Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers.

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of six amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together, these data allow us to develop a general model for activation of TpoR and explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.

Tryptophan at the transmembrane-cytosolic junction modulates thrombopoietin receptor dimerization and activation.

Dimerization of single-pass membrane receptors is essential for activation. In the human thrombopoietin receptor (TpoR), a unique amphipathic RWQFP motif separates the transmembrane (TM) and intracellular domains. Using a combination of mutagenesis, spectroscopy, and biochemical assays, we show that W515 of this motif impairs dimerization of the upstream TpoR TM helix. TpoR is unusual in that a specific residue is required for this inhibitory function, which prevents receptor self-activation. Mutations as diverse as W515K and W515L cause oncogenic activation of TpoR and lead to human myeloproliferative neoplasms. Two lines of evidence support a general mechanism in which W515 at the intracellular juxtamembrane boundary inhibits dimerization of the TpoR TM helix by increasing the helix tilt angle relative to the membrane bilayer normal, which prevents the formation of stabilizing TM dimer contacts. First, measurements using polarized infrared spectroscopy show that the isolated TM domain of the active W515K mutant has a helix tilt angle closer to the bilayer normal than that of the wild-type receptor. Second, we identify second-site R514W and Q516W mutations that reverse dimerization and tilt angle changes induced by the W515K and W515L mutations. The second-site mutations prevent constitutive activation of TpoR W515K/L, while preserving ligand-induced signaling. The ability of tryptophan to influence the angle and dimerization of the TM helix in wild-type TpoR and in the second-site revertants is likely associated with its strong preference to be buried in the headgroup region of membrane bilayers.

Synthesis, purification, and characterization of single helix membrane peptides and proteins for NMR spectroscopy.

Membrane proteins function as receptors, channels, transporters, and enzymes. These proteins are generally difficult to express and purify in a functional form due to the hydrophobic nature of their membrane spanning sequences. Studies on membrane proteins with a single membrane spanning helix have been particularly challenging. Single-pass membrane proteins will often form dimers or higher order oligomers in cell membranes as a result of sequence motifs that mediate specific transmembrane helix interactions. Understanding the structural basis for helix association provides insights into how these proteins function. Nevertheless, nonspecific association or aggregation of hydrophobic membrane spanning sequences can occur when isolated transmembrane domains are reconstituted into membrane bilayers or solubilized into detergent micelles for structural studies by solid-state or solution NMR spectroscopy. Here, we outline the methods used to synthesize, purify, and characterize single transmembrane segments for structural studies. Two synthetic strategies are discussed. The first strategy is to express hydrophobic peptides as protein chimera attached to the maltose binding protein. The second strategy is by direct chemical synthesis. Purification is carried out by several complementary chromatography methods. The peptides are solubilized in detergent for solution NMR studies or reconstituted into model membranes for solid-state NMR studies. We describe the methods used to characterize the reconstitution of these systems prior to NMR structural studies to establish if there is nonspecific aggregation.

Orientation-specific signalling by thrombopoietin receptor dimers.

Ligand binding to the thrombopoietin receptor is thought to stabilize an active receptor dimer that regulates megakaryocyte differentiation and platelet formation, as well as haematopoietic stem cell renewal. By fusing a dimeric coiled coil in all seven possible orientations to the thrombopoietin receptor transmembrane (TM)-cytoplasmic domains, we show that specific biological effects and in vivo phenotypes are imparted by distinct dimeric orientations, which can be visualized by cysteine mutagenesis and crosslinking. Using functional assays and computational searches, we identify one orientation that represents the inactive dimeric state and another similar to a physiologically activated receptor. Several other dimeric orientations are identified that induce proliferation and in vivo myeloproliferative and myelodysplastic disorders, indicating the receptor can signal from several dimeric interfaces. The set of dimeric thrombopoietin receptors with different TM orientations may offer new insights into the activation of distinct signalling pathways by a single receptor and suggests that subtle differences in cytokine receptor dimerization provide a new layer of signalling regulation that is relevant for disease.

Recombinant influenza B virus HA and NA antigens administered in equivalent amounts are immunogenically equivalent and induce equivalent homotypic and broader heterovariant protection in mice than conventional and live influenza vaccines.

Influenza B virus is an important cause of acute upper respiratory disease in humans. Vaccination is the primary method of control of influenza related disease, yet vaccine methodology and production technology have not changed in over 40 years. In this study, we compare the efficacy of recombinant baculovirus produced protein based neuraminidase containing influenza B vaccines with conventional inactivated influenza vaccine (CIV) and live-attenuated influenza vaccine (LAIV) in a murine model. All HA containing vaccines stimulated antibody and protected against an infectious challenge with homotypic virus (B/Harbin/7/94), only recombinant protein based (rHA + rNA and rNA) vaccines containing immunogenic amounts of influenza neuraminidase (NA) protected against challenge with a significantly antigenically different heterovariant virus (B/Beijing/243/1997), as measured by a reduction in mean pulmonary virus titers. This report demonstrates with influenza B virus, in a side-by-side comparison with CIV and LAIV in a murine model system the superiority of vaccines containing immunogenic NA over currently approved CIV and LAIV vaccines.

