Nanoparticle display of neuraminidase elicits enhanced antibody responses and protection against influenza A virus challenge.

Current Influenza virus vaccines primarily induce antibody responses against variable epitopes in hemagglutinin (HA), necessitating frequent updates. However, antibodies against neuraminidase (NA) can also confer protection against influenza, making NA an attractive target for the development of novel vaccines. In this study, we aimed to enhance the immunogenicity of recombinant NA antigens by presenting them multivalently on a nanoparticle carrier. Soluble tetrameric NA antigens of the N1 and N2 subtypes, confirmed to be correctly folded by cryo-electron microscopy structural analysis, were conjugated to Mi3 self-assembling protein nanoparticles using the SpyTag-SpyCatcher system. Immunization of mice with NA-Mi3 nanoparticles induced higher titers of NA-binding and -inhibiting antibodies and improved protection against a lethal challenge compared to unconjugated NA. Additionally, we explored the co-presentation of N1 and N2 antigens on the same Mi3 particles to create a mosaic vaccine candidate. These mosaic nanoparticles elicited antibody titers that were similar or superior to the homotypic nanoparticles and effectively protected against H1N1 and H3N2 challenge viruses. The NA-Mi3 nanoparticles represent a promising vaccine candidate that could complement HA-directed approaches for enhanced potency and broadened protection against influenza A virus.

Cardiotoxicity After Synthetic Cathinone Use; Two Cases, A Case Series and Scoping Review.

The cardiotoxic effects of synthetic cathinones remain largely unknown. In this study, we present two cases, a case series and a scoping review, to explore synthetic cathinone associated cardiotoxicity. Case 1 involved a 28-year-old male with non-ST-elevation myocardial infarction after ingesting a substance containing 4-methylmethcathinone (4-MMC), 3-methylmethcathinon (3-MMC), and methcathinone. Case 2 involved a 49-year-old male with ventricular fibrillation after 4-methylmethcathinone ingestion, who was diagnosed with severe three-vessel disease. A retrospective analysis was performed on self-reported synthetic cathinone poisonings reported to the Dutch Poisons Information Centre from 2012 to 2022. A total of 222 mono-intoxications with cardiotoxicity were included, mostly involving 3-methylmethcathinon (63%). Often tachycardia, hypertension, palpitations, and chest pain were reported. A comprehensive literature search was performed on PubMed to identify the studies reporting cardiac arrest, myocardial infarction, cardiac inflammation, cardiomyopathy, and life-threatening arrhythmias following synthetic cathinone use. A total of 30 articles reporting 40 cases were included. The reported complications included cardiac arrest (n = 28), ventricular tachycardia (n = 4), supraventricular tachycardia (n = 1), ST-elevation myocardial infarction (n = 2), non-ST-elevation myocardial infarction (n = 2), cardiomyopathy (n = 1), and myocarditis (n = 2). A total of ten different associated synthetic cathinones were identified. Cardiac arrest, myocardial infarction, and ventricular arrhythmias have been reported following the use of synthetic cathinones, underscoring the importance of obtaining a detailed recreational drug use history from patients presenting with syncope, chest pain, or palpitations.

Coronavirus M Protein Trafficking in Epithelial Cells Utilizes a Myosin Vb Splice Variant and Rab10.

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two-hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV-CoV) M protein with Myosin Vb (MYO5B), specifically with the alternative splice variant of cellular MYO5B including exon D (MYO5B+D), which mediates interaction with Rab10. When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with the MHV-CoV M protein, as well as with the M proteins from Porcine Epidemic Diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2). Co-expressed M proteins and MYO5B+D co-localized with endogenous Rab10 and Rab11a. We identified point mutations in MHV-CoV M that blocked the interaction with MYO5B+D in yeast 2-hybrid assays. One of these point mutations (E121K) was previously shown to block MHV-CoV virion assembly and its interaction with MYO5B+D. The E to K mutation at homologous positions in PEDV-CoV, MERS-CoV and SARS-CoV-2 M proteins also blocked colocalization with MYO5B+D. The knockdown of Rab10 blocked the co-localization of M proteins with MYO5B+D and was rescued by re-expression of CFP-Rab10. Our results suggest that CoV M proteins traffic through Rab10-containing systems, in association with MYO5B+D.

The association of amfetamines and cathinones with acute coronary syndrome - a systematic review.

