Msx1 has been studied as an antagonist of muscle differentiation, and its expression in cultured mouse myotubes induces about 5% of the cells to undergo cellularisation and viable fragmentation, but its relevance for the endogenous programme of salamander regeneration is unknown. We dissociated muscle fibers from the limb of larval salamanders and plated them in culture. Most of the fibers were activated by dissociation to mobilise their nuclei and undergo cellularisation or breakage into viable multinucleate fragments. This was followed by microinjection of a lineage tracer into single fibers and analysis of the labelled progeny cells, as well as by time-lapse microscopy. The fibers showing morphological plasticity selectively expressed Msx1 mRNA and protein. The uptake of morpholino antisense oligonucleotides directed to Msx1 led to a specific decrease in expression of Msx1 protein in myonuclei and marked inhibition of cellularisation and fragmentation. Myofibers of the salamander respond to dissociation by activation of an endogenous programme of cellularisation and fragmentation. Lineage tracing demonstrates that cycling mononucleate progeny cells are derived from a single myofiber. The induction of Msx1 expression is required to activate this programme. Our understanding of the regulation of plasticity in postmitotic salamander cells should inform strategies to promote regeneration in other contexts.The conversion of multinucleate postmitotic muscle fibers to dividing mononucleate progeny cells (cellularisation) occurs during limb regeneration in salamanders, but the cellular events and molecular regulation underlying this remarkable process are not understood. The homeobox gene  Amphibians such as the salamander can regenerate their limbs. This paper explores how multinucleate muscle cells transform into mononuclear cells and begin to proliferate during regeneration There is currently a significant focus on strategies to promote regeneration in adult mammals and therefore a renewed interest in the mechanisms that underlie regeneration in urodele amphibians. An adult salamander such as the newt or axolotl can regenerate its limbs and tail, jaws, ocular tissues such as the lens, and small sections of the heart . A key fMsx1 in mouse myotubes , which have been used interchangeably with comparable results. After removing the epidermis, the tissue was dissociated by proteolytic digestion, filtered through a sieve to remove most of the mononucleate cells, and plated in serum-free medium. The striated myofibers readily attached to the culture dish  or 48 h , while 16% of the mononucleate cells were labelled in the latter case. It is noteworthy that the absence of S-phase entry in nuclei within multinucleate cells includes the population of myofibers that undergoes the events of cellularisation or fragmentation described below.The dissociation of viable myofibers has allowed us to evaluate their plasticity after implantation into a limb blastema, a procedure that has previously been performed only on myotubes formed in cell culture. Fibers were labelled with a cell tracker dye in suspension after dissociation, and single fibers were examined to verify the absence of any adherent mononucleate cells and were drawn into a glass micropipette A. A few After 48 h in culture, some of the striated fibers remained viable, but showed no significant change in morphology and retained the appearance of the cell shown in Approximately 10% of the total population of myofibers underwent changes in nuclear localisation within the cells such that a lobulated or ‘cauliflower’ structure formed in the middle or end of the cell A and 3B.0 nuclei in myofibers, G1 nuclei in mononucleate cells, and G2/M nuclei in mononucleate cells labelled with antibody to phosphohistone H3. The histogram of DNA content for cells in the colony is comparable to that previously observed for cycling newt mononucleate cells  showed any reaction with the probe, whereas 51% of all myofibers (n = 820) showed reactivity. After treatment with taxol, 56.3% of inhibited fibers (n = 765) were positive, whereas 63% of all myofibers (n = 901) showed expression of Msx1. It is clear, therefore, that the arrest of nuclear mobilisation does not prevent the early expression of Msx1 in activated myofibers. These data are consistent with an upstream role for Msx1 in the activation of plasticity, but direct evidence for functional activity has come from antisense perturbation.In an initial investigation of this possibility, cultures were arrested as before by treatment with taxol, followed by reaction with the In order to evaluate the uptake of morpholino-substituted oligonucleotides, larval myofibers were dissociated as usual in the presence of 10 μM biotinylated morpholinos or underivatised morpholinos. The cells were cultured for 48 h and then analysed by a detection procedure involving tyramide signal amplification see . ApproxiMsx1. The resulting cultures were analysed in parallel for the proportion of active fibers and the antisense reagent reproducibly and specifically decreased this by 60%–70% , we have observed fragmentation comparable to that described here for fibers of the larval salamander, but no cellularisation. It is possible, therefore, that there is a gradation in the degree of plasticity after dissociation, and this may be related to the ability to undergo reversal during regeneration, although more work is required to investigate these comparative issues. It is interesting that the mononucleate progeny of cellularisation were observed to reenter the cell cycle, while multinucleate fragments retained the postmitotic arrest of the parental fibers. At least half of the salamander fibers were activated after dissociation and could be scored by morphological criteria as an index of plasticity, as well as by analysis of gene expression in situ. The occurrence of cellularisation did not reflect the activation of adherent mononucleate cells since the injection of a nontransferable tracer into the fibers resulted in labelling of the mononucleate progeny, and furthermore the mobilisation of nuclear aggregates occurred without any detectable S-phase reentry. It is probable that the process of enzymatic and mechanical dissociation mimics the activation events after amputation, either in terms of mechanical factors sensed by the fibers or the release of signals from the tissue or matrix. Earlier experiments on microinjected fibers in the larval tail have explored the stimuli required to trigger cellularisation and concluded that activation apparently required both ‘clipping’ at the end of the fiber as well as tissue injury in the vicinity  or axolotl (A. mexicanum) (3–5 cm size) were removed, the epidermis was peeled off, and the tissue was rinsed in serum-free amphibian MEM (AMEM)   prior tolsewhere . After aMyofibers were dissociated as above, retained in suspension in a sterile bacteriological dish , and incubated with 10 μM CellTracker Orange CMTMR  for 30 min at 25 °C. The labelling was terminated by addition of 10% AMEM, and the cells were incubated for 45 min at 25 °C to permit enzymatic activation of the dye. The cell suspension was diluted several fold to allow observation of myofibers at low density. The forelimbs of axolotl larvae (7–10 cm size) were amputated at mid humerus level under tricaine (0.1%) anaesthesia 48 h before injection of labelled myofibers see A. The anMyofibers were incubated in AMEM containing 2,3-butanedionne monoxime (BDM) (4 mM) for 30 min to prevent contraction of the myofibers  and main2, and phase contrast or variable relief contrast (VAREL) (Zeiss) images were acquired using a digital camera controlled through Image-Pro Plus software . A sequence gallery was created using Image Pro-Plus and images of interest were selected, digitally enhanced, and processed in Adobe Photoshop 6.0 .To record the coordinates of the myofibers in culture, the dish was scored underneath with a scalpel, and cells in each grid square were observed daily and images were acquired with a CCD camera . For time-lapse microscopy, myofiber cultures were placed under an Axiovert microscope fitted with an incubation chamber maintained at 26 °C and 3% CO3H] thymidine  for 24 h, fixed in 1% glutaraldehyde, and processed for autoradiography . Baseline values for nuclear DNA content in cycling axolotl mononucleate cells were measured in parallel after incorporation of 5-deoxy-2′-bromouridine (BrdU)  was routinely used in cultures to visualize and score the myofibers. The quiescent or inactive myofiber nuclei were aligned along the fiber see  and 7D, Dissociated myofibers were plated in medium containing taxol . Parallel control cultures were incubated in vehicle (DMSO) in a similar way. The cultures were fixed at 48 h after treatment and processed for tubulin immunofluorescence or in situ hybridisation.Msx1 gene (5′-CGGTCTGCATCCTCTGCTTGCTTAG-3′) by Gene Tools Inc. . Invert control oligos of Msx1 (5′-GATTGCTTCGTCTCCTAGCTCTGGC-3′) and standard control oligos (5′-CCTCTTACCTCAGTTACAATTTATA-3′) from the supplier were used as controls. 3′-Biotin-end-labelled standard control oligos were used for evaluating the uptake of morpholino oligonucleotides by myofibers. Oligo stock solutions were prepared according to guidelines from the manufacturer and stored at 4 °C. The morpholino oligos were added at a concentration of 10 μM to the dissociation cocktail and the myofibers were dissociated as described. After purification of myofibers, fresh morpholino oligos were added to the culture medium. After sequential washes to remove any adherent morpholino  coupled with enzyme-linked immunofluorescence  was employed to localize the uptake of morpholino oligos in cultured myofibers. Myofiber cultures were fixed at 48 h in 0.5% PFA containing 0.05% glutaraldehyde for 15 min on ice. The fixative was replaced with freshly made 0.1% NaBHMsx1 cDNA  was cloned into Bluescribe vector , and probes were generated as described elsewhere  probe was generated from a fragment of approximately 400 bp from Bluescribe vector after linearising with either HindIII (antisense) or EcoRI (sense), and riboprobes were synthesized using T3 and T7 RNA polymerase respectively , fixed in chilled 1% glutaraldehyde for 15 min, postfixed in 4% PFA, and washed in 0.3% PBT. In situ hybridisation was essentially as described elsewhere  was incorporated into the fixative. After fixation, the culture was treated with freshly prepared 0.1% NaBHlsewhere . For loced cells . The samz-stacks, and composite maximum projection images were generated through Leica software. Samples stained for Msx1 protein were observed under a Zeiss Axioplan microscope and images were acquired with an Axiocam digital camera. The fluorescence intensity in myofiber nuclei was measured in Axiovision software (Zeiss), and the data were analysed by one-way analysis of variance (ANOVA) followed by multiple range test using Instat .The myofiber cultures stained for β-tubulin, MHC, or titin, or cultures injected with TR–dextran were observed under confocal laser scanning microscope (Leica). The images were acquired as Figure S1The myonuclei incorporate Syto13 live nuclear stain. The myofiber was observed with VAREL optics at 24 h after plating. Scale bar, 100 μm.(4.1 MB TIF).Click here for additional data file.Figure S2Nuclear localisation of Msx1 protein (green) was detected with a rabbit polyclonal antibody generated against the full-length mouse Msx1 homeoprotein.(5.6 MB TIF).Click here for additional data file.Video S1Time-lapse sequence was begun 6 h after plating of the myofiber on to a culture dish. The images were taken at 6 min intervals under 32× VAREL objective magnification.(110 KB AVI).Click here for additional data file.


