keeps cells with defective spindles from leaving mitosis interacts with Cdc20 and prevents its stimulatory action on the cyclosome. The activity of Cdh1 is negatively regulated by phosphorylation, while the abundance of Cdc20 is cell cycle regulated, with a peak in M-phase. Cdc20 is also phosphorylated in G2/M and in mitotically arrested cells, but the role of phosphorylation remained unknown. Here we show that phosphorylation of Cdc20 by Cdk1/cyclin B abrogates its ability to activate cyclosome/APC from mitotic HeLa cells. A nonphosphorylatable derivative of Cdc20 stimulates cyclin-ubiquitin ligation in extracts from nocodazole-arrested cells to a much greater extent than does wild-type Cdc20. It is suggested that inhibitory phosphorylation of Cdc20/Fizzy may have a role in keeping the cyclosome inactive in early mitosis and under conditions of mitotic checkpoint arrest.

Mechanisms and regulation of the degradation of cyclin B.

The degradation of the cyclin B subunit of protein kinase Cdk1/cyclin B is required for inactivation of the kinase and exit from mitosis. Cyclin B is degraded by the ubiquitin pathway, a system involved in most selective protein degradation in eukaryotic cells. In this pathway, proteins are targeted for degradation by ligation to ubiquitin, a process carried out by the sequential action of three enzymes: the ubiquitin-activating enzyme E1, a ubiquitin-carrier protein E2 and a ubiquitin-protein ligase E3. In the system responsible for cyclin B degradation, the E3-like function is carried out by a large complex called cyclosome or anaphase-promoting complex (APC). In the early embryonic cell cycles, the cyclosome is inactive in the interphase, but becomes active at the end of mitosis. Activation requires phosphorylation of the cyclosome/APC by protein kinase Cdk1/cyclin B. The lag kinetics of cyclosome activation may be explained by Suc1-assisted multiple phosphorylations of partly phosphorylated complex. The presence of a Fizzy/Cdc20-like protein is necessary for maximal activity of the mitotic form of cyclosome/APC in cyclin-ubiquitin ligation.

SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27.

Degradation of the mammalian cyclin-dependent kinase (CDK) inhibitor p27 is required for the cellular transition from quiescence to the proliferative state. The ubiquitination and subsequent degradation of p27 depend on its phosphorylation by cyclin-CDK complexes. However, the ubiquitin-protein ligase necessary for p27 ubiquitination has not been identified. Here we show that the F-box protein SKP2 specifically recognizes p27 in a phosphorylation-dependent manner that is characteristic of an F-box-protein-substrate interaction. Furthermore, both in vivo and in vitro, SKP2 is a rate-limiting component of the machinery that ubiquitinates and degrades phosphorylated p27. Thus, p27 degradation is subject to dual control by the accumulation of both SKP2 and cyclins following mitogenic stimulation.