BACKGROUND: It is well known that cocaine increases the risk of acute coronary syndrome. However, it is uncertain if the use of other stimulants, such as amfetamines and cathinones, is also related to acute coronary syndrome. OBJECTIVES: To identify all reported cases of acute coronary syndrome related to the use of amfetamines and cathinones, the type of acute coronary syndrome, the atherothrombotic aetiology, and the mortality rate. METHODS: A systematic literature search in PubMed, Embase database, Cochrane library, PsycInfo and Web of Science was performed from inception until 31 August 2022. All original articles in English or Dutch describing adult patients with acute coronary syndrome after the use of amfetamines or cathinones were included. The main outcome was the occurrence of acute coronary syndrome associated with amfetamine-type stimulants or cathinones. Data were collected and reported according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. RESULTS: A total of 11,605 articles were identified, 56 of which met the inclusion criteria. A total of 160 patients presented with acute coronary syndrome after five different types of amfetamines, namely, amfetamine (n = 48), metamfetamine (n = 98), 3,4-methylenedioxymetamfetamine (n = 11), fenethylline (n = 2), and 4-fluoroamfetamine (n = 1). Khat chewing was associated with acute coronary syndrome (n = 4234), as were three different types of synthetic cathinones, namely, non-defined cathinones (n = 1), 4-methylmethcathinone (n = 1), and α-pyrrolidinopentiophenone (n = 1). In patients with a known acute coronary syndrome type (n = 157), ST-segment elevation myocardial infarction was diagnosed in 53 patients (75%) and non-ST-segment elevation myocardial infarction in 18 patients (25%). Of the ST-segment elevation myocardial infarction patients, 36% were diagnosed with significant coronary stenosis or thrombus. The mortality rate for khat-associated acute coronary syndrome, with more often male and older patients with fewer cardiovascular risk factors, was higher compared to non-khat-associated acute coronary syndrome. For amfetamine, metamfetamine, and 3,4-methylenedioxymetamfetamine, mortality associated with ST--segment elevation myocardial infarction and non-ST-segment elevation myocardial infarction was 14% and 7%, respectively. Risk factors for acute coronary syndrome were infrequently reported, and risk stratification scores were not reported. CONCLUSION: There is evidence that amfetamine-type stimulants and cathinones are associated with the occurrence of acute coronary syndrome. Khat chewing appears to be a risk factor for acute coronary syndrome. Amfetamine, metamfetamine, 3,4-methylenedioxymetamfetamine, fenethylline, 4-fluoroamfetamine, and synthetic cathinones were also reported in relation to acute coronary syndrome. However, this evidence is limited, of low quality and with a low number of reported cases. Further prospective studies need to be conducted.

The influence of position-dependency on surgical success in sleep apnea surgery - a systematic review.

PURPOSE: To evaluate the influence of position-dependency on surgical success of upper airway (UA) surgery in obstructive sleep apnea (OSA) patients. METHODS: Systematic review. RESULTS: Two prospective cohort studies and seven retrospective cohort studies were included in this review. Despite the importance of the subject, it remains unclear whether position-dependency is a predictor for surgical success. No differences were found in surgical success rate between non-positional (NPP) and positional (PP) OSA patients undergoing uvulopalatopharyngoplasty/Z-palatoplasty with or without radiofrequent thermotherapy of the tongue, isolated tongue base or multilevel surgery and hypoglossal nerve stimulation. In one study PP undergoing relocation pharyngoplasty had a greater chance of surgical success. In the majority of the remaining studies, surgical success was in favor of NPP. Furthermore, in the vast part of included studies, the effect of UA surgery was suggested to be greater in the lateral position than supine position. CONCLUSION: Although preoperative characteristics in PP (e.g., lower BMI and AHI) seem to be in favor for higher surgical success compared to NPP, it remains unclear whether position-dependency is a predictor for surgical outcome. It is suggested that the largest differences and expected preoperative and postoperative changes occur in non-supine AHI. In PP, the preoperative non-supine AHI is already lower compared to NPP suggesting a lower chance of surgical success in PP.

Recommendations on the most suitable quality-of-life measurement instruments for bariatric and body contouring surgery: a systematic review.

