Human T-cell leukemia virus (HTLV) -1 and -2 are deltaretroviruses that infect a wide range of cells. Glut1, the major vertebrate glucose transporter, has been shown to be the HTLV Env receptor. While it is well established that the extracellular surface component (SU) of the HTLV envelope glycoprotein (Env) harbors all of the determinants of interaction with the receptor, identification of SU subdomains that are necessary and sufficient for interaction with the receptor, as well as critical amino acids therein, remain to be precisely defined. Although highly divergent in the rest of their genomes, HTLV and murine leukemia virus (MLV) Env appear to be related and based on homologous motifs between the HTLV and MLV SU, we derived chimeric HTLV/MLV Env and soluble HTLV-1 and -2 truncated amino terminal SU subdomains.bona fide interaction with the HTLV receptor as isolated SU subdomains specifically interfered with HTLV Env-mediated binding, cell fusion, and cell-free as well as cell-to-cell infection. Therefore, the HTLV receptor-binding domain (RBD) lies in the amino terminus of the SU, immediately upstream of a central immunodominant proline rich region (Env residues 180 to 205), that we show to be dispensible for receptor-binding and interference. Moreover, we identified a highly conserved tyrosine residue at position 114 of HTLV-1 Env, Tyr114, as critical for receptor-binding and subsequent interference to cell-to-cell fusion and infection. Finally, we observed that residues in the vicinity of Tyr114 have lesser impact on receptor binding and had various efficiency in interference to post-binding events.Using these SU constructs, we found that the 183 and 178 amino terminal residues of the HTLV-1 and -2 Env, respectively, were sufficient to efficiently bind target cells of different species. Binding resulted from The first 160 residues of the HTLV-1 and -2 mature cleaved SU fold as autonomous domains that contain all the determinants required for binding the HTLV receptor. Indeeiquitous . A cell iquitous .The corresponding domain in MLV Env SU – located upstream of a conserved K/R L L T/N L V Q motif in the SU of the HTLV-1 and F-MLV Env ,10 – is per se, as well as interference to receptor-binding, cell-to-cell fusion, syncytium formation, and infection involve several distinct cell surface-associated parameters [Several studies using synthetic peptides and neutralizing antibodies against the HTLV Env have shown that determinants within this proline rich region homologue (PRRH) are involved in interference to Env-mediated syncytium formation -21. The rameters -29. In tAs shown in Figure 183FEnv, comprises the 183 amino terminal residues of the HTLV-1 SU ending with the PSQL residues fused to the PIGP sequence of the F-MLV PRR  or rabbit immunoglobulin Fc (rFc) carboxy terminal tag Figure . The H12y Figure . The H11e Figure .215SU and H2211SU, containing the PRRH sequence, and H2178SU lacking this PRRH were all efficiently expressed in transfected cells . In contrast, no significant cleavage was observed with the other soluble domains released in the medium (not shown) . Immunoblots of cell extracts revealed two forms of intracellular H1215SU and H2211SU  Figure . As expeU Figure ; this waU Figure .397SU) and used as a heterologous control. A single form of this truncated SU was efficiently expressed in transfected cells  [178SU was similar to H2211SU, demonstrating that the first 158 residues of the mature HTLV-2 SU, without the 20 amino acids of the amino terminal signal peptide, are sufficient for cell surface binding, and therefore that the PRRH is not required for receptor binding  , HeLa (hoblasts) , none ofoblasts)  and datag Figure .bona fide binding to the HTLV receptor, we performed an Env-specific binding interference assay. In this assay, transfection of the above described chimeric Env and SU subdomains into 293T cells resulted in interference to cell surface binding by the soluble HA-tagged H2178SU subdomain   Figure . This efn Figure . Therefo183FEnv and the HTLV SU amino terminal subdomains to interfere with HTLV Env-mediated cell fusion. Interference to cell fusion was measured using a quantitative HTLV envelope cell fusion interference assay (CFIA), as previously described [Viral envelope interference occurs when cell surface receptors are occupied by receptor-interacting Env components -35. Sincescribed .215SU subdomain in target cells, 12% ± 2% of control fusion (P < 0.001), consistent with previous observations using the H1215FEnv chimera [183FEnv chimera, which lacked a PRRH, down to 26% ± 4% of control fusion (P < 0.001)   , although unnecessary for receptor binding, modulates the efficiency of interference to HTLV Env-induced cell-to-cell fusion (P < 0.03).HTLV-1 Env-induced cell fusion was significantly diminished upon expression of the H1 chimera . SignifiInterference, as described above, was based on the inhibition of cell-to-cell fusion induced by fusogenic Env expressed in the absence of other viral proteins. We further evaluated the abilities of the Env chimeras and soluble subdomains to specifically interfere with HTLV Env-mediated infection. HTLV Env-pseudotyped MLV virions, MLV(HTLV), were produced to infect 293T target cells. Because these recombinant cell-free virions are not competent for replication, this viral pseudotype infection assay tests a single round of infection, and does not measure replication and subsequent exponential viral dissemination. Therefore, relative infection values are expressed in linear rather than logarithmic scales.397SU  infection to 324 ± 98 iu/ml, 3.3% ± 1% of control infection, and to 307 ± 129 iu/ml, 3.1% ± 1.3% of control infection, respectively  infection to 191 ± 56 iu/ml and 215 ± 122 iu/ml, 1.9% ± 0.6% and 2.2% ± 1.3% of control infection, respectively , and this was taken as 100% control infection Figure . SimilarU Figure . ExpressBecause HTLV dissemination appears to occur mostly via cell-to-cell contact, we also tested envelope interference to infection by HTLV-1 SU amino terminal domains using a cell-to-cell transmission interference assay. In this assay, cells harboring interfering chimeric Env and soluble subdomains were cocultured with cells producing MLV(HTLV) virions. Transfection of either chimeric Env or soluble subdomains into HeLa target cells decreased MLV(HTLV) infection to levels similar to those observed in the cell fusion interference assay presented in figure 94) and serine 101 (Ser101) for HTLV-1 Env which correspond to Arg90and Ser97 in HTLV-2 Env, have been shown to alter cell-to-cell fusion and infection when mutated [215SU subdomains with either Arg94 or Ser101 mutated to Ala, yielding the mutant H1(R94A)SU and H1(S101A)SU subdomains, respectively. We also evaluated mutations of Asp106, mutant H1(D106A)SU, and Tyr114, mutant H1(Y114A)SU, both residues found to be highly conserved between all human and simian T cell leukemia viruses (unpublished observations). Surprisingly, cell surface binding profiles of H1(R94A)SU and H1(S101A)SU mutants were not significantly altered when compared to binding by the parental H1215SU, whereas the H1(D106A)SU mutant presented reduced binding to HTLV receptor-bearing cells and the H1(Y114A)SU mutant showed a nearly complete abrogation of cell surface binding . Altogether, these experiments demonstrated that Tyr114, and to a lesser extent Asp106, are key residues involved in HTLV Env receptor binding.Two key residues contained in the HTLV SU RBD and conserved between HTLV-1 and -2, arginine 94 . Thus, overexpression of mutant HTLV SU fragments with highly decreased receptor binding abilities can still exert, albeit to a significantly lesser extent, interference to HTLV Env-mediated cell fusion and infection.We next tested the abilities of these mutants to interfere with HTLV Env-mediated cell fusion and infection, using the assays described above. As mentioned above, all wild-type and mutant HTLV SU subdomains were produced and secreted with a similar efficiency Figure . Express215SU or the mutant H1(S101A)SU were expressed in target cells SU mutant inhibited cell fusion and infection, its effects were significantly lower than those of the wild-type H1215SU (56% ± 8% and 32% ± 2.3%, respectively)  Figure . Thus, a114 of the HTLV-1 Env, which corresponds to Tyr110 of the HTLV-2 Env, as a key residue in HTLV Env binding and for all the aforementioned HTLV Env-mediated functional assays. We could not detect binding of H1(Y114A)SU by flow cytometry, while this mutant exerted residual, albeit significantly decreased, interference to HTLV Env-mediated cell fusion and infection. Altered folding outside of the binding domain per se, rather than direct alteration of the receptor-binding site, could also account for the lack of binding of this mutant. However, we favor the latter hypothesis, since the H1(Y114A)SU mutant was properly folded and transported to the plasma membrane and secreted in the medium as efficiently as wild type RBD, thus arguing against gross misfolding of this mutant. Accordingly, Tyr114 appears to be conserved in all known human and simian T cell leukemia viruses strains, which share the same receptor.Here, we report the generation of MLV Env with chimeric HTLV/MLV SU and truncated HTLV-1 and -2 amino terminal SU subdomains that can be expressed in and secreted from eukaryotic cell lines in functional, soluble form. Using these constructs, we demonstrated that the amino terminal 163 and 158 residues  of the mature HTLV-1 and -2 Env SU, respectively, were sufficient to exert both HTLV receptor