Inappropriate use of antibiotics by individuals worried about biological agent exposures during bioterrorism events is an important public health concern. However, little is documented about the extent to which individuals with self-identified risk of anthrax exposure approached physicians for antimicrobial prophylaxis during the 2001 bioterrorism attacks in the United States.We conducted a telephone survey of randomly selected members of the Pennsylvania Chapter of the American College of Emergency Physicians to assess patients' request for and emergency physicians' prescription of antimicrobial agents during the 2001 anthrax attacks.Ninety-seven physicians completed the survey. Sixty-four (66%) respondents had received requests from patients for anthrax prophylaxis; 16 (25%) of these physicians prescribed antibiotics to a total of 23 patients. Ten physicians prescribed ciprofloxacin while 8 physicians prescribed doxycycline.During the 2001 bioterrorist attacks, the majority of the emergency physicians we surveyed encountered patients who requested anthrax prophylaxis. Public fears may lead to a high demand for antibiotic prophylaxis during bioterrorism events. Elucidation of the relationship between public health response to outbreaks and outcomes would yield insights to ease burden on frontline clinicians and guide strategies to control inappropriate antibiotic allocation during bioterrorist events. Bacillus anthracis postexposure prophylaxis and clinical management of patients with anthrax disease [The September 11, 2001 terrorism events and the ensuing anthrax attacks in the United States were associated with widespread psychological trauma [ disease . For asy disease -6.Public health officials assured the public that emergency drug supplies would be delivered to clinical settings as needed from the national antimicrobial stockpile . DespiteA total of 250 potential study subjects were randomly selected from the 2001 membership database  of the Pennsylvania Chapter of the American College of Emergency Physicians (ACEP). Information in the database, such as contact telephone numbers and location of practice, is provided during enrollment or during membership renewals, and no active verification is done. Because the accuracy of the database was unknown, we used an 80% estimate based on a previous study of emergency physicians . During We used one-way frequency analyses to describe distributions of responses for all categorical items. Associations were quantified using odds ratios (ORs) with associated 95% confidence intervals (CIs). Statistical analyses were performed using SAS software .Forty-three of the 250 physicians in the sample were excluded from the study because of insufficient contact information or because the physician was no longer practicing medicine in the state. Of the remaining 207 physicians (24% of the estimated population of 848 subjects with accurate information), 97 were interviewed (47% response rate).Sixty-four (66%) of the 97 respondents had received patient requests for antimicrobial prophylaxis against anthrax; of the physicians who received requests, 16 (25%) prescribed antibiotics. Physician setting  was not associated with either patient request for, or physician prescription of, antimicrobial prophylaxis  prescribed ciprofloxacin while 8 physicians (50%) prescribed doxycycline Figure . FourteeB. anthracis analysis. During October-December 2001, the Pennsylvania Bureau of Laboratories received approximately 1400 specimens including white powder, environmental swabs, and letters from counties in eastern and western Pennsylvania districts. Of these, 27  came from Cambria County in the western district (PA DOH: unpublished data). There were 11,063 anthrax-related telephone inquiries received from October 8 to November 11, 2001 by the Centers for Disease Control and Prevention's (CDC) emergency operations centers; queries originated from all states and one US territory. Most of these calls were from members of the public concerning anthrax vaccines (≈58%), suggesting that search for protective measures against anthrax was widespread across the United States [Following the September 11 terrorist attacks and the ensuing anthrax outbreak, the majority of the emergency physicians we surveyed in Pennsylvania had received patient requests for anthrax prophylaxis; a quarter of these physicians prescribed antibiotics for these patients. Physicians that reported patients' requests for antimicrobial prophylaxis were distributed across the state, suggesting that patients' search for protection against anthrax was widespread. Our results are consistent with data on specimens submitted to Pennsylvania public health laboratory officials for Other studies have documented an increased use of antimicrobial agents that was temporally related to the anthrax outbreak; this use could not be ascribed to that recommended by the CDC. For example, a recent national study reported that approximately 160,000 more ciprofloxacin and 96,000 more doxycycline prescriptions were written in 2001 compared to 2000 . When otB. anthracis exposure until susceptibility results were known [B. anthracis isolates recovered from patients involved in the anthrax attacks were susceptible to other antibiotics, public health officials indicated that doxycycline might be preferable over ciprofloxacin [Of the physicians reporting that they had prescribed antibiotics for anthrax prophylaxis, the majority used ciprofloxacin. This finding is consistent with other studies ,13 and ire known ,5. When floxacin . While bfloxacin , the ratUnfortunately use of antibiotics has inherent risks and costs and optimizing benefits is especially difficult in the midst of bioterrorist events. Consequences of antibiotic treatment of unexposed individuals include adverse drug reactions, increased risk of antimicrobial resistance, depletion of antibiotics, and monetary costs . FurtherPublic health response likely influences demand for and outcomes associated with antibiotics requests during bioterrorism attacks. When we asked physicians to suggest on what health departments could do to reduce the influx of patients to the emergency departments, they cited official communications to make the public "less worried." In Illinois, a surge in environmental samples received by public health officials for anthrax tests was associated with both media reports of anthrax cases in other states and a specific announcement on October 29, 2001 by the US attorney general and the FBI director. The announcement asked US citizens and law enforcement agencies to be on the "highest alert" based on "credible information" . LessonsWe acknowledge some limitations to our results. First, as in any survey, these data are subject to non-response bias. But the 47% response rate is comparable with other telephone surveys conducted among physicians in general . In addiTaken together with other recent studies of antibiotic utilization ,13, thesACEP: American College of Emergency PhysiciansCDC: Centers for Disease Control and PreventionPA DOH: Pennsylvania Department of HealthThe author(s) declare that they have no competing interests.NMM conceived of the study and its design, and coordinated revision of the manuscript drafts with all authors. KGJ participated in data collection, study design, and contributed to the study's critical review. ARK participated in the design of the study and performed statistical analyses. RCA secured funding and participated in the study's critical review. JTR participated in the study design and coordination. EL contributed to the design of the study and participated in its critical review. All authors read, commented on, and approved of the final manuscript.The pre-publication history for this paper can be accessed here:


Approaches to limiting exposure to antiretroviral therapy (ART) drugs are an active area of HIV therapy research. Here we present longitudinal follow-up of a randomized, open-label, single-center study of the immune, viral, and safety outcomes of structured therapy interruptions (TIs) in patients with chronically suppressed HIV-1 infection as compared to equal follow-up of patients on continuous therapy and including a final therapy interruption in both arms.Forty-two chronically HIV-infected patients on suppressive ART with CD4 counts higher than 400 were randomized 1:1 to either (1) three successive fixed TIs of 2, 4, and 6 wk, with intervening resumption of therapy with resuppression for 4 wk before subsequent interruption, or (2) 40 wk of continuous therapy, with a final open-ended TI in both treatment groups. Main outcome was analysis of the time to viral rebound  during the open-ended TI. Secondary outcomes included study-defined safety criteria, viral resistance, therapy failure, and retention of immune reconstitution.n = 21; repeated TI, median [interquartile range] = 5  = 4 –8 wk, n Cycles of 2- to 6-wk time-fixed TIs in patients with suppressed HIV infection failed to confer a clinically significant benefit with regard to viral suppression off ART. Also, secondary analysis showed no difference between the two strategies in terms of safety, retention of immune reconstitution, and clinical therapy failure. Based on these findings, we suggest that further clinical research on the long-term consequences of TI strategies to decrease drug exposure is warranted. Structured treatment interruptions, or "drug holidays", in HIV patients yield no clinical benefits but don't lead to short- term treatment failure either Antiretroviral therapy (ART) has been a milestone in the treatment of HIV infection. Current treatment guidelines for HIV-1 infection in the United States recommend the initiation of ART in patients with CD4 T cell counts of less than 350 cells/μl , and theWe