OBJECTIVE: The objective of this study is to systematically assess the quality of existing patient-reported outcome measures developed and/or validated for Quality of Life measurement in bariatric surgery (BS) and body contouring surgery (BCS). METHODS: We conducted a systematic literature search in PubMed, EMBASE, PsycINFO, CINAHL, Cochrane Database Systematic Reviews and CENTRAL identifying studies on measurement properties of BS and BCS Quality of Life instruments. For all eligible studies, we evaluated the methodological quality of the studies by using the COnsensus-based Standards for the selection of health Measurement INstruments checklist and the quality of the measurement instruments by applying quality criteria. Four degrees of recommendation were assigned to validated instruments (A-D). RESULTS: Out of 4,354 articles, a total of 26 articles describing 24 instruments were included. No instrument met all requirements (category A). Seven instruments have the potential to be recommended depending on further validation studies (category B). Of these seven, the BODY-Q has the strongest evidence for content validity in BS and BCS. Two instruments had poor quality in at least one required quality criterion (category C). Fifteen instruments were minimally validated (category D). CONCLUSION: The BODY-Q, developed for BS and BCS, possessed the strongest evidence for quality of measurement properties and has the potential to be recommended in future clinical trials.

Antibodies Directed toward Neuraminidase N1 Control Disease in a Mouse Model of Influenza.

There is increasing evidence to suggest that antibodies directed toward influenza A virus (IAV) neuraminidase (NA) are an important correlate of protection against influenza in humans. Moreover, the potential of NA-specific antibodies to provide broader protection than conventional hemagglutinin (HA) antibodies has been recognized. Here, we describe the isolation of two monoclonal antibodies, N1-7D3 and N1-C4, directed toward the N1 NA. N1-7D3 binds to a conserved linear epitope in the membrane-distal, carboxy-terminal part of the NA and reacted with the NA of seasonal H1N1 isolates ranging from 1977 to 2007 and the 2009 H1N1pdm virus, as well as A/Vietnam/1194/04 (H5N1). However, N1-7D3 lacked NA inhibition (NI) activity and the ability to protect BALB/c mice against a lethal challenge with a range of H1N1 viruses. Conversely, N1-C4 bound to a conformational epitope that is conserved between two influenza virus subtypes, 2009 H1N1pdm and H5N1 IAV, and displayed potent in vitro antiviral activity mediating both NI and plaque size reduction. Moreover, N1-C4 could provide heterosubtypic protection in BALB/c mice against a lethal challenge with 2009 H1N1pdm or H5N1 virus. Glutamic acid residue 311 in the NA was found to be critical for the NA binding and antiviral activity of monoclonal antibody N1-C4. Our data provide further evidence for cross-protective epitopes within the N1 subtype and highlight the potential of NA as an important target for vaccine and therapeutic approaches.IMPORTANCE Influenza remains a worldwide burden on public health. As such, the development of novel vaccines and therapeutics against influenza virus is crucial. Human challenge studies have recently highlighted the importance of antibodies directed toward the viral neuraminidase (NA) as an important correlate of reduced influenza-associated disease severity. Furthermore, there is evidence that anti-NA antibodies can provide broader protection than antibodies toward the viral hemagglutinin. Here, we describe the isolation and detailed characterization of two N1 NA-specific monoclonal antibodies. One of these monoclonal antibodies broadly binds N1-type NAs, and the second displays NA inhibition and in vitro and in vivo antiviral activity against 2009 H1N1pdm and H5N1 influenza viruses. These two new anti-NA antibodies contribute to our understanding of the antigenic properties and protective potential of the influenza virus NA antigen.

Coronavirus Spike Protein and Tropism Changes.

Coronaviruses (CoVs) have a remarkable potential to change tropism. This is particularly illustrated over the last 15 years by the emergence of two zoonotic CoVs, the severe acute respiratory syndrome (SARS)- and Middle East respiratory syndrome (MERS)-CoV. Due to their inherent genetic variability, it is inevitable that new cross-species transmission events of these enveloped, positive-stranded RNA viruses will occur. Research into these medical and veterinary important pathogens-sparked by the SARS and MERS outbreaks-revealed important principles of inter- and intraspecies tropism changes. The primary determinant of CoV tropism is the viral spike (S) entry protein. Trimers of the S glycoproteins on the virion surface accommodate binding to a cell surface receptor and fusion of the viral and cellular membrane. Recently, high-resolution structures of two CoV S proteins have been elucidated by single-particle cryo-electron microscopy. Using this new structural insight, we review the changes in the S protein that relate to changes in virus tropism. Different concepts underlie these tropism changes at the cellular, tissue, and host species level, including the promiscuity or adaptability of S proteins to orthologous receptors, alterations in the proteolytic cleavage activation as well as changes in the S protein metastability. A thorough understanding of the key role of the S protein in CoV entry is critical to further our understanding of virus cross-species transmission and pathogenesis and for development of intervention strategies.

