Structural relationship of human interferon alpha genes and pseudogenes

We have isolated and characterized DNA segments containing IFN-alpha-related sequences from human lambda and cosmid clone banks. We describe six linkage groups comprising 18 distinct IFN-alpha-related loci, and report the nucleotide sequences of nine chromosomal IFN-alpha-genes with intact reading frames, as well as of five pseudogenes. Taking into account as yet unsequenced genes as well as clones described by others, there are now seven linkage groups and 23 loci, of which 15 correspond to potentially functional genes and six to non-functional genes; two loci remain unsequenced. Eighteen additional sequences are likely to be allelic to the above. The finding that at least two IFN-alpha genes appear to be natural hybrids of other IFN-alpha genes, and that two distinct IFN-alpha loci have completely identical coding sequences, although their flanking regions are different, is evidence for information exchange between the individual genes.     

Gal{alpha}4Gal-binding antibodies: specificity and use for the mapping of glycolipids of Burkitt lymphoma and other human tumors

The binding of four different Gal     

Antiproliferative effect of interferon on a Burkitt''s lymphoma cell line

The effect of interferon (IF) on the growth of a Burkitt's lymphoma cell line was analysed. The degree of depression of cell doublings was the same if the cells were in a steady state mode of exponential growth or in a resting state (G0) when IF was added. As IF had a lag time of 24 h before decreased growth could be observed, cells in G0 did not seem to be more sensitive when growth was estimated by cell counts expressed as cell doublings. IF inhibited cells to proceed into the cell cycle and the possibility that IF may increase the escape into a G0 loop is discussed.     

Monitoring the antiviral effect of alpha interferon on individual cells

An infectious hepatitis C virus (HCV) cDNA clone (JFH1) was generated recently. However, quantitative analysis of HCV infection and observation of infected cells have proved to be difficult because the yield of HCV in cell cultures is fairly low. We generated infectious HCV clones containing the convenient reporters green fluorescent protein (GFP) and Renilla luciferase in the NS5a-coding sequence. The new viruses responded to antiviral agents in a dose-dependent manner. Responses of individual cells containing HCV to alpha interferon (IFN-alpha) were monitored using GFP-tagged HCV and time-lapse confocal microscopy. Marked variations in the response to IFN-alpha were observed among HCV-containing cells.     

Optimization of the primary recovery of human interferon alpha2b from Escherichia coli inclusion bodies

The human interferon alpha2b (hu-IFNalpha2b) gene was cloned in Escherichia coli JM109(DE3) and the recombinant protein was expressed as cytoplasmic inclusion bodies (IB). The present work discusses the recovery of hu-IFNalpha2b IB from the E. coli cells. An optimized protocol is proposed based on the sequential evaluation of recovery steps and parameters: (i) cell disruption, (ii) IB recovery and separation from cell debris, (iii) IB washing, and (iv) IB solubilization. Parameters such as hu-IFNalpha2b purity and recovery yield were measured after each step. The optimized recovery protocol yielded 60% of hu-IFNalpha2b with a purity of up to 80%. The protein was renatured at high concentration after recovery and it was found to display biological activity.     

Erdheim Chester disease in a patient with Burkitt lymphoma: a case report and review of literature

Background

Erdheim Chester disease (ECD) is a rare, non-Langerhans cell histiocytosis characterized by widespread tissue infiltration by CD68-positive, CD1a-negative foamy histiocytes. ECD can be difficult to identify, and diagnosis relies on the presence of histiocytes with certain histologic and immunophenotypic features in an appropriate clinical and radiologic setting. Clinical signs and symptoms are variable depending on which organ systems are involved. Most patients have at least skeletal involvement with bone pain as well as fatigue. Other common manifestations include diabetes insipidus, cardiac, periaortic, or retro-orbital infiltration/fibrosis, kidney impairment, xanthelasmas, among others.

Case presentation

Herein, we describe a case of BRAF-mutation positive ECD in a patient with Burkitt lymphoma, and we review recent literature.

Conclusion

Underlying BRAF and other MAPK pathway mutations are identified in approximately 50% of cases of ECD, which aids in diagnosis as well as enables novel targeted treatments. ECD patients have an increased risk of myeloid neoplasms; however, unlike other histiocytoses, an association with lymphoproliferative disorders has not been recognized.