binding and efficient interference to diverse HTLV Env-mediated functions, including binding, cell-to-cell fusion and cell-free as well as cell-to-cell infection. Although the PRRH sequence comprising amino acid residues 180 to 215 of the HTLV-1 Env and 176 to 211 of the HTLV-2 Env was previously thought to be a receptor binding site, our data preclude a major role for this region in the binding properties described above. Indeed, whereas a synthetic peptide composed of amino acids 197 to 216 and located within the HTLV-1 PRRH, has been reported to interfere with HTLV Env-induced syncytia formation , this pe114 as a key HTLV-1 RBD residue points at this determinant as a very likely receptor-binding core. This, together with previous works relying on syncytia formation and cell-to-cell transmission [bona fide receptor binding determinants and determinants involved at a post-binding level.The receptor-binding site in MLV RBD is composed of a combination of several cysteine loops located upstream of the PRR ,39 whichsmission ,37, willAnother recently identified determinant, the Pro-His-Gln SU motif conserved among gammaretroviruses such as MLV and feline leukemia viruses (FeLV), has been determined to play a major role in viral entry during post-binding events . The mecThe recent identification of Glut1, the ubiquitous glucose transporter of vertebrates , as a reWe have generated truncated domains of the HTLV Env amino terminus, upstream of residues 183 and 178 of the HTLV-1 and -2 Env, respectively, that were sufficient to bind target cells of different species through interaction with the HTLV Env receptor. We also identified a tyrosine at position 114 and 110 in HTLV-1 and -2 Env, respectively, as a key determinant for this binding. In addition to their use for further exploration of the mechanisms involved in HTLV entry, the tagged HTLV-1 and -2 RBD subdomains described here are novel tools for the detection of Glut1 cell surface expression and intracellular trafficking. Indeed, we tracked intracellular expression of EGFP-tagged HTLV SU subdomains by time-lapse microscopy, and found that they are preferentially routed toward cell-cell contact areas (unpublished observations), where Glut1 is particularly abundant  and our MfeI restriction site in the HTLV-1 and F-MLV Env. Introduction of this site in F-MLV resulted in the substitution of a glutamine and leucine (QL) dipeptide for the parental arginine and valine (RV) residues of the GPRVPIGP motif, at the start of the MLV Env PRR. Introduction of the MfeI site in the PSQL motif of the HTLV-1 SU maintained the parental QL residues, at the start of the HTLV Env PRRH. By exchanging domains at the MfeI sites, we derived the H1183FEnv chimera containing the amino terminal 183 residues of the HTLV Env followed by the F-MLV PRR. In this chimera, the PSQL/PIGP hybrid sequence is generated at the exchange border, and the PRRH of HTLV is replaced by the F-MLV PRR  [To exchange the PRR and PRRH regions, we introduced an allelic R Figure . In cont HHproFc . The H11n vector . The HTL intron)  at the H215SU, H2211SU, H1179SU, and H2178SU subdomains, corresponding to the HTLV-1 and -2 SU amino terminus with and without their respective PRRH, were generated by PCR and subcloned into the pCSI expression vector as fusion proteins harboring a carboxy terminal rFc or HA tag  transfection protocol. Transfection medium was replaced with 3 ml of fresh culture medium twenty hours post-transfection. Forty-eight hours post-transfection cell culture medium (supernatant) was recovered and filtered through a 0.45 μm pore-size membrane to remove cell debris. Twenty μl were directly analyzed by SDS-PAGE (15% polyacrylamide gel), and the rest was aliquoted and stored at -20°C for later use in binding assays (see below). Cell extracts were collected 48 h post-transfection in 1 ml of cell lysis buffer  and clarified by two successive centrifugations at 13,000 rpm for 10 min at 4°C in a microcentrifuge. Approximately 20 μl of each extract, adjusted after normalization for protein concentration using the Bradford assay (Sigma), were subjected to electrophoresis on SDS-15% acrylamide gels, followed by transfer onto nitrocellulose . Membranes were blocked in phosphate-buffered saline (PBS) containing 5% powdered milk and 0.5% Tween 20, probed with a 1:1000 dilution of a goat anti-RLV gp70 polyclonal antibody (Viromed) followed by a horseradish peroxidase-conjugated anti-goat immunoglobulin (for detection of chimeric Env), or goat anti-rabbit-IgG-horseradish peroxidase-conjugated immunoglobulins (for detection of rFc-tagged SU subdomains). Immunoblots were subsequently washed three times with PBS-0.1% Tween 20 and revealed by chemiluminescence (ECL+, Amersham).Approximately 5 × 105 target cells were detached with a PBS-EDTA solution, collected by centrifugation, incubated for 30' at 37°C with 300 μl of rabbit Fc-tagged soluble HTLV-1, HTLV-2, or Ampho-MLV truncated SU, washed, labeled with an anti-rabbit-IgG FITC-conjugated antibody, and analyzed on a FACSCalibur (Becton Dickinson). Data analysis was performed using the CellQuest software (Becton Dickinson). For interference studies, 293T cells were transfected with 4 μg of Env or Env SU subdomain expression vectors  using the calcium-phosphate-HBS method. Under these conditions, transfection efficiencies ranged from approximately 80 to 90% of the target cells. Twenty-four and 48 hours post-transfection, cells were collected and transfected 293T cells expressing the different interfering HTLV or Ampho-MLV domains were incubated with a challenging HA-tagged soluble HTLV-2 SU amino terminal subdomain (H2178SU-HA). Cells were stained using a primary 12CA5 anti HA antibody followed by an anti-mouse-IgG FITC-conjugated antibody before detection by flow cytometry.Binding assays were performed as previously described . Briefly183FEnv, was used to interfere with challenging HTLV Env. The interfering non-fusogenic H1183FEnv and truncated HTLV SU subdomains were transiently transfected into HeLaCD4LTRLacZ, a cell line highly susceptible to HTLV Env-induced fusion that contains a stably integrated Tat-dependent LacZ expression vector [183FEnv Env and truncated HTLV SU subdomains plasmid DNA (2 to 3 μg) was transfected into HeLaCD4LTRLacZ cells, while challenging, fusogenic HTLV-1 Env plasmid (1 μg) was transfected into NIH3T3(TK-)Tat. The interfering Env or SU subdomain-presenting cells were detached 24 hours post-transfection and 1–2 × 105 cells were cocultured for 24 hours with 1–2 × 105 challenging HTLV-1 Env-presenting NIH3T3(TK-)Tat cells. Subsequently, the cocultured cells were fixed and stained for β-galactosidase expression as described previously [t test.Briefly, the HTLV/MLV Env chimera, H1n vector . These tn vector ,29. H118eviously . Transfe6 293T cells with 5 μg pCSI/Ampho or pCSIX/H2, respectively, 5 μg pCL/Gag-Pol [LacZ gene marker. pCSI/Ampho is an expression vector encoding the Ampho-MLV Env, and the HTLV-2 Env expression vector, pCSIX/H2, is described above.MLV(Ampho) and MLV(HTLV) pseudotyped virions were produced after transfection of 10/Gag-Pol , using aVirion-containing supernatants were used to infect target 293T cells expressing the chimeric Env or HTLV RBD subdomains. Transfection efficiencies of target 293T cells were >80% in all experiments. Infections were performed 36–48 hours post-transfection on cultures grown in 12 well plates (Costar) at 37°C, medium was changed 24 hours later, and confluent cell monolayers were fixed, stained for β-galactosidase activity before counting blue foci. Interference to infection was determined by infecting transfected target cells with approximately 100 and 1000 iu. Infection was evaluated as described above, and the number of LacZ-positive blue colonies counted was normalized by multiplying by the appropriate dilution factor. The resulting infection values were analyzed as iu/ml of virus containing supernatant. Subsequently the relative infection levels in cells expressing the HTLV SU domains were compared to those of mock transfected cells and were expressed as percentages of control infection (% control).HTLV Human T-cell leukemia virusSU envelope extracellular surface componentEnv envelope glycoproteinMLV murine leukemia virusF-MLV Friend-MLVRBD receptor-binding domainPRR proline-rich regionPRRH proline rich region homologueAmpho amphotropicHA influenza hemagglutininrFc rabbit immunoglobulin constant fragment397SU Ampho-MLV Env fused to a carboxy terminal rFc tagACFIA cell fusion interference assayiu/ml infectious units per ml94arginine 94Arg101serine 101Ser114tyrosine 114TyrFeLV feline leukemia virusesHBS Hepes buffered salinePBS phosphate-buffered salineSDS sodium dodecyl sulfateThe authors declare that they have no competing interests.FJK designed and realized or supervised most of the experiments and co-wrote the manuscript. NM participated to some molecular constructions, set up, realized and analyzed most binding assays and FACS analyses and participated to the redaction of the manuscript. ENG set up and performed the cell-to-cell transmission assay and performed the corresponding experiments, CV constructed some of the RBD point mutants and tested them, MS initiated the project, co-participated in the design of the study, co-coordinated its realization and co-wrote the manuscript, and JLB realized some of the molecular constructs, performed some of the experiments, co-participated in the design of the study, co-coordinated its realization and co-wrote the manuscript. All authors read and approved the final manuscript.