Characterization of the Campylobacter jejuni population in the barnacle geese reservoir.

Campylobacter spp. are the most common cause of bacterial gastroenteritis worldwide and have been isolated from a wide number of different hosts and environmental sources. Waterfowl is considered a natural reservoir for this zoonotic bacterium and may act as a potential infection source for human campylobacteriosis. In this study, faecal samples from 924 barnacle geese were tested for the presence of C. jejuni and C. coli. The resulting C. jejuni and C. coli populations were characterized by multilocus sequence typing (MLST), structure analysis by BAPS and phylogenetic analysis based on full genome sequences. The prevalences of C. jejuni in barnacle geese faeces were 11.5% and 23.1% in 2011 and 2012, respectively, and only 0.2% of the samples were positive for C. coli in both years. Furthermore, a possible adaption of the clonal complexes (CCs) ST-702 and ST-1034 to the barnacle geese reservoir was found, as these two CCs represented the majority of the typed isolates and were repeatedly isolated from different flocks at several time-points. Further core genome phylogenetic analysis using ClonalFrame revealed a formation of a distinct monophyletic lineage by these two CCs, suggesting a certain degree of clonality of the C. jejuni population adapted to barnacle geese. Therefore, although STs also commonly found in humans patients (e.g. ST-45) were among the barnacle geese C. jejuni isolates, this reservoir is probably an infrequent source for human campylobacteriosis.

Evaluation of nonspreading Rift Valley fever virus as a vaccine vector using influenza virus hemagglutinin as a model antigen.

Virus replicon particles are capable of infection, genome replication and gene expression, but are unable to produce progeny virions, rendering their use inherently safe. By virtue of this unique combination of features, replicon particles hold great promise for vaccine applications. We previously developed replicon particles of Rift Valley fever virus (RVFV) and demonstrated their high efficacy as a RVFV vaccine in the natural target species. We have now investigated the feasibility of using this nonspreading RVFV (NSR) as a vaccine vector using influenza virus hemagglutinin as a model antigen. NSR particles were designed to express either the full-length hemagglutinin of influenza A virus H1N1 (NSR-HA) or the respective soluble ectodomain (NSR-sHA). The efficacies of the two NSR vector vaccines, applied via either the intramuscular or the intranasal route, were evaluated. A single vaccination with NSR-HA protected all mice from a lethal challenge dose, while vaccination with NSR-sHA was not protective. Interestingly, whereas intramuscular vaccination elicited superior systemic immune responses, intranasal vaccination provided optimal clinical protection.

Adaptation of novel H7N9 influenza A virus to human receptors.

The emergence of the novel H7N9 influenza A virus (IAV) has caused global concerns about the ability of this virus to spread between humans. Analysis of the receptor-binding properties of this virus using a recombinant protein approach in combination with fetuin-binding, glycan array and human tissue-binding assays demonstrates increased binding of H7 to both α2-6 and α2-8 sialosides as well as reduced binding to α2-3-linked SIAs compared to a closely related avian H7N9 virus from 2008. These differences could be attributed to substitutions Q226L and G186V. Analysis of the enzymatic activity of the neuraminidase N9 protein indicated a reduced sialidase activity, consistent with the reduced binding of H7 to α2-3 sialosides. However, the novel H7N9 virus still preferred binding to α2-3- over α2-6-linked SIAs and was not able to efficiently bind to epithelial cells of human trachea in contrast to seasonal IAV, consistent with its limited human-to-human transmission.

Comparison of core sampling and visual inspection for assessment of concrete sewer pipe condition.

Sewer systems are costly to construct and even more costly to replace, requiring proper asset management. Sewer asset management relies to a large extent on available information. In sewer systems where pipe corrosion is the dominant failure mechanism, visual inspection by closed circuit television (CCTV) and core sampling are among the methods mostly applied to assess sewer pipe condition. This paper compares visual inspection and drill core analysis in order to enhance further understanding of the limitations and potentials of both methods. Both methods have been applied on a selected sewer reach in the city of The Hague, which was reportedly subject to pipe corrosion. Results show that both methods, visual inspection and core sampling, are associated with large uncertainties and that there is no obvious correlation between results of visual inspection and results of drill core analysis.