completed a randomized, controlled trial on the outcome of repeated 2- to 6-wk TIs in patients with chronic infection in which the comparator group maintained continuous therapy and then an open-ended interruption period was applied in both treatment groups. The study addressed the potential for repeated interruptions of therapy to delay time to viral rebound as a primary outcome and analyzed secondary outcomes regarding study-defined safety criteria, viral suppression and resistance, and retention of immune reconstitution.Between August 2000 and December 2003, we enrolled 42 patients infected with HIV who were older than 18 y and on ART; eligibility criteria included CD4 counts of greater than 400 cells/μl on ART with a nadir of no less than 100 cells/μl, ART-mediated suppression (< 500 copies/ml) for more than 6 mo and less than 50 copies/ml at recruitment on any antiretroviral regimen. Approval of the study protocol was obtained from the institutional review board (IRB) of the Philadelphia Field Initiating Group for HIV Trials . Written informed consent was obtained from all patients. Human experimentation guidelines of the United States Department of Health and Human Services and of the authors' institutions were followed. The study protocol, including the patient consent form, the CONSORT form, and the IRB approval, can be found in Forty-two eligible patients from the Jonathan Lax Immune Disorder Clinic in Philadelphia, Pennsylvania, were randomized via sealed envelopes in a 1:1 fashion to a first phase (phase I) of either (1) three successive TIs of 2, 4, and 6 wk, respectively, or (2) maintenance of ART for 40 wk before a final interruption of therapy in both arms (phase II) subject to therapy reinitiation criteria as described below. Phase II consisted of an open-ended interruption to allow for virological and immunological comparisons between the groups off therapy. Study visits were every 2 wk for the repeated interruptions group and every 4 wk for the continuous ART group during phase I. Both groups were followed every 2 wk during phase II. We followed a study design with step-wise increases in the length of TI cycles to address potential safety concerns (resuppression was confirmed after shorter TIs before longer interruptions were initiated) and the hypothesis that sequential viral replication intervals would stimulate viral control and a delay in time to viral rebound.Phase I procedures for the repeated interruptions group included the following. (1) Interruption of therapy was individually timed to occur after two HIV RNA measurements of less than 50 copies/ml without any viral load measurements greater than 400 copies/ml in between; these interruptions increased from 2 to 4 to 6 wk sequentially. (2) If a 0.5-log or greater reduction in viral load did not occur by 6 wk of reinitiated therapy or less than 50 copies/ml was not achieved within 20 wk of reinitiated therapy, patients were withdrawn as therapy failures and a resistance test was performed. (3) Patients were also withdrawn as therapy failures if (a) the CD4 cell number declined by more than 45% of the baseline CD4 count, (b) participants developed an opportunistic infection, even if retaining required CD4 count levels, or (c) a viral load of greater than 500,000 copies/ml occurred once, with or without development of acute retroviral syndrome as defined by fever, skin lesions, and pharyngitis.Phase I procedures for the continuous therapy arm included the following: (1) patient monitoring if detected viremia was between 50 and 999 copies/ml, with the patient withdrawn if their viral load did not return to less than 50 copies/ml immediately prior to phase II, and (2) patient study withdrawal as therapy failure if during the 40-wk ART period viral load rebounded to more than 1,000 copies/ml at two consecutive time points.Phase II procedures for both arms included the following: (1) monitoring for patient study withdrawal criteria as described in phase I, (2) determining time to primary end point of a viral load greater than 5,000 copies/ml, (3) monitoring until the time of therapy reinitiation at a viral load greater than 30,000 copies/ml for three consecutive time points, and (4) after reinitiation of therapy, follow-up on therapy to confirm resuppression to less than 50 copies/ml at 6, 10, and 14 wk on therapy. Clinical and laboratory parameters  were monitored at each visit, and venous blood was collected for additional secondary outcomes during selected study visits.In both phase I and II, participants taking non-nucleoside reverse-transcriptase inhibitors (NNRTIs) were instructed to stop them a day earlier than the remaining drugs in the regimen.HIV RNA) was measured as a secondary outcome at weeks 12 and 20 of phase II based on reinitiation-of-therapy criteria outlined above.The primary outcome was time to confirmed virological rebound during phase II. Rebound was defined as first time point with greater than 5,000 copies/ml. Viral replication magnitude as defined by mean HIV-1 plasma RNA area under the curve  safety outcomes , (2) retention of ART-mediated immune reconstitution, and (3) detection of viral resistance. Retention of immune reconstitution was analyzed by (1) same-day whole blood flow-cytometry-based analysis of CD4 and CD8 T cells, including total and naïve (CD62 l/CD45RA) and memory (CD45RO) subsets as described , and (2)escribed . Viral rescribed ,19.The sample size required was calculated using PS  softwareHIV RNA up to 12 and 20 wk. In all cases, a two-sided alpha level of 0.05 was used to define statistical significance. Unless otherwise stated, results are presented as median (interquartile range) in text and tables.The primary analysis was an intent-to-treat analysis in which dropouts were assigned a week 0 rebound time . In secondary analyses, these dropouts were excluded. The log-rank test was used to test the null hypothesis of no difference between arms in the number of weeks from initiation of the open-ended TI to reaching viral rebound as defined. Patients not reaching end point at 26 wk after the beginning of the open-ended TI were censored. Wilcoxon rank sum tests were used to compare baseline and week 0 of the open-ended interruption between groups. Wilcoxon signed rank tests were used to test for no change from baseline to week 0 of phase II. Finally, Wilcoxon rank sum tests were employed to test between groups for equality of the mean AUCn = 1; patient moved away) and virological failure during continuous therapy . In the repeated interruptions arm, 18 of 21 patients reached the open-ended interruption following three TIs of 2, 4, and 6 wk duration, with median peak rises in viral loads of 136 , 13,651 , and 18,887  copies/ml, respectively. Median time to less than 50 copies/ml after resumption of therapy was 2 (0–4), 3 (1.8–12), and 9.5 (2–12) wk, respectively, with 9, 18, and 20 wk as the maximum time needed to achieve suppression in 100% of patients before reaching the open-ended interruption. Study discontinuation in the repeated interruptions arm was due to protocol violation , loss to follow-up , and virological failure during on-therapy period .Trial patient flow is summarized in . No significant difference was found in baseline parameters between arms, with 33%–47% of patients on protease-inhibitor-containing and 61%–71% on NNRTI-containing regimens. Owing to the high participation of patients on NNRTI-based regimens and concerns about TI and safety in general, patient outcomes and treatment failure were reviewed monthly by the IRB of this study during the first 8 mo of study, quarterly for the following 4 mo, and semi-annually thereafter. The demographic and clinical characteristics of the two groups at baseline are summarized in n = 21; repeated TI, median = 5 [n = 21; p = 0.36). n = 21 per group). Exclusion of drop-outs in an as-treated analysis did not alter conclusions . Additional secondary analysis of the magnitude of viral load as shown in HIV RNA analysis at week 12  or week 20 .An intent-to-treat analysis of the time to viral rebound  in the open-ended interruption showed no difference between groups  or due to study-associated SAEs . However, four non-study-related SAEs occurred: two patients from the continuous therapy/single interruption arm were hospitalized, one for a cholecystectomy and one for acute rectal bleeding, during the 40-wk ART period; a patient from the repeated interruptions arm died of liver cancer during week 26 of the open-ended interruption after previously reaching a viral load greater than 5,000 copies/ml yet electing to stay off ART; and a patient from the repeated interruptions arm developed a transient ileitis.C. albicans was observed, confirming the absence of significant differences in the retention of baseline immune reconstitution correlates between arms. However, a significant decrease in the abundance of CD4 cells relative to other T cell types as summarized in CD4% (but not in absolute CD4 count ) was present in the repeated TI arm, corresponding to a significant increase in CD8 T cell count. In spite of fluctuations in CD4 T cell count levels between the start and end of each monitored TI, a recovery of CD4 count levels was achieved upon resuppression following each TI in conjunction with a retention of lymphoproliferative responses against C. albicans before, during, and after each TI, as illustrated in No significant difference was observed between groups in CD4 T cell counts at the start of phase II, as illustrated in p > 0.05).An intent-to-treat analysis of the combined number of patients per arm with detected resistance mutations irrespective of therapy failure in phase I and during the final TI in phase II showed no significant difference between arms  in association with self-reported