With effective treatment strategies, the focus of tuberculosis (TB) management has shifted from the prevention of mortality to the avoidance of morbidity. As such, there should be an increased focus on quality of life (QoL) experienced by individuals being treated for TB. The objective of our study was to identify areas of QoL that are affected by active TB using focus groups and individual interviews.English, Cantonese, and Punjabi-speaking subjects with active TB who were receiving treatment were eligible for recruitment into the study. Gender-based focus group sessions were conducted for the inner city participants but individual interviews were conducted for those who came to the main TB clinic or were hospitalized. Facilitators used open-ended questions and participants were asked to discuss their experiences of being diagnosed with tuberculosis, what impact it had on their lives, issues around adherence to anti-TB medications and information pertaining to their experience with side effects to these medications. All data were audio-recorded, transcribed verbatim, and analyzed using constant comparative analysis.39 patients with active TB participated. The mean age was 46.2 years (SD 18.4) and 62% were male. Most were Canadian-born being either Caucasian or Aboriginal. Four themes emerged from the focus groups and interviews. The first describes issues related to the diagnosis of tuberculosis and sub-themes were identified as 'symptoms', 'health care provision', and 'emotional impact'. The second theme discusses TB medication factors and the sub-themes identified were 'adverse effects', 'ease of administration', and 'adherence'. The third theme describes social support and functioning issues for the individuals with TB. The fourth theme describes health behavior issues for the individuals with TB and the identified sub-themes were "behavior modification" and "TB knowledge."Despite the ability to cure TB, there remains a significant impact on QOL. Since much attention is spent on preventative or curative mechanisms, the impact of this condition on QoL is often not considered. Attention to the issues experienced by patients being treated for TB may optimize adherence and treatment success. Mycobacterium tuberculosis [th Century. However, with the introduction of anti-tuberculosis medications in the 1940's and 1950's, the incidence of TB disease declined significantly [Globally, tuberculosis (TB) is a major public health problem . In 1997rculosis . Tubercuficantly . Howeverficantly ,4. Althoficantly .With the development of effective treatment strategies, the focus of TB management has shifted from the prevention of mortality to the avoidance of morbidity. As such, there is increased interest in the quality of life (QoL) experienced by individuals being treated for TB . There aFew studies have examined quality of life in patients with active TB ,19. WhilThis was a multi-site study involving three TB Centres in Vancouver, British Columbia. Patients were recruited from the TB Clinic at the BC Centre for Disease Control, Willow Chest Pavilion at Vancouver General Hospital and the Downtown TB Clinic. All these clinics are part of the Vancouver Coastal Health Authority. Ethics approval was obtained from the University of British Columbia Behavioural Research Ethics Board and each subject provided signed, informed consent to participate in the study.Subjects with active TB who were receiving treatment were eligible for recruitment into the study. Subjects who were less than or equal to 16 years of age and who did not speak English, Cantonese or Punjabi were excluded (interpreters for non-English language participants who spoke these languages were available).The initial contact was be made by the study nurse at the individual clinics. For those individuals residing in the inner-city region of Vancouver, gender-specific focus groups were planned with 6–8 participants who had active TB. Each participant was reimbursed $25 for their time. Focus group discussions are a variation of interviews designed for the purpose of gathering data about a specific topic from a group of individuals. Each focus group was led by an experienced facilitator.For participants who came to the TB Clinic at the BC Centre for Disease Control , individual interview sessions were conducted assessing similar information as obtained in the focus groups. The reason for using individual interviews rather than the focus group approach was two-fold. Firstly, as these individuals often had other commitments (such as work or family care), assessments needed to be done at the time of their appointments and could not be specially scheduled. Secondly, due to the cultural backgrounds, most of these participants did not wish to participate in a group setting in which details of their disease and their feelings were explored. Each interview was conducted by an experienced interviewer in combination, when necessary, with an interpreter fluent in Cantonese or Punjabi.Specifically, in either the focus group or interview setting, the facilitator began the session with an open-ended, standard question that began all sessions (e.g. "how did you find out you had TB?"). Using open-ended, probing questions, participants were asked to discuss their experiences of being diagnosed with tuberculosis, what impact it had on their lives, issues around adherence to anti-TB medications and information pertaining to their experience with side effects to these medications. Each participant was invited to comment on each question and provide their perspective on the content area. At the end of each session, the facilitator summarized salient points that arose during the discussion and invited further comments and discussion around these points and confirmed agreement.For all participants, data obtained were audio-recorded, transcribed verbatim, and analyzed. For participants who spoke Cantonese or Punjabi, field notes were kept by the nurse facilitators who are fluent in those languages.Constant comparative analysis was used as a method to explore and identify patterns and themes that emerged from the data . VariousWe conducted two focus group sessions, one with seven male participants and another six female participants; the rest of the participants for the study underwent individual interviews, including 4 hospitalized patients. In total, 39 persons with active TB participated in the study. The demographics of the study participants are described in Table Analysis identified four main themes comprising medication related issues, diagnosis, social support and knowledge of TB. The following text provides a summary of the content of the themes with illustrative quotes in Table This theme describes issues related to the diagnosis of tuberculosis Figure . Sub-theThirty-five quotes pertained to symptoms experienced by the participants at the time of diagnosis. Of these, 19 were related to specific symptoms whereas 16 participants expressed the view that they were asymptomatic at the time of being diagnosed. The most common symptoms experienced by the respondents were cough (n = 13), fatigue/weakness (n = 5), fever/night sweats (n = 5) and shortness of breath (n = 4). Most patients sought medical attention due to cough or "pneumonia-like" symptoms and feelings of general malaise. For example, one patient stated "I was coughing up harsh yellow stuff"; while another stated "I started to feel real tired and had a cold that just wouldn't go away".Four patients expressed that they were "unsure" how they acquired TB. For example, a woman stated "I didn't even know I had it. I was surprised 'cause I felt real good". Other illustrative quotes are shown in Table Most comments, related to the provision of health care at or around the time of diagnosis, were related to community health care providers and their initial hospitalization. Many patients expressed frustration with the health care system at their time of diagnosis due to lack of provider knowledge with respect to tuberculosis. Many patients either felt that they had a delayed diagnosis or delayed treatment due to issues related to their health care provider. For example, on male patient stated, "Family physicians should know more about this disease...where to refer patients to. This is an old disease". Another patient said, "Although my GP gave me a diagnosis, he told me to wait for treatment. We were concerned and phoned the British Columbia Lung Association who referred us to the TB clinic". Another stated, "I contracted a flu-type infection with fever. My GP said go home and take Tylenol but my symptoms continued so I went to see the locum who told me to take Advil. Then I started non-stop coughing. I asked my GP to get an X-ray but he flatly refused."Many participants reported negative experiences with their initial hospitalization after being diagnosed with TB. Specifically, they stated feelings of isolation, rejection and boredom Table . No partThirty-five quotes pertained to emotions experienced by the participants at the time of diagnosis. Of these, patients expressed a wide range of emotions from being calm, accepting or apathetic (n = 11), scared or afraid (n = 7), shocked or surprised (n = 6), "devastated" (n = 4), worried or concerned (n = 4), and depressed (n = 3). Representative quotes for these emotions are presented in Table Of the individuals expressing apathy or calmness related to the diagnosis, many expressed that TB