Infective endocarditis caused by Staphylococcus aureus in a patient with atopic dermatitis: a case report.

INTRODUCTION: Atopic dermatitis (AD) is a common condition in the United Kingdom with the prevalence varying from 21% in infants aged 0-6 months to 6.4% at the age of 16 years. Patients with AD experience high rates of colonization of their skin surfaces by Staphylococcus aureus (S. aureus). In severe AD there is a potential risk of staphylococcal bacteremia and invasive infection such as acute endocarditis. CASE PRESENTATION: We report a case of acute endocarditis with mitral valve destruction caused by S. aureus in a 30-year-old man with severe AD. The patient received intensive inpatient treatment with antibiotics and underwent successful mitral valve replacement and skin treatment for AD. CONCLUSION: Patients with severe AD are at higher risk of staphylococcal bacteremia and endocarditis. Staphylococcal endocarditis has to be considered in the differential diagnosis of febrile illness in patients with uncontrolled atopic dermatitis.

Changing perspective on immunization against influenza.

Current vaccination strategies against influenza rely on decades old technology of strain selection and prolonged labor-intensive, embryonated chicken-egg based production methods. Although, containing both major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), the immunity engendered by these vaccines is dominated by the anti-HA response. Consequently, current vaccines are susceptible to failure resulting from significant antigenic drift or shift in the time elapsing from the selection of the vaccine candidate strain and wild-type virus exposure. Therefore, immunity may be of short duration. There must be a change in vaccine strategy to include immunization with both HA and NA to broaden the immune response against influenza. Inclusion of the more slowly evolving NA in a vaccine against influenza will reduce the vulnerability to antigenic changes in a potential emerging influenza virus. Alternative production technologies such as recombinant baculovirus and yeast should be explored to decrease vaccine production times.

Variation in the divalent cation requirements of influenza A virus N1 neuraminidases.

Enzymatic kinetic parameters of influenza A virus N1 neuraminidases (NA) chromatographically purified from several vaccine candidate strains were tested. With ionic strength held constant, Ca2+ or Mg2+ increased the initial rate of enzymatic activity. The 1934 and 1943 strains had statistically significant highest initial velocities, V(max)/K(m) and V(max). There were no significant differences among the influenza virus strains from 1947 to 1991. Measured K(m) for the 1943 strain (6.2 x 10(-5) M) was significantly higher than other strains (3.1-4.7 x 10(-5) M). V(max)/K(m) varied from 0.78 M(-1) s(-1) to 0.91 M(-1) s(-1) and V(max) varied from 3.0 s(-1) to 5.5 s(-1) before the addition of a divalent cation and increased approximately 2-fold for each of these kinetic parameters for each strain after the addition of exogenous Ca2+ or Mg2+. Dialysis reduced the initial velocity and immunogenicity of each strain with significant differences found among strains. Enzymatic activity and immunogenicity were partially restored by the addition of exogenous Ca2+. Nucleic acid sequence analysis could not predict these differences. Selection of vaccine strains must include analysis of antigenic changes, but also enzymatic studies and determination of the requirement of divalent cations to maintain immunogenicity and activity during production.

Whiplash injury of the shoulder: is it a distinct clinical entity?

The pathophysiology of shoulder pain after whiplash injury remains uncertain. Patients with shoulder pain after a whiplash injury were recruited from the accident and emergency department in a prospective study to determine the nature of indirect shoulder trauma after a whiplash injury. Twenty patients fulfilled the inclusion criteria. Magnetic resonance imaging (MRI) was obtained in 18 patients. Three MRI scans confirmed acute shoulder injuries. Two patients underwent arthroscopic subacromial decompression after failure of non- operative treatment. In conclusion, whiplash injuries can result in indirect acute shoulder trauma, possibly through an acceleration-deceleration mechanism, and may be a distinct entity.

Immunization against influenza A virus: comparison of conventional inactivated, live-attenuated and recombinant baculovirus produced purified hemagglutinin and neuraminidase vaccines in a murine model system.