Multilocus sequence types of environmental Campylobacter jejuni isolates and their similarities to those of human, poultry and bovine C. jejuni isolates.

In this study, we investigated the multilocus sequence type (MLST) diversity and population genetics of Campylobacter jejuni isolates collected from the natural waters (n = 57), wild birds (n = 37) and zoo animals (n = 19) in southern Finland, the Helsinki area and the Helsinki Zoo, respectively. On average, we found C. jejuni in 20%, 10.4% or 11.5% of the samples collected from natural waters, wild birds and zoo animals, respectively. High ST diversity was detected in all three sources and 41.2% of the STs were novel, but the multi-host adapted ST-45 was the most common ST detected. The MLST data, supplemented with C. jejuni isolates from domestically acquired human infections (n = 454), poultry (n = 208) and bovines (n = 120), were utilized in a population structure study. The results indicate four groups of strains with varying ecological associations, demonstrating presence of genetically distinct lineages within each of the studied sources. We discovered that the greatest ST overlap occurs between human isolates and isolates from natural waters and poultry, which suggests that the latter two are the most important sources of C. jejuni among domestically acquired infections in Finland.

Heparan sulfate facilitates Rift Valley fever virus entry into the cell.

Rift Valley fever virus (RVFV), an emerging arthropod-borne pathogen, has a broad host and cell tropism. Here we report that the glycosaminoglycan heparan sulfate, abundantly present on the surface of most animal cells, is required for efficient entry of RVFV. Entry was significantly reduced by preincubating the virus inoculum with highly sulfated heparin, by enzymatic removal of heparan sulfate from cells and in cells genetically deficient in heparan sulfate synthesis.

Monitoring the performance of a storm water separating manifold with distributed temperature sensing.

Storm water separating manifolds in house connections have been introduced as a cost effective solution to disconnect impervious areas from combined sewers. Such manifolds have been applied by the municipality of Breda, the Netherlands. In order to investigate the performance of the manifolds, a monitoring technique (distributed temperature sensing or DTS) using fiber optic cables has been applied in the sewer system of Breda. This paper describes the application of DTS as a research tool in sewer systems. DTS proves to be a powerful tool to monitor the performance of (parts of) a sewer system in time and space. The research project showed that DTS is capable of monitoring the performance of house connections and identifying locations of inflow of both sewage and storm runoff. The research results show that the performance of storm water separating manifolds varies over time, thus making them unreliable.

Binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity.

The binding of viruses to host cells is the first step in determining tropism and pathogenicity. While avian infectious bronchitis coronavirus (IBV) infection and avian influenza A virus (IAV) infection both depend on α2,3-linked sialic acids, the host tropism of IBV is restricted compared to that of IAV. Here we investigated whether the interaction between the viral attachment proteins and the host could explain these differences by using recombinant spike domains (S1) of IBV strains with different pathogenicities, as well as the hemagglutinin (HA) protein of IAV H5N1. Protein histochemistry showed that S1 of IBV strain M41 and HA of IAV subtype H5N1 displayed sialic acid-dependent binding to chicken respiratory tract tissue. However, while HA bound with high avidity to a broad range of α2,3-linked sialylated glycans, M41 S1 recognized only one particular α2,3-linked disialoside in a glycan array. When comparing the binding of recombinant IBV S1 proteins derived from IBV strains with known differences in tissue tropism and pathogenicity, we observed that while M41 S1 displayed binding to cilia and goblet cells of the chicken respiratory tract, S1 derived from the vaccine strain H120 or the nonvirulent Beaudette strain had reduced or no binding to chicken tissues, respectively, in agreement with the reduced abilities of these viruses to replicate in vivo. While the S1 protein derived from the nephropathogenic IBV strain B1648 also hardly displayed binding to respiratory tract cells, distinct binding to kidney cells was observed, but only after the removal of sialic acid from S1. In conclusion, our data demonstrate that the attachment patterns of the IBV S proteins correlate with the tropisms and pathogenicities of the corresponding viruses.

Vaccination with a soluble recombinant hemagglutinin trimer protects pigs against a challenge with pandemic (H1N1) 2009 influenza virus.