     

Effects of recombinant human interferon alpha on human megakaryocyte and fibroblast colony formation

Effects of recombinant human interferon alpha (HuIFN-alpha) on human megakaryocyte (CFU-MK) and fibroblast (CFU-F) colony-forming cell growth were studied. Concentration-dependent inhibition of both CFU-MK and CFU-F by HuIFN-alpha was demonstrated. Statistically significant suppression of both CFU-MK and CFU-F was seen at a HuIFN-alpha concentration of 1000 U/ml or greater. No significant difference was found between HuIFN-alpha treated cultures and controls for the distribution of CFU-MK types and for the size and cell morphology of CFU-F. When a concentration of 1000 u/ml HuIFN-alpha was added at varying time points during the marrow cultures, decreased numbers of megakaryocyte and fibroblast colonies only appeared at the early days of cultures. When bone marrow cells were incubated with HuIFN-alpha for different periods of time prior to initiation of cultures, a reduction of megakaryocyte colony formation also occurred. These studies demonstrate a suppressive effect of HuIFN-alpha on human CFU-MK and CFU-F growth. This effect seems to occur at the initial stages of CFU-MK and CFU-F development.     

The inhibitory effect of hydrocortisone on interferon production by rat spleen cells

The effect of hydrocortisone on interferon r(IFN-r) production by rat spleen cells and its mechanism were studied. The results showed that hydrocortisone inhibited IFN-r production at concentrations as low as 5.52 x 10(-10) M, with complete suppression at 5.52 x 10(-8) M, and the total number and survival rate of the cultured spleen cells were not apparently affected by 5.52 x 10(-8) M hydrocortisone. The inhibitory effect was dose-dependent when the concentration was from 5.52 x 10(-10) M to 5.52 x 10(-8) M and could be blocked by RU38486, a competitive antagonist of glucocorticoid. Our results suggested that glucocorticoid may inhibit IFN-r production through a receptor-mediated mechanism.     

Treatment with natural human interferon alpha of a CML-patient with antibodies to recombinant interferon alpha-2b

A patient with Philadelphia-chromosome positive chronic myelogenous leukemia developed interferon antibodies on treatment with recombinant interferon alpha-2b. Clinically this event corresponded with progressive disease. No cross-reactivity of antibodies with human leukocyte interferon was found by Western blot. Treatment was switched to human leukocyte interferon with an obvious clinical effect: WBC was reduced and platelet count stabilized, but the effect was transient and no hematologic remission was achieved. Human leukocyte interferon may be an alternative in CML-patients with neutralizing antibodies to recombinant interferon alpha.     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.     

In vitro inhibitory effect of interferon on colony formation of myeloma stem cells

Summary The inhibitory effect of interferon on colony formation of myeloma stem cells in two layer plasma clot-soft agar cultures was studied. Human lymphoblast interferon inhibited in therapeutically attainable concentrations myeloma stem cell proliferation in 50% and human fibroblast interferon in 23% of the 14 myeloma patients in whom in vitro colony formation could be achieved. In interferon-sensitive patients the numbers of myeloma stem cell clusters and colonies were decreased to 34.4%–54.9% of control cultures. In addition, maturation of myeloma stem cells in differentiated plasma cells was reduced by interferon in most of these cases.     

Epstein-Barr virus small RNAs potentiate tumorigenicity of Burkitt lymphoma cells independently of an effect on apoptosis

The tumorigenic potential of the Burkitt lymphoma (BL) cell line Akata is dependent on the restricted latency program of Epstein-Barr virus (EBV) that is characteristically maintained in BL tumors. Within these cells, EBV-mediated inhibition of apoptosis correlates with an up-regulation of BCL-2 levels in concert with a down-regulation in c-MYC expression that occurs under growth-limiting conditions. Here we addressed whether EBV's effects on apoptosis and tumorigenicity are mediated by the EBV small RNAs EBER-1 and EBER-2. Stable expression of the EBERs in EBV-negative Akata BL cells, at levels comparable to those in EBV-positive cells, significantly enhanced the tumorigenic potential of EBV-negative BL cells in SCID mice, but did not fully restore tumorigenicity relative to EBV-positive Akata cells. Furthermore, wild-type or greater levels of EBER expression in EBV-negative Akata cells did not promote BL cell survival. These data therefore suggest that EBV can contribute to BL through at least two avenues: an EBER-dependent mechanism that enhances tumorigenic potential independent of a direct effect on apoptosis, and a second mechanism, mediated by an as-yet-unidentified EBV gene(s), that offsets the proapoptotic consequences of deregulated c-MYC in BL.     