nonadherence to therapy and detection of resistance mutations in phase I, as listed in p = 0.06). A greater number of resistance mutations was detected in the repeated interruption arm, as summarized in In patients who reached phase II in the absence of therapy failure, a total of 12 patients were identified to have resistance mutations at the first viremic time point  or clinical disease progression (as indicated by CD4 count on therapy or opportunistic infections) was not observed to be different between arms. Prospective safety outcomes in our study are in accordance with reports from a retrospective analysis of 1,290 patients who interrupted treatment at least once (< 3 mo) without an increased risk of HIV-associated morbidity or mortality (with the exception of patients in Center for Disease Control and Prevention stage C during first interruption only) . In regad TI see , nor fold TI see . The latd TI see . InteresImportantly, no evidence for an increase of viral resistance in association with therapy failure was present in the repeated interruptions arm See . We did In spite of the lack of difference in the total number of patients with resistant mutations detected on therapy during phase I and off therapy in phase II  in both arms, we do report in similarity to others a greater detection of resistance mutations in the TI arm when restricting analysis to the last off-drug period only ,30 as thDrug resistance that occurs during virological drug failure predicts virological responses to salvage treatment ,32,33. ITaken together, while our data show no clinically significant benefit for repeated TIs of less than 1.5 mo in patients with CD4 counts greater than 400 on therapy with regard to viral control as defined by time to rebound, secondary outcomes document no significant difference in levels of retention of immune reconstitution between arms and no increased incidence of virological failure as a consequence of TIs. While our data indicate that this TI strategy should not be pursued outside of a clinical trial setting, we argue that it will be important to collect additional data on the potential benefits of drug-sparing regimens (such as reduced long-term toxicity and reduced cost) and to define long-term outcomes in comparison with continuous therapy.clinicaltrials.gov under identifier NCT00051818.Registration of randomized trial at Protocol S1(614 KB DOC).Click here for additional data file.Protocol S2Current IRB approval for study at clinical site.(179 KB PDF).Click here for additional data file.Protocol S3IRB approval to receive study biological material at the Wistar Institute for research.(201 KB PDF).Click here for additional data file.Protocol S4(50 KB DOC).Click here for additional data file.Table S1(36 KB DOC).Click here for additional data file.Highly active antiretroviral therapy has revolutionized HIV treatment for patients who have access to the medications. But the drugs are expensive, have side effects, and can become ineffective when the virus develops resistance. Structured treatment interruptions (STIs), also known as “drug holidays” (because patients take a holiday from their drugs), have been suggested as possible alternatives to continuous therapy. Initially, there was fear that patients who went back on therapy after an interruption would not be able to control the virus again, but there was also hope that STIs might actually strengthen the immune system. In addition, STIs might alleviate some side effects, and they would certainly reduce costs. This study uses a particular design to examine the risks and benefits of STIs.The researchers studied 42 patients who received either continuous therapy for 40 weeks or three successive treatment interruptions of two, four, and six weeks, followed by a final open-ended interruption for both groups. The researchers then recorded how long patients were able to control the virus before their viral load reached a certain threshold and they had to restart therapy. They also examined CD4 counts and therapy failure, and looked for resistant viruses on and off therapy.In terms of being able to control the virus, it made no difference whether patients were on continuous therapy or had three STIs. In other words, when both groups stopped treatment at 40 weeks, the length of time that the patients could control the virus was the same in both groups. Eventually, all patients  re-initiated therapy because of a rising viral load, and the patients once on therapy all regained control over the virus. Resistant viruses were found in patients from both groups, but during the final interruption they were more common in the group that had received the three STIs.The study confirms that STIs do not help with viral control, consistent with other studies that found that STIs had no clinical benefit. On the other hand, no short-term adverse events were present, as all patients were able to regain control over the virus after they went back on treatment (without a drop in CD4 count), even after several rounds of interruptions and tests to detect of resistant viruses. There remains concern about whether recurrent cycles of viral replication and suppression might in themselves be harmful, and whether the presence of resistant virus is a signal for future treatment failure. Given these unanswered questions, STIs should only be undertaken within clinical trials.Possible risks and benefits of STIs in the management of therapy remain an active area of research. Evidence so far has not shown clinical benefits. Ongoing studies need to clarify whether there are long-term risks (and what they are), so that we can weigh these against the benefits of reducing costs and side effects.http://www.thebody.com/treat/sti.htmlThe Body information Web page on STIs: http://www.avert.org/conttrt.htmInformation on “continuing antriretroviral treatment” from AVERT, an international HIV and AIDS charity based in the United Kingdom: http://www.aidsmap.com/en/docs/7980314C-97B5–412F-93B1-AD8B64F51F73.aspInformation on STIs from NAM, a United Kingdom registered charity: http://www.niaid.nih.gov/factsheets/treat-hiv.htmFactsheet on HIV treatments from the United States National Institute for Allergy and Infectious Diseases: http://www.clinicaltrials.gov/search/term=%22Treatment+Interruption%22%5BCONDITION%5D+AND+HIV+%5BCONDITION%5DSearch results from Clinicaltrials.gov when searching for “HIV” and “treatment interruption” combined terms:


Hypertension and diabetes mellitus are closely interrelated and coexist in as many as two-thirds of patients with type 2 diabetes. The consequent risk of such an association is an accelerated development of atherosclerotic cardiovascular disease and nephropathy complications.In choosing an antihypertensive agent, effectiveness needs to be accompanied by favourable metabolic, cardioprotective, and nephroprotective properties. Given the multifactorial nature of hypertension, the approach that has gained widespread agreement is treatment with more than one agent. Agents with different mechanisms of action increase antihypertensive efficacy because of synergistic impacts on the cardiovascular system. Combination therapy allows the use of lower doses of each antihypertensive agent which accounts for the excellent tolerability of combination products.The aim of the present study is to quantify the efficacy of combination therapy of Eprosartan 600 mg respectively Ramipril 5 mg with low-dose Hydrochlorothiazide and Moxonidine on blood pressure levels in patients with essential hypertension and associated diabetes mellitus type 2.The use of monotherapy (Eprosartan or Ramipril) followed by addition of low-dose Hydrochlorothiazide as second agent and of Moxonidine as a third agent will be individualized to the severity of hypertension in the particular patient and to his/her degree of response to current treatment. Cliniortality . Thus, mre < 130/0 mm Hg iThe challenge clinicians are facing is to tighten blood pressure control to less than 130/80 mmHg and to adjust initiation of therapy to the severity of hypertension in the individual patient.This multicenter study will evaluate the efficacy and tolerability of monotherapy, double- and triple- antihypertensive combination therapies in a large spectrum of hypertension & diabetes patient population, as summarised in Table response to that therapy.Table 2 see  specifieThe primary objectives of hypertension management in patients with diabetes are to reduce blood pressure levels to currently recommended target level and thus to reduce the risk of cardiovascular and renal complications without adversely impacting glycemic and lipid control.Previous debate regarding the level of blood pressure reduction that optimizes cardiovascular risk reduction is currently settled. BP goal of < 130/85 mmHg promoted by the JNC-VI guidelines issued 1997  were repA widespread agreement, supported by the above mentioned organizations/societies is in place, regarding the principles governing the use of appropriate antihypertensive drug combinations to maximize hypotensive efficacy while minimizing side effects. Polypharmacy is common place and, with at least one third of patients requiring two or more agents simultaneously, a paradigm shift in the approach of initiating therapy is done by advocating use of two agents in subjects with more severe hypertension . Low-dose thiazide diuretic is favored as one of the two starting agents.In general, monotherapy is likely to be successful in mild hypertensive patients (grade 1 hypertension) without associated major risk factors for CHD. In contrast, patients with type 2 diabetes need more rigorous control of BP in an easier, simpler fashion, given the remarkable complexity of the multiple drug regimens needed to control their comorbid medical