In 2009 a new influenza A/H1N1 virus strain ("pandemic (H1N1) 2009", H1N1v) emerged that rapidly spread around the world. The virus is suspected to have originated in swine through reassortment and to have subsequently crossed the species-barrier towards humans. Several cases of reintroduction into pigs have since been reported, which could possibly create a reservoir for human exposure or ultimately become endemic in the pig population with similar clinical disease problems as current swine influenza strains. A soluble trimer of hemagglutinin (HA), derived from the H1N1v, was used as a vaccine in pigs to investigate the extent to which this vaccine would be able to protect pigs against infection with the H1N1v influenza strain, especially with respect to reducing virus replication and excretion. In a group of unvaccinated control pigs, no clinical symptoms were observed, but (histo)pathological changes consistent with an influenza infection were found on days 1 and 3 after inoculation. Live virus was isolated from the upper and lower respiratory tract, with titres up to 10(6) TCID(50) per gram of tissue. Furthermore, live virus was detected in brain samples. Control pigs were shedding live virus for up to 6 days after infection, with titres of up to 10(5) TCID(50) per nasal or oropharyngeal swab. The soluble H1N1v HA trimer diminished virus replication and excretion after a double vaccination and subsequent challenge. Live virus could not be detected in any of the samples taken from the vaccinated pigs. Vaccines based on soluble HA trimers provide an attractive alternative to the current inactivated vaccines.

Association of Campylobacter jejuni Cj0859c gene (fspA) variants with different C. jejuni multilocus sequence types.

Cj0859c variants fspA1 and fspA2 from 669 human, poultry, and bovine Campylobacter jejuni strains were associated with certain hosts and multilocus sequence typing (MLST) types. Among the human and poultry strains, fspA1 was significantly (P < 0.001) more common than fspA2. FspA2 amino acid sequences were the most diverse and were often truncated.

Decreasing trend of overlapping multilocus sequence types between human and chicken Campylobacter jejuni isolates over a decade in Finland.

We describe the long-term multilocus sequence typing (MLST) analysis of the population structure and dynamics of 454 Finnish human Campylobacter jejuni isolates, as well as 208 chicken isolates, collected during the mid-1990s to 2007. The sequence type clonal complexes (ST CC) ST-45 CC, ST-21 CC, and ST-677 CC were the most common ones found among all isolates, and they covered 73.9% of all isolates. The ST-283 CC also was found frequently among chicken isolates (8.2%). The predominant STs among all isolates were ST-45, ST-50, and ST-677. ST-137 and ST-230 were common among human isolates, and ST-267 was found more frequently among chicken isolates than human isolates. The ST-45 CC was significantly associated with chicken isolates (P < 0.01), whereas the ST-21 CC was associated with human isolates (P < 0.001). The ST-677 CC was not associated with any host (P = 0.5), and an opposite temporary trend of this complex was seen among chicken and human isolates, with an increase in the former and a decrease in the latter during the study period. Furthermore, the ST-22 and ST-48 CCs were significantly associated with human isolates (P < 0.01), but neither of the CCs was found in chicken isolates. The annual overlap between STs from human and chicken isolates decreased from 76% at the beginning of the study to 58% at the end. Our results suggest that the importance of chicken as a reservoir for strains associated with human infections has declined despite the consumption of domestic chicken meat increasing during the follow-up period by 83%.

SOCS-mediated downregulation of mutant Jak2 (V617F, T875N and K539L) counteracts cytokine-independent signaling.

Recently, mutations in the gene of Janus kinase 2 (Jak2) were discovered in patients suffering from chronic myeloproliferative disorders (MPD) and leukemia. As suppressors of cytokine signaling (SOCS) proteins are potent feedback inhibitors of Jak-mediated signaling, we investigated their role in signal transduction through constitutively active Jak2 mutants. We selected two mutants, Jak2-V617F and Jak2-K539L, found in patients with MPDs and Jak2-T875N identified in acute megakaryoblastic leukemia. We found SOCS family members to be induced through Jak2-V617F in human leukemia cell lines expressing the mutant allele and in stable HEK transfectants inducibly expressing constitutively active Jak2 mutants. SOCS proteins were recruited to the membrane and bound to the constitutively active Jaks. In contrast to wild-type Jak2, the mutant proteins were constitutively ubiquitinated and degraded through the proteasome. Taken together, we show a SOCS-mediated downregulation of the constitutively active, disease-associated mutant Jak2 proteins. Furthermore, a threshold level of mutant Jak expression has to be overcome to allow full cytokine-independent constitutive activation of signaling proteins, which may explain progression to homozygocity in MPDs as well as gene amplification in severe phenotypes and leukemia.