Variations of interferon inactivators and/or inhibitors in human serum and their relationship to interferon therapy

Anti-interferon (IFN) activities of normal human sera and sera from cases suspected of viral infection were determined. Of the normal sera tested (57), 65% presented anti-IFN activities against IFN-alpha and/or -beta preparations; 61% against IFN-beta and 32% against IFN-alpha. Of the sera from suspected virus-infected individuals (44), 82% presented anti-IFN activity to IFN-beta and 57% to IFN-alpha with the majority of sera (91%) exerting anti-IFN activities to IFN-alpha and/or -beta preparation. The observed variations of anti-IFN alpha and beta activities of human sera appear to be reproducible and should be taken into consideration in the clinical application of IFN.     

Evidence for a dopaminergic involvement in the inhibitory effect of alpha human natriuretic peptide on prolactin in man

Recently, it has been suggested that Atrial Natriuretic Peptides (ANP), as well as peripheral hormones, may have a role as central neurotransmitters or neuromodulators, and in humans it has been observed that alpha human ANP inhibits prolactin secretion. In this study, on 12 normal adult males, we evaluated the effects of ANP on the prolactin release induced by TRH and by the dopaminergic blocker sulpiride. Alpha-hANP administration was followed by a significant fall of prolactin plasma levels but did not influence TRH-induced prolactin response. Nevertheless, sulpiride-induced prolactin secretion was significantly lower after Alpha-hANP administration than after placebo pre-treatment (p values ranging between 0.01 and 0.001). Our results suggest that in man Alpha-hANP has no direct influence on lactotrope cells in inhibiting prolactin secretion, but seems to involve activation of the hypothalamic dopaminergic system.     

Out-of-hospital cardiac arrest in high-rise buildings: delays to patient care and effect on survival

Background:

The increasing number of people living in high-rise buildings presents unique challenges to care and may cause delays for 911-initiated first responders (including paramedics and fire department personnel) responding to calls for out-of-hospital cardiac arrest. We examined the relation between floor of patient contact and survival after cardiac arrest in residential buildings.

Methods:

We conducted a retrospective observational study using data from the Toronto Regional RescuNet Epistry database for the period January 2007 to December 2012. We included all adult patients (≥ 18 yr) with out-of-hospital cardiac arrest of no obvious cause who were treated in private residences. We excluded cardiac arrests witnessed by 911-initiated first responders and those with an obvious cause. We used multivariable logistic regression to determine the effect on survival of the floor of patient contact, with adjustment for standard Utstein variables.

Results:

During the study period, 7842 cases of out-of-hospital cardiac arrest met the inclusion criteria, of which 5998 (76.5%) occurred below the third floor and 1844 (23.5%) occurred on the third floor or higher. Survival was greater on the lower floors (4.2% v. 2.6%, p = 0.002). Lower adjusted survival to hospital discharge was independently associated with higher floor of patient contact, older age, male sex and longer 911 response time. In an analysis by floor, survival was 0.9% above floor 16 (i.e., below the 1% threshold for futility), and there were no survivors above the 25th floor.