problems .A large body of evidence derived from a multitude of international trials have demonstrated both the benefit of low-level, goal blood pressure, in terms of prevention of long-term complications and, the need for multiple drug combinations in order to achieve that goal ,18-20. FIn the HOT study , which rGiven the very poor BP control rate, i.e., 11% in patients with hypertension & diabetes, the use of combination therapy is an important therapeutic consideration, as it facilitates quicker and easier attainment of goal BP and should lead to a greater proportion of people with diabetes who achieve BP goal. Initiation of treatment by combination therapy was effectively tested in the VA study at the beginning of the antihypertensive treatment trial era ,26 and rSubjects will be recruited in outpatient clinics/offices of general practitioners and internal medicine/cardiology specialists from the entire spectrum of patients having coexistent hypertension and diabetes mellitus type 2. The upper limit of blood pressure values targeted (≤ 179/109 mmHg) correspond grade 2 hypertension (according to current ESC/ISH and WHO guidelines).Subjects to be recruited are supposed to have both entities  diagnosed since previously, undergoing current antihypertensive treatment and, to be eligible for the study according to the specific inclusion/exclusion criteria described below.The study consists of five distinct phases: Screening (S) (up to seven days), Placebo Run-in phase (two weeks), monotherapy , double-combination treatment , triple-combination treatment (Eprosartan/HCTZ/Moxonidine versus Ramipril/HCTZ/Moxonidine with HCTZ and Moxonidine open labelled) and Follow-up (two to seven days). Patients allocated to monotherapy will participate in the study for a four weeks period while those starting with double combination therapy will receive medication for a maximum of 12 weeks.The flowchart below captures the main events during the study conduct Fig. .Randomization will be concealed.A stratified randomization will be employed based on the following rules:1. Subjects with blood pressure in range: BP ≥ 130/80 – ≤ 150/90 mm Hg will be randomly allocated to one of the monotherapy arms (Eprosartan or Ramipril). 30 subjects will be recruited for each arm.2. Subjects with blood pressure in the range: BP > 150/90 – ≤ 179/109 mm Hg will be randomly allocated to double-combination therapy (Eprosartan/HCTZ or Ramipril/HCTZ); 190 subjects will be recruited for each arm.The randomization lists will be provided by the Department of Clinical Supplies at Solvay Pharmaceuticals BV with the program ADLS. Patients will be allocated in equal numbers to each sequence. A fixed block size of patients will be used, and only complete blocks of study medication will be provided to the centers.Within each center, randomization numbers will be used in ascending order and patients will be allocated to randomization code numbers in chronological order. The study will be unblinded when all CRFs are in house and the data on the database have been declared clean.The following drugs are to be used in the study:Eprosartan 600 mg, once daily• Ramipril 5 mg, once daily• Hydrochlorothiazide 12.5 mg, once daily• Moxonidine 0.4 mg, once daily• Placebo tablets matching Eprosartan 600 mg and Ramipril 5 mg will be used in the monotherapy, in the double- and in the triple-combination therapy phases. Hydrochlorothiazide tablets will be open labelled in the double- and triple-combination therapy phases. Moxonidine tablets will open-labelled in the triple-combination therapy phase.• Eprosartan and Ramipril and the corresponding placebos will be packaged according to the double-dummy technique. Fig. Patients eligible for participation in the study by the assessment at V2 will be randomized on the basis of the severity of their hypertension and allocated to either monotherapy or to double-combination therapy (described below).Patients with initial blood pressure values in the range ≥ 130/80 – ≤ 150/90 mmHg, will be randomly allocated to one of the monotherapy groups.By the end of the first four-week phase an assessment will be made as to whether patients have reached or not the "goal" hypertension (< 130/80 mmHg).Patients on monotherapy deemed to be responders at the end of four weeks treatment  will terminate their participation in the study and be followed up during the ensuing two to seven days after stopped monotherapy (procedure similar with Follow-up at end of study (V6).Non-responders , will receive double-combination therapy (Eprosartan/HCTZ respectively Ramipril/HCTZ) and will be followed-up for eight weeks (red-dotted line in the flowchart).Patients with initial blood pressure values in the range > 150/90 – ≤ 179/109 mmHg, will be randomly allocated to double-combination therapy. By the end of this first four-week phase an assessment will be made as to whether patients have reached or not the "goal" hypertension (< 130/80 mmHg).Responders will maintain double-combination therapy and will be followed-up for an eight week period  and retain therapy unchanged.Non-responders will receive triple-combination, as described below.Non-responder patients after four weeks of double-combination therapy will receive triple-combination (Eprosartan/HCTZ/Moxonidine vs. Ramipril/HCTZ/Moxonidine) and will be followed-up for an eight weeks period (V3 to V5).After the first four weeks of monotherapy respectively double-combination therapy (V3), patients will be reassessed for compliance, adverse events and supplied with medication for the next eight weeks .During the eight weeks triple-combination therapy all patients will be reassessed for compliance and adverse events . A 12-lead ECG will be performed by S, V3 and V5 while safety laboratory parameters will be performed by S, V2 and finally by visit V5.A Follow-up Visit will be performed on all patients with full physical examination, BP and pulse rate check within the ensuing two to seven days after study end (V6). Further follow-up and optimal treatment will be decided on a case-by-case basis by the physician in charge. Table 3 see  displays1. Males and females aged 40 to 80 years of age. Women of childbearing age will be subject to pregnancy testing and will agree to maintain adequate hormonal contraception.2. Eligible patients should have diagnosed essential hypertension  and diagnosed associated diabetes mellitus type 2, willing to accept withdrawal of any antihypertensive medication by the time of the Screening visit.A multitude of exclusion criteria, carefully listed in the study protocol, can be summarised in three different groups:1. Ineligibility based on hypertension grade 3 (BP ≥ 180/110 mmHg), any form of secondary hypertension or hypotension (SBP ≤ 90 mmHg).2. Any form of organic heart disease requiring medical treatment that might have hypotensive effect, imply need for invasive investigation or surgery.3. The patient is suffering from a severe concomitant illness related to any body organ or system, likely to affect outcome assessment. Likewise, ineligibility is declared for patient anticipated to have compliance problems, participants in another trial during the past 30 days, pregnancy and lactations and known hypersensitivity to ingredients of any of the employed agents . In addition, diabetes mellitus type 1 is exclusion criteria.lifestyle change advice regarding relevant diet and physical activity. Visits are to be scheduled in the context of the study . Any antihypertensive medication should be withdrawn latest by Screening visit and will be prohibited during the whole period of ongoing study.The study protocol calls for every patient to be treated optimally by the physician in charge and to receive comprehensive, individualized The study will be conducted in accordance with ICH GCP and the European Directive 2001/20/EC of the European Parliament and of the Council of 4 April 2001  and on the basis of ethical principles laid down in the current revision of the Declaration of Helsinki (Edinburgh 2000). In addition, Solvay Pharmaceuticals GmbH policies and procedures should also be followed.Written consent, involving provision of detailed information regarding the study objectives, design, scope of the intervention, risks and benefits, will be obtained for all patients before initiating any study procedures. Likewise, study documentation is to be subject to the scrutiny of local ethical committees in the two countries participating in the study.goal blood pressure (<130/80 mmHg) at visit V3.The primary objective is to demonstrate the superiority of combination therapy of Eprosartan/HCTZ (600/12.5 mg) versus Ramipril/HCTZ (5/12.5 mg) with the primary parameter of attention being the percentage of patients brought to 0: PE+HCTZ = PR+HCTZNull hypothesis: H1: PE+HCTZ ≠ PR+HCTZ,Alternative hypothesis: HE+HCTZ is the percentage of patients brought to goal blood pressure at visit 3 with Eprosartan/HCTZ and PR+HCTZ is the percentage of patients brought to goal blood pressure at visit V3 with Ramipril/HCTZ.Where PThe primary parameter will be analyzed using the Cochran-Mantel-Haenszel test, controlling for center effects. Statistical significance will be assessed with a two-sided test at 0.05 α level. The confirmative analysis of the primary parameter will be performed on the intent-to-treat patient sample.Secondary efficacy objectives:• To compare the mean change in sitting systolic blood pressure (sitSBP) and sitting diastolic blood pressure (sitDBP) between Eprosartan/HCTZ and Ramipril/HCTZ (V3 vs. V2).• To compare the percentage of patients brought to goal blood pressure by the triple-combination Eprosartan/HCTZ/Moxonidine vs. Ramipril/HCTZ/Moxonidine (V5 vs. V3).• To compare the mean change in sitSBP and sitDBP between triple-combination with Eprosartan/HCTZ/Moxonidine vs. Ramipril/HCTZ/Moxonidine (V5 vs. V3).