was just another disease to contend with on top of other chronic conditions. For example, one patient stated "Well, it is like HIV. It is in my system. What can you do?" Another person with terminal cancer stated "I had no reaction to the diagnosis. I am more concerned with the spread of my cancer and that I don't have long to live anyhow".Of those expressing concern, there were two distinct reasons cited for this emotion: 1) concern for themselves as they knew relatives or friends who had previously been infected with TB and had experience prolonged hospitalization or death; and 2) concern for others in terms of passing the disease on to family and friends. For example, a male patient said "I was kind of scared because the only person I knew who had TB died of it. Also, I was worried about other people catching it from me". Another woman stated "I was scared. It is like an old disease and I know when you have it, it is not very nice to have it, especially because I have a seven month old baby."The individuals who expressed shock and surprise at the diagnosis attributed these emotions to their lack of symptoms. As such, they had not expected a diagnosis such as TB when they have visited their health care provider despite having other diseases such as HIV  and itchiness due to isoniazid. Despite having adverse events, patients stated that they continued to take their medications. For example, one female patient said, "There is nothing you can do. You have to just continue". Representative quotes from these participants are included in Table Most comments related to the dose and dosing schedule pertained to the size n = 3) and number of tablets/capsules (n = 7). For example, patients felt that the large size of some of the dosage forms (such as ethambutol and rifampin) led to gagging and vomiting. In addition, many patients expressed consternation at the number of pills that they had to take with each dose. For example, one patient said, "When I looked at ten tablets, I thought, on an empty stomach, I cannot". Representative quotes from these participants are included in Table  and numbIndividuals living in the inner city of Vancouver, expressed little concern for compliance-related issues as they either picked up the anti-TB medications with their methadone or the Street-Nurses would find them daily to administer the medications. As an example, one patient stated "It comes with my methadone. When I get that, I get my TB pills". Another stated, "I never worry about it. I know [the Street Nurse's name] will bring it to me". However, despite high compliance in these patients, several identified alcohol (n = 13) or other illicit drug use (n = 8) as being the reason why they had missed doses.Those who came to the TB treatment clinic expressed high compliance due to the perceived gravity of the diagnosis. For example, one woman patient stated, "It is easy to remember because it is at the fore-front of mind. I want to get rid of it". Another person attributed compliance to the law: "I never forget to take my pills because I don't want to go to jail". Representative quotes from these participants are included in Table This theme describes social support and functioning issues for the individuals with TB Figure . SpecifiMost participants expressed that their family and friends were aware of their TB diagnosis (n = 18), while 11 stated that only their friends knew and 10 stated that only their immediate family knew. Of those who stated that only their friends were aware of their diagnosis, most of these persons residing in the inner city noted that they did not have family with whom they communicated. Representative quotes from these participants are included in Table However, one individual stated that she was "secretive because other people will feel that I am contagious". Another male participant stated a reluctance to tell his friends because "I do not want go cause mass hysteria". A school-age boy did not tell his friends out of fear of being shunned. In addition, he missed eight weeks of school and had to retake several courses. In one instance, fear of being shunned in a Punjabi speaking participant was instilled by the treating physician , although others had negative feelings such as fear (n = 7), shock or disbelief (n = 8), and anger (n = 3). One participant stated "my mom was really concerned but my friends did not believe it...they encouraged me to get the right diagnosis". Another stated that her partner had increased his reading on TB and was receiving regular skin tests although her mother and brother would not talk about the TB diagnosis or the clinic visits. Another stated that since his partner was not understanding about modifying his lifestyle, he was forced to end the relationship with her and move out. Representative quotes from these participants are included in Table This theme describes health behavior issues for the individuals with TB Figure . Sub-theWhen asked if they had done anything else beyond medications to help manage their TB, about half of the participants stated that they had done nothing in particular (n = 16). For example, a Cantonese speaking female stated "I do nothing special as TB is very common". However, of the 12 participants who stated that they had changed their health behavior, seven said that they consumed a healthier diet, four stated that they exercised more, three took vitamin supplements specifically to help their TB, and two used less illicit drugs and alcohol. Representative quotes from these participants are included in Table In response to the question "do you believe that you will be cured of TB?", most participants (n = 33) stated that they believed that they would be eventually cured. However, some individuals believed that their TB would never be cured ("I believe that I can keep it in remission but it can't be cured") while others were not sure if it could be cured. Two participants denied having TB despite being informed by health care providers and taking medication. There were comments regarding the participants' impressions of provider knowledge of which a representative sample have been included in Table This qualitative study has revealed that TB has a large impact on affected individuals' QoL through issues related to its diagnosis, treatment, social support and functioning, and health behavior. Specifically, we found that the domains of QoL that were affected by TB included those that are typically affected by most illnesses such as physical functioning and emotional/mental well-being. However, TB patients' social functioning was also affected through isolation, variable social support by family and friends, and the ability to continue with social and leisure activities. Also, the process of getting treatment for TB from the initial hospitalization to the daily medication schedules adversely affected the lives of our participants, although, almost all recognized the need for appropriate treatment.Although other studies ,23 have However, there were some important differences between our two studies. For example, these investigators included only 10 English speaking patients from the Baltimore city area and 13 health care providers whereas ours included non-English participants through the use of interpreters, a much large sample of patients (n = 39), but no health care providers. In addition, we included hospitalized and ambulatory patients from both inner city and public health clinic environments in order to assess the full spectrum of patients afflicted with this disease. Finally, in our study, all patients had active TB and were receiving treatment at the time of the interviews unlike the Baltimore study who recruited patients who were already cured and had completed treatment. We believe that our methodology of interviewing currently afflicted patients might have minimized recall bias although one potential advantage of the Baltimore approach was determining long-lasting influences of TB on patients lives (the investigators received 17 comments in this regard). Also, the use of health providers in the Baltimore study added an interesting perspective with the provision of comments that were, at times, in direct contrast to those stated by patients with respect to the effects of TB on health related quality of life. For example, most physicians underestimated the impact that TB had on the QoL of patients assuming that, because it was curable, its detrimental effects would be minimal.These differences in design between the two studies might have accounted for some different findings. For example, the Baltimore study found that the financial well-being of some of the participants was adversely affected through loss of income and health care expenses whereas participants in our study did not report this issue . Also, some of the male participants in the Baltimore study reported sexual dysfunction whereas this concern was not reported during our interviews. Finally, patients in the Baltimore study reported spirituality as an important domain which we did not identify as an important theme, perhaps due to the different ethnic/religious make-up of our sample.One surprising aspect of our results was the negative feelings associated with TB diagnosis and the initial hospitalization. Some participants expressed frustration with their primary care physicians for the lack of a prompt diagnosis or inappropriate management. There was a common perception among many of