Interpretation:

In high-rise buildings, the survival rate after out-of-hospital cardiac arrest was lower for patients residing on higher floors. Interventions aimed at shortening response times to treatment of cardiac arrest in high-rise buildings may increase survival.More than 400 000 out-of-hospital cardiac arrests occur annually in North America.^1,2 Despite considerable effort to improve resuscitation care, survival to hospital discharge in most communities remains below 10%.² Rapid defibrillation and high-quality cardiopulmonary resuscitation (CPR) are essential for survival, with an absolute decrease in survival of 7% to 10% for each 1-minute delay to defibrillation.^3–5Recently, there has been a dramatic increase in the number of people living in high-rise buildings (e.g., a 13% relative increase in Toronto from 2006 to 2011^6,7). As more high-rise buildings are constructed in urban centres across Canada, the number of 911 calls for emergency medical services in high-rise buildings will also continue to increase. Furthermore, over 40% of homeowners over the age of 65 years reside in high-rise buildings.⁸ These older residents have higher risks for a number of serious medical conditions, including cardiac arrest. Cardiac arrests that occur in high-rise buildings pose unique challenges for 911- initiated first responders. Building access issues, elevator delays and extended distance from the location of the responding vehicle on scene to the patient can all contribute to longer times to patient contact and, ultimately, longer times to initiation of resuscitation. Previous research has shown that longer 911 response times result in decreased patient survival after cardiac arrest,^9,10 but response times are traditionally measured from the time a call is received by the 911 dispatch centre to when the response vehicle arrives on scene. This measure fails to take into account the time required for 911-initiated first responders to make patient contact once they arrive on scene. This interval can contribute substantial delays to patient treatment, in some cases more than 4 minutes, and can account for up to 28% of the total time from the 911 call to arrival of the first responders at the patient’s side.^11–14There is a lack of literature describing the delay to patient contact during out-of-hospital cardiac arrests in high-rise buildings, where time-sensitive, life-saving interventions matter most. Furthermore, the effect on survival of vertical delay to patient contact is unknown. As the number of high-rise buildings continues to increase and as population density rises in major urban centres, is important to determine the effect of delays to patient care in high-rise buildings on survival after cardiac arrest and to examine potential barriers to patient care in this setting.The primary objective of this study was to compare the rate of survival to hospital discharge after out-of-hospital cardiac arrest at different vertical heights in residential buildings, specifically higher floors (≥ 3 floors) relative to lower floors (< 3 floors), with adjustment for standard Utstein variables.¹⁵The secondary objectives were to determine the delay to patient contact by 911-initiated first responders for cardiac arrests occurring on higher floors and to examine the use of automated external defibrillators by bystanders in private residences.     

Aggregation of recombinant human interferon alpha 2b in solution: Technical note

Sodium phosphate buffer increased the aggregation of rhIFN-α2b in the range of 1.55 to 1.810³ day⁻¹, as determined by SDS/PAGE under reduced and nonreduced conditions. In contrast, sodium citrate buffer decreased the aggregation rate of this cytokine, as compared with those samples in sodium phosphate buffer. Results from sodium citrate-phosphate buffer were very similar to those obtained with sodium citrate solutions. On the other hand, EDTA Na₂×2H₂O reduced the aggregation rate of rhIFN-α2b, showing an aggregation kinetic constant in the range of 0.52 to 0.75×10³ day⁻¹. Polysorbates 20 and 80 were less effective than the chelating agent in preventing this degradation pathway.     

Differential effect of interferon on the expression of tumor-associated antigens and histocompatibility antigens on human melanoma cells: relationship to susceptibility to immune lysis mediated by monoclonal antibodies

Incubation of cultured human melanoma cells with human leukocyte interferon did not change the expression of melanoma-associated antigens (MAA) recognized by monoclonal antibodies and of Ia-like antigens but significantly increased the expression of HLA-A,B antigens and of beta 2-microglobulin (beta 2-mu). The effect is dependent on the dose of interferon and on the incubation time. Interferon-treated melanoma cells showed an increased susceptibility to lysis mediated by monoclonal antibodies to HLA-A,B antigens and to human beta 2-mu; on the other hand, interferon-treated melanoma cells did not change in their susceptibility to murine natural killer (NK) cell lysis and to immune lysis mediated by monoclonal antibodies to MAA and to Ia-like antigens, and they displayed a reduced susceptibility to human NK cell lysis. Therefore, the increased susceptibility of interferon-treated melanoma cells to lysis mediated by anti HLA-A,B and anti beta 2-mu monoclonal antibodies is likely to reflect the increase in cell surface expression of the corresponding antigens.