• To compare the mean change in sitSBP and sitDBP between monotherapy with Eprosartan vs. Ramipril (V3 vs. V2)• To compare the percentage of patients brought to goal blood pressure at visit V5 between Eprosartan/HCTZ and Ramipril/HCTZ in patients not at goal blood pressure after four weeks of monotherapy.at goal) after four weeks of monotherapy (switched to double combination therapy) (V5 vs.V3); and to compare the mean change in sitSBP and sitDBP as well as the responder rate maintenance in patients who reached goal blood pressure value at the end of first four weeks of double-combination therapy and successively entered an eight weeks follow-up period (V5 vs.V3).• To compare the mean change in sitSBP and sitDBP as well as the responder rate in patients non-responders . The model will include the intercept, treatment and center as fixed effects and the baseline value as covariate. For response rates, the treatment groups will be compared using the Cochran-Mantel-Haenszel test, controlling for center effects. Comparisons of the medication regimens will be reported along with 95% confidence intervals of the relative risk ratios. These analyses will be considered as exploratory.All patients who receive at least one dose of double-blind medication will be assessed for clinical safety and tolerability. Evaluation of safety data will be based on comparisons of patient experience by treatment group. Clinical interpretation of safety will be based on reviews of standard displays of adverse events incidence, pulse rate data, and laboratory test values. Summary statistics of laboratory test values and incidence of adverse events according to treatment and time of onset will be presented.A formal sample size estimation has been done for patients with blood pressure in the range: > 150/90 mmHg and ≤ 179/109 mmHg.goal blood pressure as compared with only 40% in the Ramipril/HCTZ group, a 0.05% two-sided significance level with 80% power to detect the targeted 15% difference will imply the need for 346 patients supposed to complete the four-weeks double-combination therapy phase. Further 35 patients  will be recruited to account for drop-outs.Assuming that 55% of the patients in the Eprosartan/HCTZ group would reach In addition, 60 subjects with BP ≥ 130/80 and ≤ 150/90 mmHg will be randomly allocated to either Eprosartan or Ramipril monotherapy group at visit V2. Inclusion of monotherapy phase with a relatively low number of patients (30 subjects per arm) is justified by the intention to therapeutically target the whole spectrum of patient population having coexistent diabetes mellitus type 2 and mild to moderate hypertension, in whom the agents tested are likely to be effective.Office blood pressure will be determined by Riva-Rocci method with a mercury or a mercury calibrated sphygmomanometer throughout the study. All measurements will be made on the same arm supported at heart level, using the same cuff size and the same equipment. If the patient's arm circumference is > 32 cm, a large blood pressure cuff should be used. Diastolic blood pressure will be measured at the disappearance of Korotkoff sounds phaseV. Measurements should be taken by the same staff member at the particular visits.For an individual patient blood pressure measurements should be performed at 24 hours after the last oral dose, at the same time (± 2 hour) in the morning, between 8 and 10 am. Blood pressure will be measured in the following sequence: after the patient sits quietly for at least 5 minutes, blood pressure will be measured twice at approximately 2-minutes interval. The average of these measurements will be recorded. If the difference between measurements is in excess of 5 mmHg a third reading will be performed and the average value recorded as mean sitting systolic and diastolic blood pressure.Measurements should be performed by the same study assistant using the same device, in each of the centres involved in the study.goal blood pressure to be achieved in these patients. Further, common sense in clinical practice dictates that combination therapies should be tailored to severity of hypertension in the individual patient and that, eventual associated risk factors/comorbidities should be accounted for in the process of treatment decision making.The current evidence base is strongly in favour of combining drugs in order to achieve blood pressure goals, in particular in patients with coexistent hypertension & diabetes. Likewise, there is a widespread agreement in the scientific community as to the Patients with high blood pressure and associated impaired glucose tolerance or overt diabetes mellitus type 2, as a group, are insulin resistant,  glucose For every 5 to 10 mmHg decrease in systolic blood pressure achieved with diuretics, ARBs, ACE inhibitors, beta-blockers or calcium channel blockers in patients with diabetes, there is a 20% to 30% relative risk reduction in cardiovascular events -53.Agents belonging to the nine, most well-known different antihypertensive drug classes produce a similar reduction in systolic and diastolic blood pressure (10–15 and 5–10 mm Hg respectively). Differences in terms of magnitude of blood pressure lowering, as indicated by results from comparative efficacy studies, are usually small . HoweverComparisons between different agents in patients with hypertension & diabetes mellitus type 2, convincingly point to ACE inhibitors and ARBs as being the two classes of antihypertensive drugs that reduce the activity of the renin-angiotensin II system, and should be among the preferred first-step drugs for the treatment of these conditions . AngioteClinical trials carried out world-wide have shown that ACE inhibitors have renoprotective effects ,58 and cIn contrast, in placebo-controlled trials, the ARBs have demonstrated almost no side effects . Both ACUse of low-dose thiazide diuretic (< 25 mg) as a second agent in treatment of patients with hypertension & diabetes is well-documented and widely recommended ,70-73. ISince drug combinations may be required for many years in the age-groups in which type 2 diabetes is most prevalent, there have been calls for the use of agents devoid of adverse effects on carbohydrate and lipid metabolism. It has been suggested that such effects may account for the shortfall in reduction of coronary heart disease observed in clinical trials of diuretics and β-blockers ,81.1 receptors in the medulla, thereby reducing central sympathetic drive and attenuating peripheral vascular resistance. In addition, reduced sympathetic drive results in lower plasma concentrations of catecholamines and renin. Randomised comparative studies show that the efficacy of moxonidine as monotherapy is similar to that of other antihypertensive agents [1 receptor greatly reduces the adverse affects attributable to costimulation of medullary α2-adrenoceptors [Moxonidine stimulates imidazoline-Ie agents . Moreoveoceptors  observedIn clinical studies, moxonidine has been shown to have neutral or beneficial effects on lipid and carbohydrate metabolism ,83. A reOn the available evidence, moxonidine seems to be a logical choice as component of combination treatment of patients with hypertension and associated diabetes mellitus type 2 or impaired glucose tolerance.The poor blood pressure control in patients with hypertension & diabetes in everyday life lies, at least in part, in the emphasis in the evidence-based guidelines of the recent past towards advice on initial, single treatments as well as in their lack of clarity and transparency in recommending pre-specified blood pressure targets ,84-86.goal blood pressure and thereby to minimize long term risk of hypertension/diabetes-related complications [Previous consensual advice that combination treatments expose patients to the increased risk of adverse events has been replaced by good evidence to the contrary: use of several agents combined or of fixed-dose combinations treatments have the potential to bring patients to ications -27,87.blood pressure goal and individualized therapy on the basis of hypertension severity (and addition of a third agent in case of uncontrolled BP with two agents), comparative data to guide clinical practice is still lacking and this applies also to the comparison of ARBs versus ACE inhibitors. The present study attempts to explore this area.Despite the apparent simplicity of the paradigm shift towards clear Pater C, Berrou JP, Luszick J and Beckman K are employees of Solvay Pharmaceuticals.Study concept and design: Pater, Berrou, LuszickDrafting of manuscript: Pater, BhatnagarStatistical expertise: BeckmanTable 2 – Blood pressure-adjusted treatment stratificationClick here for fileTable 3 – Investigations ScheduleClick here for file