the participants that health care providers needed more extensive education regarding TB. We have recently commented on the need to consider TB as a diagnosis and in the appropriate setting, consider the initiation of empiric TB treatment . In addiDespite several negative comments regarding the size, dosing schedule and adverse effects of the anti-TB medications, most patients specified that they understood the need for treatment. As such, self-reported compliance was very high and participants reported a variety of different strategies to help manage adverse events. Our inner-city participants expressed gratitude for the street nurses who delivered their medications to them on a regular basis and did not report the intrusiveness and imposition on lifestyle that has been associated with similar programs (such as directly observed therapy or DOT) in other studies ,26.Although most comments were related to adverse impacts of TB on QOL, some participants stated that acquiring TB had resulted in positive health behavior modification. Many participants took the development of TB to be a "wake-up" call to change their lifestyle and improve their health behavior by either eliminating or reducing drug and alcohol intake, increasing exercise, or eating better. These findings were also reported by the Baltimore study group suggesting that the positive health behavior impacts of this disease might be widespread throughout those afflicted with TB in North America. Because many of those afflicted with TB in North America engage in other high-risk behaviors such as use of illicit drugs, the overall effects of this health behavior modification might be significant. Future studies should attempt to quantify this impact on the downstream development of other conditions. Although hospitalization for management of TB has negative aspects we have noted that this interlude in subjects with a history of substance abuse allows access to chemical dependency treatment resources while away from their usual chaotic environments.Although some studies in other countries have shown that TB can result in job loss, participants did not report that this had occurred in our sample ,28. One Our study had some limitations. We examined a self-selected group of TB patients who may not be representative of the entire population in Canada affected by TB. For example, in British Columbia, foreign-born persons account for close to 70% of all TB cases in the province. Despite this, we feel that we were able to get a representative sample of foreign-born persons  as well as a good cross section of marginalized inner city patients . In factOur study indicates that despite the ability to cure TB with medical therapy, there still remains a sizeable impact on the lives of afflicted patients. Since much of the current attention on TB is spent on preventative or curative mechanisms such as drug therapy, the impact of this condition on QoL is either underestimated or rarely considered. In order to fully evaluate the outcomes that are achieved through TB prevention and treatment, QoL of these patients must be considered. Further studies need to build upon these observations and instruments need to be developed to better characterize QoL in patients with this disease. This process will not only provide an added parameter to evaluate the effectiveness of a given program, but will also focus care providers to be attentive to the non-medication aspects of TB management.CAM conceived of the study, participated in the design, analysis and co-wrote the initial version of the manuscript. FM conceived of the study, obtained funding, participated in the interviews and focus groups, participated in the analysis, coordinated research staff, and co-wrote the initial version of the manuscript. VC participated in the interviews and focus groups and participated in the analysis. AP participated in the design of the study, and the interviews and focus groups. JMF participated in the design and analysis of the study. All authors read and approved the final manuscript.


The role of the crural diaphragm during increased intra-abdominal pressure is not exactly known. We investigated the hypothesis that the crural diaphragm undergoes reflex phasic contraction on elevation of the intra-abdominal pressure with a resulting increase of the lower esophageal pressure and prevention of gastro-esophageal reflux.The esophageal pressure and crural diaphragm electromyographic responses to straining were recorded in 16 subjects (10 men, 6 women, age 36.6 ± 11.2 SD years) during abdominal hernia repair. The electromyogram of crural diaphragm was recorded by needle electrode inserted into the crural diaphragm, and the lower esophageal pressure by a saline-perfused catheter. The study was repeated after crural anesthetization and after crural infiltration with saline.The crural diaphragm exhibited resting electromyographic activity which showed a significant increase on sudden (coughing, p < 0.001) or slow sustained (p < 0.01) straining with a mean latency of 29.6 ± 4.7 and 31.4 ± 4.5 ms, respectively. Straining led to elevation of the lower esophageal pressure which was coupled with the increased electromyographic activity of the crural diaphragm. The crural response to straining did not occur during crural diaphragm anesthetization, while was not affected by saline infiltration. The lower esophageal pressure declined on crural diaphragm anesthetization.Straining effected an increase of the electromyographic activity of the crural diaphragm and of the lower esophageal pressure. This effect is suggested to be reflex in nature and to be mediated through the "straining-crural reflex". The crural diaphragm seems to play a role in the lower esophageal competence mechanism. Further studies are required to assess the clinical significance of the current results in gastro-esophageal reflux disease and hiatus hernia. The LES contributes to the prevention of gastric reflux into the esophagus [Swallowing is a physiologic process by which the food bolus is transmitted from the pharynx to the stomach without esophagopharyngeal or gastro-esophageal reflux . A sphinculature . The LESculature ,6. Cholisophagus ; howeverThe diaphragm is believed to play a contributory role in the barrier function of the lower esophagus. This auxiliary function seems to be carried out by the crural and not the costal diaphragm. The latter contracts and relaxes with respiration. Crural diaphragm (CD) contraction effects LES pressure increase which is directly proportional to the depth of inspiration at the force of diaphragmatic contraction . PressurThe role of the CD during increased intra-abdominal pressure is not completely understood. We hypothesized that the CD, upon increase in intra-abdominal pressure by coughing, sneezing or straining, undergoes reflex phasic contraction with a resulting augmentation of the lower esophageal pressure and inhibition of stress reflux of the gastric contents into the esophagus. This hypothesis was investigated in the current communication.Sixteen subjects were enrolled in the study. Ten were men and six women with a mean age of 36.6 ± 11.2 SD years, (range 27–43). The tests were performed during operative repair of an upper abdominal ventral hernia in 9 patients and of incisional hernia after cholecystectomy for calculous cholecystitis in 7 patients. The patients did not complain of swallowing problems in the past or at the time of enrollment. They gave an informed consent after having been fully informed about the nature of the tests to be done and their role in the study.Physical examination results, including neurologic assessment, were normal. Also barium swallow studies and upper gut endoscopy yielded normal findings. The results of laboratory work including blood count, renal and hepatic function tests as well as electrocardiography were unremarkable.The study was approved by the Review Board and Ethics Committee of the Cairo University Faculty of Medicine.The EMG activity of the CD was recorded during coughing and during straining. The subjects had received general anesthesia using 5% halothane/ 95% oxygen for their above mentioned hernia operations.A concentric electromyographic needle electrode of 40 mm in length and 0.65 mm in diameter (Type 13 L 49 Disa, Copenhagen) was introduced into the CD as it encircled the lower end of the esophagus. A ground electrode was applied to the thigh.A standard electromyographic (EMG) apparatus (Type MES, Medelic, Woking, UK) was used to amplify and display the potentials recorded. Films of the potentials were taken on light-sensitive paper (Linagraph type 1895, Kodak, London, UK) from which measurements of the motor unit action potentials' duration were obtained. The electromyopraphic signals were also stored on an FM tape recorder  for further analysis as required.Before performing the experiment, the normality of the EMG activity of the CD was tested by stimulating it with a needle electrode introduced into the CD and registering the motor unit action potentials from the already inserted needle electrode. The CD had normal EMG activity in all examined subjects.2O/s. During pressure measurements, the catheter was rotated so as to record anteroposterior and lateral pressures.A manometric 6-F catheter was introduced into the esophagus to lie in the high pressure zone at its lower end. The catheter with 2 side ports and a metallic clip applied to its distal