To simulate the 2003-2004 influenza season and compare available vaccination methods, immunologically naive mice were immunized with: influenza A virus hemagglutinin (rHA) and neuraminidase (rNA) from A/Panama/2007/99 H3N2 or A/Fujian/411/2002 H3N2 expressed by recombinant baculovirus, chromatographically purified, either as single antigens (rHA or rNA) or in combination (rHArNA); conventional inactivated monovalent (CIV) vaccines from each heterotypic strain; or a live-attenuated influenza (LAV) vaccine derived from the A/Panama/2007/99 strain. HA containing vaccines were highly immunogenic for the HA antigen, with no statistically significant differences among groups in the amount of homotypic anti-HA antibody induced. Little cross-reactive anti-HA antibody was induced by any vaccine, including LAV. Statistically, the greatest amount of anti-NA antibody was induced by the purified NA alone or in combination with purified HA; the least amount of anti-NA antibody was found in mice immunized with LAV or CIV. Immunization with vaccines immunogenic for both HA and NA resulted in an immune response to both surface glycoproteins that suppressed homotypic, closely related heterotypic infection and had a greater reduction in mPVT following an infectious challenge by a distantly related heterotypic strain. These studies suggest that vaccines immunogenic for both HA and NA offer an increased level of protection from influenza.

Gastric mucosa-associated lymphoid tissue lymphoma detected by clonotypic polymerase chain reaction despite continuous pathologic remission induced by involved-field radiotherapy.

PURPOSE: Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is indolent and often associated with Helicobacter pylori bacterial infection. H pylori-independent MALT develops either in the absence of the bacteria or persists after bacterial eradication. We have previously demonstrated long-term pathologic remission after involved-field radiotherapy therapy (IFRT). We determined molecular remission status by clonotypic polymerase chain reaction (PCR). PATIENTS AND METHODS: Twenty-four consecutive patients with stage I to IIE gastric MALT lymphoma who obtained a pathologic remission after IFRT alone were evaluated. All had at least two follow-up endoscopic gastroduodenal biopsies at Memorial Sloan-Kettering Cancer Center. IFRT median dose was 30 Gy (range, 28.5 to 43.5 Gy). Post-treatment biopsies were subjected to semi-nested clonotypic PCR. RESULTS: All patients obtained a complete response based on routine immunohistochemical pathologic analysis of random post-treatment gastric biopsies. Median follow-up from completion of IFRT was 63 months (range, 19 to 117 months). Event-free survival was 96%; 23 of 34 patients remained in clinical and pathologic complete remission. Baseline DNA extraction yielded 17 clone-specific primer pairs. At the first follow-up test, 14 of 17 pairs were PCR positive. Eight remained persistently positive; and one was persistently negative. Others were intermittently positive. CONCLUSION: Despite sustained biopsy-proven remissions for as long as 117 months after radiation, the vast majority of patients remain positive by clonotypic PCR. This suggests that the malignant clone is present but missing either an internal or external signal essential to the cancer phenotype. One possibility is that radiation eradicates the polyclonal H pylori-specific T cells eliminating critical local factors necessary for proliferation of the monoclonal B cells.

Protection of mice with recombinant influenza virus neuraminidase.

Contemporary influenza vaccines are standardized with respect to their content of hemagglutinin, the major virus antigen. Although the immunizing effect of viral neuraminidase--the less abundant of the 2 major surface glycoproteins--has been well documented in experimental animals, the importance of the purified recombinant protein has not yet been adequately assessed in animals or humans. We demonstrate that different lots of a baculovirus-derived recombinant N2 protein, in the absence of other influenza virus proteins, can induce neuraminidase-specific antibodies, reduce the replication of both homologous and heterovariant virus in mice, and suppress disease, as it is manifested by total body weight loss.

Variation in the divalent cation requirements of influenza a virus N2 neuraminidases.

Influenza virus N2 neuraminidases were chromatographically purified from several vaccine candidate strains from 1957 to 1994. Enzymatic kinetic parameters and immunogenicity were tested for each strain. For each NA tested, with ionic strength held constant, Ca(2+) or Mg(2+) increased the initial rate of enzymatic activity. Earlier N2-NA strains had the highest initial velocity, V(max)/K(m) and V(max). There were significant differences among the influenza virus strains in enzymatic activity before and after addition of Ca(2+) or Mg(2+): V(max)/K(m) varied from 0.54 M(-1) s(-1) to 0.88 M(-1) s(-1) and V(max) varied from 2.45 s(-1) to 4.3 s(-1) before the addition of a divalent cation; and increased approximately 2-fold each of these kinetic parameters for each strain after the addition of exogenous Ca(2+) or Mg(2+). Exhaustive dialysis with EDTA reduced the initial velocity of each strain with significant differences found among strains, with a range of 0.1% to 8% of original activity. Activity was partially restored by the addition of exogenous Ca(2+) or Mg(2+), varying from 8% to 60% of pre-dialysis levels, but original rates were not achieved. This reduction in enzymatic activity for the tested strains (i.e., A/Japan/57 and A/Johannesburg/94) was accompanied by a parallel decrease in NA-immunogenicity, with antibody response decreasing by as much as 76% as measured by NI titer, and ELISA titer decreasing by as much as 68%. The addition of Ca(2+) or Mg(2+) to the post-dialysis sample restored immunogenicity to as much as 80% of pre-dialysis NI titers and as much as 78% of pre-dialysis ELISA titers. Dialysis had the least effect on early strains as measured by enzymatic kinetic parameters and immunogenicity studies. Zn(2+) had a slight inhibitory effect on the activity of all tested strains. Review of the nucleic acid sequence of each of these strains could not predict their enzymatic activity, immunogenicity or response to dialysis. If immunity against neuraminidase is desirable in vaccination against influenza, selection of vaccine candidate strains must include not only analysis of antigenic changes and sequence analysis but also enzymatic studies and determination of the requirement of divalent cations to maintain immunogenicity and activity during production.