Although pharmacological experiments have suggested the presence of several P2 receptor subtypes on monocytes and lymphocytes, some results are contradictory. Few physiological functions have been firmly established to a specific receptor subtype, partly because of a lack of truly selective agonists and antagonists. This stimulated us to investigate the expression of P2X and P2Y receptors in human lymphocytes and monocytes with a newly established quantitative mRNA assay for P2 receptors. In addition, we describe for the first time the expression of P2 receptors in CD344 receptor had the highest expression in lymphocytes and monocytes. Among the P2Y receptors, P2Y12 and P2Y2 had highest expression in lymphocytes, while the P2Y2 and P2Y13 had highest expression in monocytes. Several P2 receptors were expressed  in CD34+ stem and progenitor cells.Using a quantitative mRNA assay, we assessed the hypothesis that there are specific P2 receptor profiles in inflammatory cells. The P2X12 receptors in lymphocytes potentially explaining the anti-inflammatory effects of clopidogrel, P2Y13 receptors in monocytes and a previously unrecognised expression of P2X4 in lymphocytes and monocytes. In addition, for the first time P2 receptor mRNA expression patterns was studied in CD34+ stem and progenitor cells. Several P2 receptors were expressed , indicating a role in differentiation and proliferation. Thus, it is possible that specific antibodies to P2 receptors could be used to identify progenitors for monocytes, lymphocytes and megakaryocytes.The most interesting findings were the high mRNA expression of P2Y So fa, P2Y14) ; the P2X12 and P2Y1, together with the ATP receptor P2X1 [12 antagonist clopidogrel for the prevention of myocardial infarctions in patients with acute coronary syndromes [7 receptor mRNA in monocytes has been observed upon cell differentiation to macrophages [We have previously quantified P2 receptor mRNA expression in platelets (representing megakaryocyte expression), and demonstrated a selective expression of the ADP receptors P2Ytor P2X1 . This isyndromes ,10. Howeyndromes . Circularophages ,14.+ stem and progenitor cells implicating a potential role of P2 receptors in hematopoietic lineage and progenitor/stem cell function.Although pharmacological experiments have suggested the presence of several P2 receptor subtypes on monocytes and lymphocytes, some results are contradictory ,2. Few p+ stem and progenitor cells.Our previous studies of P2 receptor mRNA expression in man with real-time PCR has shown a good resemblance with pharmacological and physiological experiments in vascular smooth muscle cells, endothelial cells and platelets ,15. It i1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13 could be detected (n = 6). To illustrate expression of the P2 receptors relative to each other the P2Y1 receptor was used as calibrator for the others, i. e. the other receptors were expressed as a ratio of the P2Y1. Among the P2Y receptor subtypes the P2Y12 and P2Y2 had highest expression  and P2X7(P < 0.01).The most abundant P2X receptor in lymphocytes was the P2X7 in lymphocyte regulation. However, B lymphocytes stimulated with ATP do not undergo the typical increase in permeability up to 900 Da that is typical for the P2X7 receptor. On the other hand, P2X7 mediated effects on Ba2+ and ethidium influx, phospholipase D activity and shedding of L-selectin have been blocked by the P2X7 selective antagonist KN-62 in human lymphocytes [4 receptor was the highest expressed subtype in lymphocytes at the mRNA level. Even though we have demonstrated that more than 90% of the preparation consists of lymphocytes (see methods), it is possible that a small contamination of monocytes may have influenced the results, at least regarding P2X4 receptor mRNA expression, because of its high expression levels in monocytes. P2X4 receptors have indeed been demonstrated at the protein level in human B lymphocytes by confocal immunohistochemistry, in which P2X1, P2X4 and P2X7 were detected at the protein level [4 receptor staining was the most variable of the P2X receptors with weak to moderate levels of staining in a large proportion of cells in three patients and weak levels in only a minority of the cells from the other three patients examined [Selective pharmacological tools to discriminate between P2X receptors are scarce. Nevertheless, several studies have suggested the importance of P2Xphocytes . Thus itin level . Howeverexamined .1 expression was used as calibrator for the others, i. e. the other receptors were expressed as a ratio of the P2Y1. Among the P2Y receptors, the P2Y2, P2Y13 and P2Y11 had highest expression  and P2X7 (P < 0.01) had lower levels. Thus, unexpectedly the P2X7 receptor was not the highest expressed P2X receptor in monocytes. This is in agreement with patch-clamp experiments suggesting that other P2X receptors are involved [4 receptors, which is supported by our findings [4 and 6–7-fold more for P2X1 and P2X7). A physiological role for all three subtypes can therefore be expected.As showed in Figure involved . Interrefindings . It shou+ stem and progenitor cells are receiving an increasing attention because of their extensive self-renewal and multilineage differentiation ability making them attractive for cellular therapy [+ stem and progenitor cells. We found expression of several P2Y receptors, especially P2Y1 and P2Y2   Figure , n = 3, poptosis . Such ro4 receptor had the highest mRNA expression in lymphocytes and monocytes. Among the P2Y receptors, P2Y12 and P2Y2 had highest expression in lymphocytes, while the P2Y2 and P2Y13 had highest expression in monocytes. The most interesting findings were the high mRNA expression of P2Y12 receptors in lymphocytes potentially explaining the anti-inflammatory effects of clopidogrel, P2Y13 receptors in monocytes and a previously unrecognised expression of P2X4 in lymphocytes and monocytes. In addition, for the first time P2 receptor mRNA expression patterns have been studied in CD34+ stem and progenitor cells. Several P2 receptors were expressed , indicating a role in differentiation and proliferation. Thus, it is possible that specific antibodies to P2 receptors could be used to identify progenitors for monocytes, lymphocytes and megakaryocytes.The P2XThe studies were approved by the local Ethics Committee of the Lund University and were conducted according to the principles of the Declaration of Helsinki.Peripheral blood was drawn from each of 6 healthy volunteers (after informed consent) into heparin vials. The mononuclear cells were isolated by density gradient centrifugation on Lymphoprep™  at 605 g for 30 minutes. The lymphocytes and monocytes thus obtained were washed three times in RPMI 1640 medium with L-glutamine  and 0.1% human serum albumin , (medium), and centrifuged each time at 605 g for 5 minutes.6 cells/ml. Flow cytometry  analysis on these cells by detection of cell surface CD14 and CD45 showed that approximately 10% of the cells were monocytes. 800 μl of this cell-suspension was plated on a chamber slide 4 well glass slide  at 37°C in an atmosphere containing 5% CO2 and 96% humidity for 1 h in order for the monocytes to adhere. Nonadherent cells were removed by washing three times with medium. Flow cytometry analysis of these nonadherent cells showed that at least 90% were lymphocytes, and were therefore used as source of lymphocytes. The cells attached to the glass slides (<90% monocytes as assessed by flow cytometry) were detached by adding first PBS and then 0.5 mM EDTA-PBS for 3 min in room temperature.The fraction of lymphocytes and monocytes obtained according to this procedure was resuspended in medium with 15% normal human serum (NHS) added to a concentration of 4 × 10+ cells were isolated by 2passages through magnetic columns  by using a hapten-conjugated CD34 antibody (Qbend/10) and an antihapten antibody conjugated to magnetic beads . CD34 expression was analyzed by immunostaining with a FACSCalibur flow cytometer (Becton Dickinson) by using the CellQuest program (Becton Dickinson) and the purity of isolated populations was reproducibly > 95% [Bone marrow samples were obtained from healthy volunteers (n = 3), after informed consent, using guidelines approved by the Ethical Committee, Lund University. Mononuclear cells were isolated by density gradient centrifugation . CD34ly > 95% .Total cellular RNAs were extracted using TRIzol reagent  according to the supplier's instructions, dissolved in diethyl-pyrocarbonate (DEPC) treated water and stored at -70°C until used.TaqMan Reverse Transcription Reagents Kit was used to transcribe mRNA into cDNA. Real-time PCR were performed by means of a PRISM 7700Sequence Detector as described previously ,15,28,291, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2X1, P2X4, and P2X7 normalized to GAPDH [T method for the relative quantification of target without running standard curves on the same plate . The amount of target and endogenous reference was determined from the comparative CT method. The target gene normalized to GAPDH was expressed as ΔCT (CT of target gene minus CT of GAPDH). P2Y1 was arbitrarily chosen to be the calibrator in the comparative analysis and is expressed as ΔCTP2Y1 (CT of target minus CT of GAPDH for P2Y1). The normalized calibrated value is given by the equation 2-ΔΔCt, where ΔΔCT is ΔCT -ΔCTP2Y1. To further verify the specificity of PCR assays, the PCR was performed with non-reverse-transcribed total cellular RNA and samples lacking the DNA template. No significant amplifications were obtained in any of these samples (data not shown).Previous analysis showed that amplification efficiencies were almost identical for GADPH and the following receptor mRNAs: P2Yto GAPDH ,15. To cUnless otherwise stated, all reagents and drugs were purchased from Sigma Chemical Corp, St. Louis, MI, USA. PCR consumables were obtained from Perkin-Elmer Applied Biosystems Inc, Foster City, CA, USA.T values (ΔCT) was performed with a one-way ANOVA, followed by a multiple comparison post test (Tukey's test) using GraphPad InStat version 3.00 . Significant differences were considered at P < 0.05 (two-tailed test).Data are expressed as mean and standard error of the mean (SEM) unless otherwise stated. n indicates the number of subjects that were tested. Statistical analysis of the normalized CLW designed the study, carried out the RNA isolation and real-time PCR, and wrote the manuscript.+ stem and progenitor cells, and participated in writing the manuscript.SEWJ supervised the isolation of CD34AB supervised the isolation of monocytes and lymphocytes, and participated in writing the manuscript.DE conceived the study, guided throughout the study, and wrote the manuscript.All authors read and approved the final manuscript.