closed end for fluoroscopic control was connected to a pneumohydraulic capillary infusion system . The pump delivered saline solution continuously via the capillary tube at a rate of 0.6 ml / min. The transducer outputs were registered on a rectilinear recorder (model RS-3400, Gould Inc). Occlusion of the recording orifice produced a pressure elevation rate that was greater than 250 cm HNear the end of the operation when the effect of muscle relaxant had waned, the anesthetist was asked to induce coughing and straining via laryngeal and tracheal stimulation by moving the endotracheal tube while lying in the trachea. The EMG response of the CD to increased intra-abdominal pressure was registered. Readings were recorded during two types of straining: the sudden forcible straining as that induced by coughing, and the slow sustained straining which simulates that occurring during defecation or micturition. The latency of the crural response was measured from the stimulus (straining) to the first deflection of the muscle action potential complex. The millisecond latencies were calculated when the movement artifact associated with straining appeared on the crural EMG and then the time to the first muscle action potential was measured as an index of latency.To define whether the effect of coughing or straining on the crural diaphragm was direct or reflex action, the following lest was done. In 8 subjects (5 men and 3 women), the CD was infiltrated with 5 ml of 2% lidocaine to anesthetize the crura around the needle electrode. The crural response to sudden and slow sustained straining was recorded after 10 minutes and after 2 hours when the anesthetic effect had waned. Similarly, normal saline was injected and the crural response to straining was registered.The results were analyzed statistically using the Student's t test and values were given as the mean ± standard deviation. Differences assumed significance at p < 0.05.The CD in all of the subjects showed a basal activity with a mean of 112.3 ± 16.3 μV  was reproducible in all studied subjects. It was weaker in women than men, and in the elderly than in the young subjects, though the difference was insignificant (p > 0.05). The CD response disappeared when straining was sustained for more than 15–18 seconds (mean 16.8 ± 1.2) and was not evoked after frequent successive straining. The latency of the response recorded a mean of 29.6 ± 4.7 ms . Saline injection of the crura did not affect the crural response to straining.2O  which are responsible for pain and reflex activity ,16. The The mechanism of gastroesopageal competence is vague and incompletely understood -7. ThereMeanwhile, it is highly probable in the light of the findings of our study that the prevention of gastro-esophageal reflux is a "reflex process" rather than an anatomical entity. We have previously demonstrated that gastric distension by food or an increase in the intra-abdominal pressure would evoke the "gastroesophageal reflex" which acts to tighten the LES . The morThe current study presumably denotes that the CD shares reflexly in the competence mechanism of the gastroesophageal junction. Thus, upon increase of the intra-abdominal pressure, the straining-crural reflex seems to be evoked effecting crural contraction and increase of the lower esophageal pressure.In view of the aforementioned results and discussion, we believe that the "reflex theory" plays a more important role in gastroesophageal competence than the diaphragm pinchock, the flap valve mechanism or other possible anatomical factors.The CD appears to play a role in the lower esophageal competent mechanism. Straining effected an increase in the EMG activity of the CD and in the lower esophageal pressure. This effect is suggested to be reflex in nature and to be mediated through the "straining-crural reflex". Further studies are needed to evaluate the clinical significance of the current results in the pathogenesis and treatment of gastresophageal disease and hiatus hernia.lower esophageal sphincter (LES)crural diaphragm (CD)electromyographic (EMG)The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


Single photon emission computed tomography (SPECT) following injection of radiotracer during a seizure is known as ictal SPECT. Comparison of an ictal SPECT study to a baseline or interictal study can aid identification of a seizure focus.A young woman with encephalitis and refractory seizures underwent brain SPECT during a period of frequent seizure-like episodes, and during a seizure-free period. A focal area of increased radiotracer uptake present only when she was experiencing frequent seizure-like episodes was originally localized to the brainstem, but with later computerized co-registration of SPECT to MRI, was found to lie outside the brain, in the region of the sphenoid sinus.Low-resolution SPECT images present difficulties in interpretation, which can be overcome through co-registration to higher-resolution structural images. Radiotracers used for brain single photon emitted computed tomography (SPECT) pass the blood-brain barrier and bind intracellularly on their first pass through the circulation, providing a "snapshot" of cerebral perfusion at a particular timepoint. When injected during a focal epileptic seizure, an area of significantly increased radiotracer uptake typically corresponds to the region of maximal abnormal activity, often the seizure focus. This ictal pattern of cerebral blood flow can be compared to an interictal/baseline pattern obtained when the patient is not having a seizure, to provide unique information about the nature and location of a patient's epileptic focus, which can be used to guide therapy [See  for a reA previously-healthy young woman developed behavioral changes followed by seizures and refractory status epilepticus. She was diagnosed with encephalitis and treated with antiviral and multiple antiepileptic agents. She required nasotracheal intubation and mechanical ventilation for respiratory support. She experienced persistent episodes of facial twitching resembling seizures. These episodes were not however associated with an ictal EEG pattern on continuous video/EEG monitoring. To clarify the nature of these episodes, 99mTc-HMPAO was injected during a period of frequent twitching. Brain SPECT showed a prominent focus of increased uptake interpreted by the radiologists and clinical team as arising in the upper brainstem Figure . A repeaWith later co-registration of SPECT data to the patient's MRI using a computerized medical image registration and visualization program,  the areaThe nature of this patient's twitching episodes remains uncertain, though they were likely due to epileptic activity involving too small a volume of brain to be detectable by either EEG or SPECT. Her case is described here to illustrate a previously-unreported pitfall in the interpretation of brain SPECT studies, with the goal of emphasizing the importance of image co-registration in nuclear medicine. Diagnostic limitations inherent to low-resolution images can be overcome through use of computer software to accurately co-register brain images of different modalities acquired separately (as described here) or hybrid medical devices such as PET-CT scanners to automatically fuse simultaneously-acquired images. Both methods have been shown to be superior to visual inspection and mental integration of information by experienced radiologists ,8.SPECT: single photon emitted computed tomography; MRI: magnetic resonance imaging; EEG: electroencephalographyThe author(s) declare that they have no competing interests.TB performed image analysis and co-registration and drafted the manuscript. LJH identified the patient, conceived of the study, and drafted a preliminary version of the manuscript. JC participated in data collection and analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Persistent atrial fibrillation (AF) is difficult to treat. In the absence of class I or III antiarrhythmic drugs sinus rhythm is maintained in only 30% of patients during the first year after electrical cardioversion (ECV). One of the remodeling processes induced by AF is fibrosis, which relates to inducibility and maintenance of AF. The renin-angiotensin system may play a important role in this. The aim of this study was to investigate the role of angiotensin-converting enzyme (ACE) inhibitor use on efficacy of ECV, and occurrence of subacute recurrences.One hundred-seven consecutive patients with persistent AF underwent ECV. In twenty-eight (26%) patients ACE inhibitors had been started before initiation of the present episode of AF ('pre-treated' patients).ECV was successful in 96% of patients who were on ACE inhibitors before start of the present episode of AF compared to 80% of the patients not pre-treated (p = 0.04). After 1 month of follow-up 49% of the pre-treated patients and 50% of those not pre-treated with ACE inhibition were still in sinus rhythm (p=ns). Multivariate analysis showed that pre-treatment with ACE inhibitors and a smaller left atrial size were independent predictors of successful ECV (OR = 5.8, C.I. 1.3–26.1, and OR = 5.6, C.I. 1.2–25.3, respectively).Pre-treatment with ACE inhibitors may improve acute success of ECV but does not prevend AF recurrences. Persistent atrial fibrillation (AF) is difficult to treat. In the absence of class I or III antiarrhythmic drugs sinus rhythm is maintained in only 30–50% of patients