Recovery of cell wall-deficient organisms from blood does not distinguish between patients with sarcoidosis and control subjects.

STUDY OBJECTIVE: To determine if cell wall-deficient forms (CWDF) of mycobacteria can be grown in culture of blood from subjects with sarcoidosis. DESIGN: A special multicenter study of sarcoidosis (A Case Control Etiologic Study of Sarcoidosis), supported by the National Heart, Lung, and Blood Institute. PATIENTS AND CONTROL SUBJECTS: PATIENTS AND CONTROL SUBJECTS were recruited at 10 institutions in the United States. Control subjects (controls) were of the same gender and race, and within 5 years of age as matching patients with sarcoidosis (cases). RESULTS: Cultures were incubated from 347 blood specimens (197 cases, 150 controls). Two investigators trained to recognize CWDF mycobacteria examined material obtained from culture tubes after 3 weeks. Structures thought to be CWDF were seen with equal frequency in cases (38%) and controls (41%). Thirty-nine percent of cases and 37% of controls were read as negative for CWDF. CONCLUSION: This study fails to confirm earlier reports that CWDF mycobacteria can be grown from the blood of patients with sarcoidosis, but not from control subjects.

The total influenza vaccine failure of 1947 revisited: major intrasubtypic antigenic change can explain failure of vaccine in a post-World War II epidemic.

Although vaccine-induced immunity to influenza A virus is continually challenged by progressively selected mutations in the virus's major antigens (antigenic drift), virus strains within a subtype (e.g., H1N1) are antigenically cross-reactive. Although cross-immunity diminishes as further mutations accumulate, necessitating frequent changes in vaccine strains, older vaccines are usually partially protective. The post-World War II epidemic of 1947 is notable for the total failure of a vaccine previously effective in the 1943-44 and 1944-45 seasons. We have combined extensive antigenic characterization of the hemagglutinin and neuraminidase antigens of the 1943 and 1947 viruses with analysis of their nucleotide and amino acid sequences and have found marked antigenic and amino acid differences in viruses of the two years. Furthermore, in a mouse model, vaccination with the 1943 vaccine had no effect on infection with the 1947 strain. These findings are important, because complete lack of cross-immunogenicity has been found previously only with antigenic shift, in which antigenically novel antigens have been captured by reassortment of human and animal strains, sometimes leading to pandemics. Although the 1947 epidemic lacked the usual hallmarks of pandemic disease, including an extensive increase in mortality, it warns of the possibility that extreme intrasubtypic antigenic variation (if coupled with an increase in disease severity) could produce pandemic disease without the introduction of animal virus antigens.

Rapid confirmation by RFLP of transfer to vaccine candidate reassortment viruses of the principal 'high yield' gene of influenza A viruses.

Influenza vaccines must be revised constantly on almost a yearly basis because of the sequential mutations (antigenic drift) that occur as the virus responds to immunologic pressure. New, high yield (hy) reassortant viruses have proved essential to meet production needs for the supply of new vaccines. We have devised a method for simple, rapid and precise identification of the principal influenza A virus RNA segment (RNA 7) associated with hy and transferred from the hy donor virus, A/PR/8/34 (H1N1). The method entails the use of a single restriction enzyme, Bsgl, in analysis by restriction fragment length polymorphism (RFLP) of reverse transcriptase-polymerase chain reaction (RT-PCR)-generated DNA amplicons. The method clearly distinguishes the RNA coding for the M proteins of the donor virus from that of representative and epidemiologically significant human wild type viruses of the past 60 years. In the course of this methodological study further evidence has been found of the variability of the so-called 'invariant' and stable M1 and M2 proteins of the virus. Another finding of potentially basic significance that merits further study is the occurrence of a consistent change at the same amino acid (aa) site of the donated RNA 7 upon its transfer to reassortant viruses.