RNA interference (RNAi) is used in the lab to silence virtually any gene. Previously, antisense and ribozymes were successful in the lab, but have been disappointing in the clinic. Will RNAi succeed where these two have not? Science as the “Breakthrough of the Year” in 2002 break out of the lab and lead to novel therapies as well? Pharmaceutical giants are hoping so, and several biotech companies have bet their futures on it, but not everyone is sanguine about the future of RNAi therapy.RNA interference (RNAi) has been called “one of the most has exciting discoveries in biology in the last couple decades,” and since it was first recognized by Andrew Fire et al. in 1998, it has quickly become one of the most powerful and indispensable tools in the molecular biologist's toolkit. Using short double-stranded RNA (dsRNA) molecules, RNAi can selectively silence essentially any gene in the genome. It is an ancient mechanism of gene regulation, found in eukaryotes as diverse as yeast and mammals, and probably plays a central role in controlling gene expression in all eukaryotes. In the lab, RNAi is routinely used to reveal the genetic secrets of development, intracellular signaling, cancer, infection, and a full range of other phenomena. But can the phenomenon hailed by the journal At the heart of its promise as a powerful therapeutic drug lies the exquisite selectivity of RNAi: like the fabled “magic bullet,” an RNAi sequence seeks out and destroys its target without affecting other genes. The clinical applications appear endless: any gene whose expression contributes to disease is a potential target, from viral genes to oncogenes to genes responsible for heart disease, Alzheimer's disease, diabetes, and more.But for all its promise, RNAi therapy is a long way from entering the clinic. While it is a proven wunderkind in the lab, to date no tests have been done in humans, and only the most modest and circumscribed successes have been demonstrated in animals. The road to clinical success is littered with great ideas that have come a cropper along the way, including two other RNA-based therapies, antisense and ribozymes, both of which showed promise at the bench but have largely stumbled before reaching the bedside. Is RNAi also likely to fall short? Or is it different enough to make this third try the charm?To be a successful drug, a molecule must overcome a long set of hurdles. First, it must be able to be manufactured at reasonable cost and administered safely and conveniently. Then, and even more importantly, it must be stable enough to reach its target cells before it is degraded or excreted; it must get into those cells, link up with its intracellular target, and exert its effect; and it must exert enough of an effect to improve the health of the person taking it. And, finally, it must do all this without causing significant toxic effects in either target or nontarget tissues. No matter how good a compound looks in the lab, if it fails to clear any one of these hurdles, it is useless as a drug.For RNA-based therapies, manufacture has been seen as a soluble problem, while delivery, stability, and potency have been the most significant obstacles. “There was a lot of clinical naïveté” in the early days of antisense and ribozymes, according to Nassim Usman, Vice President for Research and Development at Sirna Therapeutics in Boulder, Colorado. “Compounds were pushed into the clinic prematurely.” Sirna began as the biotech startup Ribozyme Pharmaceuticals, which tried to develop ribozymes to treat several conditions, including hepatitis C. A ribozyme is an RNA molecule whose sequence and structure allow it to cleave specific target RNA molecules see . “The inSimilarly, despite much initial enthusiasm, attempts to develop antisense drugs have been largely disappointing. Antisense is a single strand of RNA or DNA, complementary to a target messenger RNA (mRNA) sequence; by pairing up with it, the antisense strand prevents translation of the mRNA see . At leasBut as long as patients were getting better, does it matter what the mechanism was? “It doesn't matter if you are a patient, but it does matter if you are trying to develop the next drug,” says Cy Stein, Associate Professor of Medicine and Pharmacology at Columbia University College of Physicians and Surgeons in New York City. Stein has researched antisense for more than a decade. “If you get the mechanism wrong, you're not going to be able to do it.”To date, only one antisense drug has received FDA approval. Vitravene, from Isis Pharmaceuticals in Carlsbad, California, is used to treat cytomegalovirus infections in the eye for patients with HIV. Vitravene is actually a DNA antisense drug, which binds to viral DNA, though, says Usman, “it's unclear whether it actually works by an antisense mechanism.” Stein expresses a similar skepticism about the mechanism of a second antisense drug, Genasense. Genasense is a DNA-based treatment that targets Bcl-2, a protein expressed in high levels in cancer cells, which is thought to protect them from standard chemotherapy. The FDA is currently reviewing an application for Genasense, based on promising results in the treatment of malignant melanoma.Growing disillusionment with antisense and ribozymes coincided with the discovery of RNAi and the realization that it was a far more potent way to silence gene expression. RNAi uses short dsRNA molecules whose sequence matches that of the gene of interest. Once in a cell, a dsRNA molecule is cleaved into segments approximately 22 nucleotides long, called short interfering RNAs (siRNAs) see . siRNAs The extraordinary selectivity of RNAi, combined with its potency—in theory, only a few dsRNAs are needed per cell—quickly made it the tool of choice for functional genomics (determining what a gene product does and with what other products it interacts) and for drug target discovery and validation. By “knocking down” a gene with RNAi and determining how a cell responds, a researcher can, in the course of only a few days, develop significant insight into the function of the gene and determine whether reducing its expression is likely to be therapeutically useful. But does RNAi have a better chance to succeed as a drug than antisense or ribozymes?“The fundamental difference favoring RNAi is that we're harnessing an endogenous, natural pathway,” says Nagesh Mahanthappa, Director of Corporate Development at Alnylam Pharmaceuticals in Cambridge, Massachusetts, the second of two major biotech company developing RNAi-based therapy. The exploitation of a pre-existing mechanism, he says, is the main reason RNAi is orders of magnitude more potent than either of the other two types of RNA strategies.More potent in the test tube, at least. But stability and delivery are also the major obstacles to successful RNAi therapy, obstacles that are intrinsic to the biochemical nature of RNA itself, as well as the body's defenses against infection with foreign nucleotides. “For the strongest reasons, you can't get away from this,” says Stein. “The problem is that a charged oligonucleotide will not pass through a lipid layer,” which it must do in order to enter a cell. John Rossi, Director of the Department of Molecular Biology at City of Hope Hospital in Duarte, California, who has worked on RNA-based therapies for 15 years, concurs. “The cell doesn't want to take up RNA,” he says, which makes evolutionary sense, since extracellular RNA usually signifies a viral infection. Injected into the bloodstream, unmodified RNA is rapidly excreted by the kidneys or degraded by enzymes.To solve the problem of cell penetration, most researchers have either complexed the RNA with a lipid or modified the RNA's phosphate backbone to minimize its charge. Mahanthappa thinks the complexing approach is unlikely to be a simple solution, since drug approval would require independent testing of the lipid delivery system as well. Instead, Alnylam is pursuing backbone modification. “Some minimal modification is going to be necessary” to increase cell uptake and to improve stability in the blood stream, Mahanthappa says. “What we have learned from the antisense field is that even without other delivery strategies, when you administer RNA at sufficient doses, if it's stable, it gets taken up by cells.”“Anything that can be done to increase half-life in circulation would improve delivery,” says Judy Lieberman, a Senior Investigator at the Center for Blood Research and Associate Professor of Pediatrics at Harvard Medical School in Cambridge, Massachusetts. But that may not be the only problem, she cautions. Lieberman's lab recently demonstrated the ability of RNAi to silence expression of the Fas gene in mice, protecting them from fulminant hepatitis. Fas triggers apoptosis, or programmed cell death, in response to a variety of cell insults. In her experiment, Lieberman delivered the RNA by high-pressure injection into the tail. The RNA got to the liver, silenced Fas, and protected the mice from hepatitis. However, a significant fraction of animals died of heart failure, brought on because the injection volume was about 20% of the mouse blood volume. Such a delivery scheme simply will not work in humans. “Delivery to the cell is still an obstacle,” Lieberman explains. “Unless you really focus on how to solve that problem, you're not going to get very far.”Even assuming delivery problems can be solved, other questions remain, including that of whether therapeutic levels of RNAi may be toxic. Mahanthappa says, “The conservative answer is we just don't know. The more aggressive answer is there's no reason to think so.” Rossi isn't so sure. “The target of interest may be in normal cells as well as cancer cells,” he says. “That's where you get toxicity.”But if small RNAs can be delivered to target cells efficiently and without significant toxicity, will they be effective medicines? Usman of Sirna is confident they will be. “If you can get it there, and if it's in one piece, there no doubt in our minds that it will work,” he says. To date, numerous experiments in animal models suggest RNAi can downregulate a variety of target genes effectively. However, there are still two unanswered questions about whether that will translate into effective therapy.The first is whether RNAi's exquisite specificity is really an advantage beyond the bench. “It's unclear whether highly specific drugs give you a big therapeutic effect,” says Cy Stein. For instance, he says, “most active antitumor medicines have multiple mechanisms of action. The more specific you make it, the less robust the therapeutic activity is likely to be.” Rossi agrees: “Overspecificity has never worked,” he says.The second question is what effect an excess of RNA from outside the cell will have on the normal function of the RISC, the complex at the heart of the RNAi mechanism. The number of RISCs in the cell is unknown, and one concern is that the amount of RNA needed to have a therapeutic effect may occupy all the available complexes. “We are usurping a natural cell system that is there for some other purpose, for knocking out endogenous gene function,” says Rossi. With the introduction of foreign RNA, will the system continue to perform its normal function as well, or will it become saturated? “That's the big black box in the field,” he says.Despite the questions and unsolved problems, Sirna, Alnylam, and several other companies are moving ahead to develop RNAi therapy; indeed, some outstanding questions are probably only likely to be answered in the process of therapeutic development. The first applications are likely to be in cancer (targeting out-of-control oncogenes) or viral infection . To avoid some of the problems of delivery, initial trials may deliver the RNA by direct injection into the target tissue  or ex vivo, treating white blood cells infected with HIV, for example.Having spent a decade trying to develop ribozymes, says Usman, Sirna is prepared for the rough road it faces. “We haven't solved all the problems, but we know how to proceed to work through them. It's no surprise to us—we've seen this movie before.” Usman expects Sirna to file an Investigational New Drug Application with the FDA by the end of 2004 and to have a human clinical trial in progress in 2005. “Without a doubt, there will be an RNAi-based drug within ten years,” Usman predicts.Stein isn't so sure and thinks that too much is still to be learned about RNAi and the body's reaction to it to be confident that RNA-based therapies will ultimately be successful. “The whole field was founded on the belief it was rational, but there are huge gaps in our knowledge, and so you need a bit of luck to be successful,” he says. “I think people are surprised at how complicated it is, but why should it be any other way? It's an intellectual conceit to think that nature is simple.”