during the first year after Direct Current electrical cardioversion (ECV),2. FurthIn the present study we report on the effects of ACE inhibition on the outcome of ECV and the prevention of early recurrences after ECV of persistent AF.onset of the current AF episode. Most patients on ACE inhibitors used these drugs for hypertension or congestive heart failure. To make sure that all patients were not completely remodeled at the very moment of start of the current episode of AF , only patients with at least 1 month sinus rhythm before the current episode of AF were included in this study. Duration of AF was determined as precisely as possible by previous electrocardiograms, 24-hours Holter registrations, and by the patient's history. None of the patients were on class I or III antiarrhythmic drugs neither at the moment of ECV nor during follow-up.One hundred-seven consecutive patients with persistent AF, defined as the presence of AF for at least 24 hours were included in this study. ECV wasQuantitative variables were compared between groups using a two-tailed t-test for normally distributed variables or a Wilcoxon two-sample test for skewed distributed variables. For qualitative variables , group differences were evaluated using a Fisher's exact test or a Chi-square test. Accordingly, baseline characteristics are given in mean ± SD, median and range (min-max) or percentages.To determine the predictive factors for successful ECV, an univariate logistic regression analysis was performed using the relevant baseline predictors. Variables with a p-value < 0.20 were selected for the multiple logistic regression analysis to derive a model with statistically significant predictors, by using a backward selection method. All p-values are two-sided and a p-value of < 0.05 was considered statistically significant. SAS version 6.12 (Cary, NC) was used for all statistical evaluations.before start of the current episode of AF (pre-treated patients). In the latter group, ECV was successful in 96% while in the group of patients not pre-treated with ACE inhibitors before start of the current AF episode only 80% had a successful ECV (p = 0.04).Baseline characteristics are listed in table In general, patients treated with ACE inhibitors before start of the current episode of AF had more evidence for heart disease than those who were not pre-treated. Prevalence of hypertension (46% versus 24%, respectively, p = 0.03), and left ventricular dysfunction (40% versus 14%, respectively, p = 0.01) was significantly higher in the pre-treated group in comparison to those who were not pre-treated with ACE inhibitors. . Use of beta adrenergic receptor blockers, calcium channel blockers or digoxin, started before the present episode of AF or not, did not influence success of ECV.An additional 21 patients received ACE inhibitors After 1 month of follow-up 49% of the pre-treated patients compared to 50% of those who were not pre-treated with ACE inhibition were still in sinus rhythm .This post-hoc retrospective analysis shows that use of ACE inhibitors before the onset of AF enhances acute ECV outcome but it does not improve maintenance of sinus rhythm. Furthermore, when ACE inhibitiors are instituted later, i.e. after the start of AF, it does no longer improve success of ECV.ACE inhibitors reduce the incidence of AF in patients with left ventricular dysfunction,6. This In 1995 it was shown that AF induces several electrophysiological changes, called electrical remodeling. Experimafter the start of AF. No differences were found in electrical cardioversion outcome between the two treatment groups which is in contrast to the present study. This may relate to the fact that all patients were in AF at the very moment of start of irbesartan. Two months after cardioversion it appeared that patients treated with irbesartan and amiodarone had a significantly lower AF recurrence rate compared to amiodarone alone treated patients, 15% versus 37%, respectively (p = 0.007). This difference was maintained during a median follow-up of 254 days. According to their figure 2, the benefit of irbesartan (in combination with amiodarone) was mainly achieved by reduction of recurrences after the first two weeks after cardioversion. An earlier study on the role of ACE inhibitors in patients with heart failure and AF showed a trend towards more patients maintaining sinus rhythm after ECV when instituted on lisinopril in comparison to patients not treated with lisinopril[Madrid et al. investigated the role of the angiotensin II type 1 receptor antagonist irbesartan in amiodarone treated patients. Persistisinopril. In the This was a non-randomized and post-hoc analysis. This implies that all findings can only be used to generate hypotheses. However, this is the first clinical study showing that ACE-inhibition initiated before start of AF enhances direct ECV outcome.Only a very small amount of patients was on angiotensin receptor blockers. Whether the effect of pretreatment with angiotensin receptor blockers on cardioversion outcome would be the same as the effect of pretreatment with ACE inhibitors could not be investigated in this study. However, in this context the results of the study of Madrid et al. are encouraging .Pretreatment with ACE-inhibitors significantly improves acute outcome of ECV when initiated before the onset of AF but it does not lead to better maintenance of sinus rhythm. When ACE inhibitiors are, however, instituted later, i.e. after the start of AF, it does no longer improve success of ECV.The author(s) declare that they have no competing interests.TVN carried out the study and drafted the manuscript, HC designed the study, and interpreted the data, MB participated in the draft of the manuscript and interpreted the data, DVV participated in the draft of the manuscript and interpreted the data, IVG participated in the design of the study and interpreted the data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Behavioral recovery (e.g., diminished symptoms), encompassing 1 week after unilateral labyrinthectomy, has been termed vestibular compensation. Evidence suggesting that the histamine H3 receptor  as well as three isoforms which display variable lengths of the third intracellular loop of the receptor were analyzed using in situ hybridization on brain sections containing the rat medial vestibular nucleus after unilateral labyrinthectomy. We compared these expression levels to H3 receptor binding densities.Expression levels for histamine H3 receptor mRNA levels (detected by oligo probe H3X) as well as mRNA levels of the three receptor isoforms studied (detected by oligo probes H3A, H3B, and H3C) showed a pattern of increase, which was bilaterally significant at 24 h post-lesion for both H3X and H3C, followed by significant bilateral decreases in medial vestibular nuclei occurring 48 h (H3X and H3B) and 1 week post-lesion (H3A, H3B, and H3C). Expression levels of H3B was an exception to the forementioned pattern with significant decreases already detected at 24 h post-lesion. Coinciding with the decreasing trends in H3 receptor mRNA levels was an observed increase in H3 receptor binding densities occurring in the ipsilateral medial vestibular nuclei 48 h post-lesion.Total H3 receptor may be an essential part of pre-synaptic mechanisms required for reestablishing resting activities 48 h after unilateral labyrinthectomy.Progressive recovery of the resting discharge of the deafferentated medial vestibular nuclei neurons results in functional restoration of the static postural and occulomotor deficits, usually occurring within a time frame of 48 hours in rats. Our data suggests that the H In rat, deafferentation of one labyrinth  results in a characteristic syndrome of ocular motor and postural disorders. These disorders have been divided into two categories : One category of symptoms, called static, includes head rotation in both the frontal and horizontal planes and ocular nystagmus . The oth3 receptor that was initially characterized as an autoreceptor controlling histamine synthesis and release [3 receptor was found to mediate presynaptic inhibition of release of histamine, noradrenaline, serotonin, dopamine, glutamate, GABA and tachykinins [3 receptor was recently cloned from human [3 receptor  as well as three of the known functionally active isoforms (H3A, H3B, and H3C), described by Drutel et al. [The focus of our study is the histamine H release ,9. Subsehykinins -13, preshykinins -16. The om human , monkey om human , rat [19om human , and guiom human . Moreoveom human -26 with om human ,24). Thi3 receptors) are histaminergic perikarya located exclusively in the tuberomammillary nuclei of the posterior hypothalamus [3 receptor was established by use of ligand binding [The primary source of histamine  and itsribed in ) in the 3 receptor binding densities to reveal the possible plastic changes in the H3 receptor which is responsible for significant constitutive activity also in vivo [Moreover, in this study, we compared the aforementioned trend in mRNA expression levels with H in vivo , histami3 receptor and the three H3 receptor isoforms  in ipsilateral MVe of control animals to that of test animals from the four time points  returned to normal levels. The trend observed was similar when comparing H3X mRNA levels in contralateral MVe of control animals with that of test animals from the four time points. There was no significant change in total H3 receptor mRNA levels detected in contralateral MVe 4 hours post-lesion, but we found a significant increase in H3X mRNA levels detected in contralateral MVe both 24 hours and 48 hours post-lesion. Finally, a return to normal levels in total H3X receptor mRNA level was detected in contralateral MVe 1 week post-lesion.We compared the total Hnts Fig. . Fig. 2B3A mRNA expression levels were detected when we compared ipsilateral MVe in control animals with ipsilateral MVe 4 hours, 24 hours, and 48 hours post-lesion .No significant changes were found in H3 receptor (detected by using probe H3X) and its isoforms (H3A, H3B, and H3C) occurring during the process of post-lesional plasticity in the CNS. Moreover, the probes used in this study were designed to detect unique areas in the transcripts (H3A), or junctional areas in deletion isoforms (probes H3B and H3C) which would make it highly unlikely for non-specific hybridization would occur. This study also includes a comparison of the aforementioned patterns of expression with binding densities for the H3 receptor.Knowing that there is no reliable method to determine the efficiency of a probe for its targetted sequence in an mRNA of interest, this study focuses instead on the pattern of expression for the total HThe prepositus nuclei are delineated and mentioned in legends for figures 2 receptors [3 receptors in the medial vestibular nuclei [3 receptors in the MVe: 1) On the histaminergic or other incoming fibers innervating the MVe [3 receptor mediated inhibition of GABA release from inhibitory interneurons or glutamate release from terminals of second order excitatory MVe neurons may underlie the restoration of resting activity in the deafferentated MVe; more precisely, H3 receptor action would result in disinhibition of neurons in the deafferentated MVeThere are three previously described types of neurons in the medial vestibular nucleus ,42; in veceptors ,44 sugger nuclei . Consequ the MVe -32. 2) O the MVe . 3) On t the MVe . With re3 receptors that function as autoreceptors and inhibit histamine release [3 receptors are at the next two possible locations  comes from work showing that betahistine antagonizes the excitatory effect of histamine on vestibular neurons from in vitro slice preparations of the dorsal brainstem of the rat [3 receptors mediate presynaptic inhibition of release of other neurotransmitters including: noradrenaline, serotonin, dopamine, glutamate, GABA and tachykinins [A and GABAB receptors in the vestibular nuclei and their involvement in vestibular compensation has been demonstrated by either unilateral perfusion or microinjection of GABAergic agonists and antagonists (e.g., GABA, muscimol, and bicuculline) [With respect to the first possible location, it has been established that histamine fibers are endowed with H release ,13. Supp the rat . This fihykinins -13. Unilhykinins  and GluRhykinins ) have beculline) .3 receptor antagonist such as betahistine could either facilitate GABA release from inhibitory interneurons located in the MVe that make synaptic contacts with second order neurons or facilitate glutamate release from terminals of second order MVe neurons that synapse on inhibitory interneurons in the contralateral MVe [Consequently, an Heral MVe ,45. Eitheral MVe .2 receptors on the perikarya of MVe neurons ipsilateral and contralateral to the lesion resulting in a bilateral increase in activity. This should facilitate behavioral recovery as it is thought that an imbalance in discharge of MVe neurons  an increase in translation has occurred. 2) A change in receptor trafficking between intracellular stores and cell membrane has occurred so that it can be detected as an increase in H3 receptor receptor binding densities. In either case, an increase in functional H3 receptor protein coupled with an increase in receptor activity may lead to a restoration of resting activity in the deafferentated MVe by scenarios already mentioned in this section.The trends toward bilateral increases (24 h post-lesion) followed by decreases (48 h post-lesion) in total H3 receptors at the terminals of either GABAergic inhibitory MVe interneurons or on terminals of glutamatergic second order MVe neurons with contralateral projections should result in the observed commissural disinhibition as increased H3 receptor activity would result in an inhibition of synaptic release of neurotransmitters.Our findings are significant given that normalization of resting activities in neurons located in the ipsilateral MVe has been shown to occur 48 hours after unilateral labyrinthectomy . Moreove3 receptors are involved in presynaptic mechanisms resulting in a normalization of resting activities in ipsilateral MVe neurons which would balance discharge activity in MVe on both sides.Our data would thus suggest that H2 gas and killed by decapitation 4 h (n = 4), 24 h (n = 4), 48 h (n = 4) and 1 week (n = 5) after labyrinthectomy.This study was approved by the Local Committee for Animal Experiments and the Provincial State Office of Western Finland; in addition, animal experiments were in accordance with the European Convention (1986) guidelines and approved by the Animal Ethics Committee of Abo Akademi Unviversity. Adult male Sprague Dawley rats (200–250 g) were used. Intraperitoneal (ip) injection of pentobarbital  was used as an anesthetic; in addition, local anesthetic Lidocain (Orion, Finland) was infiltrated under the skin and periosteum prior the procedure. Three steps initiated the surgical procedure to left side of the animal's head: retroauricular skin incision, opening of the middle ear bulla with a drill, and removal of the ossicular chain with the aid of a microscope. Unilateral labyrinthectomy was carried out by opening the horizontal semicircular canal duct in the temporal bone, drilling through the horizontal and anterior semicircular canal ampullae and aspirating the contents of the vestibule. 100% ethanol was injected into the opened labyrinth to finalize the procedure; finally, the wound was sutured. The sham operation entailed opening the middle ear bulla and leaving the ossicular chain intact prior to suturing the wound, as described in Cameron and Dutia ; the conAfter the mentioned decapitation, brains were removed, frozen in isopentane (-25°C), and stored at -70°C. Tissues were then cut to 20 μm thick cryosections, thaw mounted onto poly-L-lysine coated slides (Menzel-Gläser, Germany), and stored at -70°C until used.3 receptor isoform mRNAs  triphosphate (New England Nuclear, USA) at their 3' ends using terminal deoxynucleotide transferase (Promega, USA). Nonincorporated nucleotides were removed by purification through Sephadex G-50 columns.The oligonucleotide probes used for in situ hybridization were designed so that they specifically recognized the different Hribed in ); an addribed in ). Sequenublished . It is nisoforms . Howeverisoforms . As a coications ,55. All 7cpm/ml) was carried out at 50°C for 16 to 20 hours in a humidified chamber. Posthybridization washes were carried out as described previously [Before hybridization, cryosections were taken from the -70°C environment and kept at room temeprature for 10 min and treated with UV light for 5 min. The hybridization . The sections were then incubated for 1 hour at room temperature in the same buffer containing 15 pM [125I]iodoproxyfan (Amersham Pharmacia Biotech UK Limited, England). Non-specific binding was determined by incubating consecutive sections in the presence of 1 μM (R)α-methylhistamine (Sigma-Aldrich, Germany). At the completion of incubation, the tissues were washed four times (4 min each) in the same fresh ice-cold buffer, dipped into ice-cold water, and dried under a current of air. Brain sections from control animals and animals 4 h, 24 h, 48 h, and 1 week post-lesion were treated simultaneously. Dried sections, along with standards, were exposed to Kodak BioMax X-ray films (Kodak, USA) for 2 days.Autoradiographic localization of [d before . BrieflyAutoradiographic films were quantified by digitizing the film images with a computer based MCID 5+ image analysis system (Imaging Research, Canada) and by measuring gray scale pixel values. The relative optic density was converted to integrated optic density (IOD) based on a standard curve derived from standards exposed to films. Gray scale values were determined from four sections for each animal, measurements from white matter of each respective section were subtracted to obtain the final values, and the data were analyzed using either a paired t-test or one-way ANOVA combined with Bonferroni's Multiple Comparison Test as a post-hoc test. Significance was determined when p < 0.05.in situ hybridization and binding studies, performed all of the image and statistical analyses, and participated in the design of the study. AAA performed the surgeries, assisted with tissue collection, assisted with image analyses, and participated in the design of the study. KK established the method of in situ hybridization in the lab, designed the oligonucleotide probes, assisted with surgeries and tissue collection, and participated in the design of the study. HS synthesized and supplied rare chemicals required for the binding study. PP acquired funding, coordinated the study, and participated in its design. All authors read and approved the final manuscript.AL assisted with the surgeries and tissue collection, sectioned all of the brains, carried out the